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.

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.

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.

The Distribution and Composition of Vector Abundance in Hanoi City, Vietnam: Association with Livestock Keeping and Flavivirus Detection.

BACKGROUND: Dengue virus and Japanese encephalitis virus are two common flaviviruses that are spread widely by Aedes and Culex mosquitoes. Livestock keeping is vital for cities; however, it can pose the risk of increasing the mosquito population. Our study explored how livestock keeping in and around a large city is associated with the presence of mosquitoes and the risk of them spreading flaviviruses. METHODS: An entomological study was conducted in 6 districts with 233 households with livestock, and 280 households without livestock, in Hanoi city. BG-Sentinel traps and CDC light traps were used to collect mosquitoes close to animal farms and human habitats. Adult mosquitoes were counted, identified to species level, and grouped into 385 pools, which were screened for flaviviruses using a pan-flavivirus qPCR protocol and sequencing. RESULTS: A total of 12,861 adult mosquitoes were collected at the 513 households, with 5 different genera collected, of which the Culex genus was the most abundant. Our study found that there was a positive association between livestock keeping and the size of the mosquito population-most predominantly between pig rearing and Culex species (p < 0.001). One pool of Cx. tritaeniorhynchus, collected in a peri-urban district, was found to be positive for Japanese encephalitis virus. CONCLUSIONS: The risk of flavivirus transmission in urban areas of Hanoi city due to the spread of Culex and Aedes mosquitoes could be facilitated by livestock keeping.

Molecular characterization of bovine foamy virus and its association with bovine leukemia virus infection in Vietnamese cattle.

The detection of bovine foamy virus (BFV) in Vietnamese cattle was performed using conventional PCR targeting pol and gag genes. Out of 243 tested samples, ten (4.1%) and eight (3.3%) samples were positive for BFV gag and pol DNA, respectively. The prevalence of bovine leukemia virus (BLV) estimated by detection of proviral DNA using nested PCR targeting env gene was 26.7% (65/243). The results of nucleotide sequence alignment and the phylogenetic analysis suggested that Vietnamese BFV strains showed high homology to isolates belonging to either European or non-European clades. There was no significant correlation between BLV and BFV. This study provides information regarding BFV infection and confirms the existence of two BFV clades among Vietnamese cattle for the first time.

An epidemiological study of Streptococcus suis prevalence among swine at industrial swine farms in Northern Vietnam.

INTRODUCTION: Streptococcus suis is a zoonotic pathogen found in swine that may cause systemic infection in humans. S. suis is endemic in Southeast Asia and is the leading cause of adult meningitis in Vietnam. Given Vietnam's increasing centralization of the swine industry, we sought to estimate the prevalence of S. suis on large swine farms in Northern Vietnam. METHODS: A cross-sectional, one-health-oriented, surveillance study for S. suis was conducted between October 2019-March 2020. Swine oral, swine worker nasal, and bioaerosol samples were collected from four large-scale swine farms (>500 swine) in three provinces in Northern Vietnam: Lao Cai, Bac Giang, and Quang Ninh. Samples were evaluated for presence of S. suis growth on blood agar plates and confirmed with conventional polymerase chain reaction. RESULTS: The authors found that 4/174 (2.3%, 95% CI: 0.6-5.8%) of swine oral samples and 1/58 (1.7%, 95% CI: 0-9.2%) bioaerosol samples were positive for S. suis by bacterial culture and conventional PCR. S. suis was not detected in any swine worker nasal wash samples. There was no significant relationship between sampling location and month of sample collection with results of swine oral or bioaerosol samples. CONCLUSION: Compared to previous reports from slaughterhouses in Vietnam, the lower than expected prevalence of S. suis, supports the notion that that recent efforts to centralize Vietnam's pork industry through establishment of large-scale farms with better biosecurity may have been effective in limiting S. suis prevalence on these large farms.

A stochastic network-based model to simulate farm-level transmission of African swine fever virus in Vietnam.

African swine fever virus is highly contagious, and mortality rates reach up to 100% depending on the host, virus dose, and the transmission routes. The main objective of this study was to develop a network-based simulation model for the farm-level transmission of ASF virus to evaluate the impact of changes in farm connectivity on ASF spread in Vietnam. A hypothetical population of 1,000 pig farms was created and used for the network-based simulation, where each farm represented a node, and the connection between farms represented an edge. The three scenarios modelled in this way (baseline, low, and high) evaluated the impact of connectivity on disease transmission. The median number of infected farms was higher as the connectivity increased (low: 659, baseline: 968 and high: 993). In addition, we evaluated the impact of the culling strategy on the number of infected farms. A total of four scenarios were simulated depending on the timing of culling after a farm was infected. We found that the timing of culling at 16, 12, 8, and 6 weeks had resulted in a reduction of the number of median infected farms by 81.92%, 91.63%, 100%, and 100%, respectively. Finally, our evaluation of the implication of stability of ties between farms indicated that if the farms were to have the same trading partners for at least six months could significantly reduce the median number of infected farms to two (95th percentile: 413) than in the basic model. Our study showed that pig movements among farms had a significant influence on the transmission dynamics of ASF virus. In addition, we found that the either timing of culling, reduction in the number of trading partners each farm had, or decreased mean contact rate during the outbreaks were essential to prevent or stop further outbreaks.

A stochastic simulation model of African swine fever transmission in domestic pig farms in the Red River Delta region in Vietnam.

The main objectives of this study were to model various scenarios of African swine fever (ASF) virus transmission among farms in Vietnam and to evaluate the impact of control strategies using North American Animal Disease Spread Model (NAADSM). A total of 7,882 pig farms in the Red River Delta (RRD) region were obtained from the General Statistics Office, and then, random points corresponding to the number of farms in each province were generated as exact farm locations were not available. A total of 10 models were developed, including movement control scenarios. In addition, we conducted sensitivity analysis to assess the impact of indirect contact transmission probability (TP). Overall, the indirect contact exhibited an important role in transmitting the ASF virus. In order to minimize ASF transmission between farms, we found that movement restriction needed to reach a certain level (approximately between 50% and 75%) and that the restriction had to be applied in a timely manner. This study offers valuable insight into how ASF virus can be transmitted via direct and indirect contact and controlled among farms under the various simulation scenarios. Our results suggest that the enforcement of movement restriction was an effective control measure as soon as the outbreaks were reported. In addition, this study provided evidence that high standards of biosecurity can contribute to the reduction of disease spread.

Molecular characterization of full genome sequences of Newcastle disease viruses circulating among vaccinated chickens in Egypt during 2011-2013.

Although intensive vaccination programs have been implemented, Newcastle disease (ND) outbreaks, accompanied by severe economic losses, are still reported in Egypt. The genetic characterization of ND virus (NDV) strains isolated from ND-vaccinated chicken flocks provides essential information for improving ND control strategies. Therefore, here, 38 NDV strains were isolated and identified from outbreaks among vaccinated flocks of broiler chickens located in the provinces of Qena, Luxor, and Aswan of Upper Egypt during 2011-2013. The investigated broiler chicken flocks (aged 28 to 40 days) had high mortality rates of up to 80%. All NDV isolates were genetically analyzed using next-generation DNA sequencing. From these isolates, 10 representative NDV strains were selected for further genetic analyses. Phylogenetic analysis of full-length coding genes revealed that the Egyptian NDV isolates belonged to a single sub-genotype, VII.1.1. These isolates were phylogenetically distant from the vaccine strains, including La Sota or Clone 30 (genotype II), which have been commonly used to vaccinate chicken flocks. Amino acid substitution K78R was observed in the neutralizing epitopes of the F proteins; whereas several mutations were found in the neutralizing epitopes of the hemagglutinin-neuraminidase proteins, notably, E347K. Overall, our results suggested that the occurrence of neutralizing epitope variants may be one of potential reasons for ND outbreaks. Further studies are needed to determine the protective effect of current vaccines against circulating virulent NDV strains.

Sensitive detection of influenza a virus based on a CdSe/CdS/ZnS quantum dot-linked rapid fluorescent immunochromatographic test.

Prevention is the most effective management strategy for influenza A infection in humans. In this study, we developed a CdSe/CdS/ZnS quantum dot (QD) fluorescent dye for rapid and sensitive detection of two common subtypes (H1N1 and H3N2) of influenza A virus, and examined its utility. CdSe/CdS/ZnS QD was conjugated with antibody (Ab) after conjugation with latex, making QD conjugate of QD + Latex + Ab. A stable photoluminescence of QD conjugate and advantage of CdSe/CdS/ZnS QD used was characterized in this study. The performance of a rapid fluorescent immunochromatographic test (FICT) employing QD conjugate (QD-FICT) in detecting influenza A/H1N1 was 8-fold and 64-fold higher than that of a europium nanoparticle-based FICT and a rapid diagnostic test (RDT; Standard Diagnostics BIOLINE Influenza A/B), respectively. For influenza A/H3N2, QD-FICT showed 8-fold and 128-fold higher performance than europium nanoparticle-based FICT and RDT, respectively. In clinical evaluations, QD-FICT showed 93.75% clinical sensitivity [45/48; 95% confidence interval (95% CI): 82.80-98.69], 100% clinical specificity (117/117; 95% CI: 96.90-100.00), and strong correlation (kappa; 0.98) with rRT-PCR (20 ≤ Ct ≤ 40). Europium nanoparticle-linked FICT showed 79.17% clinical sensitivity (38/48; 95% CI: 65.01-89.53) and 100% clinical specificity (117/117; 95% CI: 96.90-100.00), whereas RDT showed 77.08% sensitivity (37/48; 95% CI: 62.69-87.97), 100% specificity (117/117; 95% CI: 96.90-100.00), and reasonably good correlation with rRT-PCR (kappa; 0.93). Water-soluble QDs can therefore be used as an effective material for developing fluorescent diagnostic systems for rapid detection of human influenza A virus in clinical specimens.

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.

Peptide Aptamer of Complementarity-determining Region to Detect Avian Influenza Virus.

Despite significant progress in the development of diagnostic methods for influenza, avian influenza (AI) infection continues to represent a substantial threat to human health. Among the subtypes of AI, H5 influenza is highly infectious to animals and humans; however, there are no reliable H5 subtype-specific diagnostic systems owing to a scarcity of H5 subtype-specific detection elements. In this study, a new peptide aptamer (P1:KASGYTFTSF) was developed to recognize the H5 viral subtype using an in silico bioinformatics approach for predicting complementarity-determining regions (CDRs), and the aptamer was evaluated by immunoassays. The three-dimensional structure of influenza hemagglutinin (HA) and the peptide were used in a molecular docking study, and the peptide was compared to the epitope-derived peptide aptamer (H5-P2:KPNGAINF). Interactions between the peptides and the virus were then assessed by fluorescence-linked sandwich immunosorbent assay (FLISA), immunofluorescence staining assay (IFA), and rapid fluorescent immunochromatographic assay (FICT). P1 and H5-P2 both significantly detected H5N3 at 15.6 HAU/mL (P < 0.05), and P1 detected the virus more effectively (P < 0.05), consistent with the docking result. An optical image of the peptide recognizing an H5N3-infected cell was acquired by IFA, and was consistent with the antibody-linked IFA result. FICT employing the peptide showed the ability for H5 subtype-specific diagnosis, with 2-fold higher performance than that of a conventional, antibody-linked rapid test. This work shows the potential of a CDR-predicted peptide aptamer as a probe for immunological assays that can specifically recognize AI virus.

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.

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.

Development of a smartphone-based rapid dual fluorescent diagnostic system for the simultaneous detection of influenza A and H5 subtype in avian influenza A-infected patients.

Accurate and rapid diagnosis of highly pathogenic avian influenza A H5N1 is of critical importance for the effective clinical management of patients. Here, we developed a rapid and simultaneous detection toolkit for influenza A H5 subtype viruses in human samples based on a bioconjugate of quantum dots (QDs) assembly and a smartphone-based rapid dual fluorescent diagnostic system (SRDFDS). Methods: Two types of QDs were assembled on a latex bead to enhance the detection sensitivity and specificity of influenza A infection (QD580) and H5 subtype (QD650). The dual signals of influenza A and H5 subtype of H5N1-infected patients were detected simultaneously and quantified separately by SRDFDS equipped with two emission filters. Results: Our results showed a high sensitivity of 92.86% (13/14) and 78.57% (11/14), and a specificity of 100% (38/38, P < 0.0001) and 97.37% (37/38) for influenza A and H5 subtype detection, respectively. Conclusion: Therefore, our multiplex QD bioconjugates and SRDFDS-based influenza virus detection toolkit potentially provide accurate and meaningful diagnosis information with improved detection accuracies and sensitivities for H5N1 patients.