Concurrent Infection with Clade 2.3.4.4b Highly Pathogenic Avian Influenza H5N6 and H5N1 Viruses, South Korea, 2023.

Highly pathogenic avian influenza H5N6 and H5N1 viruses of clade 2.3.4.4b were simultaneously introduced into South Korea at the end of 2023. An outbreak at a broiler duck farm consisted of concurrent infection by both viruses. Sharing genetic information and international surveillance of such viruses in wild birds and poultry is critical.

Research Note: Comparative evaluation of pathogenicity in SPF chicken between different subgroups of H5N6 high pathogenicity avian influenza viruses.

Since 2014, periodic outbreaks of high pathogenicity avian influenza (HPAI) caused by clade 2.3.4.4 H5 HPAI virus (HPAIV) have resulted in huge economic losses in the Korean poultry industry. During the winter season of 2016-2017, clade 2.3.4.4e H5N6 HPAIVs classified into 5 subgroups (C1-5) were introduced into South Korea. Interestingly, it was revealed that the subgroup C2 and C4 viruses were predominantly distributed throughout the country, whereas detection of the subgroup C3 viruses was confined in a specific local region. In the present study, we conducted comparative evaluation of the pathogenicity of viruses belonging to subgroups C2 and C3 (H15 and HN1 strains) in specific pathogen-free (SPF) chickens, and further compared them with previously determined pathogenicity of subgroup C4 (ES2 strain) virus. The HN1 strain showed lower viral replication in tissues, less transmissibility, and higher mean chicken lethal dose than the H15 and ES2 strains in SPF chickens. Considering that the HN1 strain has a different NS gene segment from the H15 and ES2 strains, the reassortment of the NS gene segment likely affects their infectivity and transmissibility in chickens. These findings emphasize the importance of monitoring the genetic characteristics and pathogenic features of HPAIVs to effectively control their outbreaks in the field.

Phylogenetic Analysis of G and P Genotypes of Bovine Group A Rotavirus Strains Isolated from Diarrheic Vietnam Cows in 2017 and 2018.

This study aimed to investigate the genetic diversity of G- and P-type bovine RVAs (BoRVAs) prevalent in Vietnam. Between 2017 and 2018, the prevalence of BoRVAs detected in diarrhea samples from 8 regions was as low as 1.9% (11/582). The prevalence of the G-type was 45.5% for G6 and 18.2% for G10; however, 36.3% remain unidentified. Interestingly, all BoRVAs were investigated as P[11], and there was no diversity within this P-type. Geographically, the G6 and G10 types were not identified in any specific area; rather, they occurred in both Northern and Southern Vietnam. G6P[11] and G10P[11], which are combined G- and P-types, were identified in 71.4% and 28.6% of BoRVA-positive samples, respectively. Phylogenetic tree analysis revealed that the G6-type detected in Vietnamese cows is similar to strains derived from China, Japan, and Korea, whereas the G10 type is closely related to the Chinese strain. In addition, the P11 strain detected in Vietnamese cows is similar to the Spanish and Chinese strains. The BoRVA-positive rate was higher in cows aged less than 2 months (3.2%, 3/94) than in those aged 2 months or more (1.6%, 8/488). In summary, we detected the presence of G6P11 and G10P11 BoVRAs on Vietnamese cow farms, and found that they were more predominant in young calves than in older cows.

Genetic Characterization and Pathogenesis of H5N1 High Pathogenicity Avian Influenza Virus Isolated in South Korea during 2021-2022.

High pathogenicity avian influenza (HPAI) viruses of clade 2.3.4.4 H5Nx have been circulating in poultry and wild birds worldwide since 2014. In South Korea, after the first clade 2.3.4.4b H5N1 HPAI viruses were isolated from wild birds in October 2021, additional HPAIV outbreaks occurred in poultry farms until April 2022. In this study, we genetically characterized clade 2.3.4.4b H5N1 HPAIV isolates in 2021-2022 and examined the pathogenicity and transmissibility of A/mandarin duck/Korea/WA585/2021 (H5N1) (WA585/21) in chickens and ducks. Clade 2.3.4.4b H5N1 HPAI viruses caused 47 outbreaks in poultry farms and were also detected in multiple wild birds. Phylogenetic analysis of HA and NA genes indicated that Korean H5N1 HPAI isolates were closely related to Eurasian viruses isolated in 2021-2022. Four distinct genotypes of H5N1 HPAI viruses were identified in poultry, and the majority were also found in wild birds. WA585/21 inoculated chickens showed virulent pathogenicity with high mortality and transmission. Meanwhile, ducks infected with the virus showed no mortality but exhibited high rates of transmission and longer viral shedding than chickens, suggesting that they may play an important role as silent carriers. In conclusion, consideration of both genetic and pathogenic traits of H5N1 HPAI viruses is required for effective viral control.

Genetic Diversity of Bovine Group A Rotavirus Strains Circulating in Korean Calves during 2014 and 2018.

The purpose of this study was to investigate annual changes in BoRVA strains by examining the VP4 and VP7 genes of rotaviruses in Korean calves. Between 2014 and 2018, 35 out of 138 samples of calf diarrhea feces collected nationwide were positive for BoRVA. Further genetic characterization of the VP7 and VP4 genes of 35 BoRVA isolates identified three different G-genotypes (G6, G8, and G10) and two different P genotypes (P[5] and P[11]). The G6 genotype was most common (94.3%) in BoRVA-positive calves, followed by the P[5] genotype (82.9%). Four genotypes comprised combinations of VP4 and VP7: 80% were G6P[5], 14.2% were G6P[11], 2.9% were G8P[5], and 2.9% were G10P[11]. Susceptibility to infection was highest in calves aged < 10 days (35%) and lowest in calves aged 30−50 days (15.4%). The data presented herein suggest that the G6P[5] genotype is the main causative agent of diarrhea in Korean calves. In addition, it is predicted that G6P[5] will continue to act as a major cause of diarrhea in Korean calves.

Emergence of clade 2.3.4.4b novel reassortant H5N1 high pathogenicity avian influenza virus in South Korea during late 2021.

High pathogenicity H5N1 avian influenza viruses pose a threat to both animal and human health worldwide. In late 2020, outbreaks of H5 high pathogenicity avian influenza viruses belonging to clade 2.3.4.4b emerged in Europe, following on from outbreaks in East Asia in earlier years. However, very recent studies show that clade 2.3.4.4b H5N1, rather than 2.3.4.4b H5N8, has become predominant in wild birds and has infected poultry in several countries. In this study, we describe isolation of a novel H5N1 virus from a captured mandarin duck in South Korea, and another H5N1 virus from a quail farm. We performed genetic analysis of these two viruses to identify their origin and to determine their relationship with the clade 2.3.4.4b H5N1 viruses currently circulating in Europe. Based on our results, it is presumed that the novel H5N1 virus isolated in Korea originated from an unknown reassortant between clade 2.3.4.4b H5N8 viruses circulating from 2020 and other Eurasian viruses, with additional reassortment of genes and point mutations that discriminate them from the recently reported H5N1 virus in Europe.

Genetic Characterization of Novel H7Nx Low Pathogenic Avian Influenza Viruses from Wild Birds in South Korea during the Winter of 2020-2021.

Zoonotic infection with avian influenza viruses (AIVs) of subtype H7, such as H7N9 and H7N4, has raised concerns worldwide. During the winter of 2020-2021, five novel H7 low pathogenic AIVs (LPAIVs) containing different neuraminidase (NA) subtypes, including two H7N3, an H7N8, and two H7N9, were detected in wild bird feces in South Korea. Complete genome sequencing and phylogenetic analysis showed that the novel H7Nx AIVs were reassortants containing two gene segments (hemagglutinin (HA) and matrix) that were related to the zoonotic Jiangsu-Cambodian H7 viruses causing zoonotic infection and six gene segments originating from LPAIVs circulating in migratory birds in Eurasia. A genomic constellation analysis demonstrated that all H7 isolates contained a mix of gene segments from different viruses, indicating that multiple reassortment occurred. The well-known mammalian adaptive substitution (E627K and D701N) in PB2 was not detected in any of these isolates. The detection of multiple reassortant H7Nx AIVs in wild birds highlights the need for intensive surveillance in both wild birds and poultry in Eurasia.

Pathogenicity of H5N8 High Pathogenicity Avian Influenza Virus in Chickens and Ducks from South Korea in 2020-2021.

During the 2020-2021 winter season, an outbreak of clade 2.3.4.4b H5N8 high pathogenicity avian influenza (HPAI) virus occurred in South Korea. Here, we evaluated the pathogenicity and transmissibility of A/mandarin duck/Korea/H242/2020 (H5N8) (H242/20(H5N8)) first isolated from this outbreak in specific pathogen-free (SPF) chickens and commercial ducks in comparison with those of A/duck/Korea/HD1/2017(H5N6) (HD1/17(H5N6)) from a previous HPAI outbreak in 2017-2018. In chickens, the 50% chicken lethal dose and mean death time of H242/20(H5N8) group were 104.5 EID50 and 4.3 days, respectively, which indicate less virulent than those of HD1/17(H5N6) (103.6 EID50 and 2.2 days). Whereas, chickens inoculated with H242/20(H5N8) survived longer and had a higher titer of viral shedding than those inoculated with HD1/17(H5N6), which may increase the risk of viral contamination on farms. All ducks infected with either HPAI virus survived without clinical symptoms. In addition, they exhibited a longer virus shedding period and a higher transmission rate, indicating that ducks may play an important role as a silent carrier of both HPAI viruses. These results suggest that the pathogenic characteristics of HPAI viruses in chickens and ducks need to be considered to effectively control HPAI outbreaks in the field.

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.

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.

Complete Genome Sequences of Two Type 3 Porcine Circoviruses, WB17KW and WB20GG, Isolated from Korean Wild Boar.

Porcine circovirus type 3 (PCV3) in domestic pigs was first reported in South Korea in 2017. Here, we report the first complete genome sequences of two PCV3 strains isolated from Korean wild boar, which enhance our understanding about the genetic relatedness of PCV3 in domestic pigs and wild boar.

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.

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.

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.

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.

Adverse Effects of Classical Swine Fever Virus LOM Vaccine and Jeju LOM Strains in Pregnant Sows and Specific Pathogen-Free Pigs.

In Jeju island of South Korea, a classical swine fever (CSF) non-vaccinated region, many pig farmers insisted on abortion and stillbirth in pregnant sows and high mortality of suckling/weaning piglets by circulating CSF virus from 2014 to 2018. We investigated whether CSF viruses isolated from pigs in Jeju Island (Jeju LOM) have recovered their pathogenicity by conducting experiments using pregnant sows and specific pathogen-free (SPF) pigs. The CSF modified live LOM vaccine (MLV-LOM) and Jeju LOM strains induced abortion and stillbirth in pregnant sows. Viral antigens were detected in the organs of fetuses and stillborn piglets in the absence of specific pathological lesions associated with the virulent CSF virus in both groups (MLV-LOM and Jeju LOM strain). However, antigen was detected in one newborn piglet from a sow inoculated with a Jeju LOM strain, suggesting that it may cause persistent infections in pigs. SPF pigs inoculated with the MLV-LOM or Jeju LOM strains were asymptomatic, but virus antigen was detected in several organ and blood samples. Virus shedding in both groups of animals was not detected in the feces or saliva until 21 days post inoculation. The serum concentration of the three major cytokines, IFN-α, TNF-α, and IL-10, known to be related to lymphocytopenia, were similar in both groups when the MLV-LOM or Jeju LOM strains were inoculated into SPF pigs. In conclusion, Jeju LOM strains exhibited most of the characteristics of the MLV-LOM in pigs and resulted in the same adverse effects as the MLV-LOM strain.

Impact of a Live Attenuated Classical Swine Fever Virus Introduced to Jeju Island, a CSF-Free Area.

Here, we examine the effects of LOM(Low virulence of Miyagi) strains isolated from pigs (Jeju LOM strains) of Jeju Island, where vaccination with a live attenuated classical swine fever (CSF) LOM vaccine strain was stopped. The circulation of the Jeju LOM strains was mainly caused by a commercial swine erysipelas (Erysipelothrix rhusiopathiae) vaccine mixed with a LOM vaccine strain, which was inoculated into pregnant sows of 20 pig farms in 2014. The Jeju LOM strain was transmitted to 91 pig farms from 2015 to 2018. A histopathogenic investigation was performed for 25 farms among 111 farms affected by the Jeju LOM strain and revealed pigs infected with the Jeju LOM strain in combination with other pathogens, which resulted in the abortion of fetuses and mortality in suckling piglets. Histopathologic examination and immunohistochemical staining identified CSF-like lesions. Our results also confirm that the main transmission factor for the Jeju LOM strain circulation is the vehicles entering/exiting farms and slaughterhouses. Probability estimates of transmission between cohabiting pigs and pigs harboring the Jeju LOM strain JJ16LOM-YJK08 revealed that immunocompromised pigs showed horizontal transmission (r = 1.22). In a full genome analysis, we did not find genetic mutation on the site that is known to relate to pathogenicity between Jeju LOM strains (2014-2018) and the commercial LOM vaccine strain. However, we were not able to determine whether the Jeju LOM strain (2014-2018) is genetically the same virus as those of the commercial LOM vaccine due to several genetic variations in structure and non-structure proteins. Therefore, further studies are needed to evaluate the pathogenicity of the Jeju LOM strain in pregnant sow and SPF pigs and to clarify the characteristics of Jeju LOM and commercial LOM vaccine strains.