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International audience; Enteric fever is a major public health concern in endemic areas, particularly in infrastructure-limited countries where Salmonella Paratyphi A has emerged in increasing proportion of cases. We aimed to evaluate a method to detect Salmonella Typhi (S. Typhi) and Salmonella Paratyphi A (S. Paratyphi A) in febrile patients in Bangladesh. We conducted a prospective study enrolling patients with fever > 38°C admitted to two large urban hospitals and two outpatient clinics located in Dhaka, Bangladesh. We developed and evaluated a method combining short culture with a new molecular assay to simultaneously detect and differentiate S. Typhi and S. Paratyphi A from other Salmonella directly from 2 to 4 mL of whole blood in febrile patients (n = 680). A total of 680 cases were enrolled from the four participating sites. An increase in the detection rate (+38.8%) in S. Typhi and S. Paratyphi A was observed with a multiplex polymerase chain reaction (PCR) assay, and absence of non-typhoidal Salmonella detection was reported. All 45 healthy controls were culture and PCR negative, generating an estimated 92.9% of specificity on clinical samples. When clinical performance was assessed in the absence of blood volume prioritization for testing, a latent class model estimates clinical performance ≥ 95% in sensitivity and specificity with likelihood ratio (LR) LR+ > 10 and LR- < 0.1 for the multiplex PCR assay. The alternative method to blood culture we developed may be useful alone or in combination with culture or serological tests for epidemiological studies in high disease burden settings and should be considered as secondary endpoint test for future vaccine trials.

We characterized 55 influenza A(H9N2) viruses isolated in Pakistan during 2014-2016 and found that the hemagglutinin gene is of the G1 lineage and that internal genes have differentiated into a variety of novel genotypes. Some isolates had up to 4-fold reduction in hemagglutination inhibition titers compared with older viruses. Viruses with hemagglutinin A180T/V substitutions conveyed this antigenic diversity and also caused up to 3,500-fold greater binding to avian-like and >20-fold greater binding to human-like sialic acid receptor analogs. This enhanced binding avidity led to reduced virus replication in primary and continuous cell culture. We confirmed that altered receptor-binding avidity of H9N2 viruses, including enhanced binding to human-like receptors, results in antigenic variation in avian influenza viruses. Consequently, current vaccine formulations might not induce adequate protective immunity in poultry, and emergence of isolates with marked avidity for human-like receptors increases the zoonotic risk.

Contains fulltext : 177503.pdf (Publisher’s version ) (Open Access) Rural farming communities in northern Vietnam do not routinely practice vaccination for influenza A viruses (IAV) for either humans or poultry, which enables us to study transmission intensity via seroepidemiology. Using samples from a longitudinal cohort of farming households, we determined the number of symptomatic and asymptomatic human infections for seasonal IAV and avian A/H9 over 2 years. As expected, we detected virologically confirmed acute cases of seasonal IAV in humans, as well as large numbers of subclinical seroconversions to A/H1pdm [55/265 (21 %)], A/H3 [95/265 (36 %)] and A/H9 [24/265 (9 %)]. Five of the A/H9 human seroconverters likely represented true infections rather than heterosubtypic immunity, because the individuals seroconverted solely to A/H9. Among co-located poultry, we found significantly higher seroprevalance for A/H5 compared to A/H9 in both chickens and ducks [for northern study sites overall, 337/1105 (30.5 %) seropositive for A/H5 and 123/1105 (11.1 %) seropositive for A/H9].

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, Highlights • We report detection of highly pathogenic avian influenza (HPAI) from healthy chickens in Live Bird Markets of Vietnam. Because all breeds of domestic chickens are extremely susceptible to HPAI, we speculate that HPAI detections from market chickens may reflect infections that occur after arrival in the market. Alternatively, shedding of HPAI from healthy birds may reflect vaccine-induced protective immunity that mitigates disease but does not block viral infection. • As many as 49% of all pooled surveillance swabs were positive for influenza A virus, corresponding to an overall Influenza A prevalence of 5.45% (95% Confidence Interval 5.4-6.0%). • Low pathogenicity avian influenza (LPAI) H9N2 accounted for the vast majority of all influenza A detections in market chickens sampled from 9 northern provinces. • To date there is no evidence to suggest an interaction effect between circulation of H5 and H9 viruses; however sampling strategies that involve pooling of surveillance swabs from multiple birds greatly complicates the assessment of co-infection rates or evaluation of epidemiological associations.

Highlights • Rhino, Influenza and Corona viruses caused ∼50% of ILI in Vietnam households. • There was no clear seasonality for ILI, or for individual viruses causing ILI. • Children did not contribute substantially to ILI burden or transmission. • Seasonality and the contribution of children were unlike temporal settings. • Seasonality and social factors may affect the involvement of children in ILI., 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.

Rapid and correct determination of Mycobacterium tuberculosis (MTB) drug susceptibility is a challenge for tuberculosis (TB) management. Phenotypic drug susceptibility testing (DST) remains the reference method but is time consuming. In this study, genotypic prediction of the first-line drug susceptibility profile obtained by whole-genome sequencing (WGS) was compared with that obtained by phenotypic DST and the line probe assay (LPA). All MTB strains isolated from patients during routine practice at the mycobacteria laboratory of Lyon University Hospital, France, between November 2016 and July 2019 were included (n = 274). Isolates were tested for the first-line drugs using phenotypic DST (Mycobacteria Growth Indicator Tube) and for genotypic prediction of the susceptibility profile with LPA and WGS. Considering phenotypic DST as the reference, WGS predicted resistance to rifampicin, isoniazid, ethambutol and pyrazinamide with sensitivities of 100%, 100%, 100% and 93.8%, respectively, and susceptibility to these drugs with specificities of 99.6%, 100%, 98.5% and 100%, respectively. Performance of the LPA was poorer, with sensitivity of 83.3% for rifampicin and 85.7% for isoniazid resistance. Five isolates were classified as susceptible according to phenotypic DST (1 for rifampicin, 4 for ethambutol) while WGS detected resistance mutations in rpoB and embB genes. WGS, used under appropriate quality-control conditions, has good performance to predict the resistance profile for the four first-line drugs and can correct phenotypic DST results. This study highlights the need for future guidelines recommending WGS as the initial tool in routine practice in areas where the prevalences of TB and drug-resistant MTB are low.

Cross-species transmission of viruses poses a sustained threat to public health. Due to increased contact between humans and other animal species the possibility exists for cross-species transmissions and ensuing disease outbreaks. By using conventional PCR amplification and next generation sequencing, we obtained 130 partial or full genome kobuvirus sequences from humans in a sentinel cohort in Vietnam and various mammalian hosts including bats, rodents, pigs, cats, and civets. The evolution of kobuviruses in different hosts was analysed using Bayesian phylogenetic methods. We estimated and compared time of origin of kobuviruses in different host orders; we also examined the cross-species transmission of kobuviruses within the same host order and between different host orders. Our data provide new knowledge of rodent and bat kobuviruses, which are most closely related to human kobuviruses. The novel bat kobuviruses isolated from bat roosts in Southern Vietnam were genetically distinct from previously described bat kobuviruses, but closely related to kobuviruses found in rodents. We additionally found evidence of frequent cross-species transmissions of kobuviruses within rodents. Overall, our phylogenetic analyses reveal multiple cross-species transmissions both within and among mammalian species, which increases our understanding of kobuviruses genetic diversity and the complexity of their evolutionary history.

Highly pathogenic avian influenza (HPAI) A(H5N1) viruses pose a significant economic burden to the poultry industry worldwide and have pandemic potential. Poultry vaccination against HPAI A(H5N1) viruses has been an important component of HPAI control measures and has been performed in Vietnam since 2005. To systematically assess antigenic matching of current vaccines to circulating field variants, we produced a panel of chicken and ferret antisera raised against historical and contemporary Vietnamese reference viruses representing clade variants that were detected between 2001 and 2014. The antisera were used for hemagglutination inhibition (HI) assays to generate data sets for analysis by antigenic cartography, allowing for a direct comparison of results from chicken or ferret antisera. HI antigenic maps, developed with antisera from both hosts, revealed varying patterns of antigenic relationships and clustering of viruses that were dependent on the clade of viruses analyzed. Antigenic relationships between existing poultry vaccines and circulating field viruses were also aligned with in vivo protection profiles determined by previously reported vaccine challenge studies. Our results establish the feasibility and utility of HPAI A(H5N1) antigenic characterization using chicken antisera and support further experimental and modeling studies to investigate quantitative relationships between genetic variation, antigenic drift and correlates of poultry vaccine protection in vivo.

Duck-targeted vaccines to protect against avian influenza are critically needed to aid in influenza disease control efforts in regions where ducks are endemic for highly pathogenic avian influenza (HPAI). Duck enteritis virus (DEV) is a promising candidate viral vector for development of vaccines targeting ducks, owing to its large genome and narrow host range. The clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 system is a versatile gene-editing tool that has proven beneficial for gene modification and construction of recombinant DNA viral vectored vaccines. Currently, there are two commonly used methods for gene insertion: non-homologous end-joining (NHEJ) and homology-directed repair (HDR). Owing to its advantages in efficiency and independence from molecular requirements of the homologous arms, we utilized NHEJ-dependent CRISPR/Cas9 to insert the influenza hemagglutinin (HA) antigen expression cassette into the DEV genome. The insert was initially tagged with reporter green fluorescence protein (GFP), and a Cre-Lox system was later used to remove the GFP gene insert. Furthermore, a universal donor plasmid system was established by introducing double bait sequences that were independent of the viral genome. In summary, we provide proof of principle for generating recombinant DEV viral vectored vaccines against the influenza virus using an integrated NHEJ-CRISPR/Cas9 and Cre-Lox system.

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.

Contains fulltext : 177468.pdf (Publisher’s version ) (Open Access) 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.

H9N2 avian influenza viruses are enzootic in poultry across Asia and North Africa, where they pose a threat to human health as both zoonotic agents and potential pandemic candidates. Poultry vaccination against H9N2 viruses has been employed in many regions; however, vaccine effectiveness is frequently compromised due to antigenic drift arising from amino acid substitutions in the major influenza virus antigen hemagglutinin (HA). Using selection with HA-specific monoclonal antibodies, we previously identified H9N2 antibody escape mutants that contained deletions of amino acids in the 220 loop of the HA receptor binding sites (RBSs). Here we analyzed the impact of these deletions on virus zoonotic infection characteristics and fitness. We demonstrated that mutant viruses with RBS deletions are able to escape polyclonal antiserum binding and are able to infect and be transmitted between chickens. We showed that the deletion mutants have increased binding to human-like receptors and greater replication in primary human airway cells; however, the mutant HAs also displayed reduced pH and thermal stability. In summary, we infer that variant influenza viruses with deletions in the 220 loop could arise in the field due to immune selection pressure; however, due to reduced HA stability, we conclude that these viruses are unlikely to be transmitted from human to human by the airborne route, a prerequisite for pandemic emergence. Our findings underscore the complex interplay between antigenic drift and viral fitness for avian influenza viruses as well as the challenges of predicting which viral variants may pose the greatest threats for zoonotic and pandemic emergence. IMPORTANCE Avian influenza viruses, such as H9N2, cause disease in poultry as well as occasionally infecting humans and are therefore considered viruses with pandemic potential. Many countries have introduced vaccination of poultry to try to control the disease burden; however, influenza viruses are able to rapidly evolve to escape immune pressure in a process known as “antigenic drift.” Previously, we experimentally generated antigenic-drift variants in the laboratory, and here, we test our “drifted” viruses to assess their zoonotic infection characteristics and transmissibility in chickens. We found that the drifted viruses were able to infect and be transmitted between chickens and showed increased binding to human-like receptors. However, the drift mutant viruses displayed reduced stability, and we predict that they are unlikely to be transmitted from human to human and cause an influenza pandemic. These results demonstrate the complex relationship between antigenic drift and the potential of avian influenza viruses to infect humans.

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.

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.

Coordinated and synchronous virological surveillance for zoonotic viruses in both human clinical cases and animal reservoirs provides an opportunity to identify interspecies virus movement. Rotavirus is an important cause of viral gastroenteritis in humans and animals. We have documented the rotavirus diversity within co-located humans and animals sampled from the Mekong delta region of Vietnam using a primer-independent, agnostic, deep sequencing approach. A total of 296 stool samples (146 from diarrhoeal human patients and 150 from pigs living in the same geographical region) were directly sequenced, generating the genomic sequences of 60 human rotaviruses (all group A) and 31 porcine rotaviruses (13 group A, 7 group B, 6 group C and 5 group H). Phylogenetic analyses showed the co-circulation of multiple distinct rotavirus group A (RVA) genotypes/strains, many of which were divergent from the strain components of licensed RVA vaccines, as well as considerable virus diversity in pigs including full genomes of rotaviruses in groups B, C and H, none of which have been previously reported in Vietnam. Furthermore the detection of an atypical RVA genotype constellation (G4-P[6]-I1-R1-C1-M1-A8-N1-T7-E1-H1) in a human patient and a pig from the same region provides some evidence for a zoonotic event

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.

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.

Highlights ► A novel real-time RT-PCR using specific locked nucleic acid probes is described. ► The assay is quantitative and distinguishes RSV subgroup A & B. ► Compared with a commercial multiplex PCR using 264 respiratory samplesin Vietnam. ► Sensitivity was significantly higher with detection a rate of 32 vs. 24%., 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 × 101 and 6.0 × 102 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.

International audience; 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.

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 (IC 50 ) titers did not exceed 10, although both IC 50 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 IC 50 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 IC 50 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.

SUMMARYPorcine 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.

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.

To the Editor: Streptococcus suis, an opportunistic pathogen of swine, is an emerging zoonotic pathogen among humans (1). In Vietnam, S. suis is the leading cause of human acute bacterial meningitis (2). Infection in humans is associated with direct exposure to infected pigs or infected raw or undercooked pork products (3). Of the 35 S. suis serotypes, only a limited number are pathogenic for pigs, and clinical cases in humans have most frequently been attributed to serotype 2 (SS2) (1). In Vietnam during September 2006–November 2007, the carrier rate of S. suis among slaughterhouse pigs was 41% (222/542); SS2 was the most frequently identified serotype in 14% (45/317) of S. suis isolations (4). Porcine respiratory and reproductive syndrome (PRRS) is a major disease affecting the swine industry globally; the severity of PRRS in pigs can be increased by co-infection with S. suis (5). In 2010, PRRS outbreaks in swine were reported in 49 of 63 Vietnamese provinces (Technical Appendix Figure) (6). To understand the potential implications of swine PRRS outbreaks for human S. suis disease, we investigated co-infections of S. suis and PRRS virus (PRRSV) in sick pigs in 3 provinces of Vietnam during the PRRS outbreaks in 2010 (Technical Appendix Figure). We sampled 108 farms reporting pigs that had a clinical syndrome consistent with PRRSV infections in the provinces of Thai Binh (May), Tien Giang (July), and Soc Trang (July). Samples were blood from sick febrile pigs and postmortem tissue from freshly culled pigs. To confirm swine PRRS outbreaks, we performed reverse transcription real-time PCR on 1 randomly selected plasma sample from each farm (7). A total of 103 (95%) plasma samples from 103 farms tested positive for PRRSV (Chinese genotype). We additionally selected 3 PRRSV-positive farms per province for comprehensive PRRSV screening of all 42 sampled pigs; 100% of samples from the 9 farms were PRRSV positive. After swine outbreaks ended, blood samples from 52 healthy pigs from 10 farms that had no recent history of PRRS were collected from Tien Giang Province (March 2011). None of the 52 plasma samples from the 10 control farms tested positive for PRRSV. We investigated the presence of SS2 in blood and tissue samples from pigs on PRRS- and non-PRRS–affected farms by bacterial culture (Technical Appendix Table). A total of 534 specimens from sick pigs yielded 9 (1.7%) SS2 isolates. One (2%) of 52 specimens from the healthy control pigs yielded a non-SS2 S. suis isolate. S. suis has been proposed to contribute to the spread of antimicrobial resistance genes to other human pathogenic streptococci (8). The antimicrobial susceptibility results of 9 SS2 isolates by disk diffusion (9) revealed a high prevalence (6/9, 66%) of resistance to tetracycline, tobramycin, enrofloxacin, and either marbofloxacin or chloramphenicol. PCR amplification of the 16SrDNA gene (10) and the cps2J gene (2) was performed on all blood samples to detect S. suis and SS2, respectively. Ninety-two (18%) of 521 sick pigs from PRRSV outbreak farms were systemically infected with S. suis. In contrast, no healthy pigs from control farms were positive for S. suis by PCR (Technical Appendix Table). The SS2-cps2J–specific PCR was positive for 58 (11%) of 521 samples, and the S. suis-16SrDNA PCR was positive for 55 (11%). Twenty-one of the 16SrDNA-positive samples also were positive for cps2J-PCR, which indicated that 34 (7%) sick pigs were infected with non-SS2 strains. Therefore, SS2 accounted for most (58 [63%] of 92) S. suis–positive detections. The bacterial load of SS2 in blood ranged from 1 × 103 CFU/mL–1 to 8.3 × 106 CFU/mL–1 (median 9.2 × 103 CFU/mL–1). Overall, SS2 was found in 58 (11%) sick pigs and on 33 (32%) PRRS outbreak farms. The higher prevalence (92 [18%]) of systemic infections of S. suis and SS2 with high bacterial load in pigs from PRRS outbreak farms compared with prevalence on nonoutbreak farms (1 [2%] of 52) suggests increased systemic S. suis infections during swine PRRS outbreaks (p = 0.001, Fisher exact test). We investigated the possible association between swine PRRS outbreaks and human S. suis infection. Case reports of confirmed human infections during 2007–2010 at the 2 tertiary referral hospitals in Hanoi and Ho Chi Minh City were reviewed. The number of human S. suis infection cases increased in August 2010 in southern Vietnam and doubled in northern Vietnam during May–August and October–November 2010 (Figure). Swine PRRS outbreaks were reported during June–September and March–December 2010 in southern and northern provinces, respectively (6) (Technical Appendix Figure). Most patients with S. suis infection during these periods resided in provinces reporting swine PRRS outbreaks. Our data suggest a possible temporal association between swine PRRS outbreaks and human S. suis infections. Figure Monthly distribution of human Streptococcus suis infections in 2 referral hospitals, Vietnam, 2007–2010. Humans infected with S. suis during 2007–2009 are presented as mean total cases per month. Dark gray and black bars represent the ... We demonstrated increased prevalence of systemic S. suis and SS2 infection in pigs co-infected with PRRSV during the 2010 swine outbreaks in Vietnam. The results indicate an increased risk for potential zoonotic transmission of S. suis to humans during outbreaks of PRRS in swine. Technical Appendix: Systemic Streptococcus suis infections in sick pigs from farms with confirmed porcine reproductive and respiratory syndrome and temporal distribution of outbreaks of porcine reproductive and respiratory syndrome in swine, 3 provinces, Vietnam, 2010. Click here to view.(151K, pdf)

The prevailing paradigm of yellow fever virus (YFV) ecology in South America is that of wandering epizootics. The virus is believed to move from place to place in epizootic waves involving monkeys and mosquitoes, rather than persistently circulating within particular locales. After a large outbreak of YFV illness in Peru in 1995, we used phylogenetic analyses of virus isolates to reexamine the hypothesis of virus movement. We sequenced a 670-nucleotide fragment of the prM/E gene region of from 25 Peruvian YFV samples collected from 1977 to 1999, and delineated six clades representing the states (Departments) of Puno, Pasco, Junin, Ayacucho, San Martin/Huanuco, and Cusco. The concurrent appearance of at least four variants during the 1995 epidemic and the genetic stability of separate virus lineages over time, indicate that Peruvian YFV is locally maintained and circulates continuously in discrete foci of enzootic transmission.

International audience; Leptospirosis is a zoonosis known to be endemic in the Mekong Delta of Vietnam, even though clinical reports are uncommon. We investigated leptospira infection in rats purchased in food markets during the rainy season (October) (n = 150), as well as those trapped during the dry season (February-March) (n = 125) in the region using RT-PCR for the lipL32 gene, confirmed by 16S rRNA, as well as by the microscopic agglutination test (MAT). Results were compared with the serovar distribution of human cases referred from Ho Chi Minh City hospitals (2004-2012) confirmed by MAT (n = 45). The MAT seroprevalence among rats was 18.3%. The highest MAT seroprevalence corresponded, in decreasing order, to: Rattus norvegicus (33.0%), Bandicota indica (26.5%), Rattus tanezumi (24.6%), Rattus exulans (14.3%), and Rattus argentiventer (7.1%). The most prevalent serovars were, in descending order: Javanica (4.6% rats), Lousiana (4.2%), Copenageni (4.2%), Cynopterie (3.7%), Pomona (2.9%), and Icterohaemorrhagiae (2.5%). A total of 16 rats (5.8%) tested positive by RT-PCR. Overall, larger rats tended to have a higher prevalence of detection. There was considerable agreement between MAT and PCR (kappa = 0.28 [0.07-0.49]), although significantly more rats were positive by MAT (McNemar 29.9 (p < 0.001). MAT prevalence was higher among rats during the rainy season compared with rats in the dry season. There are no current available data on leptospira serovars in humans in the Mekong Delta, although existing studies suggest limited overlapping between human and rat serovars. Further studies should take into account a wider range of potential reservoirs (i.e., dogs, pigs) as well as perform geographically linked co-sampling of humans and animals to establish the main sources of leptospirosis in the region.

International audience; A survey of Bartonella spp. from 275 rats purchased in food markets (n=150) and trapped in different ecosystems (rice field, forest, and animal farms) (n=125) was carried out during October, 2012-March, 2013, in the Mekong Delta of Vietnam. The overall Bartonella spp. prevalence detected by culture and PCR in blood was 14.9% (10.7-19.1%), the highest corresponding to Rattus tanezumi (49.2%), followed by Rattus norvegicus (20.7%). Trapped rats were also investigated for the presence and type of chiggers (larvae of trombiculid mites), and Bartonella spp. were investigated on chigger pools collected from each rat by RT-PCR. A total of five Bartonella spp. were identified in rats, three of which (B. elizabethae, B. rattimassiliensis, and B. tribocorum) are known zoonotic pathogens. Among trapped rats, factors independently associated with increased prevalence of Bartonella spp. included: (1) Rat species (R. tanezumi); (2) the number of Trombiculini-Blankaartia and Schoengastiini-Ascoschoengastia mites found on rats; and (3) the habitat of the rat (i.e., forest/fields vs. animal farms). The prevalence of Bartonella infection among chiggers from Bartonella spp.-positive R. tanezumi rats was 5/25 (25%), compared with 1/27 (3.7%) among Bartonella spp.-negative R. tanezumi rats (relative risk [RR]=5.4, 95% confidence interval [CI] 0.68-43.09). The finding of Bartonella spp.-positive chiggers on Bartonella spp.-negative rats is strongly suggestive of a transovarial transmission cycle. Rats are ubiquitous in areas of human activity and farms in the Mekong Delta; in addition, trapping and trading of rats for food is common. To correctly assess the human risks due to rat trapping, marketing, and carcass dressing, further studies are needed to establish the routes of transmission and cycle of infection. The widespread presence of these zoonotic pathogens in rats and the abundance of human-rat interactions suggest that surveillance efforts should be enhanced to detect any human cases of Bartonella infection that may arise.

SUMMARYA 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

Human respiratory syncytial virus (RSV) is the major cause of lower respiratory tract infections in children

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.

Active surveillance for avian influenza (AI) 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 clade 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 clade variants and the antigenic divergence previously described for these clades, current AI poultry vaccines used in Vietnam may require bivalent formulations containing representatives of both clade 1.1 and clade 2.3.2.1 viruses.

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.

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.

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., IMPORTANCE Acute central nervous system (CNS) infections cause substantial morbidity and mortality, but the etiology frequently remains unknown, which hampers development of therapeutic or preventive strategies. Hence, identification of novel pathogens is essential and is facilitated by current next-generation sequencing-based methods. Using such technology, we identified and characterized the full genome of a novel cyclovirus in cerebrospinal fluid (CSF) specimens from two Vietnamese patients with CNS infections of unknown etiology, which was subsequently detected in none of 122 CSF specimens from patients with noninfectious neurological disorders but 4% of 642 CSF specimens from Vietnamese patients with suspected or confirmed CNS infections. Similar detection rates in feces from healthy children suggested food-borne or orofecal transmission routes, while frequent detection in feces from Vietnamese pigs and poultry (average, 58%) suggested the existence of animal reservoirs for such transmission. Further studies are needed to address the epidemiology and pathogenicity of this novel, potentially zoonotic virus.

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.

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 multiple 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%), parainfluenxa virus 1-3 (6%), adenovirus (5%), and human rhinovirus A (4%). Co-infections with multiple viruses were dectected 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 of 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.

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.

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.

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., Author Summary Throughout the 18th and 19th centuries, yellow fever was one of the most dreaded of diseases in New and Old world port cities. Large-scale epidemics of yellow fever helped shape colonial expansion in both the Americas and in Africa, and the medical and scientific developments associated with control of the virus have been a favored topic of historians for many years. The most commonly cited hypothesis of the origin of yellow fever virus (YFV) in the Americas is that the virus was introduced from Africa, along with Aedes Aegypti mosquitoes, in the bilges of sailing vessels during the slave trade. Although the hypothesis of a slave trade introduction is often repeated, it has not been subject to rigorous examination using gene sequence data and modern phylogenetic techniques for estimating divergence times. Herein we have assembled a comprehensive data set of gene sequences for YFV, which we used to infer the time-scale and evolutionary history of YFV. These data show that the spread of YFV to the Americas corresponds closely with the routes and timing of the slave trade. Overall, this study demonstrates how molecular epidemiological studies can provide new insight into debates on the origin and spread of infectious disease.

Examining viral isolates collected over 66 years shows divergence into clades and potential dispersal by human migration., 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 Rondônia, 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.

Summary 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, Highlights • The determinants of influenza immunity were examined in an unvaccinated cohort. • The risk of H3N2 and B infection decreased with increasing pre-season HI titer. • Pre-season HI titer had less effect on H1N1 infection. • H1N1 immunity increased with age and seasonal H1N1 induced pandemic H1N1 immunity. • The contribution of non-HI antibodies to immunity may be relatively high for H1N1.