Differential Infectivity of Human Neural Cell Lines by a Dengue Virus Serotype-3 Genotype-III with a Distinct Nonstructural Protein 2A (NS2A) Amino Acid Substitution Isolated from the Cerebrospinal Fluid of a Dengue Encephalitis Patient.

Dengue encephalitis is considered as a severe but unusual clinical presentation of dengue infection. Limited molecular information is available on the neurotropism of dengue virus (DENV), highlighting the need for further research. During a dengue outbreak in Vietnam in 2013, two DENV-3 strains were isolated, in which one was isolated from cerebrospinal fluid (CSF) samples from a dengue encephalitis patient and another strain was isolated from a patient with classical dengue fever in Hai Phong, Vietnam. DENV serotype-3 (DENV-3) isolated from these samples belonged to genotype III, marking the first report of this genotype in the country at that time. Genetic variation between both strains was elucidated by using a full genome sequencing by next-generation sequencing (NGS). The infectivity of the isolated DENV-3 strains was further characterized using human and mouse neuronal cell lines. Phylogenetic analysis of the isolates demonstrated high homogeneity between the CSF-derived and serum-derived DENV-3, in which the full genome sequences of the CSF-derived DENV-3 presented a Thr-1339-Ile mutation in the nonstructural 2A (NS2A) protein. The CSF-derived DENV-3 isolate grew preferentially in human neuronal cells, with a significant proportion of cells that were positive for nonstructural 1 (NS1), nonstructural 4B (NS4B), and nonstructural 5 (NS5) antigens. These results suggest that NS2A may be a crucial region in the neuropathogenesis of DENV-3 and its growth in human neuronal cells. Taken together, our results demonstrate that a CSF-derived DENV-3 has unique infectivity characteristics for human neuronal cells, which might play a crucial role in the neuropathogenesis of DENV infection.

Characterization of a Novel Alphaherpesvirus Isolated from the Fruit Bat Pteropus lylei in Vietnam.

Herpesviruses exist in nature within each host animal. Ten herpesviruses have been isolated from bats and their biological properties reported. A novel bat alphaherpesvirus, which we propose to name "Pteropus lylei-associated alphaherpesvirus (PLAHV)," was isolated from urine of the fruit bat Pteropus lylei in Vietnam and characterized. The entire genome sequence was determined to be 144,008 bp in length and predicted to include 72 genes. PLAHV was assigned to genus Simplexvirus with other bat alphaherpesviruses isolated from pteropodid bats in Southeast Asia and Africa. The replication capacity of PLAHV in several cells was evaluated in comparison with that of herpes simplex virus 1 (HSV-1). PLAHV replicated better in the bat-originated cell line and less in human embryonic lung fibroblasts than HSV-1 did. PLAHV was serologically related to another bat alphaherpesvirus, Pteropodid alphaherpesvirus 1 (PtAHV1), isolated from a Pteropus hypomelanus-related bat captured in Indonesia, but not with HSV-1. PLAHV caused lethal infection in mice. PLAHV was as susceptible to acyclovir as HSV-1 was. Characterization of this new member of bat alphaherpesviruses, PLAHV, expands the knowledge on bat-associated alphaherpesvirology.IMPORTANCE A novel bat alphaherpesvirus, Pteropus lylei-associated alphaherpesvirus (PLAHV), was isolated from urine of the fruit bat Pteropus lylei in Vietnam. The whole-genome sequence was determined and was predicted to include 72 open reading frames in the 144,008-bp genome. PLAHV is circulating in a species of fruit bats, Pteropus lylei, in Asia. This study expands the knowledge on bat-associated alphaherpesvirology.

Prevalence of Zika virus neutralizing antibodies in healthy adults in Vietnam during and after the Zika virus epidemic season: a longitudinal population-based survey.

BACKGROUND: Between 2016 and 2019, 265 cases of Zika virus (ZIKV) infection were reported in Vietnam, predominantly in southern Vietnam. In 2016, a case of ZIKV-associated microcephaly was confirmed in the Central Highlands, and several members of the infant's family were confirmed to be infected with ZIKV. The study aims to determine the level of immunity to ZIKV in the general population of the ZIKV epidemic region. METHODS: A total of 879 serum samples were collected from 801 participants between January 2017 and July 2018, during and after the ZIKV epidemic in Vietnam. The samples were tested for anti-ZIKV immunoglobulin M (IgM) and immunoglobulin G (IgG), and anti-dengue virus (DENV) IgG antibodies using enzyme-linked immunosorbent assays (ELISA). Plaque-reduction neutralization test (PRNT) for ZIKV was performed on all samples, and for DENV on the samples that ZIKV neutralizing antibody positive. RESULTS: A total of 83 (10.3%) participants had anti-ZIKV IgM. Of the 83, 6 were confirmed to be ZIKV antibodies positive using PRNT and anti-ZIKV IgG ELISA. Of the 718 participants who were anti-ZIKV IgM negative, a further 3 cases were confirmed as positive for antibodies against ZIKV. Of the 9 participants with ZIKV infection, 5 lived in the same village as the infant with ZIKV-associated microcephaly and the other 4 lived in 2 neighboring communes. Repeat samples were collected from the 83 ZIKV IgM positive participants 1.5 years after the first collection. No new cases of ZIKV infection were detected. In addition, 2 of 3 participants with anti-ZIKV NS1 IgG demonstrated a 4- to 8-fold increase in ZIKV neutralizing antibody titer. CONCLUSIONS: ZIKV was present in the area around Krong Buk, with the rate of ZIKV-specific antibodies was 1.1% in the community since at least 2016. While the low levels of circulation together with low seroprevalence suggests a limited outbreak in the region, the results also reflect on low levels of protective immunity to Zika within the population. These results provide a better understanding of the current ZIKV epidemic status in the region and demonstrate a need for implementation of more effective ZIKV infection control measures.

Pre-existing cross-reactive neutralizing activity against SARS-CoV-2 and seasonal coronaviruses prior to the COVID-19 pandemic (2014-2019) with limited immunity against recent emerging SARS-CoV-2 variants, Vietnam.

OBJECTIVES: SARS-CoV-2 transmission and epidemic potential is related to the population's immunity levels. As such, assessing different regions' preexisting immune responses to SARS-CoV-2 is important to understand the transmission potential of emerging SARS-CoV-2 variants. DESIGN: In 975 serum samples from Vietnam (2014 to 2019), anti-SARS-CoV-2 Immunoglobulin G levels were determined by enzyme-linked immunosorbent assay. Plaque reduction neutralization test (PRNT) was performed using Wuhan strain and variants of concern (VOCs). Cross-reactivity was confirmed by analyzing B-cell receptor (BCR) repertoire sequences and identifying BCR repertoire sequences-derived T-cell epitopes. RESULTS: Overall, 20.9% (n = 76/364) and 9.2% (n = 7) demonstrated SARS-CoV-2 neutralizing activity (PRNT50) against the Wuhan and Alpha strain, respectively. Neutralizing activity against Beta, Gamma, and Delta strains was absent (PRNT50<5) in all samples. Cross-reactive epitopes against SARS-CoV-2 and other coronavirus spike proteins were detected in the N-terminal domain, S2, and receptor-binding domain regions. CONCLUSIONS: Following BCR and major histocompatibility complex analysis, T-cell receptor-recognized epitope motif (TREM) among pathogenic coronaviruses and coronaviruses spike proteins were the top TREM peptide, suggesting that pre-existing immunity against SARS-CoV-2 in Vietnam was due to exposure to common cold coronaviruses. With limited immunity against emerging VOCs, further monitoring, and control of the epidemic, along with COVID-19 vaccine programs against VOCs, are necessary.

The 2017 Dengue virus 1 outbreak in northern Vietnam was caused by a locally circulating virus group.

BACKGROUND: Dengue virus (DENV) is a member of insect vector-borne viruses, and it causes dengue fever. Southeast Asia is the epi-center of dengue fever in the world. The characterization of the virus is essential to identify the transmission and evolution of DENV. OBJECTIVES: In 2017, there was an outbreak of Dengue virus type 1 (DENV1) in northern Vietnam and the neighboring countries. To identify the genetic character of the outbreak virus in the area, we conducted whole-genome sequencing analysis on the samples positive for the DENV1 along with real-time PCR. STUDY DESIGN: In total, 1026 blood samples were collected from patients with suspected dengue fever in Ha Nam and Hai Duong province, nearby areas of the capital of Vietnam. After screening by real-time PCR, 40 of DENV1 positive samples were subjected to whole-genome sequencing, and 28 complete coding sequences were obtained. RESULTS: All 28 sequences were genotype I of DENV1, which is dominant in the southeast and East Asian countries. The phylogenetic analysis of the E region showed that they fell into a single cluster with the reported sequences from Vietnam between 2009 and 2016, in which the isolates from other countries are very rare. Our results suggested that the 2017 outbreak in the area was caused by locally circulating viruses.

Direct Viral RNA Detection of SARS-CoV-2 and DENV in Inactivated Samples by Real-Time RT-qPCR: Implications for Diagnosis in Resource Limited Settings with Flavivirus Co-Circulation.

The RT-qPCR method remains the gold standard and first-line diagnostic method for the detection of SARS-CoV-2 and flaviviruses, especially in the early stage of viral infection. Rapid and accurate viral detection is a starting point in the containment of the COVID-19 pandemic and flavivirus outbreaks. However, the shortage of diagnostic reagents and supplies, especially in resource-limited countries that experience co-circulation of SARS-CoV-2 and flaviviruses, are limitations that may result in lesser availability of RT-qPCR-based diagnostic tests. In this study, the utility of RNA-free extraction methods was assessed for the direct detection of SARS-CoV-2 and DENV-2 in heat-inactivated or chemical-inactivated samples. The findings demonstrate that direct real-time RT-qPCR is a feasible option in comparison to conventional real-time RT-qPCR based on viral genome extraction-based methods. The utility of heat-inactivation and direct real-time RT-qPCR for SARS-CoV-2, DENV-2 viral RNA detection was demonstrated by using clinical samples of SARS-CoV-2 and DENV-2 and spiked cell culture samples of SARS-CoV-2 and DENV-2. This study provides a simple alternative workflow for flavivirus and SARS-CoV-2 detection that includes heat inactivation and viral RNA extraction-free protocols, with aims to reduce the risk of exposure during processing of SARS-CoV-2 biological specimens and to overcome the supply-chain bottleneck, particularly in resource limited settings with flavivirus co-circulation.

Discrepancies in Infectivity of Flavivirus and SARS-CoV-2 Clinical Samples: An Improved Assay for Infectious Virus Shedding and Viremia Assessment.

Infectivity and neutralizing antibody titers of flavivirus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are frequently measured using the conventional plaque assay. While the assay is useful in the determination of infectivity, conventional plaque assays generally possess lower sensitivity and are time-consuming compared to nucleic acid amplification tests. In this study, a microcrystalline cellulose (MCC), Avicel, was evaluated as an alternative to the conventional virus overlay medium, methylcellulose, for a plaque assay. The plaque assay was performed using dengue and COVID-19 clinical samples and laboratory-established flavivirus and SARS-CoV-2 strains. In virus titration of clinical samples, the plaques were significantly larger, and the virus titers were higher when Avicel MCC-containing overlay medium was used than with conventional methylcellulose overlay medium. In addition, for some clinical samples and laboratory virus strains, infectious particles were detected as plaques in the Avicel MCC-containing medium, but not in the conventional methylcellulose medium. The results suggest that the viremia titer determined using the new overlay medium containing Avicel MCC may better reflect the innate infectious and plaque-forming capabilities of clinical samples and better reflect virus infectivity.

Circulation of influenza B lineages in northern Viet Nam, 2007-2014.

INTRODUCTION: Influenza B viruses circulate throughout Viet Nam, and their activities vary by region. There have been two antigenically distinct lineages of influenza B viruses co-circulating in the past 20 years; however, only one lineage is selected as a component of contemporary trivalent seasonal influenza vaccines. To improve the understanding of circulating influenza B lineages and influenza vaccine mismatches, we report the virus lineages circulating in northern Viet Nam over an eight-year period (2007-2014). METHODS: Lineages of 331 influenza B viruses were characterized by haemagglutination inhibition assay against standard reference ferret (Yamagata) and sheep (Victoria) antisera. Sequence analysis of the haemagglutinin gene was performed in 64 selected influenza B isolates. RESULTS: The proportion of influenza B lineages changed by year. The Yamagata lineage predominated in 2007, 2008 and 2012; the Victoria lineage predominated in 2009-2014 except 2012. The two lineages showed continuous evolution over time. The Northern Hemisphere's influenza vaccine components were mismatched with the predominant circulating viruses in 2007, 2009 and 2014. DISCUSSION: The seasonality of influenza B activity is more variable in tropical and subtropical regions than in temperate zones. Our data showed a common co-circulation of both influenza B lineages in northern Viet Nam, and it was difficult to predict which one was the predominant lineage. Quadrivalent influenza vaccines containing both lineages may improve the effectiveness of influenza vaccine programmes in the future.

Oseltamivir resistance among influenza viruses: surveillance in northern Viet Nam, 2009-2012.

INTRODUCTION: Antiviral resistance has been reported in seasonal influenza A viruses and avian influenza A(H5N1) viruses in Viet Nam, raising concerns about the efficacy of treatment. METHODS: We analysed specimens from two sources during the period 2009-2012: influenza-positive samples from influenza-like illness patients at sentinel clinics in northern Viet Nam and isolates from patients with confirmed A(H5N1) infections. Pyrosequencing was used to detect mutations: H275Y [for A(H1N1) and A(H5N1)], E119V [for A(H3N2)] and I117V [for A(H5N1)]. A neuraminidase inhibition assay was used to determine the Inhibitory Concentration 50 (IC50) values for all influenza A and B isolates. RESULTS: There were 341 influenza A positive samples identified; influenza A(H1N1)pdm09 was identified most frequently (n = 215). In 2009, oseltamivir resistance was observed in 100% (19 of 19) of seasonal A(H1N1) isolates and 1.4% (3/215) of A(H1N1)pdm09 isolates. This H275Y mutation was not found in influenza subtypes A(H5N1) or A(H3N2) isolates. DISCUSSION: In Viet Nam, seasonal and A(H5N1) influenza vaccines are not currently available; thus, effective treatment is required. The presence of oseltamivir-resistant viruses is therefore a concern. Active surveillance for oseltamivir resistance among influenza viruses circulating in Viet Nam should be continued.

Oseltamivir resistance among influenza viruses: surveillance in northern Viet Nam, 2009–2012

Introduction: Antiviral resistance has been reported in seasonal influenza A viruses and avian influenza A(H5N1) viruses in Viet Nam, raising concerns about the efficacy of treatment. Methods: We analysed specimens from two sources during the period 2009–2012: influenza-positive samples from influenza-like illness patients at sentinel clinics in northern Viet Nam and isolates from patients with confirmed A(H5N1) infections. Pyrosequencing was used to detect mutations: H275Y [for A(H1N1) and A(H5N1)], E119V [for A(H3N2)] and I117V [for A(H5N1)]. A neuraminidase inhibition assay was used to determine the Inhibitory Concentration 50 (IC50) values for all influenza A and B isolates. Results: There were 341 influenza A positive samples identified; influenza A(H1N1)pdm09 was identified most frequently (n = 215). In 2009, oseltamivir resistance was observed in 100% (19 of 19) of seasonal A(H1N1) isolates and 1.4% (3/215) of A(H1N1)pdm09 isolates. This H275Y mutation was not found in influenza subtypes A(H5N1) or A(H3N2) isolates. Discussion: In Viet Nam, seasonal and A(H5N1) influenza vaccines are not currently available; thus, effective treatment is required. The presence of oseltamivir-resistant viruses is therefore a concern. Active surveillance for oseltamivir resistance among influenza viruses circulating in Viet Nam should be continued.