Juliet E. Bryant

Out of Africa: A Molecular Perspective on the Introduction of Yellow Fever Virus into the Americas

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.

Host and viral features of human dengue cases shape the population of infected and infectious Aedes aegypti mosquitoes

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.

Viral Etiologies of Acute Respiratory Infections among Hospitalized Vietnamese Children in Ho Chi Minh City, 2004–2008

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.

Identification of a New Cyclovirus in Cerebrospinal Fluid of Patients with Acute Central Nervous System Infections

ABSTRACT 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.

Slaughterhouse Pigs Are a Major Reservoir of Streptococcus suis Serotype 2 Capable of Causing Human Infection in Southern Vietnam

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.

Early Pandemic Influenza (2009 H1N1) in Ho Chi Minh City, Vietnam: A Clinical Virological and Epidemiological Analysis

Rogier van Doorn and colleagues analyze the initial outbreak, attempts at containment, and establishment of community transmission of pandemic H1N1 influenza in Ho Chi Minh City, Vietnam.

Emergence of multiple clade 2.3.2.1 influenza A (H5N1) virus subgroups in Vietnam and detection of novel reassortants

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.

Serological Investigations of Flavivirus Prevalence in Khammouane Province, Lao People's Democratic Republic, 2007–2008

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.

A sensitive real-time PCR for detection and subgrouping of human respiratory syncytial virus.

Abstract 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.

An epidemiological investigation of Campylobacter in pig and poultry farms in the Mekong delta of Vietnam

SUMMARY 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.