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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.

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.

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. 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. 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. 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. 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.

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.

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. 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. 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. 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.

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.

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.

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. 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.

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. 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. 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.

Coordinated and synchronous surveillance for zoonotic viruses in both human clinical cases and animal reservoirs provides an opportunity to identify interspecies virus movement. Rotavirus (RV) is an important cause of viral gastroenteritis in humans and animals. In this study, we document the RV 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 sixty human rotaviruses (all group A) and thirty-one porcine rotaviruses (thirteen group A, seven group B, six group C, and five group H). Phylogenetic analyses showed the co-circulation of multiple distinct RV 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.