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.

Application of chitosan as a natural disinfectant against porcine epidemic diarrhoea virus.

Porcine epidemic diarrhoea virus (PEDV) is one of the major pathogens causing acute enteritis, which is characterised by vomiting and watery diarrhoea and commonly leads to high rates of mortality and morbidity in suckling piglets. Chitosan has been regarded as a promising natural disinfectant. In this study, the disinfectant effect and mammalian-cell toxicity of chitosan were evaluated against PEDV using Vero cells. A 0.01% solution of chitosan was determined to be an effective disinfectant. In addition, no evidence of toxicity was observed during the cell toxicity test; on the contrary, chitosan promoted cell proliferation. In conclusion, chitosan is a promising candidate for an effective and safe disinfectant against PEDV as well as other coronaviruses.

PCR-based detection and genetic characterization of porcine parvoviruses in South Korea in 2018.

BACKGROUND: with the advantage of sequencing technology, many novel porcine parvoviruses (PPV) rather than PPV1 has been reported. This study ultilized specific PCR- based method and gene- based analysis to study the presence and genetic diversity of porcine parvoviruses in South Korea in 2018. RESULTS: The present study was conducted in 2018 and found PPV1 and PPV7 in nine out of 151 field samples (organs and semen) by the PCR method. Among these, the complete genome sequences of five strains (N2, N91, N108, N133, and N141) were recovered. Phylogenic analysis revealed that the strains N2, N91, and N108 belong to the PPV1 genotype, while N133 and N141 belong to PPV7 genotype. The PPV7 strains collected in this study had deletion mutations in the VP2 gene but differed from that of PPV7 strains collected in 2017. Among the PPV1 strains, the amino acid variations in the B cell epitopes of the VP2 protein were observed between three Korean PPV1 field strains (N2, N91, and N108) and the reference PPV1 strains. Those substitutions resulted in six out of 12 predicted epitopes having significant differences in antigenic index compared to the other PPV1 strains. CONCLUSIONS: This study confirmed the presence of different genotypes of porcine parvoviruses in South Korea. The PPVs circulating in South Korea were phylogenetically classified as PPV1 and PPV7 genotypes. Three Korean PPV1 strains collected in 2018 were predicted to have antigenic alteration in VP2 compared to several reference strains of PPV1.

A descriptive survey of porcine epidemic diarrhea in pig populations in northern Vietnam.

Porcine epidemic diarrhea (PED) virus (PEDV) is a globally emerging and re-emerging epizootic swine virus that causes massive economic losses in the swine industry, with high mortality in piglets. In Vietnam, PED first emerged in 2009 and has now developed to an endemic stage. This is the first cross-sectional survey performed to evaluate the proportion of PEDV-positive swine farms in Vietnam from January 2018 to February 2019. Fecal samples from 327 pig farms in northern Vietnam were collected and tested for PEDV infection by reverse transcription-loop-mediated isothermal amplification (RT-LAMP) method. The proportion of PEDV-positive farms was 30.9% and PEDV-positive farms were distributed throughout the study area. The highest proportion of PEDV-positive farms was 70% (7/10) among nucleus production type farms (P < 0.05). Higher proportions of PEDV-positive farms were found in the Northeast and Red River Delta areas, which are the major areas of pig production (P < 0.05). The proportion of PEDV-positive farms was higher among larger farms (P < 0.05). Our findings illustrate the high proportion of PEDV-positive farms in the Vietnamese pig population and will help to better understand the epidemiological dynamics of PED infection, to estimate impact, and establish and improve prevention and control measures.

Effect of increasing levels of rice distillers' by-product on growth performance, nutrient digestibility, blood profile and colonic microbiota of weaned piglets.

OBJECTIVE: This study was conducted to evaluate the effects of diets containing different wet rice distillers' by-product (RDP) levels on growth performance, nutrient digestibility, blood profiles and gut microbiome of weaned piglets. METHODS: A total of 48 weaned castrated male crossbred pigs, initial body weight 7.54±0.97 kg, and age about 4 wks, were used in this experiment. The piglets were randomly allocated into three iso-nitrogenous diet groups that were fed either a control diet, a diet with 15% RDP, or a diet with 30% RDP for a total of 35 days. Chromium oxide was used for apparent digestibility measurements. On d 14 and d 35, half of the piglets were randomly selected for hemato-biochemical and gut microbiota evaluations. RESULTS: Increasing inclusion levels of RDP tended to linearly increase (p≤0.07) average daily gain on d 14 and d 35, and decreased (p = 0.08) feed conversion ratio on d 35. Empty stomach weight increased (p = 0.03) on d 35 while digestibility of diet components decreased. Serum globulin concentration decreased on d 14 (p = 0.003) and red blood cell count tended to decrease (p = 0.06) on d 35, parallel to increase RDP levels. Gene amplicon profiling of 16S rRNA revealed that the colonic microbiota composition of weaned pigs changed by inclusion of RDP over the period. On d 14, decreased proportions of Lachnospiraceae_ge, Ruminococcaceae_ge, Ruminococcaceae_UCG-005, and Bacteroidales_ge, and increased proportions of Prevotellaceae_ge, Prevotella_2, and Prevotella_9 were found with inclusion of RDP, whereas opposite effect was found on d 35. Additionally, the proportion of Lachnospiraceae_ge, Ruminococcaceae_ge, Ruminococcaceae_UCG-005, and Bacteroidales_ge in RDP diets decreased over periods in control diet but increased largely in diet with 30% RDP. CONCLUSION: These results indicate that RDP in a favorable way modulate gastrointestinal microbiota composition and improve piglet performance despite a negative impact on digestibility of lipids and gross energy.

Gouleako and Herbert viruses in pigs, Republic of Korea, 2013.

Several viruses in the family Bunyaviridae are pathogenic to animals and cause vector-borne zoonoses. In 2013, investigation of cause of death of 9 pigs on 1 farm in the Republic of Korea found infection with Gouleako and Herbert viruses. Subsequent investigation revealed high prevalence of these viruses among pigs throughout the country.

The phylogenetic study on Thelohanellus species (Myxosporea) in relation to host specificity and infection site tropism.

Thelohanellus kitauei (Myxobolidae) infects cyprinid fish. The evolution of species derived from common ancestors results in the sharing of biological features. To reveal the origin of T. kitauei biological features, the correlation between phylogeny and biological features of Myxobolidae was investigated by Bayesian inference tree and Bayesian tip association significance testing. The results demonstrated that host specificity and infection site tropism were correlated with the phylogeny of Myxobolidae, and that the biological features of T. kitauei originated from the ancient Myxobolidae as exhibited by the non-specific infection site tropism and the ability to infect cyprinids.

Molecular detection and genetic analysis of porcine bocavirus in Korean domestic swine herds.

Several porcine bocaviruses have been detected worldwide, and this report is the first to describe this virus in a Korean domestic swine herd. We identified porcine bocavirus in various samples, including serum, tissue, feces and saliva, which revealed that porcine bocavirus predominates in Korean domestic swine populations. The results of this study also suggested that porcine bocaviruses primarily infect weaned piglets. Phylogenetic analysis of the ORF3 gene was performed to determine the genetic relationship of the Korean strains to reference strains from other countries.

Characterization of a complete genome of a circular single-stranded DNA virus from porcine stools in Korea.

Porcine circular single-stranded DNA viruses have been just identified from swine feces in Korea. This virus was mentioned as bovine stool-associated circular DNA virus (BoSCV)-like virus discovered from porcine stools. However, the thorough characteristics of the virus were not identified. Therefore, this research focuses on finding a full genome sequence and analyzing the genetic features of the virus. The virus, now called porcine stool-associated circular DNA virus in Korea (PoSCV Kor), consists of 2,589 bases forming circular structure. It has two major ORFs inversely encoding replicase and capsid protein, with each stem-loop structure between 5' ends and 3' ends of the two putative ORFs. This characteristics is the same as PoSCV in New Zealand, but different from chimpanzee stool-associated circular virus (ChiSCVs) and BoSCV, which have one stem-loop structure. Therefore, it would be sure that PoSCV Kor is very similar to PoSCV in respect to the genetic aspect; the same number of nucleotide bases and the amino acid identity of replicase and capsid protein (96 and 93 %, respectively). This fact could be certified through the finding that PoSCV Kor and PoSCV are in the same cluster by phylogenetic analysis based on the comparison with full-sequences of other circular ssDNA viruses.

Inhibition of porcine endogenous retrovirus in PK15 cell line by efficient multitargeting RNA interference.

To effectively suppress porcine endogenous retroviruses (PERV)s, RNAi technique was utilized. RNAi is the up-to-date skill for gene knockdown which simultaneously multitargets both gag and pol genes critical for replication of PERVs. Previously, two of the most effective siRNAs (gag2, pol2) were found to reduce the expression of PERVs. Concurrent treatment of these two siRNAs (gag2+pol2) showed knockdown efficiency of up to 88% compared to negative control. However, despite the high initial knockdown efficiency 48 h after transfection caused by siRNA, it may only be a transient effect of suppressing PERVs. The multitargeting vector was designed, containing both gag and pol genes and making use of POL II miR Expression Vector, which allowed for persistent and multiple targeting. This is the latest shRNA system technique expressing and targeting like miRNA. Through antibiotics resistance characteristics utilizing this vector, miRNA-transfected PK15 cells (gag2-pol2) were selected during 10 days. An 88.1% reduction in the level of mRNA expression was found. In addition, we performed RT-activity analysis and fluorescence in situ hybridization assay, and it demonstrated the highest knockdown efficiency in multitargeting (gag2+pol2) miRNA group. Therefore, according to the results above, gene knockdown system (siRNA and shRNA) through multitargeting strategy could effectively inhibit PERVs.

PCR-Based Detection and Genetic Characterization of Parainfluenza Virus 5 Detected in Pigs in Korea from 2016 to 2018.

This study applied a molecular-based method to detect parainfluenza virus 5 (PIV5) collected from 2016 to 2018 in nine provinces of Republic of Korea. We demonstrated that PIV5 was detectable in both serum and pooled organs at an average positive rate of 1.78% (99/5566). Among these, the complete genome sequence of 15,246 nucleotides was obtained for 12 field strains. Three out of the 12 strains had the lowest genetic identity (96.20-96.68%) among the 21 porcine PIV5 genomes collected in Germany, China, India, and Republic of Korea from 1998 to 2017. By analyzing a large collection of complete genome sequences of the structural protein-coding F and HN genes, this study proposed a classification of PIV5 into two lineages, 1 and 2, and identified that group 2.2.2 within sub-lineage 2.2 was substantially divergent. The evolution of two structural protein-coding genes was largely under purifying selection. A few codons (6/9 for the F gene, 7/8 for the HN gene) had elevated dN/dS values, which were loaded on internal branches and were predicted to be related to beneficial trait(s) of the virus.

A Multiplex PCR Method for Simultaneous Detection of Infectious Laryngotracheitis Virus and Ornithobacterium rhinotracheale.

To date, many fluorescence- and gel-based multiplex polymerase chain reaction (PCR) assays have been developed for the simultaneous detection of multiple infectious agents of respiratory disease in poultry. However, PCR assays are not available for other important emerging respiratory bacteria, such as Ornithobacterium rhinotracheale (ORT). We aimed to fill this gap by establishing a new duplex PCR method for the simultaneous detection of infectious laryngotracheitis virus (ILTV) and ORT. Multiplex primer design software was used to select the compatible multiplex primer pairs. It was determined that an annealing temperature of 65 °C and an initial concentration of 2.5 pmol/µL for each primer set were the most suitable conditions for multiplex PCR. The assay was confirmed to be specific, as it only detected the target pathogens, even in the presence of six non-target agents. The limit of detection was up to 103 copies/µL of template DNA for both ILTV and ORT. In the screening of 304 field samples, 23, 88, and 44 were positive for both ILTV and ORT, solely for ILTV, and solely ORT, respectively.

A Cell-Adapted Live-Attenuated Vaccine Candidate Protects Pigs against the Homologous Strain VNUA-ASFV-05L1, a Representative Strain of the Contemporary Pandemic African Swine Fever Virus.

African swine fever (ASF) is a lethal and highly contagious transboundary animal disease with the potential for rapid international spread. Currently, there is no ASF vaccine commercially available. All infected animals must be isolated and culled immediately upon the confirmation of the presence of the virus. Studies leading to the rational development of protective ASF vaccines are urgently needed. Here, we generated a safe and efficacious live-attenuated vaccine (LAV) VNUA-ASFV-LAVL2 by serially passaging a field isolate (VNUA-ASFV-05L1, genotype II) in porcine alveolar macrophages (PAMs, 65 passages) and an immortalized porcine alveolar macrophage cell line (3D4/21, 55 passages). VNUA-ASFV-LAVL2 can efficiently replicate in both PAMs and 3D4/21 cells. It provides 100% protection, even with the low dose of 102 HAD50, to the vaccinated pigs against the challenge of contemporary pandemic ASFV field isolate. Pigs vaccinated with this LAV in a dose range of 102 to 105 HAD50 remained clinically healthy during both the 28-day observation period of immunization and the 28-day observation period of challenge. VNUA-ASFV-LAVL2 was eliminated from blood by 28 days post-inoculation (DPI), and from feces or oral fluids by 17 DPI. Although the vaccine strain in serum remained a safe and attenuated phenotype after five passages in swine, a reversion-to-virulence study using blood or tissue homogenates at peak viremia will be conducted in the future. ASFV-specific IgG antibodies and significant cellular immunity were detected in vaccinated pigs before the ASFV challenge. These results indicate that the VNUA-ASFV-LAVL2 strain is a safe and efficacious LAV against the genotype II ASFV strain responsible for current ASF outbreaks in Asia.

Identification of a novel single-stranded, circular DNA virus from bovine stool.

We report the identification of a novel single-stranded, circular DNA virus isolated from bovine stool. The virus, named bovine stool-associated circular DNA virus (BoSCV), has a genome comprising 2600 bases of circular ssDNA, with two putative ORFs encoding replicase and capsid proteins, arranged inversely. The stem-loop structure was located between the 3' ends of the two putative ORFs, as in chimpanzee stool-associated circular virus (ChimpSCV) and unlike other circular DNA viruses, including members of the families Circoviridae, Nanoviridae and Geminiviridae. BoSCV was also genetically similar to ChimpSCV, with approximately 30 % identity in the replicase and capsid proteins. A phylogenetic analysis based on the replicase protein showed that BoSCV and ChimpSCV are in the same clade. A field survey using BoSCV-specific PCRs targeting ORF1 detected BoSCV and BoSCV-like sequences in bovine and porcine stool samples. BoSCV appears to belong to a new genus of circular DNA viruses.

Population dynamics and ORF3 gene evolution of porcine circovirus type 2 circulating in Korea.

This study was conducted to investigate the status and population dynamics of porcine circovirus type 2(PCV2) in Korea and to assess the molecular evolutionary pattern of the two biologically important, overlapping open reading frames, the ORF1 and ORF3 genes. A wide range of PCV2 genomic sequences (entire genome, ORF1, ORF2 and ORF3) collected between 2001 and 2010 were analyzed using the Bayesian Markov chain Monte Carlo and maximum-likelihood approaches. These techniques identified the PCV2d genotype and the 2Ek cluster of PCV2a in Korea for the first time. Second, the genotypic shift of PCV2b dominating over PCV2a likely occurred between 2002 and 2004 due to a population expansion of PCV2b. In the context of positive Darwinian selection, the results uncovered independent evolutionary patterns in the ORF3 gene compared to the overlapping ORF1 gene and new sites in the viral ORFs/proteins that might relate to differences in the biological properties of the PCV2 groups.

Molecular-based detection, genetic characterization and phylogenetic analysis of porcine circovirus 4 from Korean domestic swine farms.

Porcine circovirus 4 (PCV4), a novel and unclassified member of the genus Circovirus, was first reported in China in 2019. Aiming to provide more evidence about the active circulation of PCV4, this study screened 335 pooled internal organs and detected the virus (i) at a rate of 3.28%, (ii) from both clinically healthy and clinically sick pigs of various age groups, and (iii) in six out of nine provinces of Korea. The complete genomic sequence of the Korean PCV4 strain (E115) was 1,770 nucleotides in length and had 98.5%-98.9% identity to three PCV4 strains currently available at GenBank. Utilizing a set of bioinformatic programs, it was revealed that the Korean PCV4 strain contained several genomic features of (i) a palindrome stem-loop structure with a conserved nonanucleotide, (ii) packed overlapping ORFs oriented in different directions and (iii) two intergenic regions in between genes encoding the putative replication-associated protein (Rep) and capsid (Cap) proteins. This study also predicted the presence of essential elements for the replication of circoviruses in all PCV4 strains, for example the origin of DNA replication, endonuclease and helicase domains of Rep, and the nuclear localization signal on the putative Cap protein. Finally, based on the phylogeny inferred from sequences of the putative Rep protein, this study further clarified the genetic relationships between PCV4 and other CRESS DNA viruses in general and circoviruses in particular.

Polymerase chain reaction-based detection of coinfecting DNA viruses in Vietnamese pigs in 2017 and 2021.

Background and Aim: Many studies have reported on the phenomenon of co-infections involving two or more pathogens (bacteria or viruses) over the past few years. However, very few studies on this issue were conducted in Vietnam. Therefore, this study aimed to determine the circulation of single and multiple porcine parvovirus (PPV) (e.g., PPV1, PPV2, PPV3, and PPV4), porcine bocavirus (PBoV), and torque teno virus (TTV) (TTV1 and TTV2) infections in Vietnamese pigs. Materials and Methods: A total of 174 porcine circovirus 2-positive samples from pigs (n = 86 for 2017 and n = 88 for 2021), including from the sera and internal organs, across 11 provinces were examined by polymerase chain reaction. Results: This study demonstrated the wide distribution of DNA viruses among pig farms in Vietnam in 2021, with the detection rate for PPV ranging from 3.4% to 27.3% among PPV1-PPV4. Moreover, the detection rates of TTV genotypes were confirmed to be 14.8% (TTV1) and 63.6% (TTV2), respectively, and the positive rate of PBoV was 65.9%. The most frequent combinations were double and triple infections. Double infection was found in 16/86 (18.6%) in 2017 and 26/88 (29.5%) in 2021, while triple infection was found at 19/86 (22.1%) in 2017 and 26/88 (29.5%) in 2021. The incidence of simultaneous detection of more than three viruses was low. Conclusion: These results provide at least partial information about the occurrence of three viruses, including PPV (including PPV1 to 4), PBoV, and TTV (TTV1 and TTV2), in pigs. Determination of particular viruses in pigs will help to prevent the porcine respiratory disease complex caused by DNA viruses in Vietnamese pigs in the future.

Experimental infection of a newly emerging Korean type I porcine reproductive and respiratory syndrome virus isolate in colostrum-deprived pigs.

BACKGROUND: Recently, new emergence of type I PRRSV has been reported in Korea by several research groups. Although specific subgroups of type I PRRSVs in Korea were observed in the previous phylogenetic analysis, there is a lack of information about the virulence of type I PRRSV recently isolated in Korea. METHODS: One type I PRRSV isolate (G2446, 3 times passaged in primarily cultured pulmonary macrophages) in Korea was experimentally infected in colostrum-deprived pigs. The pathological and serological evaluations were performed and compared to type II PRRSV strain (CP07-401-9, 5 times passaged in MARC-145 cell lines)-infected pigs, for 21 days post challenge (dpc). RESULTS: The pneumonia found in gross examination was more severe in type I PRRSV-infected pigs than type II PRRSV-infected pigs. Both groups showed bronchointerstitial pneumonia, mild multifocal perivascular lymphohistiocytic myocarditis and lymphadenopathy at 14 dpc. However, the unique histopathologic lesions were not found in the pigs experimentally infected with a Korean type I PRRSV isolate, when compared to previous data about classical pathology of PRRSV. The PRRS-specific antibodies were detected in the first week after challenge and viremia continued at least until 21 dpc in both groups. CONCLUSION: The gross and histopathologic lesion in this study indicated that Korean type I PRRSV strain (G2446) caused classical PRRSV-specific lesions. Although this study evaluated one representative strain of Korean type I PRRSV, the results may provide information regarding the pathogenicity of type I PRRSV recently emerged in Korea.

Genotyping of PCV3 based on reassembled viral gene sequences.

Porcine circovirus type 3 (PCV3) has been reported in many countries such as USA, China, Korea and many European countries during 2015-2018. The six PCV3 strains named IH, SJ, N5, N10, N13 and N62 were detected out of 220 samples by PCR methods while the prevalence our study was conducted in 2017 to 2018. The six detected strains were hard to genotype with reference viruses due to their diverse phylogenetic relationship. PCV3 capsid, ORF3 and replicase protein coding genes were reassembled at the nucleotide sequence level, then 16 new reassembled PCV3 sequences were generated. Based on the maximum likelihood mapping analysis of 303 PCV3 sequences a model with a combination of replicase, ORF3 and capsid protein coding genes was selected as the most appropriate target for genotyping, which provided the best support for the clade classification into three genotypes and several subtypes (genotype 1, genotype 2; subtype: a and b, genotype 3; subtype a, b, c, d, e, f, g, h). This study, the IH_Korea_2017 and N62_Korea_2018 strains belong to genogroup 3 (subtype a) the SJ_Korea_2017 strain genogroup 3 (subtype g) and the N5, N10, N13 Korea_ 2018 strains genogroup 3 (subtype f), respectively. In conclusion, this study may provide insights to classification of PCV3 genotypes around the world.

Genome Sequence of a Virulent African Swine Fever Virus Isolated in 2020 from a Domestic Pig in Northern Vietnam.

This study reports the genome sequence of an isolated African swine fever (ASF) virus (VNUA-ASFV-05L1/HaNam) obtained at the fourth passage on pulmonary alveolar macrophages. The virus was isolated during a typical acute ASF outbreak in pigs in a northern province of Vietnam in 2020.