Dr Juliet Bryant

Research Area: Global Health
Technology Exchange: Bioinformatics and Vaccine production and evaluation
Scientific Themes: Tropical Medicine & Global Health
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Juliet Bryant is a virologist at the Oxford University Clinical Research Unit in Hanoi, Vietnam (OUCRU-VN), where she serves as manager for the clinical diagnostic laboratory. Her primary research interests are the ecology and evolution of viral zoonotic pathogens, with a particular interest on how patterns of zoonotic transmission (and human-to-animal ‘back transmission’) are influenced by changing agricultural practices and intensification of livestock production.

Current projects include molecular genetic and antigenic characterization of influenza isolates generated by national surveillance programs, as well as longitudinal cohort studies of rural communities to explore population immunity to influenza. The community-based studies involve sampling of both humans and in-contact domestic animals (primarily poultry, pigs, and companion pets) as well as surveys of non-traditional ‘exotic’ species traded through speciality wet markets (e.g. rodents and bats). The research activities explore the linkages between human and animal health, combining laboratory and field studies as well as public engagement and outreach activities such as the Health in the Backyard project. Bryant has worked in southeast Asia for eight years, and has a background in animal science and arbovirology.

Name Department Institution Country
Professor Menno de Jong Academic Medical Centre, University of Amsterdam Netherlands
Dr Constance Schultsz Academic Medical Center, University of Amsterdam Netherlands
Professor To Long Thanh National Center for Veterinary Diagnostics, Department of Animal Health Vietnam
Dr Ho Thi Viet Thu Veterinary Virology Can Tho University Vietnam
Trung NV, Hoi LT, Thuong NTH, Toan TK, Huong TTK, Hoa TM, Fox A, Kinh NV, van Doorn HR, Wertheim HFL et al. 2017. Seroprevalence of Scrub Typhus, Typhus, and Spotted Fever Among Rural and Urban Populations of Northern Vietnam. Am J Trop Med Hyg, 96 (5), pp. 1084-1087. | Show Abstract | Read more

AbstractRickettsial infections are recognized as important causes of fever throughout southeast Asia. Herein, we determined the seroprevalence to rickettsioses within rural and urban populations of northern Vietnam. Prevalence of individuals with evidence of prior rickettsial infections (IgG positive) was surprisingly low, with 9.14% (83/908) testing positive to the three major rickettsial serogroups thought to circulate in the region. Prevalence of typhus group rickettsiae (TG)-specific antibodies (6.5%, 58/908) was significantly greater than scrub typhus group orientiae (STG)- or spotted fever group rickettsiae (SFG)-specific antibodies (P < 0.05). The majority of TG seropositives were observed among urban rather than rural residents (P < 0.05). In contrast, overall antibody prevalence to STG and SFG were both very low (1.1%, 10/908 for STG; 1.7%, 15/908 for SFG), with no significant differences between rural and urban residents. These results provide data on baseline population characteristics that may help inform development of Rickettsia serological testing criteria in future clinical studies.

Peacock TP, Benton DJ, Sadeyen JR, Chang P, Sealy JE, Bryant JE, Martin SR, Shelton H, McCauley JW, Barclay WS, Iqbal M. 2017. Variability in H9N2 haemagglutinin receptor-binding preference and the pH of fusion. Emerg Microbes Infect, 6 (3), pp. e11. | Show Abstract | Read more

H9N2 avian influenza viruses are primarily a disease of poultry; however, they occasionally infect humans and are considered a potential pandemic threat. Little work has been performed to assess the intrinsic biochemical properties related to zoonotic potential of H9N2 viruses. The objective of this study, therefore, was to investigate H9N2 haemagglutinins (HAs) using two well-known correlates for human adaption: receptor-binding avidity and pH of fusion. Receptor binding was characterized using bio-layer interferometry to measure virus binding to human and avian-like receptor analogues and the pH of fusion was assayed by syncytium formation in virus-infected cells at different pHs. We characterized contemporary H9N2 viruses of the zoonotic G1 lineage, as well as representative viruses of the zoonotic BJ94 lineage. We found that most contemporary H9N2 viruses show a preference for sulphated avian-like receptor analogues. However, the 'Eastern' G1 H9N2 viruses displayed a consistent preference in binding to a human-like receptor analogue. We demonstrate that the presence of leucine at position 226 of the HA receptor-binding site correlated poorly with the ability to bind a human-like sialic acid receptor. H9N2 HAs also display variability in their pH of fusion, ranging between pH 5.4 and 5.85 which is similar to that of the first wave of human H1N1pdm09 viruses but lower than the pH of fusion seen in zoonotic H5N1 and H7N9 viruses. Our results suggest possible molecular mechanisms that may underlie the relatively high prevalence of human zoonotic infection by particular H9N2 virus lineages.

Nguyen DT, Jang Y, Nguyen TD, Jones J, Shepard SS, Yang H, Gerloff N, Fabrizio T, Nguyen LV, Inui K et al. 2017. Shifting Clade Distribution, Reassortment, and Emergence of New Subtypes of Highly Pathogenic Avian Influenza A(H5) Viruses Collected from Vietnamese Poultry from 2012 to 2015. J Virol, 91 (5), pp. e01708-16-e01708-16. | Show Abstract | Read more

Whole-genome sequences of representative highly pathogenic avian influenza A(H5) viruses from Vietnam were generated, comprising samples from poultry outbreaks and active market surveillance collected from January 2012 to August 2015. Six hemagglutinin gene clades were characterized. Clade 1.1.2 was predominant in southern Mekong provinces throughout 2012 and 2013 but gradually disappeared and was not detected after April 2014. Clade 2.3.2.1c viruses spread rapidly during 2012 and were detected in the south and center of the country. A number of clade 1.1.2 and 2.3.2.1c interclade reassortant viruses were detected with different combinations of internal genes derived from 2.3.2.1a and 2.3.2.1b viruses, indicating extensive cocirculation. Although reassortment generated genetic diversity at the genotype level, there was relatively little genetic drift within the individual gene segments, suggesting genetic stasis over recent years. Antigenically, clade 1.1.2, 2.3.2.1a, 2.3.2.1b, and 2.3.2.1c viruses remained related to earlier viruses and WHO-recommended prepandemic vaccine strains representing these clades. Clade 7.2 viruses, although detected in only low numbers, were the exception, as indicated by introduction of a genetically and antigenically diverse strain in 2013. Clade 2.3.4.4 viruses (H5N1 and H5N6) were likely introduced in April 2014 and appeared to gain dominance across northern and central regions. Antigenic analyses of clade 2.3.4.4 viruses compared to existing clade 2.3.4 candidate vaccine viruses (CVV) indicated the need for an updated vaccine virus. A/Sichuan/26221/2014 (H5N6) virus was developed, and ferret antisera generated against this virus were demonstrated to inhibit some but not all clade 2.3.4.4 viruses, suggesting consideration of alternative clade 2.3.4.4 CVVs.IMPORTANCE Highly pathogenic avian influenza (HPAI) A(H5) viruses have circulated continuously in Vietnam since 2003, resulting in hundreds of poultry outbreaks and sporadic human infections. Despite a significant reduction in the number of human infections in recent years, poultry outbreaks continue to occur and the virus continues to diversify. Vaccination of poultry has been used as a means to control the spread and impact of the virus, but due to the diversity and changing distribution of antigenically distinct viruses, the utility of vaccines in the face of mismatched circulating strains remains questionable. This study assessed the putative amino acid changes in viruses leading to antigenic variability, underscoring the complexity of vaccine selection for both veterinary and public health purposes. Given the overlapping geographic distributions of multiple, antigenically distinct clades of HPAI A(H5) viruses in Vietnam, the vaccine efficacy of bivalent poultry vaccine formulations should be tested in the future.

Puckett EE, Park J, Combs M, Blum MJ, Bryant JE, Caccone A, Costa F, Deinum EE, Esther A, Himsworth CG et al. 2016. Global population divergence and admixture of the brown rat (Rattus norvegicus). Proc Biol Sci, 283 (1841), pp. 20161762-20161762. | Show Abstract | Read more

Native to China and Mongolia, the brown rat (Rattus norvegicus) now enjoys a worldwide distribution. While black rats and the house mouse tracked the regional development of human agricultural settlements, brown rats did not appear in Europe until the 1500s, suggesting their range expansion was a response to relatively recent increases in global trade. We inferred the global phylogeography of brown rats using 32 k SNPs, and detected 13 evolutionary clusters within five expansion routes. One cluster arose following a southward expansion into Southeast Asia. Three additional clusters arose from two independent eastward expansions: one expansion from Russia to the Aleutian Archipelago, and a second to western North America. Westward expansion resulted in the colonization of Europe from which subsequent rapid colonization of Africa, the Americas and Australasia occurred, and multiple evolutionary clusters were detected. An astonishing degree of fine-grained clustering between and within sampling sites underscored the extent to which urban heterogeneity shaped genetic structure of commensal rodents. Surprisingly, few individuals were recent migrants, suggesting that recruitment into established populations is limited. Understanding the global population structure of R. norvegicus offers novel perspectives on the forces driving the spread of zoonotic disease, and aids in development of rat eradication programmes.

Nguyen DNT, Mai LQ, Bryant JE, Hang NLK, Hoa LNM, Nadjm B, Thai PQ, Duong TN, Anh DD, Horby P et al. 2016. Epidemiology and etiology of influenza-like-illness in households in Vietnam; it's not all about the kids! J Clin Virol, 82 pp. 126-132. | Show Abstract | Read more

BACKGROUND: Household studies provide opportunities to understand influenza-like-illness (ILI) transmission, but data from (sub)tropical developing countries are scarce. OBJECTIVE: To determine the viral etiology and epidemiology of ILI in households. STUDY DESIGN: ILI was detected by active case finding amongst a cohort of 263 northern Vietnam households between 2008 and 2013. Health workers collected nose and throat swabs for virus detection by multiplex real-time RT-PCR. RESULTS: ILI was detected at least once in 219 (23.7%) of 945 household members. 271 (62.3%) of 435 nose/throat swabs were positive for at least one of the 15 viruses tested. Six viruses predominated amongst positive swabs: Rhinovirus (28%), Influenza virus (17%), Coronavirus (8%), Enterovirus (5%), Respiratory syncytial virus (3%), Metapneumovirus virus (2.5%) and Parainfluenza virus 3 (1.8%). There was no clear seasonality, but 78% of episodes occurred in Winter/Spring for Influenza compared to 32% for Rhinovirus. Participants, on average, suffered 0.49 ILI, and 0.29 virus-positive ILI episodes, with no significant effects of gender, age, or household size. In contrast to US and Australian community studies, the frequency of ILI decreased as the number of household members aged below 5 years increased (p=0.006). CONCLUSION: The findings indicate the need for tailored ILI control strategies, and for better understanding of how local childcare practices and seasonality may influence transmission and the role of children.

Thuy DM, Peacock TP, Bich VT, Fabrizio T, Hoang DN, Tho ND, Diep NT, Nguyen M, Hoa LENM, Trang HT et al. 2016. Prevalence and diversity of H9N2 avian influenza in chickens of Northern Vietnam, 2014. Infect Genet Evol, 44 pp. 530-540. | Show Abstract | Read more

Despite their classification as low pathogenicity avian influenza viruses (LPAIV), A/H9N2 viruses cause significant losses in poultry in many countries throughout Asia, the Middle East and North Africa. To date, poultry surveillance in Vietnam has focused on detection of influenza H5 viruses, and there is limited understanding of influenza H9 epidemiology and transmission dynamics. We determined prevalence and diversity of influenza A viruses in chickens from live bird markets (LBM) of 7 northern Vietnamese provinces, using pooled oropharyngeal swabs collected from October to December 2014. Screening by real time RT-PCR revealed 1207/4900 (24.6%) of pooled swabs to be influenza A virus positive; overall prevalence estimates after accounting for pooling (5 swabs/pools) were 5.8% (CI 5.4-6.0). Subtyping was performed on 468 pooled swabs with M gene Ct<26. No influenza H7 was detected; 422 (90.1%) were H9 positive; and 22 (4.7%) were H5 positive. There was no evidence was of interaction between H9 and H5 virus detection rates. We sequenced 17 whole genomes of A/H9N2, 2 of A/H5N6, and 11 partial genomes. All H9N2 viruses had internal genes that clustered with genotype 57 and were closely related to Chinese human isolates of A/H7N9 and A/H10N8. Using a nucleotide divergence cutoff of 98%, we identified 9 distinct H9 genotypes. Phylogenetic analysis suggested multiple introductions of H9 viruses to northern Vietnam rather than in-situ transmission. Further investigations of H9 prevalence and diversity in other regions of Vietnam are warranted to assess H9 endemicity elsewhere in the country.

Hoa LENM, Mai LEQ, Bryant JE, Thai PQ, Hang NLEK, Yen NT, Duong TN, Thoang DD, Horby P, Werheim HF, Fox A. 2016. Association between Hemagglutinin Stem-Reactive Antibodies and Influenza A/H1N1 Virus Infection during the 2009 Pandemic. J Virol, 90 (14), pp. 6549-6556. | Show Abstract | Read more

UNLABELLED: The discovery of influenza virus broadly neutralizing (BrN) antibodies prompted efforts to develop universal vaccines. Influenza virus stem-reactive (SR) broadly neutralizing antibodies have been detected by screening antibody phage display libraries. However, studies of SR BrN antibodies in human serum, and their association with natural infection, are limited. To address this, pre- and postpandemic sera from a prospective community cohort study in Vietnam were assessed for antibodies that inhibit SR BrN monoclonal antibody (MAb) (C179) binding to H1N1 pandemic 2009 virus (H1N1pdm09). Of 270 households, 33 with at least one confirmed H1N1pdm09 illness or at least two seroconverters were included. The included households comprised 71 infected and 41 noninfected participants. Sera were tested as 2-fold dilutions between 1:5 and 1:40. Fifty percent C179 inhibition (IC50) titers did not exceed 10, although both IC50 titers and percent C179 inhibition by sera diluted 1:5 or 1:10 correlated with hemagglutination inhibition (HI) and microneutralization (MN) titers (all P < 0.001). Thirteen (12%) participants had detectable prepandemic IC50 titers, but only one reached a titer of 10. This proportion increased to 44% after the pandemic, when 39 participants had a titer of 10, and 67% of infected compared to 44% of noninfected had detectable IC50 titers (P < 0.001). The low levels of SR antibodies in prepandemic sera were not associated with subsequent H1N1pdm09 infection (P = 0.241), and the higher levels induced by H1N1pdm09 infection returned to prepandemic levels within 2 years. The findings indicate that natural infection induces only low titers of SR antibodies that are not sustained. IMPORTANCE: Universal influenza vaccines could have substantial health and economic benefits. The focus of universal vaccine research has been to induce antibodies that prevent infection by diverse influenza virus strains. These so-called broadly neutralizing antibodies are readily detected in mice and ferrets after infection with a series of distinct influenza virus strains. The 2009 H1N1 pandemic provided an opportunity to investigate whether infection with a novel strain induced broadly neutralizing antibodies in humans. We found that broadly neutralizing antibodies were induced, but levels were low and poorly maintained. This could represent an obstacle for universal vaccine development and warrants further investigation.

Oude Munnink BB, Phan MVT, van der Hoek L, Kellam P, Cotten M. 2016. Genome Sequences of a Novel Vietnamese Bat Bunyavirus Genome Announcements, 4 (6), pp. e01366-16-e01366-16. | Show Abstract | Read more

© 2016 Oude Munnink et al. To document the viral zoonotic risks in Vietnam, fecal samples were systematically collected from a number of mammals in southern Vietnam and subjected to agnostic deep sequencing. We describe here novel Vietnamese bunyavirus sequences detected in bat feces. The complete L and S segments from 14 viruses were determined.

Lu L, Van Dung N, Bryant JE, Carrique-Mas J, Van Cuong N, Anh PH, Rabaa MA, Baker S, Simmonds P, Woolhouse ME. 2016. Evolution and phylogeographic dissemination of endemic porcine picornaviruses in Vietnam. Virus Evol, 2 (1), pp. vew001. | Show Abstract | Read more

Members of the Picornaviridae are important and often zoonotic viruses responsible for a variety of human and animal diseases. However, the evolution and spatial dissemination of different picornaviruses circulating in domestic animals are not well studied. We examined the rate of evolution and time of origin of porcine enterovirus G (EV-G) and porcine kobuvirus species C lineages (PKV-C) circulating in pig farms in Vietnam and from other countries. We further explored the spatiotemporal spread of EV-G and PKV-C in Southwest Vietnam using phylogeographic models. Multiple types of EV-G are co-circulating in Vietnam. The two dominant EV-G types among isolates from Vietnam (G1 and G6) showed strong phylogenetic clustering. Three clades of PKV-C (PKV-C1-3) represent more recent introductions into Vietnam; PKV-C2 is closely related to PKV-C from Southwest China, indicating possible cross-border dissemination. In addition, high virus lineage migration rates were estimated within four districts in Dong Thap province in Vietnam for both EV-G types (G1, G6) and all PKV-C (C1-3) clades. We found that Chau Thanh district is a primary source of both EV-G and PKV-C clades, consistent with extensive pig trading in and out of the district. Understanding the evolution and spatial dissemination of endemic picornaviruses in pigs may inform future strategies for the surveillance and control of picornaviruses.

Do LA, Bryant JE, Tran AT, Nguyen BH, Tran TT, Tran QH, Vo QB, Tran Dac NA, Trinh HN, Nguyen TT et al. 2016. Respiratory Syncytial Virus and Other Viral Infections among Children under Two Years Old in Southern Vietnam 2009-2010: Clinical Characteristics and Disease Severity. PLoS One, 11 (8), pp. e0160606. | Show Abstract | Read more

BACKGROUND: Despite a high burden of respiratory syncytial virus (RSV) infections among children, data on demographic and clinical characteristics of RSV are scarce in low and middle income countries. This study aims to describe the viral etiologies, the demographic, epidemiological, and clinical characteristics of children under two years of age who were hospitalized with a lower respiratory tract infections (LRTI), focusing on RSV (prevalence, seasonality, subgroups, viral load) and its association with disease severity. METHODS: A prospective study among children under two years of age, hospitalized with LRTI was conducted in two referral pediatric hospitals in Ho Chi Minh City, Vietnam, from May 2009 to December 2010. Socio-demographic, clinical data and nasopharyngeal swabs were collected on enrolment and discharge. Multiplex real-time RT-PCR (13 viruses) and quantitative RSV RT-PCR were used to identify viral pathogens, RSV load and subgroups. RESULTS: Among 632 cases, 48% were RSV positive. RSV infections occurred at younger age than three other leading viral infections i.e rhinovirus (RV), metapneumovirus (MPV), parainfluenza virus (PIV-3) and were significantly more frequent in the first 6 months of life. Clinical severity score of RSV infection was significantly higher than PIV-3 but not for RV or MPV. In multivariate analysis, RV infection was significantly associated with severity while RSV infection was not. Among RSV infections, neither viral load nor viral co-infections were significantly associated with severity. Young age and having fever at admission were significantly associated with both RSV and LRTI severity. A shift in RSV subgroup predominance was observed during two consecutive rainy seasons but was not associated with severity. CONCLUSION: We report etiologies, the epidemiological and clinical characteristics of LRTI among hospitalized children under two years of age and risk factors of RSV and LRTI severity.

Carrique-Mas JJ, Tue NT, Bryant JE, Saylors K, Cuong NV, Hoa NT, An NN, Hien VB, Lao PV, Tu NC et al. 2015. The baseline characteristics and interim analyses of the high-risk sentinel cohort of the Vietnam Initiative on Zoonotic InfectiONS (VIZIONS). Sci Rep, 5 (1), pp. 17965. | Show Abstract | Read more

The Vietnam Initiative for Zoonotic Infections (VIZIONS) includes community-based 'high-risk sentinel cohort' (HRSC) studies investigating individuals at risk of zoonotic infection due to occupational or residential exposure to animals. A total of 852 HRSC members were recruited between March 2013 and August 2014 from three provinces (Ha Noi, Dak Lak, and Dong Thap). The most numerous group (72.8%) corresponded to individuals living on farms, followed by slaughterers (16.3%) and animal health workers (8.5%). Nasal/pharyngeal and rectal swabs were collected from HRSC members at recruitment and after notifying illness. Exposure to exotic animals (including wild pigs, porcupine, monkey, civet, bamboo rat and bat) was highest for the Dak Lak cohort (53.7%), followed by Ha Noi (13.7%) and Dong Thap (4.0%). A total of 26.8% of individuals reported consumption of raw blood over the previous year; 33.6% slaughterers reported no use of protective equipment at work. Over 686 person-years of observation, 213 episodes of suspect infectious disease were notified, equivalent of 0.35 reports per person-year. Responsive samples were collected from animals in the farm cohort. There was noticeable time and space clustering of disease episodes suggesting that the VIZIONS set up is also suitable for the formal epidemiological investigation of disease outbreaks.

Van Dung N, Anh PH, Van Cuong N, Hoa NT, Carrique-Mas J, Hien VB, Sharp C, Rabaa M, Berto A, Campbell J et al. 2016. Large-scale screening and characterization of enteroviruses and kobuviruses infecting pigs in Vietnam. J Gen Virol, 97 (2), pp. 378-388. | Show Abstract | Read more

A recent survey of pigs in Dong Thap province, Vietnam identified a high frequency of enterovirus species G (EV-G) infection (144/198; 72.7%). Amongst these was a plethora of EV-G types (EV-G1, EV-G6 and four new types EV-G8-EV-G11). To better characterize the genetic diversity of EV-G and investigate the possible existence of further circulating types, we performed a larger-scale study on 484 pig and 45 farm-bred boar faecal samples collected in 2012 and 2014, respectively. All samples from the previous and current studies were also screened for kobuviruses. The overall EV infection frequency remained extremely high (395/484; 81.6%), but with comparable detection rates and viral loads between healthy and diarrhoeic pigs; this contrasted with less frequent detection of EV-G in boars (4/45; 8.9%). EV was most frequently detected in pigs ≤ 14 weeks old (∼ 95%) and declined in older pigs. Infections with EV-G1 and EV-G6 were most frequent, whilst less commonly detected types included EV-G3, EV-G4 and EV-G8-EV-G11, and five new types (EV-G12-EV-G16). In contrast, kobuvirus infection frequency was significantly higher in diarrhoeic pigs (40.9 versus 27.6%; P = 0.01). Kobuviruses also showed contrasting epizootiologies and age associations; a higher prevalence was found in boars (42%) compared with domestic pigs (29%), with the highest infection frequency amongst pigs >52 weeks old. Although genetically diverse, all kobuviruses identified belonged to the species Aichivirus C. In summary, this study confirms infection with EV-G was endemic in Vietnamese domestic pigs and exhibits high genetic diversity and extensive inter-type recombination.

Do LA, Wilm A, Van Doorn HR, Lam HM, Sim S, Sukumaran R, Tran AT, Nguyen BH, Tran TT, Tran QH et al. 2015. Direct whole-genome deep-sequencing of human respiratory syncytial virus A and B from Vietnamese children identifies distinct patterns of inter- and intra-host evolution. J Gen Virol, 96 (12), pp. 3470-3483. | Show Abstract | Read more

Human respiratory syncytial virus (RSV) is the major cause of lower respiratory tract infections in children ,2 years of age. Little is known about RSV intra-host genetic diversity over the course of infection or about the immune pressures that drive RSV molecular evolution. We performed whole-genome deep-sequencing on 53 RSV-positive samples (37 RSV subgroup A and 16 RSV subgroup B) collected from the upper airways of hospitalized children in southern Vietnam over two consecutive seasons. RSV A NA1 and RSV B BA9 were the predominant genotypes found in our samples, consistent with other reports on global RSV circulation during the same period. For both RSV A and B, the M gene was the most conserved, confirming its potential as a target for novel therapeutics. The G gene was the most variable and was the only gene under detectable positive selection. Further, positively selected sites inG were found in close proximity to and in some cases overlapped with predicted glycosylation motifs, suggesting that selection on amino acid glycosylation may drive viral genetic diversity. We further identified hotspots and coldspots of intra-host genetic diversity in the RSV genome, some of which may highlight previously unknown regions of functional importance.

Rabaa MA, Tue NT, Phuc TM, Carrique-Mas J, Saylors K, Cotten M, Bryant JE, Nghia HD, Cuong NV, Pham HA et al. 2015. The Vietnam Initiative on Zoonotic Infections (VIZIONS): A Strategic Approach to Studying Emerging Zoonotic Infectious Diseases. Ecohealth, 12 (4), pp. 726-735. | Show Abstract | Read more

The effect of newly emerging or re-emerging infectious diseases of zoonotic origin in human populations can be potentially catastrophic, and large-scale investigations of such diseases are highly challenging. The monitoring of emergence events is subject to ascertainment bias, whether at the level of species discovery, emerging disease events, or disease outbreaks in human populations. Disease surveillance is generally performed post hoc, driven by a response to recent events and by the availability of detection and identification technologies. Additionally, the inventory of pathogens that exist in mammalian and other reservoirs is incomplete, and identifying those with the potential to cause disease in humans is rarely possible in advance. A major step in understanding the burden and diversity of zoonotic infections, the local behavioral and demographic risks of infection, and the risk of emergence of these pathogens in human populations is to establish surveillance networks in populations that maintain regular contact with diverse animal populations, and to simultaneously characterize pathogen diversity in human and animal populations. Vietnam has been an epicenter of disease emergence over the last decade, and practices at the human/animal interface may facilitate the likelihood of spillover of zoonotic pathogens into humans. To tackle the scientific issues surrounding the origins and emergence of zoonotic infections in Vietnam, we have established The Vietnam Initiative on Zoonotic Infections (VIZIONS). This countrywide project, in which several international institutions collaborate with Vietnamese organizations, is combining clinical data, epidemiology, high-throughput sequencing, and social sciences to address relevant one-health questions. Here, we describe the primary aims of the project, the infrastructure established to address our scientific questions, and the current status of the project. Our principal objective is to develop an integrated approach to the surveillance of pathogens circulating in both human and animal populations and assess how frequently they are exchanged. This infrastructure will facilitate systematic investigations of pathogen ecology and evolution, enhance understanding of viral cross-species transmission events, and identify relevant risk factors and drivers of zoonotic disease emergence.

Le Viet T, Choisy M, Bryant JE, Vu Trong D, Pham Quang T, Horby P, Nguyen Tran H, Tran Thi Kieu H, Nguyen Vu T, Nguyen Van K et al. 2015. A dengue outbreak on a floating village at Cat Ba Island in Vietnam. BMC Public Health, 15 (1), pp. 940. | Show Abstract | Read more

BACKGROUND: A dengue outbreak in an ecotourism destination spot in Vietnam, from September to November 2013, impacted a floating village of fishermen on the coastal island of Cat Ba. The outbreak raises questions about how tourism may impact disease spread in rural areas. METHODS: Epidemiological data were obtained from the Hai Phong Preventive Medical Center (PMC), including case histories and residential location from all notified dengue cases from this outbreak. All household addresses were geo-located. Knox test, a spatio-temporal analysis that enables inference dengue clustering constrained by space and time, was performed on the geocoded locations. From the plasma available from two patients, positive for Dengue serotype 3 virus (DENV3), the Envelope (E) gene was sequenced, and their genetic relationships compared to other E sequences in the region. RESULTS: Of 192 dengue cases, the odds ratio of contracting dengue infections for people living in the floating villages compared to those living on the island was 4.9 (95 % CI: 3.6-6.7). The space-time analyses on 111 geocoded dengue residences found the risk of dengue infection to be the highest within 4 days and a radius of 20 m of a given case. Of the total of ten detected clusters with an excess risk greater than 2, the cluster with the highest number of cases was in the floating village area (24 patients for a total duration of 31 days). Phylogenetic analysis revealed a high homology of the two DENV3 strains (genotype III) from Cat Ba with DENV3 viruses circulating in Hanoi in the same year (99.1 %). CONCLUSIONS: Our study showed that dengue transmission is unlikely to be sustained on Cat Ba Island and that the 2013 epidemic likely originated through introduction of viruses from the mainland, potentially Hanoi. These findings suggest that prevention efforts should be focused on mainland rather than on the island.

Anh PH, Van Cuong N, Son NT, Tue NT, Kosoy M, Woolhouse ME, Baker S, Bryant JE, Thwaites G, Carrique-Mas JJ, Rabaa MA. 2015. Diversity of Bartonella spp. in Bats, Southern Vietnam. Emerg Infect Dis, 21 (7), pp. 1266-1267. | Read more

Tu LT, Hoang NV, Cuong NV, Campbell J, Bryant JE, Hoa NT, Kiet BT, Thompson C, Duy DT, Phat VV et al. 2015. High levels of contamination and antimicrobial-resistant non-typhoidal Salmonella serovars on pig and poultry farms in the Mekong Delta of Vietnam. Epidemiol Infect, 143 (14), pp. 3074-3086. | Show Abstract | Read more

We investigated the prevalence, diversity, and antimicrobial resistance (AMR) profiles of non-typhoidal Salmonella (NTS) and associated risk factors on 341 pig, chicken, and duck farms in Dong Thap province (Mekong Delta, Vietnam). Sampling was stratified by species, district (four categories), and farm size (three categories). Pooled faeces, collected using boot swabs, were tested using ISO 6575: 2002 (Annex D). Isolates were serogrouped; group B isolates were tested by polymerase chain reaction to detect S. Typhimurium and (monophasic) serovar 4,[5],12:i:- variants. The farm-level adjusted NTS prevalence was 64·7%, 94·3% and 91·3% for chicken, duck and pig farms, respectively. Factors independently associated with NTS were duck farms [odds ratio (OR) 21·2], farm with >50 pigs (OR 11·9), pig farm with 5-50 pigs (OR 4·88) (vs. chickens), and frequent rodent sightings (OR 2·3). Both S. Typhimurium and monophasic S. Typhimurium were more common in duck farms. Isolates had a high prevalence of resistance (77·6%) against tetracycline, moderate resistance (20-30%) against chloramphenicol, sulfamethoxazole-trimethoprim, ampicillin and nalidixic acid, and low resistance (<5%) against ciprofloxacin and third-generation cephalosporins. Multidrug resistance (resistance against ⩾3 classes of antimicrobial) was independently associated with monophasic S. Typhimurium and other group B isolates (excluding S. Typhimurium) and pig farms. The unusually high prevalence of NTS on Mekong Delta farms poses formidable challenges for control.

Huong VT, Thanh LV, Phu VD, Trinh DT, Inui K, Tung N, Oanh NT, Trung NV, Hoa NT, Bryant JE et al. 2016. Temporal and spatial association of Streptococcus suis infection in humans and porcine reproductive and respiratory syndrome outbreaks in pigs in northern Vietnam. Epidemiol Infect, 144 (1), pp. 35-44. | Show Abstract | Read more

Porcine reproductive and respiratory syndrome (PRRS) outbreaks in pigs are associated with increased susceptibility of pigs to secondary bacterial infections, including Streptococcus suis - an important zoonotic pathogen causing bacterial meningitis in humans. This case-control study examined the association between human S. suis infection and PRRS outbreaks in pigs in northern Vietnam. We included 90 S. suis case-patients and 183 non-S. suis sepsis controls from a referral hospital in Hanoi in 2010, a period of major PRRS epizootics in Vietnam. PRRS exposure was determined using data from the National Centre of Veterinary Diagnosis. By univariate analysis, significantly more S. suis patients were reported residing in or adjacent to a PRRS district compared to controls [odds ratio (OR) 2·82, 95% confidence interval (CI) 1·35-5·89 and OR 3·15, 95% CI 1·62-6·15, respectively]. Only residency in adjacent districts remained significantly associated with risk of S. suis infection after adjusting for sex, occupation, and eating practices. SaTScan analysis showed a possible cluster of S. suis infection in humans around PRRS confirmed locations during the March-August period. The findings indicate an epidemiological association between PRRS in pigs and S. suis infections in humans. Effective strategies to strengthen control of PRRS in pigs may help reduce transmission of S. suis infection to humans.

Trang NV, Choisy M, Nakagomi T, Chinh NT, Doan YH, Yamashiro T, Bryant JE, Nakagomi O, Anh DD. 2015. Determination of cut-off cycle threshold values in routine RT-PCR assays to assist differential diagnosis of norovirus in children hospitalized for acute gastroenteritis. Epidemiol Infect, 143 (15), pp. 3292-3299. | Show Abstract | Read more

Norovirus (NV) is an important cause of acute gastroenteritis in children, but is also frequently detected in asymptomatic children, which complicates the interpretation of NV detection results in both the clinical setting and population prevalence studies. A total of 807 faecal samples from children aged <5 years hospitalized for acute gastroenteritis were collected in Thai Binh, Vietnam, from January 2011 to September 2012. Real-time RT-PCR was used to detect and quantify NV-RNA in clinical samples. A bimodal distribution of cycle threshold (Ct) values was observed in which the lower peak was assumed to represent cases for which NV was the causal agent of diarrhoea, whereas the higher peak was assumed to represent cases involving an alternative pathogen other than NV. Under these assumptions, we applied finite-mixture modelling to estimate a threshold of Ct <21·36 (95% confidence interval 20·29-22·46) to distinguish NV-positive patients for which NV was the likely cause of diarrhoea. We evaluated the validity of the threshold through comparisons with NV antigen ELISA results, and comparisons of Ct values in patients co-infected with rotavirus. We conclude that the use of an appropriate cut-off value in the interpretation of NV real-time RT-PCR results may improve differential diagnosis of enteric infections, and could contribute to improved estimates of the burden of NV disease.

Tu LTP, Hoang NVM, Cuong NV, Campbell J, Bryant JE, Hoa NT, Kiet BT, Thompson C, Duy DT, Phat VV et al. 2015. High levels of contamination and antimicrobial-resistant non-typhoidal Salmonella serovars on pig and poultry farms in the Mekong Delta of Vietnam Epidemiology and Infection, 143 (14), pp. 3074-3086. | Show Abstract | Read more

Copyright © 2015 Cambridge University Press. We investigated the prevalence, diversity, and antimicrobial resistance (AMR) profiles of non-typhoidal Salmonella (NTS) and associated risk factors on 341 pig, chicken, and duck farms in Dong Thap province (Mekong Delta, Vietnam). Sampling was stratified by species, district (four categories), and farm size (three categories). Pooled faeces, collected using boot swabs, were tested using ISO 6575: 2002 (Annex D). Isolates were serogrouped; group B isolates were tested by polymerase chain reaction to detect S. Typhimurium and (monophasic) serovar 4,[5],12:i:- variants. The farm-level adjusted NTS prevalence was 64.7%, 94.3% and 91.3% for chicken, duck and pig farms, respectively. Factors independently associated with NTS were duck farms [odds ratio (OR) 21.2] , farm with > 50 pigs (OR 11.9), pig farm with 5-50 pigs (OR 4.88) (vs. chickens), and frequent rodent sightings (OR 2.3). Both S. Typhimurium and monophasic S. Typhimurium were more common in duck farms. Isolates had a high prevalence of resistance (77.6%) against tetracycline, moderate resistance (20-30%) against chloramphenicol, sulfamethoxazole-trimethoprim, ampicillin and nalidixic acid, and low resistance ( < 5%) against ciprofloxacin and third-generation cephalosporins. Multidrug resistance (resistance against ≥3 classes of antimicrobial) was independently associated with monophasic S. Typhimurium and other group B isolates (excluding S. Typhimurium) and pig farms. The unusually high prevalence of NTS on Mekong Delta farms poses formidable challenges for control.

Hoa LN, Bryant JE, Choisy M, Nguyet LA, Bao NT, Trang NH, Chuc NT, Toan TK, Saito T, Takemae N et al. 2015. Population susceptibility to a variant swine-origin influenza virus A(H3N2) in Vietnam, 2011-2012. Epidemiol Infect, 143 (14), pp. 2959-2964. | Show Abstract | Read more

A reassortant swine-origin A(H3N2) virus (A/swine/BinhDuong/03-9/2010) was detected through swine surveillance programmes in southern Vietnam in 2010. This virus contains haemagglutinin and neuraminidase genes from a human A(H3N2) virus circulating around 2004-2006, and the internal genes from triple-reassortant swine influenza A viruses (IAVs). To assess population susceptibility to this virus we measured haemagglutination inhibiting (HI) titres to A/swine/BinhDuong/03-9/2010 and to seasonal A/Perth/16/2009 for 947 sera collected from urban and rural Vietnamese people during 2011-2012. Seroprevalence (HI ⩾ 40) was high and similar for both viruses, with 62·6% [95% confidence interval (CI) 59·4-65·7] against A/Perth/16/2009 and 54·6% (95% CI 51·4-57·8%) against A/swine/BinhDuong/03-9/2010, and no significant differences between urban and rural participants. Children aged <5 years lacked antibodies to the swine origin H3 virus despite high seroprevalence for A/Perth/16/2009. These results reveal vulnerability to infection to this contemporary swine IAV in children aged <5 years; however, cross-reactive immunity in adults would likely limit epidemic emergence potential.

Van Cuong N, Carrique-Mas J, Vo Be H, An NN, Tue NT, Anh NL, Anh PH, Phuc NT, Baker S, Voutilainen L et al. 2015. Rodents and risk in the Mekong Delta of Vietnam: seroprevalence of selected zoonotic viruses in rodents and humans. Vector Borne Zoonotic Dis, 15 (1), pp. 65-72. | Show Abstract | Read more

In the Mekong Delta in southern Vietnam, rats are commonly traded in wet markets and sold live for food consumption. We investigated seroprevalence to selected groups of rodent-borne viruses among human populations with high levels of animal exposure and among co-located rodent populations. The indirect fluorescence antibody test (IFAT) was used to determine seropositivity to representative reference strains of hantaviruses (Dobrava virus [DOBV], Seoul virus [SEOV]), cowpox virus, arenaviruses (lymphocytic choriomeningitis virus [LCMV]), flaviviruses (tick-borne encephalitis virus [TBEV]), and rodent parechoviruses (Ljungan virus), using sera from 245 humans living in Dong Thap Province and 275 rodents representing the five common rodent species sold in wet markets and present in peridomestic and farm settings. Combined seropositivity to DOBV and SEOV among the rodents and humans was 6.9% (19/275) and 3.7% (9/245), respectively; 1.1% (3/275) and 4.5% (11/245) to cowpox virus; 5.4% (15/275) and 47.3% (116/245) for TBEV; and exposure to Ljungan virus was 18.8% (46/245) in humans, but 0% in rodents. Very little seroreactivity was observed to LCMV in either rodents (1/275, 0.4%) or humans (2/245, 0.8%). Molecular screening of rodent liver tissues using consensus primers for flaviviruses did not yield any amplicons, whereas molecular screening of rodent lung tissues for hantavirus yielded one hantavirus sequence (SEOV). In summary, these results indicate low to moderate levels of endemic hantavirus circulation, possible circulation of a flavivirus in rodent reservoirs, and the first available data on human exposures to parechoviruses in Vietnam. Although the current evidence suggests only limited exposure of humans to known rodent-borne diseases, further research is warranted to assess public health implications of the rodent trade.

Nguyen DT, Bryant JE, Davis CT, Nguyen LV, Pham LT, Loth L, Inui K, Nguyen T, Jang Y, To TL et al. 2014. Prevalence and distribution of avian influenza a(H5N1) virus clade variants in live bird markets of Vietnam, 2011-2013. Avian Dis, 58 (4), pp. 599-608. | Show Abstract | Read more

Active surveillance for avian influenza (Al) viruses in poultry sold at live bird markets (LBMs) was conducted in 44 of 63 provinces throughout Vietnam over two periods from September 2011 to February 2012 and October 2012 to June 2013. The study objectives were to assess the prevalence of avian influenza type A, H5, and H5N1 subtype viruses and characterize the geographical and temporal distribution of H5N1 virus genetic variants across the country. Monthly sampling was conducted in 394 LBMs located in 372 communes. A total of 9790 oropharyngeal swabs from poultry were screened for influenza A virus by real-time reverse-transcriptase PCR Virus isolation was attempted on all positive samples in embryonated chicken eggs, and the HA1 region of each H5 virus isolate was sequenced. Market prevalence of H5 subtype virus was 32.2% (127/394) over the cumulative 15 mo of surveillance. Phylogenetic analyses indicated that clade 1.1 viruses persisted in the south, whereas three genetically distinct subgroups of dade 2.3.2.1 were found simultaneously in northern, central, and southern Vietnam. Clade 2.3.2.1c viruses first appeared in July 2012 and spread rapidly to the center and south of Vietnam in late 2012, where they were predominant among clade 2.3.2.1 viruses and were detected in both active LBM surveillance and poultry outbreaks. Given the overlapping geographic distribution of dade variants and the antigenic divergence previously described for these dades, current AI poultry vaccines used in Vietnam may require bivalent formulations containing representatives of both dade 1.1 and dade 2.3.2.1 viruses.

My PVT, Rabaa MA, Donato C, Cowley D, Phat VV, Dung TTN, Anh PH, Vinh H, Bryant JE, Kellam P et al. 2014. Novel porcine-like human G26P[19] rotavirus identified in hospitalized paediatric diarrhea patients in Ho Chi Minh City, Vietnam Journal of General Virology, 95 pp. 2727-2733. | Show Abstract | Read more

© 2014 The Authors. During a hospital-based diarrhoeal disease study conducted in Ho Chi Minh City, Vietnam from 2009 to 2010, we identified four symptomatic children infected with G26P[19] rotavirus (RV)-an atypical variant that has not previously been reported in human gastroenteritis. To determine the genetic structure and investigate the origin of this G26P[19] strain, the whole genome of a representative example was characterized, revealing a novel genome constellation: G26-P[19]-I5-R1-C1-M1-A8-N1-T1-E1-H1. The genome segments were most closely related to porcine (VP7, VP4, VP6 and NSP1) and Wa-like porcine RVs (VP1-3 and NSP2-5). We proposed that this G26P[19] strain was the product of zoonotic transmission coupled with one or more reassortment events occurring in human and/or animal reservoirs. The identification of such strains has potential implications for vaccine efficacy in south-east Asia, and outlines the utility of whole-genome sequencing for studying RV diversity and zoonotic potential during disease surveillance.

Nhung NT, Cuong NV, Campbell J, Hoa NT, Bryant JE, Truc VN, Kiet BT, Jombart T, Trung NV, Hien VB et al. 2015. High levels of antimicrobial resistance among escherichia coli isolates from livestock farms and synanthropic rats and shrews in the Mekong Delta of Vietnam. Appl Environ Microbiol, 81 (3), pp. 812-820. | Show Abstract | Read more

In Mekong Delta farms (Vietnam), antimicrobials are extensively used, but limited data are available on levels of antimicrobial resistance (AMR) among Escherichia coli isolates. We performed a structured survey of AMR in E. coli isolates (n = 434) from 90 pig, chicken, and duck farms. The results were compared with AMR among E. coli isolates (n = 234) from 66 small wild animals (rats and shrews) trapped on farms and in forests and rice fields. The isolates were susceptibility tested against eight antimicrobials. E. coli isolates from farmed animals were resistant to a median of 4 (interquartile range [IQR], 3 to 6) antimicrobials versus 1 (IQR, 1 to 2) among wild mammal isolates (P < 0.001). The prevalences of AMR among farmed species isolates (versus wild animals) were as follows: tetracycline, 84.7% (versus 25.6%); ampicillin, 78.9% (versus 85.9%); trimethoprim-sulfamethoxazole, 52.1% (versus 18.8%); chloramphenicol, 39.9% (versus 22.5%); amoxicillin-clavulanic acid, 36.6% (versus 34.5%); and ciprofloxacin, 24.9% (versus 7.3%). The prevalence of multidrug resistance (MDR) (resistance against three or more antimicrobial classes) among pig isolates was 86.7% compared to 66.9 to 72.7% among poultry isolates. After adjusting for host species, MDR was ∼8 times greater among isolates from wild mammals trapped on farms than among those trapped in forests/rice fields (P < 0.001). Isolates were assigned to unique profiles representing their combinations of susceptibility results. Multivariable analysis of variance indicated that AMR profiles from wild mammals trapped on farms and those from domestic animals were more alike (R(2) range, 0.14 to 0.30) than E. coli isolates from domestic animals and mammals trapped in the wild (R(2) range, 0.25 to 0.45). The results strongly suggest that AMR on farms is a key driver of environmental AMR in the Mekong Delta.

Huong VT, Hoa NT, Horby P, Bryant JE, Van Kinh N, Toan TK, Wertheim HF. 2014. Raw pig blood consumption and potential risk for Streptococcus suis infection, Vietnam. Emerg Infect Dis, 20 (11), pp. 1895-1898. | Show Abstract | Read more

We assessed consumption of raw pig blood, which is a risk factor for Streptococcus suis infection in Vietnam, by using a mix-method design. Factors associated with consumption included rural residency, age, sex, occupation, income, and marital status. We identified risk groups and practices and perceptions that should be targeted by communication programs.

Fox A, Mai LEQ, Thanh LET, Wolbers M, Le Khanh Hang N, Thai PQ, Thi Thu Yen N, Minh Hoa LEN, Bryant JE, Duong TN et al. 2015. Hemagglutination inhibiting antibodies and protection against seasonal and pandemic influenza infection. J Infect, 70 (2), pp. 187-196. | Show Abstract | Read more

OBJECTIVES: Hemagglutination inhibiting (HI) antibodies correlate with influenza vaccine protection but their association with protection induced by natural infection has received less attention and was studied here. METHODS: 940 people from 270 unvaccinated households participated in active ILI surveillance spanning 3 influenza seasons. At least 494 provided paired blood samples spanning each season. Influenza infection was confirmed by RT-PCR on nose/throat swabs or serum HI assay conversion. RESULTS: Pre-season homologous HI titer was associated with a significantly reduced risk of infection for H3N2 (OR 0.61, 95%CI 0.44-0.84) and B (0.65, 95%CI 0.54-0.80) strains, but not H1N1 strains, whether re-circulated (OR 0.90, 95%CI 0.71-1.15), new seasonal (OR 0.86, 95%CI 0.54-1.36) or pandemic H1N1-2009 (OR 0.77, 95%CI 0.40-1.49). The risk of seasonal and pandemic H1N1 decreased with increasing age (both p < 0.0001), and the risk of pandemic H1N1 decreased with prior seasonal H1N1 (OR 0.23, 95%CI 0.08-0.62) without inducing measurable A/California/04/2009-like titers. CONCLUSIONS: While H1N1 immunity was apparent with increasing age and prior infection, the effect of pre-season HI titer was at best small, and weak for H1N1 compared to H3N2 and B. Antibodies targeting non-HI epitopes may have been more important mediators of infection-neutralizing immunity for H1N1 compared to other subtypes in this setting.

My PV, Rabaa MA, Donato C, Cowley D, Phat VV, Dung TT, Anh PH, Vinh H, Bryant JE, Kellam P et al. 2014. Novel porcine-like human G26P[19] rotavirus identified in hospitalized paediatric diarrhoea patients in Ho Chi Minh City, Vietnam. J Gen Virol, 95 (Pt 12), pp. 2727-2733. | Show Abstract | Read more

During a hospital-based diarrhoeal disease study conducted in Ho Chi Minh City, Vietnam from 2009 to 2010, we identified four symptomatic children infected with G26P[19] rotavirus (RV)--an atypical variant that has not previously been reported in human gastroenteritis. To determine the genetic structure and investigate the origin of this G26P[19] strain, the whole genome of a representative example was characterized, revealing a novel genome constellation: G26-P[19]-I5-R1-C1-M1-A8-N1-T1-E1-H1. The genome segments were most closely related to porcine (VP7, VP4, VP6 and NSP1) and Wa-like porcine RVs (VP1-3 and NSP2-5). We proposed that this G26P[19] strain was the product of zoonotic transmission coupled with one or more reassortment events occurring in human and/or animal reservoirs. The identification of such strains has potential implications for vaccine efficacy in south-east Asia, and outlines the utility of whole-genome sequencing for studying RV diversity and zoonotic potential during disease surveillance.

Pham HA, Carrique-Mas JJ, Nguyen VC, Ngo TH, Nguyet LA, Do TD, Vo BH, Phan VT, Rabaa MA, Farrar J et al. 2014. The prevalence and genetic diversity of group A rotaviruses on pig farms in the Mekong Delta region of Vietnam. Vet Microbiol, 170 (3-4), pp. 258-265. | Show Abstract | Read more

Group A rotaviruses (ARoVs) are a common cause of severe diarrhea among children worldwide and the cause of approximately 45% of pediatric hospitalizations for acute diarrhea in Vietnam. ARoVs are known to cause significant economic losses to livestock producers by reducing growth performance and production efficiencies, however little is known about the implications of asymptomatic endemic circulation of ARoV. We aimed to determine the prevalence and predominant circulating genotypes of ARoVs on pig farms in a southern province of Vietnam. We found overall animal-level and farm-level prevalence of 32.7% (239/730) and 74% (77/104), respectively, and identified six different G types and 4 P types in various combinations (G2, G3, G4, G5, G9, G11 and P[6], P[13], P[23], and P[34]). There was no significant association between ARoV infection and clinical disease in pigs, suggesting that endemic asymptomatic circulation of ARoV may complicate rotavirus disease attribution during outbreaks of diarrhea in swine. Sequence analysis of the detected ARoVs suggested homology to recent human clinical cases and extensive genetic diversity. The epidemiological relevance of these findings for veterinary practitioners and to ongoing pediatric ARoV vaccine initiatives in Vietnam merits further study.

Cuong NV, Carrique-Mas J, Thu HTV, Hien ND, Hoa NT, Nguyet LA, Anh PH, Bryant JE. 2014. Serological and virological surveillance for porcine reproductive and respiratory syndrome virus, porcine circovirus type 2, and influenza A viruses among smallholder swine farms of the Mekong Delta, Vietnam JOURNAL OF SWINE HEALTH AND PRODUCTION, 22 (5), pp. 224-231. | Show Abstract

Objectives: To evaluate the feasibility and utility of oral-luids collection for surveillance of porcine viruses in the Mekong Delta, Vietnam, and to establish baseline serological and virological prevalence estimates for porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2(PCV2), and influenza A virus (IAV) among smallholder farms. Materials and methods: Paired serum and oral-luids samples from 68 farms (sows, boars, weaners, and growers) were tested during 2011 by reverse transcriptase polymerase chain reaction and enzyme-linked immunosorbent assay for PRRSV, PCV2, and IAV. Results: Low numbers of PRRSV-positive and IAV-positive pigs were detected (1.6% PRRSV viremic, two of 124; 0.8% IAV in oral luids, one of 124). However, PCV2 detection rates were high in both serum and oral fluids (54.8% and 61.3%, respectively). Overall proportions of pigs seropositive for IAV and PRRSV were 37.9% and 33.9%, respectively. Proportions of pigs seropositive for PRRSV were 48.6% (17 of 35) and 12.1% (four of 33) on vaccinated and unvaccinated farms, respectively. Oral luids and serum samples yielded comparable prevalence estimates for molecular detection of PCV2, and detected one sample PCR-positive for hemagglutinin of influenza A/H1N1/pdm09. There was no evidence of PRRSV shedding in oral luids. Implications: Antibody prevalence estimates based on testing oral luids may pro vide an acceptable and useful surrogate for testing serum in future field studies if optimized assays are employed.

Hong Anh P, Carrique-Mas JJ, Van Cuong N, Hoa NT, Lam Anh N, Duy DT, Hien VB, Vu Tra My P, Rabaa MA, Farrar J et al. 2014. The prevalence and genetic diversity of group A rotaviruses on pig farms in the Mekong Delta region of Vietnam Veterinary Microbiology, 170 (3-4), pp. 258-265. | Show Abstract | Read more

Group A rotaviruses (ARoVs) are a common cause of severe diarrhea among children worldwide and the cause of approximately 45% of pediatric hospitalizations for acute diarrhea in Vietnam. ARoVs are known to cause significant economic losses to livestock producers by reducing growth performance and production efficiencies, however little is known about the implications of asymptomatic endemic circulation of ARoV. We aimed to determine the prevalence and predominant circulating genotypes of ARoVs on pig farms in a southern province of Vietnam. We found overall animal-level and farm-level prevalence of 32.7% (239/730) and 74% (77/104), respectively, and identified six different G types and 4 P types in various combinations (G2, G3, G4, G5, G9, G11 and P[6], P[13] , P[23], and P[34] ). There was no significant association between ARoV infection and clinical disease in pigs, suggesting that endemic asymptomatic circulation of ARoV may complicate rotavirus disease attribution during outbreaks of diarrhea in swine. Sequence analysis of the detected ARoVs suggested homology to recent human clinical cases and extensive genetic diversity. The epidemiological relevance of these findings for veterinary practitioners and to ongoing pediatric ARoV vaccine initiatives in Vietnam merits further study. © 2014 The Authors.

Khampapongpane B, Lewis HC, Ketmayoon P, Phonekeo D, Somoulay V, Khamsing A, Phengxay M, Sisouk T, Vongphrachanh P, Bryant JE. 2014. National dengue surveillance in the Lao People's Democratic Republic, 2006-2012: epidemiological and laboratory findings. Western Pac Surveill Response J, 5 (1), pp. 7-13. | Show Abstract | Read more

Although dengue has been a public health problem for several decades in the Lao People's Democratic Republic, the magnitude of the disease burden and epidemiological trends remain poorly understood. We analysed national dengue surveillance and laboratory data from 2006 to 2012 by person, place and time. Between 2006 and 2012, the annual dengue notification rate ranged between 62 and 367 cases per 100 000 population with an apparent geographical expansion of transmission throughout the country in recent years and concurrent co-circulation of all four dengue virus subtypes. An electronic database, called Lao People's Democratic Republic Early Warning Alert and Response Network, was introduced in 2008 to provide automated early warning for outbreaks and epidemics. Village outbreaks continue to be notified primarily through event-based surveillance, whereas the weekly indicator-based system provides systematic assessment of annual epidemic cycles. The dengue case data indicate a high and increasing burden of disease. Efforts now need to focus on using available data to prompt more effective outbreak response and to guide the design and implementation of intervention strategies.

Le VT, de Jong MD, Nguyen VK, Nguyen VT, Taylor W, Wertheim HF, van der Ende A, van der Hoek L, Canuti M, Crusat M et al. 2014. Limited geographic distribution of the novel cyclovirus CyCV-VN. Sci Rep, 4 (1), pp. 3967. | Show Abstract | Read more

A novel cyclovirus, CyCV-VN, was recently identified in cerebrospinal fluid (CSF) from patients with central nervous system (CNS) infections in central and southern Vietnam. To explore the geographic distribution of this novel virus, more than 600 CSF specimens from patients with suspected CNS infections in northern Vietnam, Cambodia, Nepal and The Netherlands were screened for the presence of CyCV-VN but all were negative. Sequence comparison and phylogenetic analysis between CyCV-VN and another novel cyclovirus recently identified in CSF from Malawian patients indicated that these represent distinct cycloviral species, albeit phylogenetically closely related. The data suggest that CyCV-VN has a limited geographic distribution within southern and central Vietnam. Further research is needed to determine the global distribution and diversity of cycloviruses and importantly their possible association with human disease.

Van Dung N, Anh PH, Van Cuong N, Hoa NT, Carrique-Mas J, Hien VB, Campbell J, Baker S, Farrar J, Woolhouse ME et al. 2014. Prevalence, genetic diversity and recombination of species G enteroviruses infecting pigs in Vietnam. J Gen Virol, 95 (Pt 3), pp. 549-556. | Show Abstract | Read more

Picornaviruses infecting pigs, described for many years as 'porcine enteroviruses', have recently been recognized as distinct viruses within three distinct genera (Teschovirus, Sapelovirus and Enterovirus). To better characterize the epidemiology and genetic diversity of members of the Enterovirus genus, faecal samples from pigs from four provinces in Vietnam were screened by PCR using conserved enterovirus (EV)-specific primers from the 5' untranslated region (5' UTR). High rates of infection were recorded in pigs on all farms, with detection frequencies of approximately 90% in recently weaned pigs but declining to 40% in those aged over 1 year. No differences in EV detection rates were observed between pigs with and without diarrhoea [74% (n = 70) compared with 72% (n = 128)]. Genetic analysis of consensus VP4/VP2 and VP1 sequences amplified from a subset of EV-infected pigs identified species G EVs in all samples. Among these, VP1 sequence comparisons identified six type 1 and seven type 6 variants, while four further VP1 sequences failed to group with any previously identified EV-G types. These have now been formally assigned as EV-G types 8-11 by the Picornavirus Study Group. Comparison of VP1, VP4/VP2, 3D(pol) and 5' UTRs of study samples and those available on public databases showed frequent, bootstrap-supported differences in their phylogenies indicative of extensive within-species recombination between genome regions. In summary, we identified extremely high frequencies of infection with EV-G in pigs in Vietnam, substantial genetic diversity and recombination within the species, and evidence for a much larger number of circulating EV-G types than currently described.

Carrique-Mas JJ, Bryant JE. 2013. A review of foodborne bacterial and parasitic zoonoses in Vietnam EcoHealth, 10 (4), pp. 465-489. | Show Abstract | Read more

Vietnam has experienced unprecedented economic and social development in recent years, and the livestock sector is undergoing significant transformations. Although food animal production is still dominated by small-scale 'backyard' enterprises with mixed crop-livestock or livestock-aquatic systems, there is a trend towards more intensive and vertically integrated operations. Changes in animal production, processing and distribution networks for meat and animal products, and the shift from wet markets to supermarkets will undoubtedly impact food safety risks in Vietnam in unforeseen and complex ways. Here, we review the available published literature on bacterial and parasitic foodborne zoonoses (FBZ) in Vietnam. We report on clinical disease burden and pathogen prevalence in animal reservoirs for a number of important FBZ, and outline opportunities for future research. © 2013 International Association for Ecology and Health.

Carrique-Mas JJ, Bryant JE. 2013. A review of foodborne bacterial and parasitic zoonoses in Vietnam. Ecohealth, 10 (4), pp. 465-489. | Show Abstract | Read more

Vietnam has experienced unprecedented economic and social development in recent years, and the livestock sector is undergoing significant transformations. Although food animal production is still dominated by small-scale 'backyard' enterprises with mixed crop-livestock or livestock-aquatic systems, there is a trend towards more intensive and vertically integrated operations. Changes in animal production, processing and distribution networks for meat and animal products, and the shift from wet markets to supermarkets will undoubtedly impact food safety risks in Vietnam in unforeseen and complex ways. Here, we review the available published literature on bacterial and parasitic foodborne zoonoses (FBZ) in Vietnam. We report on clinical disease burden and pathogen prevalence in animal reservoirs for a number of important FBZ, and outline opportunities for future research.

Carrique-Mas JJ, Bryant JE, Cuong NV, Hoang NV, Campbell J, Hoang NV, Dung TT, Duy DT, Hoa NT, Thompson C et al. 2014. An epidemiological investigation of Campylobacter in pig and poultry farms in the Mekong delta of Vietnam. Epidemiol Infect, 142 (7), pp. 1425-1436. | Show Abstract | Read more

Campylobacter are zoonotic pathogens commonly associated with gastroenteritis. To assess the relevance of Campylobacter in Vietnam, an economically transitioning country in SE Asia, we conducted a survey of 343 pig and poultry farms in the Mekong delta, a region characterized by mixed species farming with limited biosecurity. The animal-level prevalence of Campylobacter was 31·9%, 23·9% and 53·7% for chickens, ducks and pigs, respectively. C. jejuni was predominant in all three host species, with the highest prevalence in pigs in high-density production areas. Campylobacter isolates demonstrated high levels of antimicrobial resistance (21% and 100% resistance against ciprofloxacin and erythromycin, respectively). Multilocus sequence type genotyping showed a high level of genetic diversity within C. jejuni, and predicted C. coli inter-species transmission. We suggest that on-going intensification of animal production systems, limited biosecurity, and increased urbanization in Vietnam is likely to result in Campylobacter becoming an increasingly significant cause of human diarrhoeal infections in coming years.

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Creanga A, Thi Nguyen D, Gerloff N, Thi Do H, Balish A, Dang Nguyen H, Jang Y, Thi Dam V, Thor S, Jones J et al. 2013. Emergence of multiple clade 2.3.2.1 influenza A (H5N1) virus subgroups in Vietnam and detection of novel reassortants Virology, 444 (1-2), pp. 12-20. | Show Abstract | Read more

Phylogenetic analyses of 169 influenza A(H5N1) virus genomes were conducted for samples collected through active surveillance and outbreak responses in Vietnam between September 2010 and September 2012. While clade 1.1 viruses persisted in southern regions, three genetically distinct subgroups of clade 2.3.2.1 were found in northern and central Vietnam. The identification of each subgroup corresponded with detection of novel reassortants, likely due to their overlapping circulation throughout the country. While the previously identified clade 1.1 and A/Hubei/1/2010-like 2.3.2.1 genotypes remained the predominant viruses detected, four viruses were found to be reassortants between A/Hubei/1/2010-like (HA, NA, PB2, PB1, PA, NP) and A/duck/Vietnam/NCVD-885/2010-like (M, NS) viruses and one virus was identified as having A/duck/Vietnam/NCVD-885/2010-like HA, NA, PB1, and NP with A/Hubei/1/2010-like PB2 and PA genes. Additionally, clade 2.3.2.1 A/Hong Kong/6841/2010-like viruses, first detected in mid-2012, were identified as reassortants comprised of A/Hubei/1/2010-like PB2 and PA and A/duck/Vietnam/NCVD-885/2010-like PB1, NP, NA, M, NS genes. © 2013.

South East Asia Infectious Disease Clinical Research Network. 2013. Effect of double dose oseltamivir on clinical and virological outcomes in children and adults admitted to hospital with severe influenza: double blind randomised controlled trial. BMJ, 346 (may30 2), pp. f3039. | Show Abstract | Read more

OBJECTIVE: To investigate the validity of recommendations in treatment guidelines to use higher than approved doses of oseltamivir in patients with severe influenza. DESIGN: Double blind randomised trial. SETTING: Thirteen hospitals in Indonesia, Singapore, Thailand, and Vietnam. PARTICIPANTS: Patients aged ≥ 1 year admitted to hospital with confirmed severe influenza. INTERVENTIONS: Oral oseltamivir at double dose (150 mg twice a day/paediatric equivalent) versus standard dose (75 mg twice a day/paediatric equivalent). MAIN OUTCOME MEASURE: Viral status according to reverse transcriptase polymerase chain reaction (RT-PCR) for influenza RNA in nasal and throat swabs on day five. RESULTS: Of 326 patients (including 246 (75.5%) children aged <15), 165 and 161 were randomised to double or standard dose oseltamivir, respectively. Of these, 260 (79.8%) were infected with influenza virus A (133 (40.8%) with A/H3N2, 72 (22.1%) with A/H1N1-pdm09, 38 (11.7%) with seasonal A/H1N1, 17 (5.2%) with A/H5N1) and 53 (16.2%) with influenza virus B. A further 3.9% (13) were false positive by rapid antigen test (negative by RT-PCR and no rise in convalescent haemagglutination inhibition titers). Similar proportions of patients were negative for RT-PCR on day five of treatment: 115/159 (72.3%, 95% confidence interval 64.9% to 78.7%) double dose recipients versus 105/154 (68.2%, 60.5% to 75.0%) standard dose recipients; difference 4.2% (-5.9 to 14.2); P=0.42. No differences were found in clearance of virus in subgroup analyses by virus type/subtype, age, and duration of illness before randomisation. Mortality was similar: 12/165 (7.3%, 4.2% to 12.3%) in double dose recipients versus 9/161 (5.6%, 3.0% to 10.3%) in standard dose recipients. No differences were found between double and standard dose arms in median days on supplemental oxygen (3 (interquartile range 2-5) v 3.5 (2-7)), in intensive care (4.5 (3-6) v 5 (2-11), and on mechanical ventilation (2.5 (1-16) v 8 (1-16)), respectively. No important differences in tolerability were found. CONCLUSIONS: There were no virological or clinical advantages with double dose oseltamivir compared with standard dose in patients with severe influenza admitted to hospital. REGISTRATION: Clinical Trials NCT00298233.

Nguyet MN, Duong TH, Trung VT, Nguyen TH, Tran CN, Long VT, Dui LET, Nguyen HL, Farrar JJ, Holmes EC et al. 2013. Host and viral features of human dengue cases shape the population of infected and infectious Aedes aegypti mosquitoes. Proc Natl Acad Sci U S A, 110 (22), pp. 9072-9077. | Show Abstract | Read more

Dengue is the most prevalent arboviral disease of humans. The host and virus variables associated with dengue virus (DENV) transmission from symptomatic dengue cases (n = 208) to Aedes aegypti mosquitoes during 407 independent exposure events was defined. The 50% mosquito infectious dose for each of DENV-1-4 ranged from 6.29 to 7.52 log10 RNA copies/mL of plasma. Increasing day of illness, declining viremia, and rising antibody titers were independently associated with reduced risk of DENV transmission. High early DENV plasma viremia levels in patients were a marker of the duration of human infectiousness, and blood meals containing high concentrations of DENV were positively associated with the prevalence of infectious mosquitoes 14 d after blood feeding. Ambulatory dengue cases had lower viremia levels compared with hospitalized dengue cases but nonetheless at levels predicted to be infectious to mosquitoes. These data define serotype-specific viremia levels that vaccines or drugs must inhibit to prevent DENV transmission.

Hoa NT, Chieu TT, Do Dung S, Long NT, Hieu TQ, Luc NT, Nhuong PT, Huong VT, Trinh DT, Wertheim HF et al. 2013. Streptococcus suis and porcine reproductive and respiratory syndrome, Vietnam. Emerg Infect Dis, 19 (2), pp. 331-333. | Read more

Hoa NT, Chieu TTB, Dung SD, Long NT, Hieu TQ, Luc NT, Nhuong PT, Huong VTL, Trinh DT, Wertheim HFL et al. 2013. Streptococcus suis and porcine reproductive and respiratory syndrome, vietnam Emerging Infectious Diseases, 19 (2), pp. 331-333. | Read more

Creanga A, Thi Nguyen D, Gerloff N, Thi Do H, Balish A, Dang Nguyen H, Jang Y, Thi Dam V, Thor S, Jones J et al. 2013. Emergence of multiple clade 2.3.2.1 influenza A (H5N1) virus subgroups in Vietnam and detection of novel reassortants. Virology, 444 (1-2), pp. 12-20. | Show Abstract | Read more

Phylogenetic analyses of 169 influenza A(H5N1) virus genomes were conducted for samples collected through active surveillance and outbreak responses in Vietnam between September 2010 and September 2012. While clade 1.1 viruses persisted in southern regions, three genetically distinct subgroups of clade 2.3.2.1 were found in northern and central Vietnam. The identification of each subgroup corresponded with detection of novel reassortants, likely due to their overlapping circulation throughout the country. While the previously identified clade 1.1 and A/Hubei/1/2010-like 2.3.2.1 genotypes remained the predominant viruses detected, four viruses were found to be reassortants between A/Hubei/1/2010-like (HA, NA, PB2, PB1, PA, NP) and A/duck/Vietnam/NCVD-885/2010-like (M, NS) viruses and one virus was identified as having A/duck/Vietnam/NCVD-885/2010-like HA, NA, PB1, and NP with A/Hubei/1/2010-like PB2 and PA genes. Additionally, clade 2.3.2.1 A/Hong Kong/6841/2010-like viruses, first detected in mid-2012, were identified as reassortants comprised of A/Hubei/1/2010-like PB2 and PA and A/duck/Vietnam/NCVD-885/2010-like PB1, NP, NA, M, NS genes.

Tan LEV, van Doorn HR, Nghia HD, Chau TT, Tu LETP, de Vries M, Canuti M, Deijs M, Jebbink MF, Baker S et al. 2013. Identification of a new cyclovirus in cerebrospinal fluid of patients with acute central nervous system infections. MBio, 4 (3), pp. e00231-e00213. | Show Abstract | Read more

Acute central nervous system (CNS) infections cause substantial morbidity and mortality, but the etiology remains unknown in a large proportion of cases. We identified and characterized the full genome of a novel cyclovirus (tentatively named cyclovirus-Vietnam [CyCV-VN]) in cerebrospinal fluid (CSF) specimens of two Vietnamese patients with CNS infections of unknown etiology. CyCV-VN was subsequently detected in 4% of 642 CSF specimens from Vietnamese patients with suspected CNS infections and none of 122 CSFs from patients with noninfectious neurological disorders. Detection rates were similar in patients with CNS infections of unknown etiology and those in whom other pathogens were detected. A similar detection rate in feces from healthy children suggested food-borne or orofecal transmission routes, while high detection rates in feces from pigs and poultry (average, 58%) suggested the existence of animal reservoirs for such transmission. Further research is needed to address the epidemiology and pathogenicity of this novel, potentially zoonotic virus.

van Doorn HR, Kinh NV, Tuan HM, Tuan TA, Minh NN, Bryant JE, Hang VT, Uyen LETT, Thinh LEQ, Anh TT et al. 2012. Clinical validation of a point-of-care multiplexed in vitro immunoassay using monoclonal antibodies (the MSD influenza test) in four hospitals in Vietnam. J Clin Microbiol, 50 (5), pp. 1621-1625. | Show Abstract | Read more

Point-of-care (POC) diagnostic tests for influenza can considerably shorten the time to clinical decision making. An investigational POC test based on a multiplexed immunoassay was developed by Meso Scale Diagnostics, LLC (MSD), with the objective to make a more sensitive rapid test that can also subtype influenza A viruses (1977 H1, H3, and H5). Between February and November 2010, we conducted a prospective multicenter study at four hospitals in Vietnam and compared the performance of this test to that of the WHO/CDC real-time reverse transcriptase PCR (RT-PCR) on nasal and throat swab specimens from patients presenting with influenza-like illness. Five hundred sixty-three adults and children with a median age of 25 months were enrolled. Sensitivity and specificity of the test with combined results from nasal and throat swab samples were 74.0% (131/177) and 99.7% (351/352), respectively, compared to RT-PCR. The POC test was as sensitive for influenza virus B as for influenza virus A (74.4% [64/86] versus 73.6% [67/91]). The positivity rate was associated with lower cycle threshold values (a marker for higher viral loads), sample type (73.6% for nasal swab versus 52.4% for throat swab), and younger age. A total of 210 (18.7%) out of 1,126 MSD tests failed, and for 34 (6%) of patients, both test samples failed (these were excluded from the performance analysis). Subtyping could be assessed only for influenza virus A/H3N2, as 1977 H1N1 was not circulating at the time and no H5N1-infected patients were enrolled, and was successful only in 9/54 patients infected with H3 influenza virus who had a positive POC test result for influenza virus A. This novel POC test provided highly sensitive detection of influenza viruses A and B compared to the reported sensitivities of other rapid tests. However, 18.7% of tests failed for technical reasons and subtyping for H3 was poor. Drawbacks to the technology include the requirement for a dedicated reader instrument and the need for continual updating of subtyping antibodies within the test array.

Do LAH, van Doorn HR, Bryant JE, Nghiem MN, Nguyen Van VC, Vo CK, Nguyen MD, Tran TH, Farrar J, De Jong MD. 2012. A sensitive real-time PCR for detection and subgrouping of human respiratory syncytial virus Journal of Virological Methods, 179 (1), pp. 250-255. | Show Abstract | Read more

Improved diagnostic tools for rapid detection, quantitation, and subgrouping of human respiratory syncytial virus (RSV) are needed to aid the development and evaluation of novel intervention strategies. A quantitative real-time RT-PCR using specific locked nucleic acid (LNA) probes was developed to identify RSV and to distinguish RSV subgroups A and B (RSV LNA assay). RSV subgroup diversity and the relationship between viral load and disease severity in confirmed RSV infections were also explored. 264 archived respiratory specimens from pediatric patients were tested in parallel using the commercial multiplex Seeplex™ RV detection kit (Seegene) and the novel RSV LNA assay. The LNA assay demonstrated a significantly higher sensitivity than Seeplex, improving overall detection rates from 24% (64/264) to 32% (84/264). Detection limits of 9.0×10 1 and 6.0×10 2 copies/mL were observed for RSV A and B, respectively. RSV A was detected in 53/84 (63%) cases, and 31/84 (37%) were positive for RSV B. This novel method offers a rapid, quantitative, highly specific and sensitive approach to laboratory diagnosis of RSV. © 2011 Elsevier B.V.

Do LA, van Doorn HR, Bryant JE, Nghiem MN, Nguyen Van VC, Vo CK, Nguyen MD, Tran TH, Farrar J, de Jong MD. 2012. A sensitive real-time PCR for detection and subgrouping of human respiratory syncytial virus. J Virol Methods, 179 (1), pp. 250-255. | Show Abstract | Read more

Improved diagnostic tools for rapid detection, quantitation, and subgrouping of human respiratory syncytial virus (RSV) are needed to aid the development and evaluation of novel intervention strategies. A quantitative real-time RT-PCR using specific locked nucleic acid (LNA) probes was developed to identify RSV and to distinguish RSV subgroups A and B (RSV LNA assay). RSV subgroup diversity and the relationship between viral load and disease severity in confirmed RSV infections were also explored. 264 archived respiratory specimens from pediatric patients were tested in parallel using the commercial multiplex Seeplex™ RV detection kit (Seegene) and the novel RSV LNA assay. The LNA assay demonstrated a significantly higher sensitivity than Seeplex, improving overall detection rates from 24% (64/264) to 32% (84/264). Detection limits of 9.0×10(1) and 6.0×10(2)copies/mL were observed for RSV A and B, respectively. RSV A was detected in 53/84 (63%) cases, and 31/84 (37%) were positive for RSV B. This novel method offers a rapid, quantitative, highly specific and sensitive approach to laboratory diagnosis of RSV.

Long NT, Thanh TT, van Doorn HR, Vu PP, Dung PT, Dung TT, Tien TN, Thao DT, Hung P, Quang NV et al. 2011. Recent avian influenza virus A/H5N1 evolution in vaccinated and unvaccinated poultry from farms in Southern Vietnam, January-March 2010. Transbound Emerg Dis, 58 (6), pp. 537-543. | Show Abstract | Read more

We report 15 new avian influenza virus A/H5N1 haemagglutinin (HA) sequences sampled from visibly sick domestic poultry in southern Vietnam, between 1 January 2010 and 6 March 2010. These HA sequences form a new sub-clade of the clade 1 H5N1 viruses that have been circulating in Vietnam since 2003/2004. The viruses are characterized by a change from isoleucine to valine at position 514 (I514V) and are 1.8% divergent at the nucleotide level from HA sequences sampled in Vietnam in 2007. Five new amino acid changes were observed at previously identified antigenic sites, and three were located within structural elements of the receptor-binding domain. One new mutation removed a potential N-linked glycosylation site, and a methionine insertion was observed in one virus at the polybasic cleavage site. Five of these viruses were sampled from farms where poultry were vaccinated against H5N1, but there was no association between observed amino acid changes and flock vaccination status. Despite the current lack of evidence for antigenic drift or immune escape in Vietnamese H5N1 viruses, continued surveillance remains a high priority.

Long NT, Thanh TT, van Doorn HR, Vu PP, Dung PT, Dung TTK, Tien TN, Thao DTT, Hung P, Quang NV et al. 2011. Recent avian influenza virus A/H5N1 evolution in vaccinated and unvaccinated poultry from farms in Southern Vietnam, January-March 2010 Transboundary and Emerging Diseases, 58 (6), pp. 537-543. | Show Abstract | Read more

We report 15 new avian influenza virus A/H5N1 haemagglutinin (HA) sequences sampled from visibly sick domestic poultry in southern Vietnam, between 1 January 2010 and 6 March 2010. These HA sequences form a new sub-clade of the clade 1 H5N1 viruses that have been circulating in Vietnam since 2003/2004. The viruses are characterized by a change from isoleucine to valine at position 514 (I514V) and are 1.8% divergent at the nucleotide level from HA sequences sampled in Vietnam in 2007. Five new amino acid changes were observed at previously identified antigenic sites, and three were located within structural elements of the receptor-binding domain. One new mutation removed a potential N-linked glycosylation site, and a methionine insertion was observed in one virus at the polybasic cleavage site. Five of these viruses were sampled from farms where poultry were vaccinated against H5N1, but there was no association between observed amino acid changes and flock vaccination status. Despite the current lack of evidence for antigenic drift or immune escape in Vietnamese H5N1 viruses, continued surveillance remains a high priority. © 2011 Blackwell Verlag GmbH.

Ngo TH, Tran TB, Tran TT, Nguyen VD, Campbell J, Pham HA, Huynh HT, Nguyen VV, Bryant JE, Tran TH et al. 2011. Slaughterhouse pigs are a major reservoir of Streptococcus suis serotype 2 capable of causing human infection in southern Vietnam. PLoS One, 6 (3), pp. e17943. | Show Abstract | Read more

Streptococcus suis is a pathogen of major economic significance to the swine industry and is increasingly recognized as an emerging zoonotic agent in Asia. In Vietnam, S. suis is the leading cause of bacterial meningitis in adult humans. Zoonotic transmission is most frequently associated with serotype 2 strains and occupational exposure to pigs or consumption of infected pork. To gain insight into the role of pigs for human consumption as a reservoir for zoonotic infection in southern Vietnam, we determined the prevalence and diversity of S. suis carriage in healthy slaughterhouse pigs. Nasopharyngeal tonsils were sampled from pigs at slaughterhouses serving six provinces in southern Vietnam and Ho Chi Minh City area from September 2006 to November 2007. Samples were screened by bacterial culture. Isolates of S. suis were serotyped and characterized by multi locus sequence typing (MLST) and pulse field gel electrophoresis (PFGE). Antibiotic susceptibility profiles and associated genetic resistance determinants, and the presence of putative virulence factors were determined. 41% (222/542) of pigs carried S. suis of one or multiple serotypes. 8% (45/542) carried S. suis serotype 2 which was the most common serotype found (45/317 strains, 14%). 80% of serotype 2 strains belonged to the MLST clonal complex 1,which was previously associated with meningitis cases in Vietnam and outbreaks of severe disease in China in 1998 and 2005. These strains clustered with representative strains isolated from patients with meningitis in PFGE analysis, and showed similar antimicrobial resistance and virulence factor profiles. Slaughterhouse pigs are a major reservoir of S. suis serotype 2 capable of causing human infection in southern Vietnam. Strict hygiene at processing facilities, and health education programs addressing food safety and proper handling of pork should be encouraged.

Cited:

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Ngo TH, Tran Chieu TB, Tran TT, Nguyen VD, Campbell J, Pham HA, Huynh HT, Nguyen VVC, Bryant JE, Tran TH et al. 2011. Slaughterhouse pigs are a major reservoir of Streptococcus suis serotype 2 capable of causing human infection in Southern Vietnam PLoS ONE, 6 (3), | Show Abstract | Read more

Streptococcus suis is a pathogen of major economic significance to the swine industry and is increasingly recognized as an emerging zoonotic agent in Asia. In Vietnam, S. suis is the leading cause of bacterial meningitis in adult humans. Zoonotic transmission is most frequently associated with serotype 2 strains and occupational exposure to pigs or consumption of infected pork. To gain insight into the role of pigs for human consumption as a reservoir for zoonotic infection in southern Vietnam, we determined the prevalence and diversity of S. suis carriage in healthy slaughterhouse pigs. Nasopharyngeal tonsils were sampled from pigs at slaughterhouses serving six provinces in southern Vietnam and Ho Chi Minh City area from September 2006 to November 2007. Samples were screened by bacterial culture. Isolates of S. suis were serotyped and characterized by multi locus sequence typing (MLST) and pulse field gel electrophoresis (PFGE). Antibiotic susceptibility profiles and associated genetic resistance determinants, and the presence of putative virulence factors were determined. 41% (222/542) of pigs carried S. suis of one or multiple serotypes. 8% (45/542) carried S. suis serotype 2 which was the most common serotype found (45/317 strains, 14%). 80% of serotype 2 strains belonged to the MLST clonal complex 1,which was previously associated with meningitis cases in Vietnam and outbreaks of severe disease in China in 1998 and 2005. These strains clustered with representative strains isolated from patients with meningitis in PFGE analysis, and showed similar antimicrobial resistance and virulence factor profiles. Slaughterhouse pigs are a major reservoir of S. suis serotype 2 capable of causing human infection in southern Vietnam. Strict hygiene at processing facilities, and health education programs addressing food safety and proper handling of pork should be encouraged. © 2011 Hoa et al.

Do AH, van Doorn HR, Nghiem MN, Bryant JE, Hoang TH, Do QH, Van TL, Tran TT, Wills B, Nguyen VC et al. 2011. Viral etiologies of acute respiratory infections among hospitalized Vietnamese children in Ho Chi Minh City, 2004-2008. PLoS One, 6 (3), pp. e18176. | Show Abstract | Read more

BACKGROUND: The dominant viral etiologies responsible for acute respiratory infections (ARIs) are poorly understood, particularly among hospitalized children in resource-limited tropical countries where morbidity and mortality caused by ARIs are highest. Improved etiological insight is needed to improve clinical management and prevention. OBJECTIVES: We conducted a three-year prospective descriptive study of severe respiratory illness among children from 2 months to 13 years of age within the largest referral hospital for infectious diseases in southern Vietnam. METHODS: Molecular detection for 15 viral species and subtypes was performed on three types of respiratory specimens (nose, throat swabs and nasopharyngeal aspirates) using a multiplex RT-PCR kit (Seeplex™ RV detection, Seegene) and additional monoplex real-time RT-PCRs. RESULTS: A total of 309 children were enrolled from November 2004 to January 2008. Viruses were identified in 72% (222/309) of cases, including respiratory syncytial virus (24%), influenza virus A and B (17%), human bocavirus (16%), enterovirus (9%), human coronavirus (8%), human metapneumovirus (7%), parainfluenza virus 1-3 (6%), adenovirus (5%), and human rhinovirus A (4%). Co-infections with multiple viruses were detected in 20% (62/309) of patients. When combined, diagnostic yields in nose and throat swabs were similar to nasopharyngeal aspirates. CONCLUSION: Similar to other parts in the world, RSV and influenza were the predominant viral pathogens detected in Vietnamese hospitalized children. Combined nasal and throat swabs are the specimens of choice for sensitive molecular detection of a broad panel of viral agents. Further research is required to better understand the clinical significance of single versus multiple viral coinfections and to address the role of bacterial (co-)infections involved in severe respiratory illness.

Hiscox A, Winter CH, Vongphrachanh P, Sisouk T, Somoulay V, Phompida S, Kaul S, Sananikhom P, Nguyen TY, Paul RE et al. 2010. Serological investigations of flavivirus prevalence in Khammouane Province, Lao People's Democratic Republic, 2007-2008. Am J Trop Med Hyg, 83 (5), pp. 1166-1169. | Show Abstract | Read more

A large-scale cross-sectional seroprevalence study of dengue (DEN) and Japanese encephalitis (JE) was conducted in Khammouane province, Lao PDR, as part of the initial baseline health impact assessment of the Nam Theun 2 hydroelectric dam construction project. Health surveys were performed between May 2007 and February 2008 with serum samples collected from healthy individuals involved in the resettlement program of 16 villages (total surveyed population 4,369). Hemagglutination inhibition assay using flavivirus antigens (DENV1, DENV3, and JEV) performed on 1,708 plasma specimens revealed 30.4% (519) cross-reactive positives, and 10% (172) and 1.3% (22) positives to JEV or DENV, respectively. Entomological surveys conducted during the rainy season of 2008 indicated the presence of competent flavivirus vectors (Culex vishnui group and Aedes albopictus), although Aedes aegypti was not found. Continued surveillance and investigation is warranted to assess the clinical disease burden of flaviviruses in this area that is undergoing rapid ecological and demographic change.

Vongphrachanh P, Simmerman JM, Phonekeo D, Pansayavong V, Sisouk T, Ongkhamme S, Bryce GT, Corwin A, Bryant JE. 2010. An early report from newly established laboratory-based influenza surveillance in Lao PDR. Influenza Other Respir Viruses, 4 (2), pp. 47-52. | Show Abstract | Read more

BACKGROUND: Prior to 2007, little information was available about the burden of influenza in Laos. We report data from the first laboratory-based influenza surveillance system established in the Lao People's Democratic Republic. METHODS: Three hospitals in the capital city of Vientiane began surveillance for influenza-like illness (ILI) in outpatients in 2007 and expanded to include hospitalized pneumonia patients in 2008. Nasal/throat swab specimens were collected and tested for influenza and other respiratory viruses by multiplex ID-Tag respiratory viral panel (RVP) assay on a Luminex 100x MAP IS instrument (Qiagen, Singapore). RESULTS: During January 2007 to December 2008, 287 of 526 (54.6%) outpatients with ILI were positive for at least one respiratory virus. Influenza was most commonly identified, with 63 (12.0%) influenza A and 92 (17.5%) influenza B positive patients identified. In 2008, six of 79 (7.6%) hospitalized pneumonia patients were positive for influenza A and four (5.1%) were positive for influenza B. Children <5 years represented 19% of viral infections in outpatients and 38% of pneumonia inpatients. CONCLUSION: Our results provide the first documentation of influenza burden among patients with febrile respiratory illness and pneumonia requiring hospitalization in Laos. Implementing laboratory-based influenza surveillance requires substantial investments in infrastructure and training. However, continuing outbreaks of avian influenza A/H5N1 in poultry and emergence of the 2009 influenza A(H1N1) pandemic strain further underscore the importance of establishing and maintaining influenza surveillance in developing countries.

Hien TT, Boni MF, Bryant JE, Ngan TT, Wolbers M, Nguyen TD, Truong NT, Dung NT, Ha DOQ, Hien VM et al. 2010. Early pandemic influenza (2009 H1N1) in Ho Chi Minh City, Vietnam: a clinical virological and epidemiological analysis. PLoS Med, 7 (5), pp. e1000277. | Show Abstract | Read more

BACKGROUND: To date, little is known about the initial spread and response to the 2009 pandemic of novel influenza A ("2009 H1N1") in tropical countries. Here, we analyse the early progression of the epidemic from 26 May 2009 until the establishment of community transmission in the second half of July 2009 in Ho Chi Minh City (HCMC), Vietnam. In addition, we present detailed systematic viral clearance data on 292 isolated and treated patients and the first three cases of selection of resistant virus during treatment in Vietnam. METHODS AND FINDINGS: Data sources included all available health reports from the Ministry of Health and relevant health authorities as well as clinical and laboratory data from the first confirmed cases isolated at the Hospital for Tropical Diseases in HCMC. Extensive reverse transcription (RT)-PCR diagnostics on serial samples, viral culture, neuraminidase-inhibition testing, and sequencing were performed on a subset of 2009 H1N1 confirmed cases. Virological (PCR status, shedding) and epidemiological (incidence, isolation, discharge) data were combined to reconstruct the initial outbreak and the establishment of community transmission. From 27 April to 24 July 2009, approximately 760,000 passengers who entered HCMC on international flights were screened at the airport by a body temperature scan and symptom questionnaire. Approximately 0.15% of incoming passengers were intercepted, 200 of whom tested positive for 2009 H1N1 by RT-PCR. An additional 121 out of 169 nontravelers tested positive after self-reporting or contact tracing. These 321 patients spent 79% of their PCR-positive days in isolation; 60% of PCR-positive days were spent treated and in isolation. Influenza-like illness was noted in 61% of patients and no patients experienced pneumonia or severe outcomes. Viral clearance times were similar among patient groups with differing time intervals from illness onset to treatment, with estimated median clearance times between 2.6 and 2.8 d post-treatment for illness-to-treatment intervals of 1-4 d, and 2.0 d (95% confidence interval 1.5-2.5) when treatment was started on the first day of illness. CONCLUSIONS: The patients described here represent a cross-section of infected individuals that were identified by temperature screening and symptom questionnaires at the airport, as well as mildly symptomatic to moderately ill patients who self-reported to hospitals. Data are observational and, although they are suggestive, it is not possible to be certain whether the containment efforts delayed community transmission in Vietnam. Viral clearance data assessed by RT-PCR showed a rapid therapeutic response to oseltamivir.

Vallée J, Dubot-Pérès A, Ounaphom P, Sayavong C, Bryant JE, Gonzalez JP. 2009. Spatial distribution and risk factors of dengue and Japanese encephalitis virus infection in urban settings: the case of Vientiane, Lao PDR. Trop Med Int Health, 14 (9), pp. 1134-1142. | Show Abstract | Read more

OBJECTIVES: To evaluate the prevalence of flavivirus infection in Vientiane city (Lao PDR), to describe the spatial distribution of infection within this city, and to explore the link between flavivirus seroprevalence and urbanization levels of residential neighbourhoods. METHODS: A seroprevalence survey was carried out in 2006 including 1990 adults (>or=35 years) and 1568 children (>or=6 months and <6 years) randomly selected. RESULTS: The prevalence of individuals with previous flavivirus infection (i.e. negative for both DEN and JE IgM but positive for DEN IgG) was 57.7%, with a significantly (P < 0.001) higher prevalence among adults (84.6%; 95% confidence interval (CI) = 82.4-86.8) than children (9.4%; 95% CI = 7.2-11.6). The prevalence of individuals with recent flavivirus infection (i.e. positive for DEN and/or JE IgM) was 6.5% and also significantly (P < 0.001) higher among adults (10.0%; 95% CI = 8.3-11.7) than children (2.5%; 95% CI = 1.5-3.5). In terms of spatial distribution, IgG prevalence was significantly (P < 0.001) higher among individuals living in the central city (60.1%; 95% CI = 56.2-64.1) than among those living in the periphery (44.3%; 95% CI = 41.5-47.2). In contrast, seroprevalence of recent flavivirus infections was significantly (P < 0.001) higher among individuals living in the periphery (8.8%; 95% CI = 6.9-10.7) than in the central city (4.0%; 95% CI = 2.9-5.2). This association was also statistically consistent (P < 0.01) in multivariate logistic regression after controlling for individual risk factors. CONCLUSIONS: Our findings indicate that the level of urbanization of residential neighbourhoods influences the risk of flavivirus infection. The spatial distribution of flavivirus infection varies, even within a small city of less than 300,000 habitants such as Vientiane.

Bryant JE, Calvert AE, Mesesan K, Crabtree MB, Volpe KE, Silengo S, Kinney RM, Huang CY, Miller BR, Roehrig JT. 2007. Glycosylation of the dengue 2 virus E protein at N67 is critical for virus growth in vitro but not for growth in intrathoracically inoculated Aedes aegypti mosquitoes. Virology, 366 (2), pp. 415-423. | Show Abstract | Read more

To determine the importance of dengue 2 virus (DEN2V) envelope (E) protein glycosylation, virus mutants in one or both of the N-linked glycosylation motifs were prepared. We found that while the E2 mutant virus (N153Q) replicated in mammalian and mosquito cells, the E1 (N67Q) and E1/2 (N67Q and N153Q) mutant viruses were unable to grow in mammalian cells. Infection of C6/36 mosquito cells with either the E1 or E1/2 mutants resulted in the introduction of a compensatory mutation, K64N, restoring glycosylation in the area. All mutants replicated similarly in inoculated Aedes aegypti mosquitoes, with no change in their mutations. These results suggest that N-linked glycosylation of the E protein is not necessary for DEN2V replication in mosquitoes, however N-linked glycosylation at amino acid N67 (or nearby N64) is critical for the survival of the virus in either mammalian or insect cell culture.

Bryant JE, Holmes EC, Barrett AD. 2007. Out of Africa: a molecular perspective on the introduction of yellow fever virus into the Americas. PLoS Pathog, 3 (5), pp. e75. | Show Abstract | Read more

Yellow fever virus (YFV) remains the cause of severe morbidity and mortality in South America and Africa. To determine the evolutionary history of this important reemerging pathogen, we performed a phylogenetic analysis of the largest YFV data set compiled to date, representing the prM/E gene region from 133 viral isolates sampled from 22 countries over a period of 76 years. We estimate that the currently circulating strains of YFV arose in Africa within the last 1,500 years and emerged in the Americas following the slave trade approximately 300-400 years ago. These viruses then spread westwards across the continent and persist there to this day in the jungles of South America. We therefore illustrate how gene sequence data can be used to test hypotheses of viral dispersal and demographics, and document the role of human migration in the spread of infectious disease.

Bryant JE, Crabtree MB, Nam VS, Yen NT, Duc HM, Miller BR. 2005. Isolation of arboviruses from mosquitoes collected in northern Vietnam. Am J Trop Med Hyg, 73 (2), pp. 470-473. | Show Abstract

In response to recent increases in cases of pediatric encephalitis with unknown etiology in northern Vietnam, surveillance for arbovirus activity was conducted in four provinces surrounding the city of Hanoi during June 2002 and July-August 2004. A total of 20,615 mosquitoes consisting of 19 species in 1,122 pools were processed for virus isolation; virus isolates were obtained from 44 pools. Sagiyama virus (11 isolates), Getah virus (15 isolates), Oya virus (13 isolates), and Akabane virus (4 isolates) were identified by immunofluorescence assay and sequence analysis of reverse transcription-polymerase chain reaction fragments. Surprisingly, no isolates of Japanese encephalitis (JE) virus were obtained. Isolation of Akabane virus, Oya virus, Getah virus, and Sagiyama virus is reported for the first time from Vietnam.

Bryant JE, Vasconcelos PF, Rijnbrand RC, Mutebi JP, Higgs S, Barrett AD. 2005. Size heterogeneity in the 3' noncoding region of South American isolates of yellow fever virus. J Virol, 79 (6), pp. 3807-3821. | Show Abstract | Read more

The 3' noncoding region (3' NCR) of flaviviruses contains secondary and tertiary structures essential for virus replication. Previous studies of yellow fever virus (YFV) and dengue virus have found that modifications to the 3' NCR are sometimes associated with attenuation in vertebrate and/or mosquito hosts. The 3' NCRs of 117 isolates of South American YFV have been examined, and major deletions and/or duplications of conserved RNA structures have been identified in several wild-type isolates. Nineteen isolates (designated YF-XL isolates) from Brazil, Trinidad, and Venezuela, dating from 1973 to 2001, exhibited a 216-nucleotide (nt) duplication, yielding a tandem repeat of conserved hairpin, stem-loop, dumbbell, and pseudoknot structures. YF-XL isolates were found exclusively within one subclade of South American genotype I YFV. One Brazilian isolate exhibited, in addition to the 216-nt duplication, a deletion of a 40-nt repeated hairpin (RYF) motif (YF-XL-DeltaRYF). To investigate the biological significance of these 3' NCR rearrangements, YF-XL-DeltaRYF and YF-XL isolates, as well as other South American YFV isolates, were evaluated for three phenotypes: growth kinetics in cell culture, neuroinvasiveness in suckling mice, and ability to replicate and produce disseminated infections in Aedes aegypti mosquitoes. YF-XL-DeltaRYF and YF-XL isolates showed growth kinetics and neuroinvasive characteristics comparable to those of typical South American YFV isolates, and mosquito infectivity trials demonstrated that both types of 3' NCR variants were capable of replication and dissemination in a laboratory-adapted colony of A. aegypti.

Vasconcelos PF, Bryant JE, da Rosa TP, Tesh RB, Rodrigues SG, Barrett AD. 2004. Genetic divergence and dispersal of yellow fever virus, Brazil. Emerg Infect Dis, 10 (9), pp. 1578-1584. | Show Abstract | Read more

An analysis of 79 yellow fever virus (YFV) isolates collected from 1935 to 2001 in Brazil showed a single genotype (South America I) circulating in the country, with the exception of a single strain from Rondonia, which represented South America genotype II. Brazilian YFV strains have diverged into two clades; an older clade appears to have become extinct and another has become the dominant lineage in recent years. Pairwise nucleotide diversity between strains ranged from 0% to 7.4%, while amino acid divergence ranged from 0% to 4.6%. Phylogenetic analysis indicated traffic of virus variants through large geographic areas and suggested that migration of infected people may be an important mechanism of virus dispersal. Isolation of vaccine virus from a patient with a fatal case suggests that vaccine-related illness may have been misdiagnosed in the past.

Bryant JE, Barrett AD. 2003. Comparative phylogenies of yellow fever isolates from Peru and Brazil. FEMS Immunol Med Microbiol, 39 (2), pp. 103-118. | Show Abstract | Read more

We recently reported phylogenetic evidence to support the presence of enzootic transmission foci of yellow fever virus (YFV) in Peru [Bryant et al., Emerg. Infect. Dis. (2003)]. Because the prevailing paradigm of YFV transmission in Brazil is that of 'wandering epizootics' rather than discrete enzootic foci, we have now compared the molecular phylogenies of YFV isolates from Peru and Brazil, and re-examined the question of virus mobility by mapping the spatio-temporal distribution of genetic variants from these areas. Sequences were obtained for two genomic regions from 50 strains of YFV collected between 1954 and 2000 comprising 223 codons of the structural proteins (premembrane and envelope genes, 'prM/E'), and a distal region spanning the carboxy terminus of NS5 and part of the 3' non-coding region ('EMF'). Peruvian and Brazilian isolates formed two monophyletic clades with no evidence to support recombination between lineages. Variation within both coding and non-coding regions revealed similar substitution rates and overall levels of diversity within each clade. The branching structure of the prM/E and EMF trees of Brazilian sequences showed strong agreement of intra-lineage relationships; in contrast, the EMF sequences of Peruvian isolates failed to fully support the subclade structure of the prM/E phylogeny. These phylogenies suggest that transmission cycles of YFV in Peru and Brazil may sometimes be locally maintained within specific locales, but have also on occasion become very widely dispersed.

Mutebi JP, Wang H, Li L, Bryant JE, Barrett AD. 2001. Phylogenetic and evolutionary relationships among yellow fever virus isolates in Africa. J Virol, 75 (15), pp. 6999-7008. | Show Abstract | Read more

Previous studies with a limited number of strains have indicated that there are two genotypes of yellow fever (YF) virus in Africa, one in west Africa and the other in east and central Africa. We have examined the prM/M and a portion of the E protein for a panel of 38 wild strains of YF virus from Africa representing different countries and times of isolation. Examination of the strains revealed a more complex genetic relationship than previously reported. Overall, nucleotide substitutions varied from 0 to 25.8% and amino acid substitutions varied from 0 to 9.1%. Phylogenetic analysis using parsimony and neighbor-joining algorithms identified five distinct genotypes: central/east Africa, east Africa, Angola, west Africa I, and west Africa II. Extensive variation within genotypes was observed. Members of west African genotype II and central/east African genotype differed by 2.8% or less, while west Africa genotype I varied up to 6.8% at the nucleotide level. We speculate that the former two genotypes exist in enzootic transmission cycles, while the latter is genetically more heterogeneous due to regular human epidemics. The nucleotide sequence of the Angola genotype diverged from the others by 15.7 to 23.0% but only 0.4 to 5.6% at the amino acid level, suggesting that this genotype most likely diverged from a progenitor YF virus in east/central Africa many years ago, prior to the separation of the other east/central African strains analyzed in this study, and has evolved independently. These data demonstrate that there are multiple genotypes of YF virus in Africa and suggest independent evolution of YF virus in different areas of Africa.

Kahn J, Mehraban F, Ingle G, Xin X, Bryant JE, Vehar G, Schoenfeld J, Grimaldi CJ, Peale F, Draksharapu A et al. 2000. Gene expression profiling in an in vitro model of angiogenesis. Am J Pathol, 156 (6), pp. 1887-1900. | Show Abstract | Read more

In the present study we have used a novel, comprehensive mRNA profiling technique (GeneCalling) for determining differential gene expression profiles of human endothelial cells undergoing differentiation into tubelike structures. One hundred fifteen cDNA fragments were identified and shown to represent 90 distinct genes. Although some of the genes identified have previously been implicated in angiogenesis, potential roles for many new genes, including OX-40, white protein homolog, KIAA0188, a homolog of angiopoietin-2, ADAMTS-4 (aggrecanase-1), and stanniocalcin were revealed. Support for the biological significance was confirmed by the abrogation of the changes in the expression of angiogenesis inhibitors and in situ hybridization studies. This study has significantly extends the molecular fingerprint of the changes in gene expression that occur during endothelial differentiation and provides new insights into the potential role of a number of new molecules in angiogenesis.

Miller DN, Bryant JE, Madsen EL, Ghiorse WC. 1999. Evaluation and optimization of DNA extraction and purification procedures for soil and sediment samples. Appl Environ Microbiol, 65 (11), pp. 4715-4724. | Show Abstract

We compared and statistically evaluated the effectiveness of nine DNA extraction procedures by using frozen and dried samples of two silt loam soils and a silt loam wetland sediment with different organic matter contents. The effects of different chemical extractants (sodium dodecyl sulfate [SDS], chloroform, phenol, Chelex 100, and guanadinium isothiocyanate), different physical disruption methods (bead mill homogenization and freeze-thaw lysis), and lysozyme digestion were evaluated based on the yield and molecular size of the recovered DNA. Pairwise comparisons of the nine extraction procedures revealed that bead mill homogenization with SDS combined with either chloroform or phenol optimized both the amount of DNA extracted and the molecular size of the DNA (maximum size, 16 to 20 kb). Neither lysozyme digestion before SDS treatment nor guanidine isothiocyanate treatment nor addition of Chelex 100 resin improved the DNA yields. Bead mill homogenization in a lysis mixture containing chloroform, SDS, NaCl, and phosphate-Tris buffer (pH 8) was found to be the best physical lysis technique when DNA yield and cell lysis efficiency were used as criteria. The bead mill homogenization conditions were also optimized for speed and duration with two different homogenizers. Recovery of high-molecular-weight DNA was greatest when we used lower speeds and shorter times (30 to 120 s). We evaluated four different DNA purification methods (silica-based DNA binding, agarose gel electrophoresis, ammonium acetate precipitation, and Sephadex G-200 gel filtration) for DNA recovery and removal of PCR inhibitors from crude extracts. Sephadex G-200 spin column purification was found to be the best method for removing PCR-inhibiting substances while minimizing DNA loss during purification. Our results indicate that for these types of samples, optimum DNA recovery requires brief, low-speed bead mill homogenization in the presence of a phosphate-buffered SDS-chloroform mixture, followed by Sephadex G-200 column purification.

Van Dung N, Anh PH, Van Cuong N, Hoa NT, Carrique-Mas J, Hien VB, Campbell J, Baker S, Farrar J, Woolhouse ME et al. 2014. Prevalence, genetic diversity and recombination of species G enteroviruses infecting pigs in Vietnam. J Gen Virol, 95 (Pt 3), pp. 549-556. | Show Abstract | Read more

Picornaviruses infecting pigs, described for many years as 'porcine enteroviruses', have recently been recognized as distinct viruses within three distinct genera (Teschovirus, Sapelovirus and Enterovirus). To better characterize the epidemiology and genetic diversity of members of the Enterovirus genus, faecal samples from pigs from four provinces in Vietnam were screened by PCR using conserved enterovirus (EV)-specific primers from the 5' untranslated region (5' UTR). High rates of infection were recorded in pigs on all farms, with detection frequencies of approximately 90% in recently weaned pigs but declining to 40% in those aged over 1 year. No differences in EV detection rates were observed between pigs with and without diarrhoea [74% (n = 70) compared with 72% (n = 128)]. Genetic analysis of consensus VP4/VP2 and VP1 sequences amplified from a subset of EV-infected pigs identified species G EVs in all samples. Among these, VP1 sequence comparisons identified six type 1 and seven type 6 variants, while four further VP1 sequences failed to group with any previously identified EV-G types. These have now been formally assigned as EV-G types 8-11 by the Picornavirus Study Group. Comparison of VP1, VP4/VP2, 3D(pol) and 5' UTRs of study samples and those available on public databases showed frequent, bootstrap-supported differences in their phylogenies indicative of extensive within-species recombination between genome regions. In summary, we identified extremely high frequencies of infection with EV-G in pigs in Vietnam, substantial genetic diversity and recombination within the species, and evidence for a much larger number of circulating EV-G types than currently described.

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Creanga A, Thi Nguyen D, Gerloff N, Thi Do H, Balish A, Dang Nguyen H, Jang Y, Thi Dam V, Thor S, Jones J et al. 2013. Emergence of multiple clade 2.3.2.1 influenza A (H5N1) virus subgroups in Vietnam and detection of novel reassortants Virology, 444 (1-2), pp. 12-20. | Show Abstract | Read more

Phylogenetic analyses of 169 influenza A(H5N1) virus genomes were conducted for samples collected through active surveillance and outbreak responses in Vietnam between September 2010 and September 2012. While clade 1.1 viruses persisted in southern regions, three genetically distinct subgroups of clade 2.3.2.1 were found in northern and central Vietnam. The identification of each subgroup corresponded with detection of novel reassortants, likely due to their overlapping circulation throughout the country. While the previously identified clade 1.1 and A/Hubei/1/2010-like 2.3.2.1 genotypes remained the predominant viruses detected, four viruses were found to be reassortants between A/Hubei/1/2010-like (HA, NA, PB2, PB1, PA, NP) and A/duck/Vietnam/NCVD-885/2010-like (M, NS) viruses and one virus was identified as having A/duck/Vietnam/NCVD-885/2010-like HA, NA, PB1, and NP with A/Hubei/1/2010-like PB2 and PA genes. Additionally, clade 2.3.2.1 A/Hong Kong/6841/2010-like viruses, first detected in mid-2012, were identified as reassortants comprised of A/Hubei/1/2010-like PB2 and PA and A/duck/Vietnam/NCVD-885/2010-like PB1, NP, NA, M, NS genes. © 2013.

Nguyet MN, Duong TH, Trung VT, Nguyen TH, Tran CN, Long VT, Dui LET, Nguyen HL, Farrar JJ, Holmes EC et al. 2013. Host and viral features of human dengue cases shape the population of infected and infectious Aedes aegypti mosquitoes. Proc Natl Acad Sci U S A, 110 (22), pp. 9072-9077. | Show Abstract | Read more

Dengue is the most prevalent arboviral disease of humans. The host and virus variables associated with dengue virus (DENV) transmission from symptomatic dengue cases (n = 208) to Aedes aegypti mosquitoes during 407 independent exposure events was defined. The 50% mosquito infectious dose for each of DENV-1-4 ranged from 6.29 to 7.52 log10 RNA copies/mL of plasma. Increasing day of illness, declining viremia, and rising antibody titers were independently associated with reduced risk of DENV transmission. High early DENV plasma viremia levels in patients were a marker of the duration of human infectiousness, and blood meals containing high concentrations of DENV were positively associated with the prevalence of infectious mosquitoes 14 d after blood feeding. Ambulatory dengue cases had lower viremia levels compared with hospitalized dengue cases but nonetheless at levels predicted to be infectious to mosquitoes. These data define serotype-specific viremia levels that vaccines or drugs must inhibit to prevent DENV transmission.

Hoa NT, Chieu TT, Do Dung S, Long NT, Hieu TQ, Luc NT, Nhuong PT, Huong VT, Trinh DT, Wertheim HF et al. 2013. Streptococcus suis and porcine reproductive and respiratory syndrome, Vietnam. Emerg Infect Dis, 19 (2), pp. 331-333. | Read more

Long NT, Thanh TT, van Doorn HR, Vu PP, Dung PT, Dung TT, Tien TN, Thao DT, Hung P, Quang NV et al. 2011. Recent avian influenza virus A/H5N1 evolution in vaccinated and unvaccinated poultry from farms in Southern Vietnam, January-March 2010. Transbound Emerg Dis, 58 (6), pp. 537-543. | Show Abstract | Read more

We report 15 new avian influenza virus A/H5N1 haemagglutinin (HA) sequences sampled from visibly sick domestic poultry in southern Vietnam, between 1 January 2010 and 6 March 2010. These HA sequences form a new sub-clade of the clade 1 H5N1 viruses that have been circulating in Vietnam since 2003/2004. The viruses are characterized by a change from isoleucine to valine at position 514 (I514V) and are 1.8% divergent at the nucleotide level from HA sequences sampled in Vietnam in 2007. Five new amino acid changes were observed at previously identified antigenic sites, and three were located within structural elements of the receptor-binding domain. One new mutation removed a potential N-linked glycosylation site, and a methionine insertion was observed in one virus at the polybasic cleavage site. Five of these viruses were sampled from farms where poultry were vaccinated against H5N1, but there was no association between observed amino acid changes and flock vaccination status. Despite the current lack of evidence for antigenic drift or immune escape in Vietnamese H5N1 viruses, continued surveillance remains a high priority.

Long NT, Thanh TT, van Doorn HR, Vu PP, Dung PT, Dung TTK, Tien TN, Thao DTT, Hung P, Quang NV et al. 2011. Recent avian influenza virus A/H5N1 evolution in vaccinated and unvaccinated poultry from farms in Southern Vietnam, January-March 2010 Transboundary and Emerging Diseases, 58 (6), pp. 537-543. | Show Abstract | Read more

We report 15 new avian influenza virus A/H5N1 haemagglutinin (HA) sequences sampled from visibly sick domestic poultry in southern Vietnam, between 1 January 2010 and 6 March 2010. These HA sequences form a new sub-clade of the clade 1 H5N1 viruses that have been circulating in Vietnam since 2003/2004. The viruses are characterized by a change from isoleucine to valine at position 514 (I514V) and are 1.8% divergent at the nucleotide level from HA sequences sampled in Vietnam in 2007. Five new amino acid changes were observed at previously identified antigenic sites, and three were located within structural elements of the receptor-binding domain. One new mutation removed a potential N-linked glycosylation site, and a methionine insertion was observed in one virus at the polybasic cleavage site. Five of these viruses were sampled from farms where poultry were vaccinated against H5N1, but there was no association between observed amino acid changes and flock vaccination status. Despite the current lack of evidence for antigenic drift or immune escape in Vietnamese H5N1 viruses, continued surveillance remains a high priority. © 2011 Blackwell Verlag GmbH.

Ngo TH, Tran TB, Tran TT, Nguyen VD, Campbell J, Pham HA, Huynh HT, Nguyen VV, Bryant JE, Tran TH et al. 2011. Slaughterhouse pigs are a major reservoir of Streptococcus suis serotype 2 capable of causing human infection in southern Vietnam. PLoS One, 6 (3), pp. e17943. | Show Abstract | Read more

Streptococcus suis is a pathogen of major economic significance to the swine industry and is increasingly recognized as an emerging zoonotic agent in Asia. In Vietnam, S. suis is the leading cause of bacterial meningitis in adult humans. Zoonotic transmission is most frequently associated with serotype 2 strains and occupational exposure to pigs or consumption of infected pork. To gain insight into the role of pigs for human consumption as a reservoir for zoonotic infection in southern Vietnam, we determined the prevalence and diversity of S. suis carriage in healthy slaughterhouse pigs. Nasopharyngeal tonsils were sampled from pigs at slaughterhouses serving six provinces in southern Vietnam and Ho Chi Minh City area from September 2006 to November 2007. Samples were screened by bacterial culture. Isolates of S. suis were serotyped and characterized by multi locus sequence typing (MLST) and pulse field gel electrophoresis (PFGE). Antibiotic susceptibility profiles and associated genetic resistance determinants, and the presence of putative virulence factors were determined. 41% (222/542) of pigs carried S. suis of one or multiple serotypes. 8% (45/542) carried S. suis serotype 2 which was the most common serotype found (45/317 strains, 14%). 80% of serotype 2 strains belonged to the MLST clonal complex 1,which was previously associated with meningitis cases in Vietnam and outbreaks of severe disease in China in 1998 and 2005. These strains clustered with representative strains isolated from patients with meningitis in PFGE analysis, and showed similar antimicrobial resistance and virulence factor profiles. Slaughterhouse pigs are a major reservoir of S. suis serotype 2 capable of causing human infection in southern Vietnam. Strict hygiene at processing facilities, and health education programs addressing food safety and proper handling of pork should be encouraged.

Do AH, van Doorn HR, Nghiem MN, Bryant JE, Hoang TH, Do QH, Van TL, Tran TT, Wills B, Nguyen VC et al. 2011. Viral etiologies of acute respiratory infections among hospitalized Vietnamese children in Ho Chi Minh City, 2004-2008. PLoS One, 6 (3), pp. e18176. | Show Abstract | Read more

BACKGROUND: The dominant viral etiologies responsible for acute respiratory infections (ARIs) are poorly understood, particularly among hospitalized children in resource-limited tropical countries where morbidity and mortality caused by ARIs are highest. Improved etiological insight is needed to improve clinical management and prevention. OBJECTIVES: We conducted a three-year prospective descriptive study of severe respiratory illness among children from 2 months to 13 years of age within the largest referral hospital for infectious diseases in southern Vietnam. METHODS: Molecular detection for 15 viral species and subtypes was performed on three types of respiratory specimens (nose, throat swabs and nasopharyngeal aspirates) using a multiplex RT-PCR kit (Seeplex™ RV detection, Seegene) and additional monoplex real-time RT-PCRs. RESULTS: A total of 309 children were enrolled from November 2004 to January 2008. Viruses were identified in 72% (222/309) of cases, including respiratory syncytial virus (24%), influenza virus A and B (17%), human bocavirus (16%), enterovirus (9%), human coronavirus (8%), human metapneumovirus (7%), parainfluenza virus 1-3 (6%), adenovirus (5%), and human rhinovirus A (4%). Co-infections with multiple viruses were detected in 20% (62/309) of patients. When combined, diagnostic yields in nose and throat swabs were similar to nasopharyngeal aspirates. CONCLUSION: Similar to other parts in the world, RSV and influenza were the predominant viral pathogens detected in Vietnamese hospitalized children. Combined nasal and throat swabs are the specimens of choice for sensitive molecular detection of a broad panel of viral agents. Further research is required to better understand the clinical significance of single versus multiple viral coinfections and to address the role of bacterial (co-)infections involved in severe respiratory illness.

Vongphrachanh P, Simmerman JM, Phonekeo D, Pansayavong V, Sisouk T, Ongkhamme S, Bryce GT, Corwin A, Bryant JE. 2010. An early report from newly established laboratory-based influenza surveillance in Lao PDR. Influenza Other Respir Viruses, 4 (2), pp. 47-52. | Show Abstract | Read more

BACKGROUND: Prior to 2007, little information was available about the burden of influenza in Laos. We report data from the first laboratory-based influenza surveillance system established in the Lao People's Democratic Republic. METHODS: Three hospitals in the capital city of Vientiane began surveillance for influenza-like illness (ILI) in outpatients in 2007 and expanded to include hospitalized pneumonia patients in 2008. Nasal/throat swab specimens were collected and tested for influenza and other respiratory viruses by multiplex ID-Tag respiratory viral panel (RVP) assay on a Luminex 100x MAP IS instrument (Qiagen, Singapore). RESULTS: During January 2007 to December 2008, 287 of 526 (54.6%) outpatients with ILI were positive for at least one respiratory virus. Influenza was most commonly identified, with 63 (12.0%) influenza A and 92 (17.5%) influenza B positive patients identified. In 2008, six of 79 (7.6%) hospitalized pneumonia patients were positive for influenza A and four (5.1%) were positive for influenza B. Children <5 years represented 19% of viral infections in outpatients and 38% of pneumonia inpatients. CONCLUSION: Our results provide the first documentation of influenza burden among patients with febrile respiratory illness and pneumonia requiring hospitalization in Laos. Implementing laboratory-based influenza surveillance requires substantial investments in infrastructure and training. However, continuing outbreaks of avian influenza A/H5N1 in poultry and emergence of the 2009 influenza A(H1N1) pandemic strain further underscore the importance of establishing and maintaining influenza surveillance in developing countries.

Hien TT, Boni MF, Bryant JE, Ngan TT, Wolbers M, Nguyen TD, Truong NT, Dung NT, Ha DOQ, Hien VM et al. 2010. Early pandemic influenza (2009 H1N1) in Ho Chi Minh City, Vietnam: a clinical virological and epidemiological analysis. PLoS Med, 7 (5), pp. e1000277. | Show Abstract | Read more

BACKGROUND: To date, little is known about the initial spread and response to the 2009 pandemic of novel influenza A ("2009 H1N1") in tropical countries. Here, we analyse the early progression of the epidemic from 26 May 2009 until the establishment of community transmission in the second half of July 2009 in Ho Chi Minh City (HCMC), Vietnam. In addition, we present detailed systematic viral clearance data on 292 isolated and treated patients and the first three cases of selection of resistant virus during treatment in Vietnam. METHODS AND FINDINGS: Data sources included all available health reports from the Ministry of Health and relevant health authorities as well as clinical and laboratory data from the first confirmed cases isolated at the Hospital for Tropical Diseases in HCMC. Extensive reverse transcription (RT)-PCR diagnostics on serial samples, viral culture, neuraminidase-inhibition testing, and sequencing were performed on a subset of 2009 H1N1 confirmed cases. Virological (PCR status, shedding) and epidemiological (incidence, isolation, discharge) data were combined to reconstruct the initial outbreak and the establishment of community transmission. From 27 April to 24 July 2009, approximately 760,000 passengers who entered HCMC on international flights were screened at the airport by a body temperature scan and symptom questionnaire. Approximately 0.15% of incoming passengers were intercepted, 200 of whom tested positive for 2009 H1N1 by RT-PCR. An additional 121 out of 169 nontravelers tested positive after self-reporting or contact tracing. These 321 patients spent 79% of their PCR-positive days in isolation; 60% of PCR-positive days were spent treated and in isolation. Influenza-like illness was noted in 61% of patients and no patients experienced pneumonia or severe outcomes. Viral clearance times were similar among patient groups with differing time intervals from illness onset to treatment, with estimated median clearance times between 2.6 and 2.8 d post-treatment for illness-to-treatment intervals of 1-4 d, and 2.0 d (95% confidence interval 1.5-2.5) when treatment was started on the first day of illness. CONCLUSIONS: The patients described here represent a cross-section of infected individuals that were identified by temperature screening and symptom questionnaires at the airport, as well as mildly symptomatic to moderately ill patients who self-reported to hospitals. Data are observational and, although they are suggestive, it is not possible to be certain whether the containment efforts delayed community transmission in Vietnam. Viral clearance data assessed by RT-PCR showed a rapid therapeutic response to oseltamivir.

Bryant JE, Calvert AE, Mesesan K, Crabtree MB, Volpe KE, Silengo S, Kinney RM, Huang CY, Miller BR, Roehrig JT. 2007. Glycosylation of the dengue 2 virus E protein at N67 is critical for virus growth in vitro but not for growth in intrathoracically inoculated Aedes aegypti mosquitoes. Virology, 366 (2), pp. 415-423. | Show Abstract | Read more

To determine the importance of dengue 2 virus (DEN2V) envelope (E) protein glycosylation, virus mutants in one or both of the N-linked glycosylation motifs were prepared. We found that while the E2 mutant virus (N153Q) replicated in mammalian and mosquito cells, the E1 (N67Q) and E1/2 (N67Q and N153Q) mutant viruses were unable to grow in mammalian cells. Infection of C6/36 mosquito cells with either the E1 or E1/2 mutants resulted in the introduction of a compensatory mutation, K64N, restoring glycosylation in the area. All mutants replicated similarly in inoculated Aedes aegypti mosquitoes, with no change in their mutations. These results suggest that N-linked glycosylation of the E protein is not necessary for DEN2V replication in mosquitoes, however N-linked glycosylation at amino acid N67 (or nearby N64) is critical for the survival of the virus in either mammalian or insect cell culture.

Bryant JE, Holmes EC, Barrett AD. 2007. Out of Africa: a molecular perspective on the introduction of yellow fever virus into the Americas. PLoS Pathog, 3 (5), pp. e75. | Show Abstract | Read more

Yellow fever virus (YFV) remains the cause of severe morbidity and mortality in South America and Africa. To determine the evolutionary history of this important reemerging pathogen, we performed a phylogenetic analysis of the largest YFV data set compiled to date, representing the prM/E gene region from 133 viral isolates sampled from 22 countries over a period of 76 years. We estimate that the currently circulating strains of YFV arose in Africa within the last 1,500 years and emerged in the Americas following the slave trade approximately 300-400 years ago. These viruses then spread westwards across the continent and persist there to this day in the jungles of South America. We therefore illustrate how gene sequence data can be used to test hypotheses of viral dispersal and demographics, and document the role of human migration in the spread of infectious disease.

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