Long-Term Epidemiology and Evolution of Swine Influenza Viruses, Vietnam.

Influenza A viruses are a One Health threat because they can spill over between host populations, including among humans, swine, and birds. Surveillance of swine influenza virus in Hanoi, Vietnam, during 2013-2019 revealed gene pool enrichment from imported swine from Asia and North America and showed long-term maintenance, persistence, and reassortment of virus lineages. Genome sequencing showed continuous enrichment of H1 and H3 diversity through repeat introduction of human virus variants and swine influenza viruses endemic in other countries. In particular, the North American H1-δ1a strain, which has a triple-reassortant backbone that potentially results in increased human adaptation, emerged as a virus that could pose a zoonotic threat. Co-circulation of H1-δ1a viruses with other swine influenza virus genotypes raises concerns for both human and animal health.

SARS-CoV-2 Infection in a Hippopotamus, Hanoi, Vietnam.

While investigating the death of a hippopotamus at a zoo in Hanoi, Vietnam, we isolated SARS-CoV-2 and sequenced the RNA-dependent RNA polymerase gene from different organs. Phylogenetic analysis showed that the SARS-CoV-2 strain was closely related to 3 human SARS-CoV-2 strains in Vietnam.

Duck circovirus in northern Vietnam: genetic characterization and epidemiological analysis.

In the present study, tissue samples collected from 130 ducks from clinically suspected commercial flocks and diseased birds in six provinces of northern Vietnam were tested for duck circovirus (DuCV) infection. The DuCV genome was detected in 56 out of 130 (43.08%) duck samples by PCR. Of 38 tested farms, 26 (68.42%) were positive for the DuCV genome. The rate of the DuCV genome detection in ducks at 3-4 weeks of age (54.17%) was significantly higher (p < 0.05) than that at <3 (32.43%) and >7 (33.33%) weeks of age and insignificantly higher than that at 5-7 weeks of age (43.33%) (p = 0.11). The genomes of six Vietnamese DuCV isolates were determined. They ranged in length from 1,988 to 1,995 nucleotides, and their nucleotide sequences were 83.24% to 99.69% identical to each other. Phylogenetic analysis based on the complete genome sequences indicated that the DuCV strains circulating in northern Vietnam can be divided into two main genotypes (I and II) and several subgenotypes. The Vietnamese DuCV isolates were closely related to Chinese, Taiwanese, and Korean strains. One positively selected site was detected in the capsid protein.

Prevalence and phylogenetic analysis of hepatitis E virus in pigs in Vietnam.

BACKGROUND: Hepatitis E virus (HEV) is a zoonotic disease and has been reported around the world. The main objective of this study was to evaluate the sero-prevalence and phylogenetic analysis of HEV in Vietnam. Pig blood and fecal pooled samples were collected to assess the prevalence of HEV. We assessed the true prevalence (TP) of HEV from apparent prevalence (AP) by taking into account the sensitivity and specificity of diagnostic tests using a Bayesian approach. For phylogenetic analysis, the data compared with worldwide HEV reference strains including all eight genotypes (G1-G8) which were identified in previous study. RESULTS: A total of 475 sera and 250 fecal pooled samples were collected at slaughterhouses and pig farms from five provinces, in Viet Nam. Overall, the sero-AP of HEV was 58.53% (95% confidence interval: 53.95-62.70) while the sero-TP was slightly higher (65.43, 95% credible interval: 47.19-84.70). In terms of pooled samples, overall, the RNA-AP was 6.80% (95% confidence interval: 4.01-10.66). One strain in Hanoi, two strains in Dak Lak, seven strains in An Giang, four strains in Son La and two strains in Nghe An were isolated. The phylogenetic tree demonstrated that 19 Vietnamese strains were clustered into HEV 3 and 4. CONCLUSIONS: This study provided evidence that HEV is circulating in domestic pigs in Vietnam. From a public health perspective, it is very important to raise public awareness for high-risk groups (e.g. slaughterhouse workers, pig traders, farmers and market sellers) who have more opportunities to come in contact with pig and contaminated meats.

Seroprevalences of multi-pathogen and description of farm movement in pigs in two provinces in Vietnam.

BACKGROUND: In Vietnam, lack of animal health information is considered a major challenge for pig production. The main objective of this study was to assess the seroprevalences of five pathogens [porcine circovirus type 2 (PCV2), porcine reproductive and respiratory syndrome virus (PRRSV), mycoplasma hyopneumoniae (M. hyo), Japanese encephalitis virus (JEV) and leptospirosis] and to better characterize the farm movements through a survey. RESULTS: A total of 600 samples were collected from 120 farms from Bac Giang and Nghe An. Among unvaccinated herds, the highest seroprevalence was found for JE with 73.81% (95% CI: 68.39-78.74) in Bac Giang and 53.51% (95% CI 47.68-59.27) in Nghe An. Seroprevalences for PCV2 and M.hyo were 49.43% (95% CI: 45.06-53.80) and 46.06% (95% CI: 41.48-50.69) among unvaccinated animals. Accumulative co-infections for JE (86.25%) showed the highest level followed by M. hyo (66.25%) and PCV2 (62.50%). Three co-infections with JE had the highest positive rate (28.75%) followed by four co-infections (25.0%). Medium farms had relatively higher herd prevalences for all pathogens, except from leptospirosis. Overall, farmers exported/imported their pigs at the most 1-2 times every 6 months. Some respondents (5% for exportation and 20% for importation) had moved pigs more than 6 times over the last 6 months. CONCLUSIONS: Our study provided another pool of evidence that showed that PCV2, PRRS and H. hyo are endemic in pigs in Vietnam. Given the economic impacts of these pathogens elsewhere, the findings confirm the need for studies to evaluate the association between antibody response and clinical relevance as well as to assess the economic impact of co-infections at farm level. We also found that high seroprevalences of JE and leptospirosis were detected in pigs. From a pubic health point of view, it is crucial to raise public awareness especially for high risk occupations (mainly pig farm workers).

Chicken anemia virus in northern Vietnam: molecular characterization reveals multiple genotypes and evidence of recombination.

Chicken anemia virus (CAV) has a ubiquitous and worldwide distribution in the chicken production industry. Our group previously reported a high seroprevalence of CAV in chickens from northern Vietnam. In the present study, tissue samples collected from a total of 330 broiler and breeder commercial chickens in eleven provinces of northern Vietnam were tested for CAV infection. All samples were collected from clinically suspected flocks and diseased birds. The CAV genome was detected in 157 out of 330 (47.58%) chicken samples by real-time PCR. The rate of CAV genome detection in young chickens at 2-3 weeks of age (61.43%), which had not been previously reported in Vietnam, was significantly higher than that in older chickens at 4-11 (44.83%) and 12-28 (35.71%) weeks of age. For nine representative CAV strains from broiler chickens, analysis of the entire protein-coding region of the viral genome was conducted. Phylogenetic analysis of the VP1 gene indicated that the CAVs circulating in northern Vietnam were divided into three distinct genotypes: II, III, and V. Only one of the nine Vietnamese CAV strains clustered with a vaccine strain (Del-Ros), whereas the other eight strains did not cluster with any vaccine strains. Among the three genotypes, genotype III was most widely found in northern Vietnam and this included three sub-genotypes (IIIa, IIIb, and IIIc). The Vietnamese CAV strains were closely related to the Chinese, Taiwanese, and USA strains. One strain was defined to be of genotype V, which is a newly reported CAV genotype. Moreover, recombination analysis suggests that this novel genotype V was generated by recombination between genotype II and sub-genotype IIIc.

Application of the SureSelect target enrichment system for next-generation sequencing to obtain the complete genome sequence of bovine leukemia virus.

In this study, the SureSelect target enrichment system for Illumina Multiplexed Sequencing was applied to proviral DNA sequencing of bovine leukemia virus (BLV). The complete genomic DNA sequences of four Vietnamese BLV strains were successfully obtained with high read depth values and a genome coverage of 100% across all sequenced samples, in less than one week. This study provides the first complete Vietnamese BLV genome sequences. Their genetic variability and phylogenetic relationship were also analyzed and compared with those of 28 whole BLV genome sequences from different parts of the world. The results obtained provided new insights into the genetic diversity of the BLV tax gene, and further enabled us to identify nucleotide mutations in the gene that might not have been detected with the commercial detection kit that is currently available.

Correction to: Application of the SureSelect target enrichment system for next-generation sequencing to obtain the complete genome sequence of bovine leukemia virus.

Unfortunately, Figure 1 was incorrectly published in the original publication and the correct version is updated here.

Evaluation of the replication and pathogenicity of a variant avian paramyxovirus serotype 6 in mice.

Avian paramyxoviruses (APMVs) have been evaluated for their potential use as vaccine vectors, sparking research efforts leading to a better understanding of APMVs' replication and pathogenicity. However, within APMV serotypes, significant genetic diversity exists, and the infectivity of variant strains in mammals has not been studied. We utilized a mouse model to evaluate the pathogenicity of a variant strain of APMV-6 (APMV-6/red-necked stint/Japan/8KS0813/2008) in comparison with the prototype APMV-6 strain (APMV-6/duck/Hong Kong/18/199/1977). Although the two viruses differ substantially, both genetically and antigenically, we found that the variant and prototype strains could similarly replicate in respiratory tissues of infected mice and induce respiratory disease, sometimes resulting in death of the mice. Both viruses induced a humoral immune response that could be clearly detected by ELISA but which was poorly recognized by the hemagglutination inhibition test.

Genetic characterization of H5N2 influenza viruses isolated from wild birds in Japan suggests multiple reassortment.

Low-pathogenic avian influenza viruses (LPAIVs) of the H5 subtype can mutate to highly pathogenic forms, potentially destabilizing the poultry industry. Wild migratory birds are considered a natural reservoir of LPAIVs capable of dispersing both high- and low-pathogenic forms of the virus. Therefore, surveillance and characterization of AIV in wild birds are essential. Here, we report on the isolation and genetic characterization of 10 AIVs of the H5N2 subtype obtained through surveillance in Hokkaido, Japan, during 2009 and 2011. Full-genome sequencing revealed that the H5 and N2 genes of these isolates are all closely related to each other, belonging to the Eurasian avian-like lineage, but they are unrelated to H5 highly pathogenic strains of clade 2.3.4.4. The internal genes of the isolates were found to be diverse, consistent with our hypothesis that these H5N2 strains have undergone multiple reassortment events. Even though all of the H5N2 isolates were characterized as LPAIV based on the amino acid sequences at the HA cleavage site, this analysis demonstrates a diverse pool of precursors that may seed future outbreaks in poultry and possible human transmissions, suggesting the need for high-quality surveillance.

Parrot bornavirus-2 and -4 RNA detected in wild bird samples in Japan are phylogenetically adjacent to those found in pet birds in Japan.

Bornaviruses (family Bornaviridae) are non-segmented negative-strand RNA viruses. Avian bornaviruses (ABVs), which are causative agents of proventricular dilatation disease, are a genetically diverse group with at least 15 genotypes, including parrot bornaviruses (PaBVs) and aquatic bird bornavirus 1(ABBV-1). Borna disease virus 1(BoDV-1), which infects mammals and causes neurological diseases, has also been reported to infect avian species, although the numbers of the cases have been markedly fewer than those of ABVs. In this study, we conducted genetic surveillance to detect ABVs (PaBV-1 to -5 and ABBV-1) and BoDV-1 in wild birds in Japan. A total of 2078 fecal or cloacal swab samples were collected from wild birds in 2006, 2007, 2008, and 2011, in two regions of Japan. The results demonstrated the presence of PaBV-2 and -4 RNA, while no positive results for other PaBVs, ABBV-1, and BoDV-1 were obtained. PaBV-2 and -4 RNA were detected in 18 samples (0.9 %) of the genera Anas, Grus, Larus, Calidris, Haliaeetus, and Emberiza, in which either PaBV-2 RNA or PaBV-4 RNA, or both PaBV-2 and -4 RNA were detected in 15 (0.7 %), 5 (0.2 %), and 2 (0.1 %) samples, respectively. The nucleotide sequences of PaBV-2 and -4 detected in these samples from wild birds are phylogenetically close to those found in samples from pet birds in Japan, with identities ranging from 99.8 to 100 % and from 98.2 to 99.4 %, respectively. To the best of our knowledge, this is the first report on the detection of PaBV-2 and -4 RNA detected in samples from wild birds.

Genetic characterization of a rare H12N3 avian influenza virus isolated from a green-winged teal in Japan.

This study reports on the genetic characterization of an avian influenza virus, subtype H12N3, isolated from an Eurasian green-winged teal (Anas crecca) in Japan in 2009. The entire genome sequence of the isolate was analyzed, and phylogenetic analyses were conducted to characterize the evolutionary history of the isolate. Phylogenetic analysis of the hemagglutinin and neuraminidase genes indicated that the virus belonged to the Eurasian-like avian lineage. Molecular dating indicated that this H12 virus is likely a multiple reassortant influenza A virus. This is the first reported characterization of influenza A virus subtype H12N3 isolated in Japan and these data contribute to the accumulation of knowledge on the genetic diversity and generation of novel influenza A viruses.

Characterization of a genetic and antigenic variant of avian paramyxovirus 6 isolated from a migratory wild bird, the red-necked stint (Calidris ruficollis).

A hemagglutinating virus (8KS0813) was isolated from a red-necked stint. Hemagglutination inhibition and neutralization tests indicated that 8KS0813 was antigenically related to a prototype strain, APMV-6/duck/Hong Kong/18/199/77, but with an 8- and 16-fold difference, respectively, in their titers. The full genome sequence of 8KS0813 showed 98.6 % nucleotide sequence identity to that of APMV-6/duck/Italy/4524-2/07, which has been reported to belong to an APMV-6 subgroup, and showed less similarity to that of the prototype strain (70.6 % similarity). The growth of 8KS0813 and the prototype strain in four different cell cultures was greatly enhanced by adding trypsin. Interestingly, this virus induced syncytia only in Vero cells. 8KS0813 was identified as APMV-6/red-necked stint/Japan/8KS0813/08, but it is antigenically and genetically distinguishable from the prototype strain, suggesting that variant APMV-6 is circulating in migratory birds.

Genetic characterization of an H5N1 avian influenza virus from a vaccinated duck flock in Vietnam.

This study reports the genetic characterization of a highly pathogenic avian influenza virus subtype H5N1 isolated from a moribund domestic duck in central Vietnam during 2012. In the moribund duck's flock, within 6 days after vaccination with a commercial H5N1 vaccine (Re-5) to 59-day-old birds, 120 out of 2,000 ducks died. Genetic analysis revealed a substantial number of mutations in the HA gene of the isolate in comparison with the vaccine strains, Re-1 and Re-5. Similar mutations were also found in selected Vietnamese H5N1 strains isolated since 2009. Mutations in the HA gene involved positions at antigenic sites associated with antibody binding and also neutralizing epitopes, with some of the mutations resulting in the modification of N-linked glycosylation of the HA. Those mutations may be related to the escape of virus from antibody binding and the infection of poultry, interpretations which may be confirmed through a reverse genetics approach. The virus also carried an amino acid substitution in the M2, which conferred a reduced susceptibility to amantadine, but no neuraminidase inhibitor resistance markers were found in the viral NA gene. Additional information including vaccination history in the farm and the surrounding area is needed to fully understand the background of this outbreak. Such understanding and expanded monitoring of the H5N1 influenza viruses circulating in Vietnam is an urgent need to provide updated information to improve effective vaccine strain selection and vaccination protocols, aiding disease control, and biosecurity to prevent H5N1 infection in both poultry and humans.

Transcriptome profiles of organ tissues from pigs experimentally infected with African swine fever virus in early phase of infection.

African swine fever, caused by African swine fever virus (ASFV), is a highly contagious and fatal disease that poses a significant threat to the global pig industry. The limited information on ASFV pathogenesis and ASFV-host interactions has recently prompted numerous transcriptomic studies. However, most of these studies have focused on elucidating the transcriptome profiles of ASFV-infected porcine alveolar macrophages in vitro. Here, we analyzed dynamic transcriptional patterns in vivo in nine organ tissues (spleen, submandibular lymph node, mesenteric lymph node, inguinal lymph node, tonsils, lungs, liver, kidneys, and heart) obtained from pigs in the early stages of ASFV infection (1 and 3 d after viremia). We observed rapid spread of ASFV to the spleen after viremia, followed by broad transmission to the liver and lungs and subsequently, the submandibular and inguinal lymph nodes. Profound variations in gene expression patterns were observed across all organs and at all time-points, providing an understanding of the distinct defence strategies employed by each organ against ASFV infection. All ASFV-infected organs exhibited a collaborative response, activating immune-associated genes such as S100A8, thereby triggering a pro-inflammatory cytokine storm and interferon activation. Functional analysis suggested that ASFV exploits the PI3K-Akt signalling pathway to evade the host immune system. Overall, our findings provide leads into the mechanisms underlying pathogenesis and host immune responses in different organs during the early stages of infection, which can guide further explorations, aid the development of efficacious antiviral strategies against ASFV, and identify valuable candidate gene targets for vaccine development.

Genetic diversity and mutation of avian paramyxovirus serotype 1 (Newcastle disease virus) in wild birds and evidence for intercontinental spread.

Avian paramyxovirus serotype 1 (APMV-1), or Newcastle disease virus, is the causative agent of Newcastle disease, one of the most economically important diseases for poultry production worldwide and a cause of periodic epizootics in wild birds in North America. In this study, we examined the genetic diversity of APMV-1 isolated from migratory birds sampled in Alaska, Japan, and Russia and assessed the evidence for intercontinental virus spread using phylogenetic methods. Additionally, we predicted viral virulence using deduced amino acid residues for the fusion protein cleavage site and estimated mutation rates for the fusion gene of class I and class II migratory bird isolates. All 73 isolates sequenced as part of this study were most closely related to virus genotypes previously reported for wild birds; however, five class II genotype I isolates formed a monophyletic clade exhibiting previously unreported genetic diversity, which met criteria for the designation of a new sub-genotype. Phylogenetic analysis of wild-bird isolates provided evidence for intercontinental virus spread, specifically viral lineages of APMV-1 class II genotype I sub-genotypes Ib and Ic. This result supports migratory bird movement as a possible mechanism for the redistribution of APMV-1. None of the predicted deduced amino acid motifs for the fusion protein cleavage site of APMV-1 strains isolated from migratory birds in Alaska, Japan, and Russia were consistent with those of previously identified virulent viruses. These data therefore provide no support for these strains contributing to the emergence of avian pathogens. The estimated mutation rates for fusion genes of class I and class II wild-bird isolates were faster than those reported previously for non-virulent APMV-1 strains. Collectively, these findings provide new insight into the diversity, spread, and evolution of APMV-1 in wild birds.

Pathobiological analysis of african swine fever virus contact-exposed pigs and estimation of the basic reproduction number of the virus in Vietnam.

BACKGROUND: African swine fever (ASF), caused by African swine fever virus (ASFV), is a fatal disease affecting wild and domestic pigs. Since China reported the first ASF outbreak in August 2018, ASFV has swept over the neighbouring Asian countries. However, studies involving experimental pig-to-pig ASFV transmission in Vietnam are lacking. The main objective of this experimental study was to demonstrate the pathobiological characteristics of ASFV contact-exposed pigs and estimate their basic reproduction number (R0) in Vietnam. Fifteen pigs were randomly divided into two groups: experimental (n = 10) and negative control (n = 5) groups. One pig in the experimental group was intramuscularly inoculated with ASFV strain from Vietnam in 2020 and housed with the uninoculated pigs during the study period (28 days). RESULTS: The inoculated pig died 6 days post-inoculation, and the final survival rate was 90.0%. We started observing viremia and excretion of ASFV 10 days post-exposure in contact-exposed pigs. Unlike the surviving and negative control pigs, all necropsied pigs showed severe congestive splenomegaly and moderate-to-severe haemorrhagic lesions in the lymph nodes. The surviving pig presented with mild haemorrhagic lesions in the spleen and kidneys. We used Susceptible-Infectious-Removed models for estimating R0. The R0 values for exponential growth (EG) and maximum likelihood (ML) were calculated to be 2.916 and 4.015, respectively. In addition, the transmission rates (ß) were estimated to be 0.729 (95% confidence interval [CI]: 0.379-1.765) for EG and 1.004 (95% CI: 0.283-2.450) for ML. CONCLUSIONS: This study revealed pathobiological and epidemiological information in about pig-to-pig ASFV transmission. Our findings suggested that culling infected herds within a brief period of time may mitigate the spread of ASF outbreaks.

Spatiotemporal Analysis and Assessment of Risk Factors in Transmission of African Swine Fever Along the Major Pig Value Chain in Lao Cai Province, Vietnam.

African swine fever (ASF) is a contagious and lethal hemorrhagic disease with a case fatality rate approaching 100% in domestic pigs. The main objectives of this study were to describe the spatiotemporal analysis as well as to assess the potential risk factors along the pig value chain in Lao Cai province, Vietnam. A total of 925 outbreaks were reported from 2019 to 2020. The three clusters (primary, secondary and 5th) were observed near the Chinese border. The most temporal clusters were detected between May and August during the study period. In addition, we evaluated the association between ASF outbreak locations to the nearest main roads and elevation. For ASF outbreak locations to the nearest main roads, compared with the reference (<5,000 m), <1,000 m (10.22 times) and 1,000-2,000 m (1.98 times) were significantly higher occurrences of ASF. For elevation, compared to the reference (>1,500 m), the farm locations with <500 m (55.31 times) showed a significantly increased risk of ASF outbreaks. Farmers perceived that the highest risk of ASF transmission may come from collectors and slaughterers, intermediaries inside and outside the commune, feed agents and maize agents in the commune, and pig retailers. Both commercial and household pig producers considered minimizing the number of people going in and out of pig stables and improving healthcare and husbandry procedures to be both very important and feasible. There is a need for compliance by all pig producers and other actors in the pig value chain to adopt biosecurity practices. Therefore, awareness, knowledge and understanding of infection and risks of ASF need to be improved. Veterinary officials at the provincial and district levels need to improve capacity and resources to perform laboratory analysis for ASF and need to coordinate with local actors on the control and prevention of ASF in the community.

Characterization of Lumpy skin disease virus isolated from a giraffe in Vietnam.

While investigating a giraffe's death in a Vietnamese zoo, we successfully identified and isolated Lumpy skin disease virus (LSDV) from skin nodule biopsies and ruptured nodule wound swab samples. Phylogenetic analysis indicated that the isolate obtained in this study was closely related to the previous Vietnamese and Chinese LSDV strains from cattle. This is the first report on the genome detection and isolation of LSDV in a diseased giraffe in Vietnam. Further study is needed to better understand the epidemiology of this disease in wildlife.

Zoonotic Flavivirus Exposure in Peri-Urban and Suburban Pig-Keeping in Hanoi, Vietnam, and the Knowledge and Preventive Practices of Pig Farmers.

Mosquito-borne diseases (MBDs), including those caused by flaviviruses, remain human health problems for developing and urbanizing economies. This cross-sectional study examined risks of flavivirus exposure through a survey regarding knowledge and practices of pig farmers, and serological analysis of pigs in peri-urban and suburban Hanoi city. A total of 636 pig sera from 179 pig farms in 4 districts, namely, Chuong My, Dan Phuong, Ha Dong, and Bac Tu Liem, were analyzed by a competitive ELISA designed for flavivirus antibody detection. The results indicated a low level of awareness about MBDs among pig farmers, and a high seroprevalence in pigs at 88.5% (95%CI = 85.8-90.9%). Moreover, common practices of pig owners to prevent mosquitoes at home and farm did not show a significant reduction in flavivirus exposure in pigs. At animal level, significant associations between seropositive pigs and the farms with more than 60 pigs, and the district location were found. Farm-level multivariable analysis did not identify significant risk factors for flavivirus exposure. The study suggests that improving awareness of pig owners about MBDs in Hanoi city may be warranted to reduce the risk for MBD flavivirus infections in both humans and pigs.