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.

Genomic Characterization of a Novel Alphacoronavirus Isolated from Bats, Korea, 2020.

Coronavirus, an important zoonotic disease, raises concerns of future pandemics. The bat is considered a source of noticeable viruses resulting in human and livestock infections, especially the coronavirus. Therefore, surveillance and genetic analysis of coronaviruses in bats are essential in order to prevent the risk of future diseases. In this study, the genome of HCQD-2020, a novel alphacoronavirus detected in a bat (Eptesicus serotinus), was assembled and described using next-generation sequencing and bioinformatics analysis. The comparison of the whole-genome sequence and the conserved amino acid sequence of replicated proteins revealed that the new strain was distantly related with other known species in the Alphacoronavirus genus. Phylogenetic construction indicated that this strain formed a separated branch with other species, suggesting a new species of Alphacoronavirus. Additionally, in silico prediction also revealed the risk of cross-species infection of this strain, especially in the order Artiodactyla. In summary, this study provided the genetic characteristics of a possible new species belonging to Alphacoronavirus.

Application of nano-graphene oxide as nontoxic disinfectant against alpha and betacoronaviruses.

New viruses are continuously emerging and recently there have been many great concerns on severe acute respiratory syndrome coronavirus (SARS-CoV-2). Nanographene oxide (nanoGO) has received much attention and is widely investigated to be utilised in therapy for infectious diseases by viruses. Thus, antiviral activity of nanoGO was evaluated using the porcine epidemic diarrhoea virus (PEDV), bovine coronavirus (BCoV), and SARS-CoV-2, which are all Alpha- and Beta-coronavirus. In a virus inhibition assay, the three viruses were inhibited by nanoGO in a dose-dependent manner, including attempts in the presence of high serum solution which partially mimicked biological fluid.

Genetic analysis of porcine parvoviruses detected in South Korean wild boars.

Porcine parvovirus 1 (PPV1) is a major cause of reproductive failure in pigs. To date, six additional porcine parvoviruses (PPV2-PPV7) have been identified. In this study, we detected 11 PPV1 strains, five PPV3 strains, three PPV4 strains, six PPV5 strains, five PPV6 strains, and one PPV7 strain in Korean wild boars. PPV1, -3, and -5, and PPV6 from Korean wild boars harbor conserved motifs within the Ca2+ binding loop and the catalytic center of the PLA1 motif. Intra-species recombination among PPV7 strains was also identified. Genetic characterization revealed that PPV1 from Korean wild boars may be similar to virulent PPV strains.

Torque teno virus from Korean domestic swine farms, 2017-2018.

BACKGROUND: Torque teno viruses (TTVs) have been detected worldwide, from a wide range of animals. Up to date, few studies focused on the prevalence of TTVs in general and swine torque teno viruses (TTSuVs) in particular in Korean swine farms. OBJECTIVE: This study aimed to investigate the appearance of TTSuVs and TTVs in sick pigs during the 2017-2018 period. MATERIALS AND METHODS: Molecular-based method using TTSuV1-, TTSuV2- and TTV3-specific primers was used to screen for the viruses from either sera or pooled internal organs of sick pigs. For genetic characterization, genomic sequences of TTVs were sequenced by a primer walking method. Several bioinformatic tools have been utilized to investigate the genomic organization and genetic relationship of TTVs. RESULTS: Two years of prevalence survey reveal that the prevalence of TTSuV2 is about twice that of TTSuV1. Furthermore, we identified TTV of genogroup 3 in swine pooled organ samples. The genome of two strains, M265_Korea_2017 and N119_Korea_2018, are 3,817 bp in size; M265_2017 has three open reading frames (ORFs); and N119_2018 strain has four ORFs. The complete genome nucleotide sequencing of the two strains shows 98.4% homology, and the phylogenetic analysis of Open reading frame (ORF)1 indicates that the strains are located close to TUPB strain subgroup C of genogroup 3. CONCLUSION: Our study provided the information of TTSuVs prevalence in swine farms in Korea and highlighted the presence of TTV genogroup 3 strains in pigs.

Comparative Analysis of the Productivity and Immunogenicity of an Attenuated Classical Swine Fever Vaccine (LOM) and an Attenuated Live Marker Classical Swine Fever Vaccine (Flc-LOM-BErns) from Laboratory to Pig Farm.

Herein, we compared the productivity of pigs inoculated with one of two classical swine fever (CSF) vaccines (low virulent of Miyagi (LOM) or Flc-LOM-BErns) plus the swine erysipelothrix rhusiopathiae (SE) vaccine. The feed intake and weight increase of the pigs inoculated with Flc-LOM-BErns + SE were normal. However, the feed intake of the pigs inoculated with LOM + SE dropped sharply from four days post-vaccination (dpv). In addition, the slaughter date was an average of eight days later than that of the pigs inoculated with Flc-LOM-BErns + SE. All pigs inoculated with the Flc-LOM-BErns + SE vaccine were completely differentiated at 14 days against CSF Erns antibody and at approximately 45 days against the bovine viral diarrhea virus (BVDV) Erns antibody; the titers were maintained until slaughter. Leucopenia occurred temporarily in the LOM + SE group, but not in the Flc-LOM-BErns + SE group. Expression of tumor necrosis factor (TNF)-α and IFN-γ was significantly (p < 0.05) higher in the LOM + SE group than in the mock (no vaccine) group. When conducting the same experiment on a breeding farm, the results were similar to those of the laboratory experiments. In conclusion, the biggest advantage of replacing the CSF LOM vaccine with the Flc-LOM-BErns vaccine is improved productivity.

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.

Characteristics of Aerococcus viridans isolated from porcine fetuses in Korean farms.

Swine abortion caused by viruses as well as bacteria has caused many economic losses in domestic farms over the years; however, bacterial abortion has not yet been studied in Korea. Several bacterial species were isolated from aborted fetuses (n = 103) for which the cause of death was not viral abortion. Among them, we focused on Aerococcus viridans, which had the highest positive rate within three provinces (Gangwon, Jeonnam and Gyeongnam). A total of 16 isolates were identified as A. viridans by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), and 13 were characterized by both antibiotic resistance and 16S rRNA gene analysis. Based on antibiotic susceptibility testing result, eight antimicrobials could not effectively eliminate the present isolation (more than 40% of isolates can resist these antibiotics), while all except two strains were susceptible to trimethoprim/sulfamethoxazole. Molecular analysis indicated genetic variation among these strains. This study is the first report detecting A. viridans from aborted fetuses in Korean domestic farms.

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.

Prevalence and Genetic Diversity of Atypical Porcine Pestivirus (APPV) Detected in South Korean Wild Boars.

Atypical porcine pestivirus (APPV), currently classified as pestivirus K, causes congenital tremor (CT) type A-II in piglets. Eighteen APPV strains were identified from 2297 South Korean wild boars captured in 2019. Phylogenetic analysis of the structural protein E2 and nonstructural proteins NS3 and Npro classified the APPV viruses, including reference strains, into Clades I, II and III. Clade I was divided into four subclades; however, the strains belonging to the four subclades differed slightly, depending on the tree analysis, the NS3, E2, and Npro genes. The maximum-likelihood method was assigned to South Korean wild boar APPV strains to various subclades within the three trees: subclades I.1 and I.2 in the E2 tree, subclade I.1 in the Npro tree, and subclades I.1 and I.4 in the NS3 ML tree. In conclusion, APPV among South Korean wild boars belonging to Clade I may be circulating at a higher level than among the South Korean domestic pig populations.

Protection of Piglets with Maternally Derived Antibodies from Sows Inoculated with an Attenuated Live Marker Classical Swine Fever Vaccine (Flc-LOM-BErns).

Here, we investigated the protective efficacy provided by passive immunity induced by a classical swine fever (Flc-LOM-BErns) vaccine with the newly developed DIVA (Differentiating Infected from Vaccinated Animals) function. Ten pigs (aged 40-60 days) with maternally derived antibodies (MDAs) obtained from sows inoculated with the Flc-LOM-BErns vaccine were challenged with virulent classical swine fever virus (CSFV). Pigs with an MDA titer of 6 log2 induced by the Flc-LOM-BErns vaccine were fully protected against virulent CSFV challenge but not the pigs with an MDA titer under 5 log2. In addition, Flc-LOM-BErns vaccine-derived MDAs successfully differentiated vaccinated pigs by bovine viral diarrhea virus (BVDV) Erns/CSFV Erns antibody detection, functioning as a DIVA.

Molecular Characterization of Porcine Epidemic Diarrhea Virus and Its New Genetic Classification Based on the Nucleocapsid Gene.

Porcine epidemic diarrhea virus (PEDV) causes continuous, significant damage to the swine industry worldwide. By RT-PCR-based methods, this study demonstrated the ongoing presence of PEDV in pigs of all ages in Korea at the average detection rate of 9.92%. By the application of Bayesian phylogenetic analysis, it was found that the nucleocapsid (N) gene of PEDV could evolve at similar rates to the spike (S) gene at the order of 10-4 substitutions/site/year. Based on branching patterns of PEDV strains, three main N gene-base genogroups (N1, N2, and N3) and two sub-genogroups (N3a, N3b) were proposed in this study. By analyzing the antigenic index, possible antigenic differences also emerged in both the spike and nucleocapsid proteins between the three genogroups. The antigenic indexes of genogroup N3 strains were significantly lower compared with those of genogroups N1 and N2 strains in the B-cell epitope of the nucleocapsid protein. Similarly, significantly lower antigenic indexes in some parts of the B-cell epitope sequences of the spike protein (COE, S1D, and 2C10) were also identified. PEDV mutants derived from genetic mutations of the S and N genes may cause severe damage to swine farms by evading established host immunities.

Genetic Diversity of Porcine Circovirus Isolated from Korean Wild Boars.

In Korea, three genotypes of porcine circovirus type 2 (PCV2a, PCV2b, and PCV2d) have been identified on domestic pig farms, while two genotypes (PCV2a and PCV2b) have been identified in wild boar populations. Here, we investigated genotype diversity and genotypic shift in 91 PCV2 isolates from 1340 wild boars captured in South Korea between 2013 and 2017. Phylogenetic analyses based on the complete ORF2 showed that the 91 PCV2 strains were detected as four genotypes by qPCR screening assay: PCV2a (2.2%, 2/91), PCV2b (16.5%, 15/91), PCV2d (80.2%, 73/91), and PCV2h (1.1%, 1/91). Only one intergenotype recombinant event was detected between PCV2 ORF2 in wild boars (PCV2b) and domestic pigs (PCV2a). Amino acid positions 86-89 within ORF2, which distinguishes the different genotypes, were conserved in all PCV2 genotypes isolated from South Korean wild boars, including TNKI in PCV2a/PCV2h, SNPR in PCV2b, and SNPL in PCV2d. The estimated nucleotide substitution rates in the ORF2 region of viruses from South Korean wild boars and domestic pigs were 5.8145 × 10-4 and 4.5838 × 10-4 substitutions per site per year (s/s/y), respectively. The times to the most recent common ancestor (tMRCA) for South Korean domestic pig PCV2 were 1937 (PCV2a), 1972 (PCV2b), 1999 (PCV2d-1), and 2000 (PCV2d-2). By contrast, the tMRCA for South Korean wild boar PCV2b and PCV2d were 1989 and 2001, respectively. Thus, the PCV2d genotype is prevalent among South Korean wild boars and domestic pigs.

A Novel Synonymous Mutation of SARS-CoV-2: Is This Possible to Affect Their Antigenicity and Immunogenicity?

The S glycoprotein of coronaviruses is important for viral entry and pathogenesis with most variable sequences. Therefore, we analyzed the S gene sequences of SARS-CoV-2 to better understand the antigenicity and immunogenicity of this virus in this study. In phylogenetic analysis, two subtypes (SARS-CoV-2a and -b) were confirmed within SARS-CoV-2 strains. These two subtypes were divided by a novel synonymous mutation of D614G. This may play a crucial role in the evolution of SARS-CoV-2 to evade the host immune system. The region containing this mutation point was confirmed as a B-cell epitope located in the S1 domain, and SARS-CoV-2b strains exhibited severe reduced antigenic indexes compared to SARS-CoV-2a in this area. This may allow these two subtypes to have different antigenicity. If the two subtypes have different serological characteristics, a vaccine for both subtypes will be more effective to prevent COVID-19. Thus, further study is urgently required to confirm the antigenicity of these two subtypes.

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.

Rapid Spread of Classical Swine Fever Virus among South Korean Wild Boars in Areas near the Border with North Korea.

There has been a rapid increase in the number of classical swine fever (CSF) sero-positive wild boars captured near the demilitarized zone (DMZ), located the border with North Korea. In 2015-2016, few CSFV-positive antibody boars were detected; however, the number has increased steeply since 2017. Most occurred in the northern region of Gyeonggi before spreading slowly to Gangwon (west to east) in 2018-2019. Multi-distance spatial cluster analysis provided an indirect estimate of the time taken for CSFV to spread among wild boars: 46.7, 2.6, and 2.49 days/km. The average CSF serum neutralization antibody titer was 4-10 (log 2), and CSFV Ab B-ELISA PI values ranged from 65.5 to 111.5, regardless of the age and sex of wild boars. Full genome analysis revealed that 16 CSFV strains isolated from wild boars between 2017 and 2019 were identical to the YC16CS strain (sub-genotype 2.1d) isolated from an outbreak in breeding pigs near the border with North Korea in 2016. The rapid increase in CSF in wild boars may be due to a continuously circulating infection within hub area and increased population density. The distribution pattern of CSFV in Korean wild boars moves from west to southeast, affected by external factors, including small-scale hunting, geographical features and highways.

Pathogenicity and Genetic Characterization of Vietnamese Classical Swine Fever Virus: 2014-2018.

Here, we examined the pathogenicity and genetic differences between classical swine fever viruses (CSFV) isolated on pig farms in North Vietnam from 2014-2018. Twenty CSFV strains from 16 pig farms were classified as genotype 2 (sub-genotypes 2.1b, 2.1c, and 2.2). The main sub-genotype, 2.1c, was classified phylogenetically as belonging to the same cluster as viruses isolated from the Guangdong region in South China. Strain HY58 (sub-genotype 2.1c), isolated from pigs in Vietnam, caused higher mortality (60%) than the Vietnamese ND20 strain (sub-genotype 2.2). The Vietnamese strain of sub-genotype 2.1b was estimated to have moderate virulence; indeed, genetic analysis revealed that it belongs to the same cluster as Korean CSFV sub-genotype 2.1b. Most CSFVs circulating in North Vietnam belong to sub-genotype 2.1c. Geographical proximity means that this genotype might continue to circulate in both North Vietnam and Southern China (Guangdong, Guangxi, and Hunan).

Efficacy of orally administered porcine epidemic diarrhea vaccine-loaded hydroxypropyl methylcellulose phthalate microspheres and RANKL-secreting L. lactis.

Here, we examined the efficacy of are combinant subunit antigen-based oral vaccine for preventing porcine epidemic diarrhea virus (PEDV). First, we generated a soluble recombinant partial spike S1 protein (aP2) from PEDV in E. coli and then evaluated the utility of aP2 subunit vaccine-loaded hydroxypropyl methylcellulose phthalate microspheres (HPMCP) and RANKL-secreting L. lactis (LLRANKL) as a candidate oral vaccine in pregnant sows. Pregnant sows were vaccinated twice (with a 2 week interval between doses) at 4 weeks before farrowing. Titers of virus-specific IgA antibodies in colostrum, and neutralizing antibodies in serum, of sows vaccinated with HPMCP (aP2) plus LL RANKL increased significantly at 4 weeks post-first vaccination. Furthermore, the survival rate of newborn suckling piglets delivered by sows vaccinated with HPMCP (aP2) plus LL RANKL was similar to that of piglets delivered by sows vaccinated with a commercial killed porcine epidemic diarrhea virus (PED) vaccine. The South Korean government promotes a PED vaccine program (live-killed-killed) to increase the titers of IgA and IgG antibodies in pregnant sows and prevent PEDV. The oral vaccine strategy described herein, which is based on a safe and efficient recombinant subunit antigen, is an alternative PED vaccination strategy that could replace the traditional strategy, which relies on attenuated live oral vaccines or artificial infection with virulent PEDV.

Detection of subtypes (3a, 4a and 4d) and high prevalence of hepatitis E virus in Korean wild boar.

The hepatitis E virus (HEV) is known to have 4 genotypes but only one serotype. Genotype 1 and 2 infect humans only and genotype 3 and 4 infect humans, pigs and other animal species. Pig and wild boar are also known as reservoirs of HEV infection. Of the 2736 wild boars captured from 2011 to 2016 to investigate the HEV prevalence among Korean wild boars, 1041 serum samples were high seropositive (38.1%; 95% CI: 35.5-40.5) for HEV, which were detected using the anti-HEV antibody ELISA and the highest prevalence rate was 40.6% (684/1683) in 2016. Twenty four HEV strains were also identified from 1859 wild boar bloods captured between 2015 and 2016. The phylogenetic tree constructed based on the partial ORF2 gene revealed that the 23 Korean wild boar HEV strains belonged to genotype 4 (4a and 4d) showing the nucleotide sequences identities 83.4-100 %. The one Korean wild boar HEV strain belonged to genotype 3, segregated into subgenotype 3a. This suggested that major circulating in Korean wild boars is genotype 4a whereas genotype 3a and -4d is minor. It is important to the human public health that HEV with wild boar have potential high risk factor for transmission to human due to eating culture of Korean people with undercooked wild boar gallbladder.

Molecular-based investigation and genetic characterization of porcine stool-associated RNA virus (posavirus) lineages 1 to 3 in pigs in South Korea from 2017 to 2019.

Recent results on the detection and genetic characterization of stool-associated RNA viruses from different species have increased the knowledge about the extreme genetic diversity of picornaviruses. This study aimed to investigate the presence of unclassified porcine stool-associated RNA viruses (posaviruses) in South Korea and to elucidate the molecular evolution of the viruses. By RT-PCR, posaviruses 1 and 3 were exclusively found in fecal samples and consistently detected in three consecutive years in six of eight provinces, with 148/697 (21.2%) and 33/84 (39.3%) positive samples and farms, respectively. Every age group but the older age groups (finisher, sow) had significantly higher positive rates of posavirus 1 than posavirus 3. An analysis of the RNA-dependent RNA polymerase sequences by likelihood mapping and maximum-likelihood-based phylogenetic analysis revealed that stool-associated RNA viruses formed four supergroups that were well separated from all recognized families of the order Picornavirales. Five genomes of Korean posaviruses generated in this study were phylogenetically grouped with posavirus 1 and posavirus 3 and were predicted to have the typical genome organization of picornaviruses.