Detection of Recombinant African Swine Fever Virus Strains of p72 Genotypes I and II in Domestic Pigs, Vietnam, 2023.

African swine fever virus (ASFV) genotype II is endemic to Vietnam. We detected recombinant ASFV genotypes I and II (rASFV I/II) strains in domestic pigs from 6 northern provinces in Vietnam. The introduction of rASFV I/II strains could complicate ongoing ASFV control measures in the region.

A multiplex real-time RT-qPCR assay for simultaneous detection of porcine epidemic diarrhea virus, porcine deltacoronavirus, and swine acute diarrhea syndrome coronavirus.

Porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), and swine acute diarrhea syndrome coronavirus (SADS-CoV) cause intestinal diseases with similar manifestations in suckling piglets. In this study, we developed a multiplex real-time PCR for differential diagnosis of PEDV, PDCoV, and SADS-CoV. The assay demonstrated high specificity with a detection limit of 5 copies/µl for each virus. The assay specifically detected PEDV, PDCoV, and SADS-CoV and excluded all other swine pathogens circulating in pigs. Furthermore, the assay exhibited satisfactory performance in analyzing clinical samples. The data indicate that the newly developed multiplex real-time PCR method can be applied for differential diagnosis of porcine enteric coronaviruses.

Magnetic-bead-based DNA-capture-assisted real-time polymerase chain reaction and recombinase polymerase amplification for the detection of African swine fever virus.

African swine fever (ASF) is a deadly disease in swine caused by African swine fever virus (ASFV). The global spread of ASFV has resulted in significant economic losses worldwide. Improved early detection has been the most important first line of defense for preventing ASF outbreaks and for activating control measures. Despite the availability of rapid amplification methods, nucleic acid extraction from specimens still needs to be performed in a laboratory. To facilitate this step, we exploited the strong affinity of biotin-streptavidin binding by functionalizing streptavidin-coated magnetic beads with biotinylated oligonucleotide capture probes to efficiently capture genotype II ASFV DNA directly from crude clinical samples. The captured DNA is suitable for detection using real-time quantitative PCR (qPCR) and recombinase polymerase amplification (RPA). In this study, ASFV DNA was efficiently captured from swine feces, serum, and tissue samples. Both DNA-capture-assisted qPCR and RPA-based detection methods have a limit of detection (LOD) of 102 copies/µl, which is comparable to those of commercially available kits. In addition, an RPA-SYBR Green I method was developed for the immediate visual detection of ASFV DNA, which is time-saving and efficient for resource-limited field settings. In summary, a rapid, versatile, sequence-specific DNA capture method was developed to efficiently capture ASFV DNA from swine clinical samples and subsequent detection by qPCR and RPA, which has the potential to be used for robust screening and surveillance of ASFV and in point-of-care (POC) diagnostics.

Instrument-free, visual and direct detection of porcine reproductive and respiratory syndrome viruses in resource-limited settings.

Porcine reproductive and respiratory syndrome (PRRS) caused by PRRS virus (PRRSV) is one of the most complicated and dangerous diseases in pigs with high mortality since it modulates the immune system of the lungs and has been closely associated with secondary infection of other lethal bacteria and viruses. The gold standard of molecular diagnosis for PRRSV, reverse transcription (RT)-PCR, is time-consuming, expensive and requires transportation of samples to a specialized laboratory. In this study, a direct colorimetric RT-loop-mediated isothermal amplification (RT-LAMP) method was developed to specifically and rapidly detect PRRSV. The RT-LAMP outcomes can be visualized by the naked eye after 45 min of incubation at 65˚C without any cross-reactivity recorded with the bacteria and other viruses tested. In particular, the mobile, non-instrumented, commercial pocket hand warmers were demonstrated to su-ccessfully provide constant temperature for consistent nucleic acid amplification throughout the RT-LAMP reactions. The limit of detection of the assay was defined as the genomic RNA concentration extracted from a known viral titer of 10-2.5 TCID50/ml. The direct use of clinical serum samples required a simple dilution to maintain the performance of the colorimetric RT-LAMP assay. Therefore, the direct colorimetric RT-LAMP assay developed is well-qualified for producing a ready-to-use kit for PRRSV diagnosis in the field. Keywords: porcine reproductive and respiratory syndrome; rapid testing; RT-LAMP; colorimetric; direct detection; instrument-free.

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.

Multiple variants of African swine fever virus circulating in Vietnam.

African swine fever (ASF) is a contagious and deadly viral disease affecting swine of all ages. ASF was first reported in Vietnam in February 2019, and it is now considered endemic in Vietnam. In this study, 122 ASF-positive samples collected from domestic pigs in 28 different provinces of northern, central, and southern Vietnam during outbreaks in 2019-2021 were genetically characterized. The findings confirmed that all ASF virus (ASFV) strains circulating in Vietnam belonged to p72 genotype II, p54 genotype II, CD2v serogroup 8, and CVR gene variant type I. However, further analysis based on the tandem repeat sequences located between I73R and I329L genes revealed that there were three different variants of ASFV, IGR I, II, and III, circulating in the domestic pig population in Vietnam. The IGR II variants were the most prevalent (117/122 strains) and were detected in pigs in all of the provinces tested, followed by IGR III (4/122 strains) and IGR I (1/122 strains). This study confirms for the first time the presence of IGR III variants in Vietnam.

Molecular and histopathological characterization of lumpy skin disease in cattle in northern Vietnam during the 2020-2021 outbreaks.

Lumpy skin disease (LSD) is a serious emerging infectious disease in cattle caused by a virus of the family Poxviridae. According to the Department of Animal Health, LSD first occurred in Vietnam at the end of October 2020 in Cao Bang and Lang Son provinces. So far, the disease has infected over 63,000 animals, resulting in 9170 deaths occurring in 32 different provinces in northern and central Vietnam. In this study, skin samples from lumpy skin disease virus (LSDV)-infected cattle from the northern provinces of Vietnam displaying clinical symptoms including fever (> 40 °C), runny nose, drooling, and skin lesions were used for genetic characterization and histopathology. Genetic analysis of the partial P32 (LSDV074), partial F (LSDV117), complete RPO30 (LSDV035), and complete G-protein-coupled-chemokine-like receptor (GPCR) (LSDV011) genes showed that all Vietnamese LSDV strains belonged to the genus Capripoxvirus and were closely related to LSDV strains isolated in China. Microscopic examination of the skin lesions showed thickening of the epidermal layer of the skin and hair follicles, hyperplasia of sebaceous glands, intracytoplasmic inclusion bodies, and hemorrhages in the mesoderm.

Molecular profile of African swine fever virus (ASFV) circulating in Vietnam during 2019-2020 outbreaks.

African swine fever (ASF) is a highly infectious disease of pigs caused by African swine fever virus (ASFV). In order to identify potential genetic variations among ASFV strains circulating in Vietnam, 26 ASFV isolates from organs and blood samples collected from domestic pigs from 23 different provinces of northern, central and southern Vietnam during 2019-2020 ASF outbreaks were genetically characterized. Nucleotide sequences were determined for a portion of the B646L (p72) gene, the complete E183L (p54) gene, the variable region of EP402R (CD2v), the central variable region (CVR) of pB602L, and a tandem repeat sequence (TRS) between the I73R and I329L genes. Analysis of the partial B646L (p72) and EP402R (CD2v) gene sequences and the full-length E183L (p54) gene sequence showed that all 26 ASFV isolates belonged to genotype II and serotype VIII and that they were identical to the strain Georgia/2007/1 and all ASFV strains sequenced in China. The TRS between the I73R and I329L genes contained a 10-nucleotide insertion that was observed in the Chinese ASFV strain CN201801 isolated from domestic pigs in 2018, but not in the Georgia/2007/1 and China/Jilin/2018/boar strains isolated from wild boar in China. This is the first intra-epidemic genome analysis reported for the ASFV strains circulating in Vietnam.

Reassortant Highly Pathogenic H5N6 Avian Influenza Virus Containing Low Pathogenic Viral Genes in a Local Live Poultry Market, Vietnam.

Sites of live poultry trade and marketing are hot spots for avian influenza virus (AIV) transmission. We conducted active surveillance at a local live poultry market (LPM) in northern Vietnamese provinces in December 2016. Feces samples from the market were collected and tested for AIV. A new reassorted AIV strain was isolated from female chickens, named A/chicken/Vietnam/AI-1606/2016 (H5N6), and was found to belong to group C of clade 2.3.4.4 H5N6 highly pathogenic (HP) AIVs. The neuraminidase gene belongs to the reassortant B type. The viral genome also contained polymerase basic 2 and polymerase acidic, which were most closely related to domestic-duck-origin low pathogenic AIVs in Japan (H3N8) and Mongolia (H4N6). The other six genes were most closely related to poultry-origin H5N6 HP AIVs in Vietnam and had over 97% sequence identity with human AIV isolate A/Guangzhou/39715/2014 (H5N6). The new reassorted AIV isolate A/chicken/Vietnam/AI-1606/2016 (H5N6) identified in this study exemplifies AIVs reassortment and evolution through contact among wild birds, poultry farms, and LPMs. Therefore, active surveillance of AIVs is necessary to prevent potential threats to human and animal health.

O/SEA/Mya-98 lineage foot-and-mouth disease virus was responsible for an extensive epidemic that occurred in late 2018 in Vietnam.

Since late 2018, foot-and-mouth disease (FMD) has reemerged and rapidly swept through pig farms in North and Central Vietnam, despite widespread use of commercial FMD vaccines. To investigate the FMD virus (FMDV) strains responsible for the current epidemics, 40 FMDV samples were collected from 17 provinces during November-December 2018, and the VP1 coding genes were sequenced and analyzed. Phylogenetic analysis and sequence comparisons revealed that all of the reemerging Vietnamese FMDVs belonged to the Mya-98 lineage of the O/Southeast Asia topotype (O/SEA/Mya-98) and shared high nucleotide (99.06-100% identity) and amino acid (97.65-100% identity) sequence similarity with each other. The study results suggested that the reemerging FMDVs originated from local Vietnamese strains. Field viruses had different amino acids in the antigenic sites of VP1 when compared to the strains used in the vaccines. The present study provides an important basis for vaccine selection in the battle against FMD in Vietnam.

Outbreak of African Swine Fever, Vietnam, 2019.

African swine fever is one of the most dangerous diseases of swine. We confirmed the 2019 outbreak in Vietnam by real-time reverse transcription PCR. The causative strain belonged to p72 genotype II and was 100% identical with viruses isolated in China (2018) and Georgia (2007). International prevention and control collaboration is needed.

Molecular epidemiology of canine parvovirus type 2 in Vietnam from November 2016 to February 2018.

BACKGROUND: Canine parvovirus type 2 (CPV-2) was first identified in the late 1970s; it causes intestinal hemorrhage with severe bloody diarrhea in kennels and dog shelters worldwide. Since its emergence, CPV-2 has been replaced with new genetic variants (CPV-2a, CPV-2b, and CPV-2c). Currently, information about the genotype prevalence of CPV-2 in Vietnam is limited. In the present study, we investigated the genotype prevalence and distribution of CPV-2 in the three regions of Vietnam. METHODS: Rectal swabs were collected from 260 dogs with suspected CPV-2 infection from northern, central, and southern Vietnam from November 2016 to February 2018. All samples were identified as parvovirus positive by real-time PCR, and further genotyping was performed using a SimpleProbe® real-time PCR assay. RESULTS: Of the 260 Vietnamese CPV-2 isolates, 6 isolates (2.31%) were identified as CPV-2a, 251 isolates (96.54%) were identified as CPV-2c and 3 isolates (1.15%) were untypable using the SimpleProbe® real-time PCR assay. In northern Vietnam, the percentages of CPV-2a and CPV-2c were 2.97% (3/101) and 97.3% (98/101), respectively. In central Vietnam, the percentages of CPV-2a and CPV-2c were 1.11% (1/90) and 98.89% (89/90), respectively. In southern Vietnam, the percentages of CPV-2a and CPV-2c were 3.03% (2/66) and 96.97% (64/66), respectively. CPV-2b was not observed in this study. The VP2 genes of CPV-2c in Vietnam are more genetically similar to those of CPV-2c strains in China and Taiwan than to those of prototype CPV-2c strains (FJ222821) or the first Vietnamese CPV-2c (AB120727). CONCLUSION: The present study provides evidence that CPV-2c is the most prevalent variant in Vietnam. Phylogenetic analysis demonstrated that the recent Vietnamese CPV-2c isolates share a common evolutionary origin with Asian CPV-2c strains.

Genetic characterization of bovine coronavirus in Vietnam.

A maximum clade credibility tree constructed using the full-length spike (S) and hemagglutinin-esterase genes revealed that Vietnamese Bovine coronavirus (BCoV) strains belong to a single cluster (C1); therefore, they might share a common origin with Cuban and Chinese BCoV strains. The omega values of cluster 1 (C1) and cluster 2 (C2) were 0.15734 and 0.11613, respectively, and naive empirical bayes analysis identified two amino acid positions (179 and 501) in the S protein in C1 and three amino acid positions (113, 501, and 525) in that of C2 that underwent positive selection (p > 99%). The evolutionary rate of C1 was estimated to be 7.6206 × 10-4 substitutions/site/year, and the most recent common ancestor (tMRCA) of Vietnamese BCoVs was estimated to date back to 1962 (95% HPD 1950-1973). The effective population sizes of C1 and C2 underwent a rapid reduction after 2000 and 2004, respectively.

Host Cell Mimic Polymersomes for Rapid Detection of Highly Pathogenic Influenza Virus via a Viral Fusion and Cell Entry Mechanism.

Highly pathogenic avian influenza virus (HPAIV) infections have occurred continuously and crossed the species barrier to humans, leading to fatalities. A polymerase chain reaction based molecular test is currently the most sensitive diagnostic tool for HPAIV; however, the results must be analyzed in centralized diagnosis systems by a trained individual. This requirement leads to delays in quarantine and isolation. To control the spread of HPAIV, rapid and accurate diagnostics suitable for field testing are needed, and the tests must facilitate a differential diagnosis between HPAIV and low pathogenic avian influenza virus (LPAIV), which undergo cleavage specifically by trypsin- or furin-like proteases, respectively. In this study, a differential avian influenza virus rapid test kit is developed and evaluated in vitro and using clinical specimens from HPAIV H5N1-infected animals. It is demonstrated that this rapid test kit provides highly sensitive and specific detection of HPAIV and LPAIV and is thus a useful field diagnostic tool for H5N1 HPAIV outbreaks and for rapid quarantine control of the disease.

A novel strain of porcine deltacoronavirus in Vietnam.

Two porcine deltacoronavirus (PDCoV) strains (Binh21 and HaNoi6) were isolated from two pig farms in North Vietnam. Phylogenetic analysis of the complete genomes and the Spike and Membrane genes revealed that the two Vietnam PDCoVs belong to the same lineage as PDCoVs from Thailand and Laos; however, the N genes belonged to the same lineage as PDCoVs from the USA, Korea, China, and Hong Kong. The recombination detection program subsequently identified the major parent (S5011 strain) and minor parent (HKU15-44 strain) of the two Vietnam PDCoV strains (p < 0.01).

Molecular characterization of the spike and ORF3 genes of porcine epidemic diarrhea virus in the Philippines.

Porcine epidemic diarrhea virus (PEDV) is a highly contagious enteric pathogen of swine. In the present study, we analyzed the spike genes and ORF3 genes of seven PEDV strains detected in Philippine pigs in June 2014. There are four major epitope regions in the spike glycoprotein: a CO-26K equivalent (COE) domain, SS2 and SS6 epitopes, and an epitope region recognized by the 2C10 monoclonal antibody. Analysis of Philippine strains revealed amino acid substitutions in the SS6 epitope region (LQDGQVKI to SQSGQVKI) of the S1 domain. Substitutions were also detected in the 2C10 epitope region (GPRLQPY to GPRFQPY) in the cytoplasmic domain. Phylogenetic analysis of the complete spike gene sequences from the seven strains revealed that they clustered within the G2 group but were distantly related to the North American and INDELs clusters. Interestingly, these strains were close to Vietnamese PEDVs on the ORF3 genetic tree and showed high (97.0-97.6 %) sequence identity to ORF3 genes at the nucleotide level.

Evolutionary phylodynamics of foot-and-mouth disease virus serotypes O and A circulating in Vietnam.

BACKGROUND: Foot-and-mouth disease virus (FMDV) is one of the highest risk factors that affects the animal industry of the country. The virus causes production loss and high ratio mortality in young cloven-hoofed animals in Vietnam. The VP1 coding gene of 80 FMDV samples (66 samples of the serotype O and 14 samples of the serotype A) collected from endemic outbreaks during 2006-2014 were analyzed to investigate their phylogeny and genetic relationship with other available FMDVs globally. RESULTS: Phylogenetic analysis indicated that the serotype O strains were clustered into two distinct viral topotypes (the SEA and ME-SA), while the serotype A strains were all clustered into the genotype IX. Among the study strains, the amino acid sequence identities were shared at a level of 90.1-100, 92.9-100, and 92.8-100% for the topotypes SEA, ME-SA, and genotype IX, respectively. Substitutions leading to changes in the amino acid sequence, which are critical for the VP1 antigenic sites were also identified. Our results showed that the studied strains are most closely related to the recent FMDV isolates from Southeast Asian countries (Myanmar, Thailand, Cambodia, Malaysia, and Laos), but are distinct from the earlier FMDV isolates within the genotypes. CONCLUSIONS: This study provides important evidence of recent movement of FMDVs serotype O and A into Vietnam within the last decade and their genetic accumulation to be closely related to strains causing FMD in surrounding countries.

Molecular evolution of type 2 porcine reproductive and respiratory syndrome viruses circulating in Vietnam from 2007 to 2015.

BACKGROUND: Porcine respiratory and reproductive syndrome (PRRS) virus is one of the most economically significant pathogens in the Vietnamese swine industry. ORF5, which participates in many functional processes, including virion assembly, entry of the virus into the host cell, and viral adaptation to the host immune response, has been widely used in molecular evolution and phylogeny studies. Knowing of molecular evolution of PRRSV fields strains might contribute to PRRS control in Vietnam. RESULTS: The results showed that phylogenetic analysis indicated that all strains belonged to sub-lineages 8.7 and 5.1. The nucleotide and amino acid identities between strains were 84.5-100% and 82-100%, respectively. Furthermore, the results revealed differences in nucleotide and amino acid identities between the 2 sub-lineage groups. N-glycosylation prediction identified 7 potential N-glycosylation sites and 11 glycotypes. Analyses of the GP5 sequences, revealed 7 sites under positive selective pressure and 25 under negative selective pressure. CONCLUSIONS: Phylogenetic analysis based on ORF5 sequence indicated the diversity of PRRSV in Vietnam. Furthermore, the variance of N-glycosylation sites and position under selective pressure were demonstrated. This study expands existing knowledge on the genetic diversity and evolution of PRRSV in Vietnam and assists the effective strategies for PRRS vaccine development in Vietnam.

A novel strain of porcine epidemic diarrhea virus in Vietnamese pigs.

Porcine epidemic diarrhea virus (PEDV) causes severe diarrhea and dehydration in suckling pigs and has caused high rates of death among piglets and substantial economic loss in Vietnam since 2009. To investigate the genotypes of prevailing PEDVs, intestinal and fecal samples from piglets from central and northern Vietnam were collected and analyzed. Phylogenetic analysis of the nucleotide sequences of complete spike genes of PEDVs from Vietnam resulted in the identification of two divergent groups. PEDVs (HUA-PED45 and HUA-PED47) belonged to the G2b group, along with Chinese, US, and Korean strains occurring at the end of 2010, in May 2013 and in November 2013, respectively. Six strains from the Quang Tri region were assigned to the G1b group, along with Chinese and US strains. The Vietnamese PEDVs detected in infected piglets had a nationwide distribution and belonged to the G2b and G1b genotypes.

Stixis scandens leaf extract-loading ZnO nanoparticles for porcine epidemic diarrhea virus (PEDV) treatment.

Porcine epidemic diarrhea (PED) is one of the diseases that causes great losses for livestock farmers. Because vaccines against the disease are not very effective, there is a great demand for biological products with effective resistance to PED virus (PEDV). One of the most important trends today is the use of active ingredients from nature in animal husbandry. This study aimed to create an effective agent against PEDV from the extract of Stixis scandens, which has been shown to inhibit PEDV. The aqueous (denoted as TCN) and ethanolic extracts (denoted as TCC) of Stixis scandens leaves were first prepared and then qualitatively analyzed for their chemical compositions. The TCN was used to synthesize ZnO nanoparticles (NPs) at various sizes from 20 to 120 nm. Subsequently, TCC was loaded on ZnO NPs to form ZnO-extract nanoformulations with an extract loading content of 5.8-7.6%. Total polyphenols (TP) and total alkaloids (TA) in TCC were 38.51 ± 0.25 µg GAE per mg and 22.37 ± 0.41 µg AtrE per mg, respectively. TP was less loaded but more released from the nanoformulations than TA. The A1T nanoformulation, containing only 7.6% extract, had a minimum PEDV inhibitory concentration of 3.9 µg mL-1, which was comparable to that of TCC. The experiments confirmed that the nanoformulations are promising for PEDV inhibition applications.