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Antimicrobial resistance genes (ARGs) are associated with mobile genetic elements (MGEs) that conscript useful genes into the human–microbe and microbe–microbe battlefields. Thus, under intense selective pressure, ARGs have been constantly adapting and evolving, spreading among microbes. tmexCD-toprJ gene clusters, which encode resistance–nodulation–cell division (RND)-type efflux pumps, confer multidrug-resistance to clinically important antimicrobials, including tigecycline. Noteworthily, these gene clusters have emerged in gram-negative bacteria in humans, animals, and the environment worldwide by MGE-mediated transfer. Here we show a hidden MGE, strand-biased circularizing integrative element (SE), that is recently recognized to mediate transpositions of ARGs, associated with the spread of tmexCD-toprJ gene clusters. We identified multidrug-resistant isolates of Aeromonas species in a water environment in Vietnam that harbored multiple copies of tmexCD-toprJ in their chromosomes that were associated with SEs. In particular, Aeromonas hydrophila NUITM-VA1 was found to harbor two copies of a novel variant of tmexC3.3D3.3-topJ1 within cognate SEs, whereas Aeromonas caviae NUITM-VA2 harbored four copies of a novel variant of tmexC2D2.3-topJ2 within cognate SEs. Based on the nature of SE to incorporate a neighboring sequence into the circular form and reinsert it into target sites during transposition, we identified the order of intragenomic movements of tmexCD-toprJ gene clusters. Altogether, our findings suggest that most known subgroups of tmexCD-toprJ and their subvariants underwent transpositions among bacterial chromosomes and plasmids via SEs. Hence, a tmexCD-toprJ gene cluster ancestor may have been initially mobilized via SE, subsequently spreading among bacteria and evolving in new hosts.

The emergence of the mobile resistance-nodulation-division (RND)-type efflux pump tmexCD-toprJ gene cluster that confers multidrug resistance (MDR), including tigecycline resistance, in gram-negative bacteria poses a global public health threat. However, the spread of such clinically important antimicrobial resistance genes (ARGs) in the natural environment has not yet been well investigated. In this study, we investigated MDR aquatic bacteria in Vietnam. A carbapenem- and tigecycline-resistant Shewanella xiamenensis isolate NUITM-VS2 was obtained from urban drainage in Hanoi, Vietnam, in October 2021. S. xiamenensis NUITM-VS2 showed resistance to most antimicrobials tested, including tigecycline, tetracyclines, carbapenems, cephalosporins, fluoroquinolone, and aminoglycosides. Whole-genome analysis was performed by long- and short-read sequencing, resulting in the complete genome sequence consisting of one chromosome and five plasmid sequences. ARGs and plasmid replicons in the genome were detected using ResFinder with the custom ARG database, including all known tigecycline resistance genes, and PlasmidFinder, respectively. A 152.2-kb IncC plasmid, pNUITM-VS2_2, co-carried two mobile tigecycline resistance genes, tet(X4) and tmexC3.2D3.2-toprJ1. In addition, a 24.8-kb untypeable plasmid, pNUITM-VS2_4, carried the carbapenemase gene blaNDM-1. pNUITM-VS2_2 was transferred to Escherichia coli by conjugation, which simultaneously conferred high-level resistance against many antimicrobials, including tigecycline. To the best of our knowledge, this is the first report of the detection of the mobile RND-type efflux pump gene cluster tmexCD-toprJ in Shewanella species. Our results provide genetic evidence of the complexity of the dynamics of clinically important ARGs among aquatic bacteria, which could be important reservoirs for ARGs in the natural environment.

Approximately 200 O-serogroups of Vibrio cholerae have already been identified; however, only 2 serogroups, O1 and O139, are strongly related to pandemic cholera. The study of non-O1 and non-O139 strains has hitherto been limited. Nevertheless, there are other clinically and epidemiologically important serogroups causing outbreaks with cholera-like disease. Here, we report a comprehensive genome analysis of the whole set of V. cholerae O-serogroup reference strains to provide an overview of this important bacterial pathogen. It revealed structural diversity of the O-antigen biosynthesis gene clusters located at specific loci on chromosome 1 and 16 pairs of strains with almost identical O-antigen biosynthetic gene clusters but differing in serological patterns. This might be due to the presence of O-antigen biosynthesis-related genes at secondary loci on chromosome 2.

The dissemination and abundances of Vibrio species in aquatic environments are of interest, as some species cause emerging diseases in humans and in aquatic organisms like fish. It is suggested that Vibrio cholerae non-O1 infections of Plecoglossus altivelis ('ayu') were spread to various parts of Japan through the annual transplantation of juvenile fish. To investigate this, we used genome-aided tracing of 17 V. cholerae strains isolated from ayu between the 1970s and 1990s in different Japanese freshwater systems. The strains formed a genomic clade distinct from all known clades, which we designate as the Ayu clade. Two clonal genomic groups identified within the clade, Ayu-1 and Ayu-2, persisted for a few years (between 1977 to 1979 and 1987 to 1990, respectively), and clonal replacement of Ayu-1 by Ayu-2 took place over an 8-year period. Despite the high similarity between Ayu-1 and Ayu-2 (> 99.9% identity and > 97% fraction of genomes shared), differences in their gene repertoires were found, raising the possibility that they are phenotypically distinct. These results highlight the importance of genome-based studies for understanding the long-term dynamics of populations over the timescale of years.

In Vietnam, data on the risk factors for diarrhea at the community level remain sparse. This study aimed to provide an overview of diarrheal diseases in an agricultural community in Vietnam, targeting all age groups. Specifically, we investigated the incidence of diarrheal disease at the community level and described the potential risk factors associated with diarrheal diseases. In this prospective cohort study, a total of 1508 residents were enrolled during the 54-week study period in northern Vietnam. The observed diarrheal incidence per person-year was 0.51 episodes. For children aged

Tigecycline is a last-resort antimicrobial that exhibits promising activity against carbapenemase-producing Enterobacterales (CPE). However, mobile tigecycline resistance genes, tet(X) and tmexCD-toprJ, have emerged in China and have spread possibly worldwide. Tet(X) family proteins, Tet(X3) to Tet(X14), function as tigecycline-inactivating enzymes, and TMexCD-TOprJ complexes function as efflux pumps for tigecycline. Here, we report a CPE isolate co-harboring both emerging tigecycline resistance factors for the first time. A carbapenem- and tigecycline-resistant Klebsiella aerogenes NUITM-VK5 was isolated from an urban drainage in Vietnam in 2021 and a plasmid pNUITM-VK5_mdr co-carrying tet(X4) and tmexCD3-toprJ3 along with the carbapenemase gene blaNDM-4 was identified in NUITM-VK5. pNUITM-VK5_mdr was transferred to Escherichia coli by conjugation and simultaneously conferred high-level resistance against multiple antimicrobials, including carbapenems and tigecycline. An efflux pump inhibitor canceled TMexCD3-TOprJ3-mediated tigecycline resistance, suggesting that both tigecycline resistance factors independently and additively contribute to the high-level resistance. The plasmid had the IncX3 and IncC replicons and was estimated to be a hybrid of plasmids with different origins. Unlike IncX3 plasmids, IncC plasmids are stably maintained in an extremely broad range of bacterial hosts in humans, animals, and environment. Thus, future global spread of multidrug-resistance plasmids such as pNUITM-VK5_mdr poses a public health crisis.

Objectives The aim of this study was to understand the natural bacterial hosts of antimicrobial resistance genes (ARGs) and their impact on the processes of evolution, spread and positive selection of acquired ARGs. Methods Environmental carbapenem-resistant Gram-negative bacteria in Vietnam were screened for based on a One Health approach. Whole-genome sequencing (WGS) and comparative genomic analysis of the isolates were performed. WGS of three carbapenem-resistant Shewanella xiamenensis strains (SxND_W2_2018, SxND_W5_2018 and SxND_W9_2018) isolated from canals in Truc Ninh District and Nghia Hung District, Nam Dinh Province, Vietnam, in 2018 was performed using an Illumina MiniSeq system. ARGs in the draft genome sequences were detected using ResFinder, and comparison of genomic regions was performed using BLASTn and Easyfig. Results TheblaOXA-48-like carbapenem-hydrolysing class D β-lactamase genes blaOXA-48, blaOXA-252 and blaOXA-547 were detected in chromosomal contigs of SxND_W2_2018, SxND_W5_2018 and SxND_W9_2018, respectively. Comparative analysis of the surrounding regions of the blaOXA-48-like genes, including both 10 kb upstream and 10 kb downstream of the genes, showed that the genomic regions were highly conserved in all three isolates. Conclusion This study analysed the draft genome sequences of carbapenem-resistantS. xiamenensis strains isolated from a water environment in Vietnam. All of the strains carried blaOXA-48-like gene variants in their chromosomes. This information will contribute to highlight the evolution of blaOXA-48 family carbapenemase genes in nature and the importance of S. xiamenensis as a natural reservoir of important ARGs in the environment in Vietnam.

We analyzed 2 clusters of 12 patients in Vietnam with severe acute respiratory syndrome coronavirus 2 infection during January–February 2020. Analysis indicated virus transmission from a traveler from China. One asymptomatic patient demonstrated virus shedding, indicating potential virus transmission in the absence of clinical signs and symptoms.

While conducting rotavirus gastroenteritis surveillance in Vietnam, two G3P[8] rotavirus A specimens possessing an identical short RNA electropherotype were detected. They were RVA/Human-wt/VNM/0232/2016/G3P[8] and RVA/Human-wt/VNM/0248/2016/G3P[8], and recovered from 9 and 23 months old boys, respectively. The patients developed diarrhoea within one-week interval in March 2016 but in places >100 km apart in northern Vietnam. Whole genome sequencing of the two G3P[8] rotavirus A strains revealed that their genomic RNA sequences were identical across the 11 genome segments, suggesting that they derived from a single clone. The backbone gene constellation was I2-R2-C2-M2-A2-N2-T2-E2-H2. The backbone genes and the VP4 gene had a virtually identical nucleotide sequences with identities ranging from 99.2 to 100% to the corresponding genes of RVA/Human-wt/VNM/1149/2014/G8P[8]; the prototype of recently-emerging bovine-like G8P[8] reassortant strains in Vietnam. On the other hand, the VP7 gene was 98.8% identical with that of RVA/Human-wt/CHN/E2451/2011/G3P[9], and they were clustered together in the lineage represented by RVA/Cat-tc/JPN/FRV-1/1986/G3P[9]. The observations led us to hypothesise that one of the bovine-like G8P[8] strains bearing the DS-1-like backbone genes reassorted with a locally circulating FRV-1-like strain to gain the G3 VP7 gene and to emerge as a thus-far undescribed feline-like G3P[8] reassortant strain. The identification of feline-like G3P[8] strains bearing the DS-1-like backbone genes exemplifies the strength and necessity of the whole genome sequencing approach in monitoring, describing and understanding the evolutionary changes that are occurring in emerging strains and their interactions with co-circulating strains.

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.

Giardia spp. is detected frequently in humans and animals. Although many studies have been conducted on the epidemiology of giardiasis, there is a scarcity of information on the genetic diversity and the dynamics of transmission of Giardia spp. in Vietnam. The zoonotic potential of Giardia spp. remains elusive. The objective of this study was to determine the genetic diversity of Giardia spp. in both humans and livestock to assess the existence of a route of infection between livestock and humans. Our goal was to assess the role animals play in the epidemiology of human infection in northern Vietnam. In Hien Khanh commune in northern Vietnam, 311 households with 1508 residents were randomly selected for a diarrheal cohort study. Of these, 2120 human diarrheal samples were collected from 1508 residents in 2014 and 2017. Of these, non-diarrheal samples were cross-sectionally collected from 471 residents. At the same site, livestock samples from buffalo, dairy and beef cattle, pigs, and dogs were collected. All stool samples were examined for Giardia spp. by Direct Immunofluorescence Assay (DFA) using fluorescent microscope. DNA extraction, PCR analysis of the 3 genes (bg, gdh, tpi), and sequencing analysis were continuously carried out. A total of 23 animal stool samples, 8 human non-diarrheal samples, and 36 human diarrheal samples were Giardia spp. were positive by PCR using the bg and gdh genes. Giardia spp. assemblage AII and E were detected in both animal samples and human samples in this study site. The detection of assemblage E in human stool samples suggests the first human case report in Vietnam. We assume that the unexpected human infection of all Giardia assemblages including A, B, and E may be due to an environment contaminated with animal and human feces in this village.

The purpose of this study was to investigate the occurrence of Cryptosporidium infection and the potential for transmission of Cryptosporidium spp. between animals and humans in northern Vietnam. A total of 2715 samples (2120 human diarrheal samples, 471 human non-diarrheal samples, and 124 animal stool samples) were collected through our community survey in an agricultural area. All samples were tested for Cryptosporidium spp. by direct immunofluorescence assay (DFA) using a fluorescent microscope. DNA extraction, PCR amplification of three genes (COWP, SSU-rRNA, and GP60), and sequencing analysis were performed to identify Cryptosporidium spp. Of 2715 samples, 15 samples (10 diarrheal samples, 2 non-diarrheal samples, and 3 animal stool samples) tested positive by PCR for the COWP gene. Three species of Cryptosporidium spp. were identified as C. canis (from six human diarrheal samples, two human non-diarrheal samples, and one dog sample), C. hominis (from four human diarrheal samples), and C. suis (from two pig samples) by sequencing the amplified COWP and/or SSU-rRNA genes. In terms of C. hominis, the GP60 subtype IeA12G3T3 was detected in all four human diarrheal samples. Although the number of positive samples was very small, our epidemiological data showed that the emerging pattern of each of the three species (C. canis, C. hominis, and C. suis) was different at this study site. While C. hominis and C. suis were only detected in human and pig samples, respectively, C. canis was detected in samples from both dogs and humans. We suspect that C. canis infections in humans at this study site may be due to environmental contamination with animal and human feces.

BackgroundCryptosporidium spp. is a protozoan parasite with worldwide distribution that causes cryptosporidiosis in humans and other animals. In the countryside of northern Vietnam, where free-roaming livestock are widespread, cryptosporidiosis is an important zoonotic disease. However, there have been few studies of cryptosporidiosis in Southeast Asia from the perspective of zoonotic disease epidemiology. The purpose of this study was to investigate the occurrence of Cryptosporidium infection in both humans and animals and to gain an awareness of the potential threat posed by this zoonotic infection in northern Vietnam.Methods We conducted a community survey to collect information about cases of diarrhea in an agricultural area of northern Vietnam. For this study, a total of 2715 samples (2120 human diarrheal samples, 471 non-diarrheal human samples, and 124 animal stool samples) were collected. A direct immunofluorescence assay (DFA) was used to detect Cryptosporidium spp. oocysts in concentrated stool samples by observation under a fluorescent microscope. DNA extraction, PCR amplification of the three genes (COWP, SSU-rRNA, and GP60), and sequencing analysis were performed to identify Cryptosporidium spp.Results Of 2715 samples, 15 samples (10 diarrheal samples, 2 non-diarrheal samples, and 3 animal stool samples) tested positive by PCR for the COWP gene. Three species of Cryptosporidium spp. were detected; C. canis (from six human diarrheal samples, two human non-diarrheal samples, and one dog sample); C. hominis (from four human diarrheal samples); and C. suis (from two pig samples). In terms of C. hominis, the GP60 subtype IeA12G3T3 was detected in all four human diarrheal samples.Conclusions Although the number of positive samples was very small, our epidemiological data showed that the emerging pattern of each of the three species (C. canis, C. hominis, and C. suis) was different at this study site. For example, zoonotic transmission of C. canis, between dogs and humans was suspected. Further studies are needed to assess the risk of oocyst contamination in the wider environment, including water, in this study area.

Colistin is one of the antibiotics of last resort for human health. However, the dissemination of the plasmid-mediated colistin resistance gene mcr-1 is of great concern globally. In the One Health framework, the environment is an important component for managing antimicrobial resistance. However, little information is available concerning the prevalence of mcr-1 in water environments. We aimed to reveal the prevalence of mcr-1 in different water environments in Hanoi, Vietnam. Quantitative PCR was applied to detect mcr-1 in four urban drainages receiving untreated domestic wastewater, three rivers, five lakes and two groundwater samples. Urban drainages contained higher concentrations of mcr-1, suggesting that urban residents carry the gene. The class 1 integron-integrase gene was identified as a good surrogate of antibiotic resistance genes including mcr-1. A significant correlation was found between the levels of mcr-1 and the human-specific cross-assembly phage, which is an indicator of human faecal pollution. These results indicated that the primary source of mcr-1 in urban water environments is human faeces, which is consistent with the fact that most domestic wastewater is untreated in Hanoi. The control of untreated wastewater is critical for alleviating the spread of mcr-1 in water environments in Vietnam.

Giardia spp. is detected frequently in humans and animals. Although many studies have been conducted on the epidemiology of giardiasis, there is a scarcity of information on the genetic diversity and the dynamics of transmission of Giardia spp. in Vietnam. The zoonotic potential of Giardia spp. remains elusive. The objective of this study was to determine the genetic diversity of Giardia spp. in both humans and livestock to assess the existence of a route of infection between livestock and humans. Our goal was to assess the role animals play in the epidemiology of human infection in northern Vietnam. In Hien Khanh commune in northern Vietnam, 311 households with 1508 residents were randomly selected for a diarrheal cohort study. Of these, 2120 human diarrheal samples were collected from 1508 residents in 2014 and 2017. Of these, non-diarrheal samples were cross-sectionally collected from 471 residents. At the same site, livestock samples from buffalo, dairy and beef cattle, pigs, and dogs were collected. All stool samples were examined for Giardia spp. by Direct Immunofluorescence Assay (DFA) using fluorescent microscope. DNA extraction, PCR analysis of the 3 genes (bg, gdh, tpi), and sequencing analysis were continuously carried out. A total of 23 animal stool samples, 8 human non-diarrheal samples, and 36 human diarrheal samples were Giardia spp. were positive by PCR using the bg and gdh genes. Giardia spp. assemblage AII and E were detected in both animal samples and human samples in this study site. The detection of assemblage E in human stool samples suggests the first human case report in Vietnam. We assume that the unexpected human infection of all Giardia assemblages including A, B, and E may be due to an environment contaminated with animal and human feces in this village., Graphical abstract Unlabelled Image, Highlights • Giardia spp. assemblage A and E have been detected in buffalo, cattle and pigs, as well as in humans, while assemblage B appeared only in humans. • This was the first report of assemblage E in humans in Vietnam. • Zoonotic transmission of assemblage AII and E could not be excluded in our study site. • In addition to the ‘animal-to-human’ route, there potentially might be transmission routes from ‘humans-to-humans’ and/or ‘humans-to-animals’ in this study site. • The novel genotype belonging to assemblage AIII has been isolated only in buffalo.

Development of multivalent hand, foot, and mouth disease (HFMD) vaccines against enterovirus A71 (EV-A71) and several non-EV-A71 enteroviruses is needed for this life-threatening disease with a huge economic burden in Asia-Pacific countries. Comprehensive studies on the molecular epidemiology and genetic and antigenic characterization of major causative enteroviruses will provide information for rational vaccine design. Compared with molecular studies on EV-A71, that for non-EV-A71 enteroviruses remain few and limited in Vietnam. Therefore, we conducted a 10-year study on the circulation and genetic characterization of coxsackievirus A16 (CV-A16) and CV-A6 isolated from patients with HFMD in Northern Vietnam between 2008 and 2017. Enteroviruses were detected in 2228 of 3212 enrolled patients. Of the 42 serotypes assigned, 28.4% and 22.4% accounted for CV-A6 and CV-A16, being the second and the third dominant serotypes after EV-A71 (31.7%), respectively. The circulation of CV-A16 and CV-A6 showed a wide geographic distribution and distinct periodicity. Phylogenetic analyses revealed that the majority of Vietnamese CV-A6 and CV-A16 strains were located within the largest sub-genotypes or sub-genogroups. These comprised strains isolated from patients with HFMD worldwide during the past decade and the Vietnamese strains have been evolving in a manner similar to the strains circulating worldwide. Amino acid sequences of the putative functional loops on VP1 and other VPs among Vietnamese CV-A6 and CV-A16 isolates were highly conserved. Moreover, the functional loop patterns of VP1 were similar to the dominant patterns found worldwide, except for the T164K substitution on the EF loop in Vietnamese CV-A16. The findings suggest that the development of a universal HFMD vaccine, at least in Vietnam, must target CV-A6 and CV-A16 as two of the three major HFMD-causing serotypes. Vietnamese isolates or their genome sequences can be considered for rational vaccine design.

The dissemination and abundances of Vibrio species in aquatic environments are of interest, as some species cause emerging diseases in humans and in aquatic organisms like fish. It is suggested that Vibrio cholerae non-O1 infections of Plecoglossus altivelis ('ayu') were spread to various parts of Japan through the annual transplantation of juvenile fish. To investigate this, we used genome-aided tracing of 17 V. cholerae strains isolated from ayu between the 1970s and 1990s in different Japanese freshwater systems. The strains formed a genomic clade distinct from all known clades, which we designate as the Ayu clade. Two clonal genomic groups identified within the clade, Ayu-1 and Ayu-2, persisted for a few years (between 1977 to 1979 and 1987 to 1990, respectively), and clonal replacement of Ayu-1 by Ayu-2 took place over an 8-year period. Despite the high similarity between Ayu-1 and Ayu-2 ( 99.9% identity and 97% fraction of genomes shared), differences in their gene repertoires were found, raising the possibility that they are phenotypically distinct. These results highlight the importance of genome-based studies for understanding the long-term dynamics of populations over the timescale of years.

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., BMC infectious diseases, 20(1), art.no.332; 2020

Cell entry by HIV-1 is mediated by its principal receptor, CD4, and a coreceptor, either CCR5 or CXCR4, with viral envelope glycoprotein gp120. Generally, CCR5-using HIV-1 variants, called R5, predominate over most of the course of infection, while CXCR4-using HIV-1 variants (variants that utilize both CCR5 and CXCR4 [R5X4, or dual] or CXCR4 alone [X4]) emerge at late-stage infection in half of HIV-1-infected individuals and are associated with disease progression. Although X4 variants also appear during acute-phase infection in some cases, these variants apparently fall to undetectable levels thereafter. In this study, replication-competent X4 variants were isolated from plasma of drug treatment-naive individuals infected with HIV-1 strain CRF01_AE, which dominantly carries viral RNA (vRNA) of R5 variants. Next-generation sequencing (NGS) confirmed that sequences of X4 variants were indeed present in plasma vRNA from these individuals as a minor population. On the other hand, in one individual with a mixed infection in which X4 variants were dominant, only R5 replication-competent variants were isolated from plasma. These results indicate the existence of replication-competent variants with different coreceptor usage as minor populations. IMPORTANCE The coreceptor switch of HIV-1 from R5 to CXCR4-using variants (R5X4 or X4) has been observed in about half of HIV-1-infected individuals at late-stage infection with loss of CD4 cell count and disease progression. However, the mechanisms that underlie the emergence of CXCR4-using variants at this stage are unclear. In the present study, CXCR4-using X4 variants were isolated from plasma samples of HIV-1-infected individuals that dominantly carried vRNA of R5 variants. The sequences of the X4 variants were detected as a minor population using next-generation sequencing. Taken together, CXCR4-using variants at late-stage infection are likely to emerge when replication-competent CXCR4-using variants are maintained as a minor population during the course of infection. The present study may support the hypothesis that R5-to-X4 switching is mediated by the expansion of preexisting X4 variants in some cases.

Vibrio cholerae O1 causes cholera, and cholera toxin, the principal mediator of massive diarrhea, is encoded by ctxAB in the cholera toxin (CTX) prophage. In this study, the structures of the CTX prophage region of V. cholerae strains isolated during the seventh pandemic wave 1 in Asian countries were determined and compared. Eighteen strains were categorized into eight groups by CTX prophage region-specific restriction fragment length polymorphism and PCR profiles and the structure of the region of a representative strain from each group was determined by DNA sequencing. Eight representative strains revealed eight distinct CTX prophage regions with various combinations of CTX-1, RS1 and a novel genomic island on chromosome I. CTX prophage regions carried by the wave 1 strains were diverse in structure. V. cholerae strains with an area specific CTX prophage region are believed to circulate in South-East Asian countries; additionally, multiple strains with distinct types of CTX prophage region are co-circulating in the area. Analysis of a phylogenetic tree generated by single nucleotide polymorphism differences across 2483 core genes revealed that V. cholerae strains categorized in the same group based on CTX prophage region structure were segregated in closer clusters. CTX prophage region-specific recombination events or gain and loss of genomic elements within the region may have occurred at much higher frequencies and contributed to producing a panel of CTX prophage regions with distinct structures among V. cholerae pathogenic strains in lineages with close genetic backgrounds in the early wave 1 period of the seventh cholera pandemic.

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/β 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.

Vibrio cholerae O1 El Tor, the pathogen responsible for the current cholera pandemic, became pathogenic by acquiring virulent factors including Vibrio seventh pandemic islands (VSP)-I and -II. Diversity of VSP-II is well recognized; however, studies addressing attachment sequence left (attL) sequences of VSP-II are few. In this report, a wide variety of V. cholerae strains were analyzed for the structure and distribution of VSP-II in relation to their attachment sequences. Of 188 V. cholerae strains analyzed, 81% (153/188) strains carried VSP-II; of these, typical VSP-II, and a short variant was found in 36% (55/153), and 63% (96/153), respectively. A novel VSP-II was found in two V. cholerae non-O1/non-O139 strains. In addition to the typical 14-bp attL, six new attL-like sequences were identified. The 14-bp attL was associated with VSP-II in 91% (139/153), whereas the remaining six types were found in 9.2% (14/153) of V. cholerae strains. Of note, six distinct types of the attL-like sequence were found in the seventh pandemic wave 1 strains; however, only one or two types were found in the wave 2 or 3 strains. Interestingly, 86% (24/28) of V. cholerae seventh pandemic strains harboring a 13-bp attL-like sequence were devoid of VSP-II. Six novel genomic islands using two unique insertion sites to those of VSP-II were identified in 11 V. cholerae strains in this study. Four of those shared similar gene clusters with VSP-II, except integrase gene.

Vibrio cholerae inhabits aquatic environments worldwide and has over 200 recognized serogroups classified by O-polysaccharide specificity. Here, we report that V. cholerae selects either of two genetic traits during their evolution. Sequencing of the specific gene locus MS6_A0927 revealed that 339 of 341 strains of V. cholerae and closely related Vibrio species originating from 34 countries over a century carried either metY (M) (~1,269 bp) or luxR-hchA (LH) (~1,600 bp) genes, and consequently those vibrios were separated into two clusters, M (45.4%) and LH (54.6%). Only two strains contained both M and LH in the same locus. Moreover, extensive polymorphisms in those genes were detected in M and LH with 79 and 46 sequence variations, respectively. V. cholerae O1 strains isolated from cholera outbreaks worldwide, and some non-O1 strains evolving from O1 via exchange of genes encoding cell surface polysaccharides possessed LH alleles. Analysis of polymorphisms in the gene locus implicated a high degree of genetic diversity and identical subpopulations among the V. cholerae species.

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., International Journal of Environmental Research and Public Health, 18(18), art. no. 9845; 2021

Cholera epidemics have been recorded periodically in Vietnam during the seventh cholera pandemic. Since cholera is a water-borne disease, systematic monitoring of environmental waters for Vibrio cholerae presence is important for predicting and preventing cholera epidemics. We conducted monitoring, isolation, and genetic characterization of V. cholerae strains in Nam Dinh province of Northern Vietnam from Jul 2013 to Feb 2015. In this study, four V. cholerae O1 strains were detected and isolated from 110 analyzed water samples (3.6%); however, none of them carried the cholera toxin gene, ctxA, in their genomes. Whole genome sequencing and phylogenetic analysis revealed that the four O1 isolates were separated into two independent clusters, and one of them diverged from a common ancestor with pandemic strains. The analysis of pathogenicity islands (CTX prophage, VPI-I, VPI-II, VSP-I, and VSP-II) indicated that one strain (VNND_2014Jun_6SS) harbored an unknown prophage-like sequence with high homology to vibriophage KSF-1 phi and VCY phi, identified from Bangladesh and the USA, respectively, while the other three strains carried tcpA gene with a distinct sequence demonstrating a separate clonal lineage. These results suggest that the aquatic environment can harbor highly divergent V. cholera strains and serve as a reservoir for multiple V. cholerae virulence-associated genes which may be exchanged via mobile genetic elements. Therefore, continuous monitoring and genetic characterization of V. cholerae strains in the environment should contribute to the early detection of the sources of infection and prevention of cholera outbreaks as well as to understanding the natural ecology and evolution of V. cholerae., Infection, Genetics and Evolution, 54, pp.146-151; 2017

While conducting sentinel surveillance of hand, foot, and mouth disease (HFMD) in Vietnam, we found a sudden increase in the prevalence of coxsackievirus A10 (CV-A10) in 2016 and CV-A2 and CV-A4 in 2017, the emergence of which has been reported recently to be associated with various clinical manifestations in other countries. However, there have been only a limited number of molecular studies on those serotypes, with none being conducted in Vietnam. Therefore, we sequenced the entire VP1 genes of CV-A10, CV-A4, and CV-A2 strains associated with HFMD in Vietnam between 2012 and 2017. Phylogenetic analysis revealed a trend of endemic circulation of Vietnamese CV-A10, CV-A4, and CV-A2 strains and the emergence of thus-far undescribed HFMD-causing lineages of CV-A4 and CV-A2. The Vietnamese CV-A10 strains belonged to a genotype comprising isolates from patients with HFMD from several other countries; however, most of the Vietnamese strains were grouped into a local lineage. Recently, emerging CV-A4 strains in Vietnam were grouped into a unique lineage within a genotype comprising strains isolated from patients with acute flaccid paralysis from various countries. New substitutions were detected in the putative BC and HI loops in the Vietnamese CV-A4 strains. Except for one strain, Vietnamese CV-A2 isolates were grouped into a unique lineage of a genotype that includes strains from various countries that are associated with other clinical manifestations. Enhanced surveillance is required to monitor their spread and to specify their roles as etiological agents of HFMD or "HFMD-like" diseases, especially for CV-A4 and CV-A2. Further studies including whole-genome sequencing should be conducted to fully understand the evolutionary changes occurring in these newly emerging strains.

A rapid increase and dominance of G9P[8] Rotavirus A strains occurred in northern Vietnam between 2016 and 2018, during which period there appeared three G9P[8] strains possessing short RNA patterns. To understand how the first-ever G9P[8] strains possessing short RNA patterns were formed, next generation sequencing technology was used to examine the whole genomes of the three strains, i.e., RVA/Human-wt/VNM/RVN16.1024/2016/G9P[8], RVA/Human-wt/VNM/RVN17.0879/2017/G9P[8], and RVA/Human-wt/VNM/RVN18.0197/2018/G9P[8], and those of seven representative G9P[8] strains possessing long RNA patterns. The VP7 genes of the short and long G9P[8] strains were > 99% identical, indicating that the origin was in the co-circulating, dominant, long G9P[8] strains. On the other hand, the VP4 genes likely derived from recently-emerging G1/G3/G8P[8] strains possessing the DS-1 backbone. At the lineage level, however, the backbone genes of any one strain differed from that of the other two in the VP1, VP3 or NSP4 gene. Moreover, even at the nucleotide sequence level of the backbone genes belonging to the same lineage, the identities between the three strains were lower than those expected for the strains deriving from an immediate, common ancestor. Thus, the three strains were likely formed by independent reassortment events in which the VP7 gene of the currently dominant G9P[8] strains was incorporated into co-circulating G1/G3/G8P[8] strains possessing similar yet distinct DS-1-like backbone genes. The observation that all of the three reassortant G9P[8] strains were detected only once among the prevalent, ordinary G9P[8] strains suggests that acquisition of the DS-1-like backbone genes unlikely provided selective advantage over the parental Wa-like G9P[8] strains.

engue remains a major public health problem in the Philippines. In this study, we determined the circulating dengue serotypes in the Philippines during the 2015?2017 outbreaks using a total of 678 serum samples from 537 individual dengue patients. Following an increase in the number of DENV-4 patients in recent years, we conducted a comprehensive molecular and epidemiology analysis on the DENV-4 strains isolated recently in the Philippines. Two genotypes of DENV-4 have been isolated in the Philippines since 1956: GI and GIIa. The GIIa DENV strains that were isolated in the present study were closely related to a distinct group of GIIa strains that were isolated from the Philippines in 2004. A majority of the isolates of this sub-group have been identified in the Philippines, suggesting that this lineage may have been introduced in the Philippines, and evolved to form the distinct sub-group within GIIa strains. The increase in DENV-4 activity also coincided with the appearance of the GIIa sub-group and the phasing-out of the GI lineage in the Philippines. Overall, our study demonstrates a shift in DENV-4 genotype and epidemic dynamics in a hyperendemic region, suggesting the importance of DENV genetic evolution in establishing and sustaining transmission., Japanese Journal of Infectious Diseases, 72(6), pp.413-419; 2019

Background There are an estimated one million patients with scrub typhus in the Asia-Pacific region. There are few reports describing the incidence of scrub typhus in Vietnam. Methods Blood samples collected from 63 patients clinically diagnosed as having scrub typhus from July 2015 to September 2016 were subjected to genotyping of Orientia tsutsugamushi. Results and conclusions Of these patients, 42 (67%) tested positive for O. tsutsugamushi, and the most common genotype was identified to be Karp (55%). Other genotypes, TA763, Gilliam type in Japan variant, and Kato were also found in 17%, 17% and 12% of patients, respectively. To better understand the epidemiological landscape of scrub typhus in Vietnam, a countrywide study is needed. Accession numbers LC110330-LC110333, LC110336-LC110351 and LC214804-LC214825.

Vibrio cholerae O1 causes cholera, and cholera toxin, the principal mediator of massive diarrhea, is encoded by ctxAB in the cholera toxin (CTX) prophage. In this study, the structures of the CTX prophage region of V. cholerae strains isolated during the seventh pandemic wave 1 in Asian countries were determined and compared. Eighteen strains were categorized into eight groups by CTX prophage region‐specific restriction fragment length polymorphism and PCR profiles and the structure of the region of a representative strain from each group was determined by DNA sequencing. Eight representative strains revealed eight distinct CTX prophage regions with various combinations of CTX‐1, RS1 and a novel genomic island on chromosome I. CTX prophage regions carried by the wave 1 strains were diverse in structure. V. cholerae strains with an area specific CTX prophage region are believed to circulate in South‐East Asian countries; additionally, multiple strains with distinct types of CTX prophage region are co‐circulating in the area. Analysis of a phylogenetic tree generated by single nucleotide polymorphism differences across 2483 core genes revealed that V. cholerae strains categorized in the same group based on CTX prophage region structure were segregated in closer clusters. CTX prophage region‐specific recombination events or gain and loss of genomic elements within the region may have occurred at much higher frequencies and contributed to producing a panel of CTX prophage regions with distinct structures among V. cholerae pathogenic strains in lineages with close genetic backgrounds in the early wave 1 period of the seventh cholera pandemic.

Vibrio cholerae O1 El Tor, the pathogen responsible for the current cholera pandemic, became pathogenic by acquiring virulent factors including Vibrio seventh pandemic islands (VSP)‐I and −II. Diversity of VSP‐II is well recognized; however, studies addressing attachment sequence left (attL) sequences of VSP‐II are few. In this report, a wide variety of V. cholerae strains were analyzed for the structure and distribution of VSP‐II in relation to their attachment sequences. Of 188 V. cholerae strains analyzed, 81% (153/188) strains carried VSP‐II; of these, typical VSP‐II, and a short variant was found in 36% (55/153), and 63% (96/153), respectively. A novel VSP‐II was found in two V. cholerae non‐O1/non‐O139 strains. In addition to the typical 14‐bp attL, six new attL‐like sequences were identified. The 14‐bp attL was associated with VSP‐II in 91% (139/153), whereas the remaining six types were found in 9.2% (14/153) of V. cholerae strains. Of note, six distinct types of the attL‐like sequence were found in the seventh pandemic wave 1 strains; however, only one or two types were found in the wave 2 or 3 strains. Interestingly, 86% (24/28) of V. cholerae seventh pandemic strains harboring a 13‐bp attL‐like sequence were devoid of VSP‐II. Six novel genomic islands using two unique insertion sites to those of VSP‐II were identified in 11 V. cholerae strains in this study. Four of those shared similar gene clusters with VSP‐II, except integrase gene.

Human immunodeficiency virus type 1 (HIV-1) infection of most human cells is dependent on cyclophilin A (CypA); however, the opposite phenomenon, known as CypA-dependent inhibition, is also observed in the combination of some capsid (CA) mutations and cell lines. Here, we identified a CA N121K mutant whose infection of 293T, Jurkat, and HeLa cells was impaired by CypA. The N121K mutant could be a useful tool for analyzing the mechanisms underlying CypA-dependent restriction.

HIV-1 passage in cell culture in the presence of chemokine receptor antagonists can result in selection of viruses with env mutations that confer resistance to these inhibitors. In the present study, we examined the effect of HIV-1env mutations that confer resistance to CXCR4 antagonists on envelope (Env) sensitivity to neutralizing antibodies (NAbs). Serial passage of CXCR4-tropic HIV-1 NL4-3 in PM1/CCR5 cells under CXCR4 antagonists KRH-3955, AMD3100 and AMD070 yielded two KRH-3955-resistant, one AMD3100-resistant and one AMD070-resistant viruses. These viruses had multiple env mutations including the Env gp120 V3 region. The majority of viruses having these CXCR4 antagonist-resistant Envs showed higher sensitivity to NAbs 447-52D, b12 and 2F5 targeting the V3 region, the gp120 CD4-binding site and the gp41 membrane proximal region, respectively, compared to NL4-3 WT virus. Recombinant NL4-3 viruses with the V3-coding region replaced with those derived from the CXCR4 antagonist-resistant viruses showed increased sensitivity to NAbs b12, 2F5 and 447-52D. Molecular dynamics simulations of Env gp120 outer domains predicted that the V3 mutations increased levels of fluctuations at the tip and stem of the V3 loop. These results indicate that mutations in the V3-coding region that result in loss of viral sensitivity to CXCR4 antagonists increase viral sensitivity to NAbs, providing insights into our understanding of the interplay of viral Env accessibility to chemokine receptors and sensitivity to NAbs.

金沢大学医薬保健研究域医学系, To investigate the prevalence of subtypes A and C, and the existence of recombinants of both subtypes in the southeast of the Democratic Republic of Congo (DRC), blood samples were collected from 27 HIV-infected individuals in Likasi, located in an area bordering close to Zambia, and analyzed phylogenetically. Out of the 24 strains with a positive PCR profile for pol-IN and env-C2V3, 15 (62.5%) had a discordant subtype or CRF designation: one subtype A/G (pol/env), four A/U (unclassified), three G/A, one G/CRF01, three H/A, one J/C, one CRF02 (G)/A, and one U/A. Nine (37.5%) strains had a concordant subtype or CRF designation: five subtype A, two C, one D, and one CRF02/G. The remaining three samples negative for PCR with env-C2V3 primers used in this study were further analyzed with env-gp41 primers and revealed the presence of two profiles: two J/J (pol-IN/env-gp41) and one C/G. These data highlight the presence of a high proportion (16/27, 59.3%) of recombinant strains and a low prevalence (4.1 and 7.4%) of subtype C based on env-C2V3 and pol-IN analyses, respectively, in Likasi. In addition, this is the first report that CRF02_AG exists in DRC, though the epidemiological significance of the existence of CRF02_AG in DRC remains unknown.