A single amino acid substitution in the NS4B protein of Dengue virus confers enhanced virus growth and fitness in human cells in vitro through IFN-dependent host response.

Dengue virus (DENV) replication between mosquito and human hosts is hypothesized to be associated with viral determinants that interact in a differential manner between hosts. However, the understanding of inter-host viral determinants that drive DENV replication and growth between hosts is limited. Through the use of clinical isolates, we identified an amino acid variation of Ala, Met and Val at position 116 of DENV-1 NS4B. While the proportion of virus with the NS4B-116V variant remained constantly high in serial passages in a mosquito cell line, populations of the NS4B-116M and NS4B-116A variants became dominant after serial passages in mammalian cell lines. Using recombinant DENV-1 viruses, the Val to Ala or Met alteration at position NS4B-116 (rDENV-1-NS4B-116A and rDENV-1-NS4B-116M) resulted in enhanced virus growth in human cells in comparison to the clone with Val at NS4B-116 (rDENV-1-NS4B-116V). However, the reverse phenomenon was observed in a mosquito cell line. Additionally, in a human cell line, differential levels of IFN-α/ß and IFN-stimulated gene expressions (IFIT3, IFI44L, OAS1) suggested that the enhanced viral growth was dependent on the ability of the NS4B protein to hamper host IFN response during the early phase of infection. Overall, we identified a novel and critical viral determinant at the pTMD3 of NS4B region that displayed differential effects on DENV replication and fitness in human and mosquito cell lines. Taken together, the results suggest the importance of the NS4B protein in virus replication and adaptation between hosts.

Effect of an asparagine-to-serine mutation at position 294 in neuraminidase on the pathogenicity of highly pathogenic H5N1 influenza A virus.

Like the histidine-to-tyrosine substitution at position 274 in neuraminidase (NA H274Y), an asparagine-to-serine mutation at position 294 in this protein (NA N294S) confers oseltamivir resistance to highly pathogenic H5N1 influenza A viruses. However, unlike viruses with the NA H274Y mutation, the properties of viruses possessing NA N294S are not well understood. Here, we assessed the effect of the NA N294S substitution on the replication and pathogenicity of human H5N1 viruses and on the efficacy of the NA inhibitors oseltamivir and zanamivir in mouse and ferret models. Although NA N294S-possessing H5N1 viruses were attenuated in mice and ferrets compared to their oseltamivir-sensitive counterparts, one of the infected ferrets died from systemic infection, demonstrating the potential lethality in ferrets of oseltamivir-resistant H5N1 viruses with the NA N294S substitution. The efficacy of oseltamivir, but not that of zanamivir, against an NA N294S-possessing virus was substantially impaired both in ferrets and in vitro. These results demonstrate the considerable pathogenicity of NA N294S substitution-possessing H5N1 viruses and underscore the importance of monitoring the emergence of the NA N294S mutation in circulating H5N1 viruses.

Characterization of oseltamivir-resistant 2009 H1N1 pandemic influenza A viruses.

Influenza viruses resistant to antiviral drugs emerge frequently. Not surprisingly, the widespread treatment in many countries of patients infected with 2009 pandemic influenza A (H1N1) viruses with the neuraminidase (NA) inhibitors oseltamivir and zanamivir has led to the emergence of pandemic strains resistant to these drugs. Sporadic cases of pandemic influenza have been associated with mutant viruses possessing a histidine-to-tyrosine substitution at position 274 (H274Y) in the NA, a mutation known to be responsible for oseltamivir resistance. Here, we characterized in vitro and in vivo properties of two pairs of oseltaimivir-sensitive and -resistant (possessing the NA H274Y substitution) 2009 H1N1 pandemic viruses isolated in different parts of the world. An in vitro NA inhibition assay confirmed that the NA H274Y substitution confers oseltamivir resistance to 2009 H1N1 pandemic viruses. In mouse lungs, we found no significant difference in replication between oseltamivir-sensitive and -resistant viruses. In the lungs of mice treated with oseltamivir or even zanamivir, 2009 H1N1 pandemic viruses with the NA H274Y substitution replicated efficiently. Pathological analysis revealed that the pathogenicities of the oseltamivir-resistant viruses were comparable to those of their oseltamivir-sensitive counterparts in ferrets. Further, the oseltamivir-resistant viruses transmitted between ferrets as efficiently as their oseltamivir-sensitive counterparts. Collectively, these data indicate that oseltamivir-resistant 2009 H1N1 pandemic viruses with the NA H274Y substitution were comparable to their oseltamivir-sensitive counterparts in their pathogenicity and transmissibility in animal models. Our findings highlight the possibility that NA H274Y-possessing oseltamivir-resistant 2009 H1N1 pandemic viruses could supersede oseltamivir-sensitive viruses, as occurred with seasonal H1N1 viruses.

The HA and NS genes of human H5N1 influenza A virus contribute to high virulence in ferrets.

Highly pathogenic H5N1 influenza A viruses have spread across Asia, Europe, and Africa. More than 500 cases of H5N1 virus infection in humans, with a high lethality rate, have been reported. To understand the molecular basis for the high virulence of H5N1 viruses in mammals, we tested the virulence in ferrets of several H5N1 viruses isolated from humans and found A/Vietnam/UT3062/04 (UT3062) to be the most virulent and A/Vietnam/UT3028/03 (UT3028) to be avirulent in this animal model. We then generated a series of reassortant viruses between the two viruses and assessed their virulence in ferrets. All of the viruses that possessed both the UT3062 hemagglutinin (HA) and nonstructural protein (NS) genes were highly virulent. By contrast, all those possessing the UT3028 HA or NS genes were attenuated in ferrets. These results demonstrate that the HA and NS genes are responsible for the difference in virulence in ferrets between the two viruses. Amino acid differences were identified at position 134 of HA, at positions 200 and 205 of NS1, and at positions 47 and 51 of NS2. We found that the residue at position 134 of HA alters the receptor-binding property of the virus, as measured by viral elution from erythrocytes. Further, both of the residues at positions 200 and 205 of NS1 contributed to enhanced type I interferon (IFN) antagonistic activity. These findings further our understanding of the determinants of pathogenicity of H5N1 viruses in mammals.

Occurrence of the Omicron variant of SARS-CoV-2 in northern Viet Nam in early 2022.

The Omicron variant caused a surge of infections with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in Viet Nam in early 2022, signalling community transmission. We report on active whole-genome sequencing surveillance of positive SARS-CoV-2 samples collected at that time in northern Viet Nam from international arrivals and community clusters. We used an amplicon protocol developed with 14 polymerase chain reaction products and the Illumina iSeq 100 platform. Overall, 213 nasopharyngeal or throat swabs were analysed, of which 172 samples were identified with the Omicron variant. Of these, 80 samples were collected from community cases in February 2022, among which 59 samples were sublineage BA.2 and one sample was the recombinant XE variant. Our results indicated that Omicron had replaced Delta as the dominant variant in a very short period of time and that continuously conducting active whole-genome sequencing surveillance is necessary in monitoring the evolution and genomic diversity of SARS-CoV-2 in Viet Nam.

Cytokine production by primary human macrophages infected with highly pathogenic H5N1 or pandemic H1N1 2009 influenza viruses.

Highly pathogenic H5N1 avian influenza viruses have caused infection in humans, with a high mortality rate, since 1997. While the pathogenesis of this infection is not completely understood, hypercytokinaemia and alveolar macrophages are thought to play a role. To gain further insight into the cytokine-mediated pathogenesis of this infection in humans, we measured various cytokines produced by primary human macrophages infected with H5N1, pandemic H1N1 or seasonal influenza viruses. We found that many cytokines were produced at higher levels on infection with the H5N1 strains tested compared with seasonal influenza viruses. Interestingly, the extent of cytokine induction varied among the H5N1 strains and did not correlate with replicative ability in macrophages. Further, a pandemic H1N1 virus induced higher levels of several cytokines compared with seasonal viruses and some H5N1 strains. Our results demonstrate that high cytokine induction is not a universal feature of all H5N1 viruses.

Virological characteristics of cases of COVID-19 in northern Viet Nam, January-May 2020.

Background: Viet Nam confirmed its first case of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection on 23 January 2020 among travellers from Wuhan, China, and experienced several clusters of community transmission until September. Viet Nam implemented an aggressive testing, isolation, contact tracing and quarantine strategy in response to all laboratory-confirmed cases. We report the results of SARS-CoV-2 testing during the first half of 2020 in northern Viet Nam. Methods: Between January and May 2020, 15 650 upper respiratory tract specimens were collected from 14 470 suspected cases and contacts in northern Viet Nam. All were tested for SARS-CoV-2 by real-time RT-PCR. Individuals with positive specimens were tested every three days until two tests were negative. Positive specimens from 81 individuals were cultured. Results: Among 14 470 tested individuals, 158 (1.1%) cases of SARS-CoV-2 infection were confirmed; 89 were imported and 69 were associated with community transmission. Most patients (122, 77%) had negative results after two tests, while 11 and 4 still tested positive when sampled a third and fourth time, respectively. SARS-CoV-2 was isolated from 29 of 81 specimens (36%) with a cycle threshold (Ct) value < 30. Seven patients who tested positive again after testing negative had Ct values > 30 and negative cultures. Conclusion: Early, widespread testing for SARS-CoV-2 in northern Viet Nam identified very few cases, which, when combined with other aggressive strategies, may have dramatically contained the epidemic. We observed rapid viral clearance and very few positive results after clearance. Large-scale molecular diagnostic testing is a critical part of early detection and containment of COVID-19 in Viet Nam and will remain necessary until vaccination is widely implemented.

Association of public health interventions and COVID-19 incidence in Vietnam, January to December 2020.

BACKGROUND: Vietnam implemented various public health interventions such as contact tracing and testing, mandatory quarantine, and lockdowns in response to coronavirus disease 2019 (COVID-19). However, the effects of these measures on the epidemic remain unclear. METHODS: This article describes the public health interventions in relation to COVID-19 incidence. Maximum likelihood estimations were used to assess containment delays (time between symptom onset and start of isolation) and multivariable regression was employed to identify associated factors between interventions and COVID-19 incidence. The effective reproductive numbers (Rt) were calculated based on transmission pairs. RESULTS: Interventions were introduced periodically in response to the epidemic. Overall, 817 (55.4%) among 1474 COVID-19 cases were imported. Based on a serial interval of 8.72 ± 5.65 days, it was estimated that Rt decreased to below 1 (lowest at 0.02, 95% CI 0-0.12) during periods of strict border control and contact tracing, and increased ahead of new clusters. The main method to detect cases shifted over time from passive notification to active case-finding at immigration or in lockdown areas, with containment delays showing significant differences between modes of case detection. CONCLUSIONS: A combination of early, strict, and consistently implemented interventions is crucial to control COVID-19. Low-middle income countries with limited capacity can contain COVID-19 successfully using non-pharmaceutical interventions.

Sensitivity of influenza rapid diagnostic tests to H5N1 and 2009 pandemic H1N1 viruses.

Simple and rapid diagnosis of influenza is useful for making treatment decisions in the clinical setting. Although many influenza rapid diagnostic tests (IRDTs) are available for the detection of seasonal influenza virus infections, their sensitivity for other viruses, such as H5N1 viruses and the recently emerged swine origin pandemic (H1N1) 2009 virus, remains largely unknown. Here, we examined the sensitivity of 20 IRDTs to various influenza virus strains, including H5N1 and 2009 pandemic H1N1 viruses. Our results indicate that the detection sensitivity to swine origin H1N1 viruses varies widely among IRDTs, with some tests lacking sufficient sensitivity to detect the early stages of infection when the virus load is low.

Antibiotic-resistant Escherichia coli isolated from urban rodents in Hanoi, Vietnam.

Antimicrobial resistance (AMR) is a global public health concern for both clinical and veterinary medicine. Rodent feces are one of the major infectious sources of zoonotic pathogens including AMR bacteria. So far, there are limited studies reported focused on Escherichia coli isolated in rodent feces from rural and suburban areas in Vietnam. In this study, we investigated the prevalence of antimicrobial resistance in E. coli isolated from feces samples of 144 urban rodents caught in Hanoi, Vietnam. A total of 59 AMR E. coli was isolated from urban rodents of which 42 were multidrug-resistant (MDR) isolates (resistance to at least three classes of antimicrobial agents), four were extended-spectrum ß-lactamase (ESBL) producing isolates and five were colistin-resistant isolates. The highest prevalence of the resistance was against ampicillin (79.7%: 47/59), followed by tetracycline (78.0%: 46/59), nalidixic acid (67.8%: 40/59), sulfamethoxazole-trimethoprim (59.3%: 35/59), chloramphenicol (45.8%: 27/59), ciprofloxacin (44.1%: 26/59), cefotaxime (30.5%: 18/59), cefodizime (23.7%: 14/59), amoxicillin-clavulanate (22.0%: 13/59), and gentamicin (22.0%: 13/59). With regard to the virulence genes associated with diarrheagenic E. coli (DEC), only aaiC gene found in one AMR isolate. In general, the use of antimicrobials does not aim to treat rodents except for companion animals. However, our findings show the carriage of AMR and MDR E. coli in urban rodents and highlight the potential risk of rodents in Hanoi acting as a reservoir of transferable MDR E. coli, including ESBL-producing, colistin-resistant E. coli, and virulence-associated with DEC.

Neutralization Potency of Sera from Vietnamese Patients with Japanese Encephalitis (JE) against Genotypes I and V JE Viruses.

Japanese encephalitis virus (JEV) is classified into 5 genotypes (GI, GII, GIII, GIV, and GV), and the GI and GIII strains are the most widely distributed in JE endemic areas. In recent years, GV JEV has been detected in China and Korea, suggesting that GV JEV may invade other JE endemic areas, including Vietnam, and that more attention should be paid to the JEV strains circulating in these areas. In this study, we investigated the neutralization ability of the sera collected from 22 Vietnamese patients with JE who lived in northern Vietnam against the GI and GV JEV strains. In most cases, the ratios of the titer against GV to that against GI (GV:GI) were equal to or less than 1:4. However, the titer against GV JEV was equivalent (1:1) to that against GI JEV in only a few cases, and no serum had a ratio higher than 1:1. Thus, our results did not show convincing evidence that GV JEV was emerging in northern Vietnam in 2014.

Molecular epidemiology of Leptospira interrogans in Rattus norvegicus in Hanoi, Vietnam.

Leptospirosis is a zoonotic disease that is caused by pathogenic spirochaetes of Leptospira spp. and it has become a public health concern in urban localities in the tropics. Rats are important reservoir animals for the transmission of leptospirosis in urban areas. Leptospirosis is considered endemic in Vietnam. However, information on the causative Leptospira genotypes and serotypes in the country is limited. We investigated the carrier status of Leptospira spp. in rats captured in Hanoi by culturing and DNA detection. Isolates were characterized using a serological method and multiple-locus variable-number tandem repeat analysis (MLVA). We captured 144 rats (1 Rattus argentiventer, 135 R. norvegicus, and 8 R. rattus) and obtained 17 L. interrogans, determined by rrs sequencing, from R. norvegicus (12.6%). Sixteen of the isolates were serogroup Bataviae. Five of the 16 isolates exhibited an MLVA type identical to that of the serovar Bataviae reference strain Van Tienen, while there were nine repeats for the other 11 isolates at VNTR31 compared with the reference strain. The remaining isolate grew poorly, and we were unable to determine its serogroup. However, it had an MLVA type matching those of serogroup Pomona strains isolated from R. norvegicus in Japan. Three different flaB sequences were detected in 23 out of 81 R. norvegicus kidney tissue samples (28.4%) using nested PCR followed by DNA sequencing. Two of the sequences were identical with those of serogroups Bataviae and Pomona, and no strain with another sequence was detected in the present study. The present study reveals a high prevalence rate of L. interrogans among R. norvegicus in Hanoi, Vietnam, indicating a potential risk of rat-borne leptospirosis in the area. The present study also demonstrates that a fastidious L. interrogans strain circulates among rats and that molecular detection is crucial in facilitating the accurate determination of reservoir animals.

Multiple polymerase gene mutations for human adaptation occurring in Asian H5N1 influenza virus clinical isolates.

The role of the influenza virus polymerase complex in host range restriction has been well-studied and several host range determinants, such as the polymerase PB2-E627K and PB2-D701N mutations, have been identified. However, there may be additional, currently unknown, human adaptation polymerase mutations. Here, we used a database search of influenza virus H5N1 clade 1.1, clade 2.3.2.1 and clade 2.3.4 strains isolated from 2008-2012 in Southern China, Vietnam and Cambodia to identify polymerase adaptation mutations that had been selected in infected patients. Several of these mutations acted either alone or together to increase viral polymerase activity in human airway cells to levels similar to the PB2-D701N and PB2-E627K single mutations and to increase progeny virus yields in infected mouse lungs to levels similar to the PB2-D701N single mutation. In particular, specific mutations acted synergistically with the PB2-D701N mutation and showed synergistic effects on viral replication both in human airway cells and mice compared with the corresponding single mutations. Thus, H5N1 viruses in infected patients were able to acquire multiple polymerase mutations that acted cooperatively for human adaptation. Our findings give new insight into the human adaptation of AI viruses and help in avian influenza virus risk assessment.

An Updated Loop-Mediated Isothermal Amplification Method for Rapid Diagnosis of H5N1 Avian Influenza Viruses.

We designed a new set of primers for reverse transcriptase loop-mediated isothermal amplification (RTLAMP) to specifically amplify the HA gene of avian influenza viruses subtype H5N1. By testing nine H5N1 virus strains and 41 clinical samples collected in Northern Vietnam, we found that the new primers showed higher sensitivity and specificity than the previously published RT-LAMP primers and were comparable to the RT-PCR method currently recommended by WHO. These results suggest that our RT-LAMP assay may be a better choice as a diagnostic tool for current H5N1 influenza virus infection.

A cross-reactive neutralizing monoclonal antibody protects mice from H5N1 and pandemic (H1N1) 2009 virus infection.

A novel influenza (H1N1) virus caused an influenza pandemic in 2009, while highly pathogenic H5N1 avian influenza viruses have continued to infect humans since 1997. Influenza, therefore, remains a serious health threat. Currently, neuraminidase (NA) inhibitors are the mainstay for influenza therapy; however, drug-resistant mutants of seasonal H1N1 and H5N1 viruses have emerged highlighting the need for alternative therapeutic approaches. One such approach is antibody immunotherapy. Here, we show that the monoclonal antibody C179, which recognizes a neutralizing epitope common among H1, H2, H5, and H6 hemagglutinins (HAs), protected mice from a lethal challenge with various H5N1 and pandemic (H1N1) 2009 viruses when administered either intraperitoneally or intranasally. The protective efficacy of intranasally inoculated C179 was comparable to that of intraperitoneal administration. Our results suggest that direct administration of this anti-influenza antibody to viral replication sites is an effective strategy for prophylaxis and therapy.