CA-CAS-01-A: A Permissive Cell Line for Isolation and Live Attenuated Vaccine Development Against African Swine Fever Virus.

African swine fever virus (ASFV) is the causative agent of the highly lethal African swine fever disease that affects domestic pigs and wild boars. In spite of the rapid spread of the virus worldwide, there is no licensed vaccine available. The lack of a suitable cell line for ASFV propagation hinders the development of a safe and effective vaccine. For ASFV propagation, primary swine macrophages and monocytes have been widely studied. However, obtaining these cells can be time-consuming and expensive, making them unsuitable for mass vaccine production. The goal of this study was to validate the suitability of novel CA-CAS-01-A (CAS-01) cells, which was identified as a highly permissive cell clone for ASFV replication in the MA-104 parental cell line for live attenuated vaccine development. Through a screening experiment, maximum ASFV replication was observed in the CAS-01 cell compared to other sub-clones of MA-104 with 14.89 and log10 7.5 ± 0.15 Ct value and TCID50/ml value respectively. When CAS-01 cells are inoculated with ASFV, replication of ASFV was confirmed by Ct value for ASFV DNA, HAD50/ml assay, TCID50/ml assay, and cytopathic effects and hemadsoption were observed similar to those in primary porcine alveolar macrophages after 5th passage. Additionally, we demonstrated stable replication and adaptation of ASFV over the serial passage. These results suggest that CAS-01 cells will be a valuable and promising cell line for ASFV isolation, replication, and development of live attenuated vaccines.

The African Swine Fever Virus Virulence Determinant DP96R Suppresses Type I IFN Production Targeting IRF3.

DP96R of African swine fever virus (ASFV), also known as uridine kinase (UK), encodes a virulence-associated protein. Previous studies have examined DP96R along with other genes in an effort to create live attenuated vaccines. While experiments in pigs have explored the impact of DP96R on the pathogenicity of ASFV, the precise molecular mechanism underlying this phenomenon remains unknown. Here, we describe a novel molecular mechanism by which DP96R suppresses interferon regulator factor-3 (IRF3)-mediated antiviral immune responses. DP96R interacts with a crucial karyopherin (KPNA) binding site within IRF3, disrupting the KPNA-IRF3 interaction and consequently impeding the translocation of IRF3 to the nucleus. Under this mechanistic basis, the ectopic expression of DP96R enhances the replication of DNA and RNA viruses by inhibiting the production of IFNs, whereas DP96R knock-down resulted in higher IFNs and IFN-stimulated gene (ISG) transcription during ASFV infection. Collectively, these findings underscore the pivotal role of DP96R in inhibiting IFN responses and increase our understanding of the relationship between DP96R and the virulence of ASFV.

Emergence and Prevalence of an African Swine Fever Virus Variant in Wild Boar Populations in South Korea from 2019 to 2022.

African swine fever (ASF), a viral disease caused by the African swine fever virus (ASFV), is associated with high mortality rates in domestic pigs and wild boars. ASF has been spreading since its discovery in wild boars in Korea in October 2019. Genomic analyses have provided insights into the genetic diversity of the ASFV isolated from various regions, enabling a better understanding of the virus origin and transmission patterns. We conducted a genome analysis to evaluate the diversity and mutations of ASFV spreading among wild boars in Korea during 2019-2022. We compared the genomes of ASFV strains isolated from Korean wild boars and publicly available ASFV genomes. Genomic analysis revealed several single-nucleotide polymorphisms within multigene families (MGFs) 360-1La and 360-4L in Korean ASFV. MGF 360-1La and 360-4L variations were not observed in other ASFV strains, including those of genotype II. Finally, we partially analyzed MGFs 360-1La and 360-4L in ASFV-positive samples between 2019 and 2022, confirming the geographical distribution of the variants. Our findings can help identify new genetic markers for epidemiological ASFV analysis and provide essential information for effective disease management.

African Swine Fever Virus EP364R and C129R Target Cyclic GMP-AMP To Inhibit the cGAS-STING Signaling Pathway.

African swine fever virus (ASFV) is a highly pathogenic swine DNA virus with high mortality that causes African swine fever (ASF) in domestic pigs and wild boars. For efficient viral infection, ASFV has developed complex strategies to evade key components of antiviral innate immune responses. However, the immune escape mechanism of ASFV remains unclear. Upon ASFV infection, cyclic GMP-AMP (2',3'-cGAMP) synthase (cGAS), a cytosolic DNA sensor, recognizes ASFV DNA and synthesizes the second messenger 2',3'-cGAMP, which triggers interferon (IFN) production to interfere with viral replication. In this study, we demonstrated a novel immune evasion mechanism of ASFV EP364R and C129R, which blocks cellular cyclic 2',3'-cGAMP-mediated antiviral responses. ASFV EP364R and C129R with nuclease homology inhibit IFN-mediated responses by specifically interacting with 2',3'-cGAMP and exerting their phosphodiesterase (PDE) activity to cleave 2',3'-cGAMP. Particularly notable is that ASFV EP364R had a region of homology with the stimulator of interferon genes (STING) protein containing a 2',3'-cGAMP-binding motif and point mutations in the Y76S and N78A amino acids of EP364R that impaired interaction with 2',3'-cGAMP and restored subsequent antiviral responses. These results highlight a critical role for ASFV EP364R and C129R in the inhibition of IFN responses and could be used to develop ASFV live attenuated vaccines. IMPORTANCE African swine fever (ASF) is a highly contagious hemorrhagic disease in domestic pigs and wild boars caused by African swine fever virus (ASFV). ASF is a deadly epidemic disease in the global pig industry, but no drugs or vaccines are available. Understanding the pathogenesis of ASFV is essential to developing an effective live attenuated ASFV vaccine, and investigating the immune evasion mechanisms of ASFV is crucial to improve the understanding of its pathogenesis. In this study, for the first time, we identified the EP364R and C129R, uncharacterized proteins that inhibit type I interferon signaling. ASFV EP364R and C129R specifically interacted with 2',3'-cGAMP, the mammalian second messenger, and exerted phosphodiesterase activity to cleave 2',3'-cGAMP. In this study, we discovered a novel mechanism by which ASFV inhibits IFN-mediated antiviral responses, and our findings can guide the understanding of ASFV pathogenesis and the development of live attenuated ASFV vaccines.

Severe Fever with Thrombocytopenia Syndrome Virus in Ticks in the Republic of Korea.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is a zoonotic, tick-borne RNA virus of the genus Bandavirus (Family Phenuiviridae), mainly reported in China, Japan, and the Republic of Korea (Korea). For the purpose of this study, a total of 3,898 adult and nymphal ticks of species Haemaphysalis longicornis (94.2%), Haemaphysalis flava (5.0%), Ixodes nipponensis (0.8%), and 1 specimen of Ixodes ovatus, were collected from the Deogyusan National Park, Korea, between April 2016 and June 2018. A single-step reverse transcriptase-nested PCR was performed, targeting the S segment of the SFTSV RNA. Total infection rate (IR) of SFTSV in individual ticks was found to be 6.0%. Based on developmental stages, IR was 5.3% in adults and 6.0% in nymphs. The S segment sequences obtained from PCR were divided into 17 haplotypes. All haplotypes were phylogenetically clustered into clades B-2 and B-3, with 92.7% sequences in B-2 and 7.3% in B-3. These observations indicate that the Korean SFTSV strains were closer to the Japanese than the Chinese strains. Further epidemiological studies are necessary to better understand the characteristics of the Korean SFTSV and its transmission cycle in the ecosystem.

Complete genome analysis of the African swine fever virus isolated from a wild boar responsible for the first viral outbreak in Korea, 2019.

African swine fever (ASF), a highly contagious and severe hemorrhagic viral disease in swine, is emerging as a major threat not only in Korea but also worldwide. The first confirmed case of ASF in Korea was reported in 2019. Despite the occurrence of ASF in Korea, only a few studies have genetically characterized the causative ASF virus (ASFV). In this study, we aimed to genetically characterize the ASFV responsible for the 2019 outbreak in Korea. The genome of the ASFV isolated during the first outbreak in Korea was analyzed. The Korea/YC1/2019 strain has 188,950 base pairs, with a GC content of 38.4%. The complete genome sequence was compared with other ASFV genomes annotated in the NCBI database. The Korea/YC1/2019 strain shared the highest similarity with Georgia 2007, Belgium 2018/1, and ASFV-wbBS01 strains. This study expands our knowledge of the genetic diversity of ASFV, providing valuable information for epidemiology, diagnostics, therapies, and vaccine development.

Relationship among bats, parasitic bat flies, and associated pathogens in Korea.

BACKGROUND: Bats are hosts for many ectoparasites and act as reservoirs for several infectious agents, some of which exhibit zoonotic potential. Here, species of bats and bat flies were identified and screened for microorganisms that could be mediated by bat flies. METHODS: Bat species were identified on the basis of their morphological characteristics. Bat flies associated with bat species were initially morphologically identified and further identified at the genus level by analyzing the cytochrome c oxidase subunit I gene. Different vector-borne pathogens and endosymbionts were screened using PCR to assess all possible relationships among bats, parasitic bat flies, and their associated organisms. RESULTS: Seventy-four bat flies were collected from 198 bats; 66 of these belonged to Nycteribiidae and eight to Streblidae families. All Streblidae bat flies were hosted by Rhinolophus ferrumequinum, known as the most common Korean bat. Among the 74 tested bat flies, PCR and nucleotide sequencing data showed that 35 (47.3%) and 20 (27.0%) carried Wolbachia and Bartonella bacteria, respectively, whereas tests for Anaplasma, Borrelia, Hepatozoon, Babesia, Theileria, and Coxiella were negative. Phylogenetic analysis revealed that Wolbachia endosymbionts belonged to two different supergroups, A and F. One sequence of Bartonella was identical to that of Bartonella isolated from Taiwanese bats. CONCLUSIONS: The vectorial role of bat flies should be checked by testing the same pathogen and bacterial organisms by collecting blood from host bats. This study is of great interest in the fields of disease ecology and public health owing to the bats' potential to transmit pathogens to humans and/or livestock.

Subclinical Infection and Transmission of Clade 2.3.4.4 H5N6 Highly Pathogenic Avian Influenza Virus in Mandarin Duck (Aix galericulata) and Domestic Pigeon (Columbia livia domestica).

Since 2014, H5Nx clade 2.3.4.4 highly pathogenic avian influenza viruses (HPAIV) have caused outbreaks in wild birds and poultry in multiple continents, including Asia, Europe, Africa, and North America. Wild birds were suspected to be the sources of the local and global spreads of HPAIV. This study evaluated the infectivity, pathogenicity, and transmissibility of clade 2.3.4.4 H5N6 HPAIV in mandarin ducks (Aixgalericulata) and domestic pigeons (Columbia livia domestica). None of the birds used in this study, 20 mandarin ducks or 8 pigeons, showed clinical signs or mortality due to H5N6 HPAI infection. Two genotypes of H5N6 HPAIV showed replication and transmission by direct and indirect contact between mandarin ducks. H5N6 HPAIV replicated and transmitted by direct contact between pigeons, although the viral shedding titer and duration were relatively lower and shorter than those in mandarin ducks. Influenza virus antigen was detected in various internal organs of infected mandarin ducks and pigeons, indicating systemic infection. Therefore, our results indicate mandarin ducks and pigeons can be subclinically infected with clade 2.3.4.4 H5N6 HPAIV and transfer the virus to adjacent birds. The role of mandarin ducks and pigeons in the spread and prevalence of clade 2.3.4.4 H5N6 viruses should be carefully monitored.

Distribution and genotypic analysis of Enterocytozoon bieneusi from wild boars in Korea.

Enterocytozoon bieneusi, an important microsporidian fungus, causes chronic diarrhea in humans and animals worldwide. Out of the 502 fecal samples from wild boars, 13 were positive for the E. bieneusi internal transcribed spacer region, with a prevalence of 2.6%. Six E. bieneusi genotypes, D, EbpC, and four novel KWB1-KWB4, were identified with zoonotic potential. Genotypes D (subgroup 1a) and EbpC (subgroup 1d) were first reported in Korean swine and Korea, respectively; KWB1-KWB4 (subgroup 1e) were most prevalent in this study. Because zoonotic genotypes have been identified, E. bieneusi transmission through wild boars must be closely monitored for proper prevention and treatment, despite their low prevalence. LAY SUMMARY: Enterocytozoon bieneusi is an important microsporidian fungus. Its sequences from wild boars were identified with zoonotic potential. Genotypes D and EbpC were first reported in Korean swine and Korea, respectively. E. bieneusi should be closely monitored to properly prevent and treat animals.

Rapid emergence of African swine fever virus variants with different numbers of a tandem repeat sequence in South Korea.

African swine fever virus variants with different numbers of a 10-bp tandem repeat were isolated in South Korea soon after being identified in wild boar. The short emergence periods and sympatric distributions within a narrow geographical region suggest that the variants were sporadically generated in the pre-existing viral population.

Genetic Diversity and Zoonotic Potential of Blastocystis in Korean Water Deer, Hydropotes inermis argyropus.

Blastocystis is a protozoan parasite commonly detected in the intestinal tract of humans and animals. It has been actively studied worldwide; however, information on Blastocystis is limited in Korea. Because there is an increasing concern about the contact between wildlife and domestic animals or humans, we assessed the infection status and zoonotic potential of Blastocystis in Korean water deer (KWD, Hydropotes inermis argyropus) using genotyping and phylogenetic analysis. A total of 125 fresh fecal samples were collected from KWD which were killed by vehicles on highways or roadsides in this study. Among the 125 samples, 51 (40.8%) were PCR positive. We performed nucleotide sequencing and phylogenetic analysis of 26 of the 51 PCR-positive samples. By analyzing Blastocystis 18S rRNA, two subtypes (ST4 and ST14) were identified in this study. Of the 26 samples analyzed, 25 were identified as ST14 and one as ST4. Infection of ST14 in humans has not been reported. Although only one ST4 sample was detected in this study, ST4 has zoonotic potential without showing ruminant specificity. Thus, continuous attention should be provided to the potential of transmission between wildlife and domestic animals and humans.

Wild boar harbouring African swine fever virus in the demilitarized zone in South Korea, 2019.

The African swine fever virus (ASFV) was first detected in wild boar in the Demilitarized Zone, a bordered area between South and North Korea, on 2 October 2019. Phylogenetic analyses of ASFV genes encoding p72 and CD2v indicated that the causative strain belongs to genotype II and serogroup 8, respectively, and contained additional tandem repeat sequences between the I73R and the I329L protein genes.

Molecular Detection and Subtyping of Blastocystis Detected in Wild Boars (Sus scrofa) in South Korea.

Blastocystis is a genus of parasitic protozoans that live in humans, mammals, and birds and which has been widely studied due to its low host specificity. Limited data are available, however, regarding its presence in wildlife, particularly in South Korea. Contact between wild boars (Sus scrofa) and livestock or humans has steadily increased as wild boars venture down from the mountains to farms and residential areas. In this study, we examined the status and subtypes (STs) of Blastocystis in wild boars in South Korea and confirmed its zoonotic potential. From March 2016 to November 2018, we collected 433 fecal samples throughout the country from trapped or road-killed wild boars. The 18S rRNA gene was used for molecular identification and subtyping and the proportion of PCR-positive samples was 10.4%. We then assessed positive samples for associations with sex, region, and seasonal infection; however, no statistical significance was observed for any variable other than season. Phylogenetic analyses revealed that all sequences belonged to subtype 5 and had 99.5-99.9% identity with sequences obtained from Japanese cattle (Bos taurus) and 97.1% identity with sequences obtained from Chinese. Subtype 5 has been implicated in zoonoses, indicating that Korean wild boars could transmit Blastocystis to humans and other livestock. Our results, in accordance with the One Health concept, strongly support continued interest and efforts by public health and disease control organizations toward transmission prevention.

Phylogenetic analysis of severe fever with thrombocytopenia syndrome virus in Korean water deer (Hydropotes inermis argyropus) in the Republic of Korea.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging zoonotic tick-borne disease caused by SFTS virus, which circulates among ticks and their host animals, including wildlife. However, few studies have examined SFTS virus infection in wildlife present in the Republic of Korea (ROK). We evaluated SFTS virus infection in tissue samples from Korean water deer (Hydropotes inermis argyropus), one of the most common wild ungulates in ROK. In this study, we evaluated tissue samples of 129 water deer carcasses collected in 2017 and detected SFTS viral RNA by conventional PCR. SFTS viral RNA was found in 3 of the 129 carcasses, showing a prevalence of 2.3 %; 2 of which were collected in Gyeongsangnam-do and 1 of which was in the Gangwon-do region. Among the 6 internal organs studied, only the spleen samples were positive. Phylogenetic analysis revealed close relationships between deer- and human-derived strains. The medium segments of the three positive cases clustered with genotype B, which is the predominant genotype in ROK. In the small segment, two cases clustered with genotype B, samples 17WD044 and 17WD065. The third sample, 17WD068 from Gangwon-do province, showed genotype A, which circulates mainly in China. The disagreement in the genotypes of the two tested segments suggests a potential reassortment between genotype A and B, resulting in genetic recombination as observed in sample 17WD068, which may be co-circulating in China and Korea. Further studies in wildlife and humans are necessary to understand the genetic characteristics of SFTS viruses circulating in ROK.

Complete Genome Sequences of Two Severe Fever with Thrombocytopenia Syndrome Virus Strains Isolated from a Human and a Dog in the Republic Of Korea.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is tick-borne and causes this disease (SFTS) in humans. We determined the complete genome sequences of two SFTSV strains isolated from serum from a human with SFTS and a dog with asymptomatic infection using reverse transcription and rapid amplification of cDNA ends PCR.

Complete genome analysis of a SARS-like bat coronavirus identified in the Republic of Korea.

Bats have been widely known as natural reservoir hosts of zoonotic diseases, such as severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) caused by coronaviruses (CoVs). In the present study, we investigated the whole genomic sequence of a SARS-like bat CoV (16BO133) and found it to be 29,075 nt in length with a 40.9% G+C content. Phylogenetic analysis using amino acid sequences of the ORF 1ab and the spike gene showed that the bat coronavirus strain 16BO133 was grouped with the Beta-CoV lineage B and was closely related to the JTMC15 strain isolated from Rhinolophus ferrumequinum in China. However, 16BO133 was distinctly located in the phylogenetic topology of the human SARS CoV strain (Tor2). Interestingly, 16BO133 showed complete elimination of ORF8 regions induced by a frame shift of the stop codon in ORF7b. The lowest amino acid identity of 16BO133 was identified at the spike region among various ORFs. The spike region of 16BO133 showed 84.7% and 75.2% amino acid identity with Rf1 (SARS-like bat CoV) and Tor2 (human SARS CoV), respectively. In addition, the S gene of 16BO133 was found to contain the amino acid substitution of two critical residues (N479S and T487 V) associated with human infection. In conclusion, we firstly carried out whole genome characterization of the SARS-like bat coronavirus discovered in the Republic of Korea; however, it presumably has no human infectivity. However, continuous surveillance and genomic characterization of coronaviruses from bats are necessary due to potential risks of human infection induced by genetic mutation.

Genetic Characterization of Canine Parvovirus Type 2 Detected in Wild Raccoon Dogs ( Nyctereutes procyonoides) in the Republic of Korea.

Canine parvovirus (CPV) was detected in three of 136 samples from dead raccoon dogs ( Nyctereutes procyonoides) in the Republic of Korea (South Korea) during 2016-17. By sequence and phylogenetic analysis of the complete VP2 gene, the strain belonged to CPV-2 and would be distinct from the previous reported CPV-2a and CPV-2b strains from Korean domestic dogs ( Canis lupus familiaris). The results indicated that the CPV strains from raccoon dogs and domestic dogs might be not circulated between wild and domestic carnivores in Korea.

Prevalence of Severe Fever with Thrombocytopenia Syndrome Virus in Ticks Collected from National Parks in Korea.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging viral disease in East Asian countries, including China, Japan, and the Republic of Korea (ROK). The causative agent of SFTS is the SFTS virus (SFTSV), which is transmitted by ticks. To investigate the prevalence of SFTSV in the ROK, a total of 9744 ticks were collected from vegetation in five national parks between July and November 2015. Of the collected adult and nymph ticks, Haemaphysalis longicornis (68.44%) was the most abundant, followed by Haemaphysalis flava (29.66%), Ixodes nipponensis (1.56%), and Amblyomma testudinarium (0.34%). Collected larval ticks were of the genera Haemaphysalis (99.61%) and Ixodes (0.39%). One-step RT-PCR and nested PCR were used to detect SFTSV-specific genes from each individual adult and nymph tick and pooled larval ticks. SFTSV was detected in 4.77% (48/1006) in H. longicornis, 1.15% (5/436) in H. flava, 0% (0/23) in I. nipponensis, and 20% (1/5) in A. testudinarium. The infection rate of SFTSV in adult and nymph ticks was 3.61%. The prevalence of SFTSV in adult and nymph ticks was relatively high, compared with previous reports. In larval ticks, the minimum infection rate was 0.31%. SFTSV was detected in ticks collected from both trail and nontrail areas in the national parks, and up to 800 meters above sea level. The sequences obtained showed 99.4-99.7% homology with SFTS virus S segment sequences from Chinese and Japanese ticks.

Genetic diversity and phylogenetic analysis of newly discovered bat astroviruses in Korea.

Bats have been identified as a natural reservoir for several potentially zoonotic viruses. Recently, astroviruses have been reported in bats in many countries, but not Korea. We collected 363 bat samples from thirteen species at twenty-nine sites in Korea across 2016 and tested them for astrovirus. The detection of the RNA-dependent RNA polymerase (RdRp) gene in bat astroviruses was confirmed in thirty-four bats across four bat species in Korea: twenty-five from Miniopterus fuliginosusi, one from Myotis macrodactylus, four from M. petax, and four from Rhinolophus ferrumequinum. The highest detection rates for astrovirus were found in Sunchang (61.5%, 8/13 bats), and in the samples collected in April (63.2%, 12/19 bats). The amino acid identity of astroviral sequences identified from bat samples was ≥ 46.6%. More specifically, the amino acid identity within multiple clones from individual bats was ≥ 50.8%. Additionally, the phylogenetic topology between astroviruses from different bat families showed a close relationship. Furthermore, phylogenetic analysis of the partial ORF2 sequence of bat astroviruses was found to have a maximum similarity of 73.3-74.8% with available bat astrovirus sequences. These results indicate potential multiple-infection by several bat astrovirus species in individual bats, or hyperpolymorphism in the astrovirus strains, as well as the transmission of astroviruses across bat families; furthermore, our phylogenetic analysis of the partial ORF2 implied that a novel astrovirus may exist. However, the wide diversity of astroviral sequences appeared to have no significant correlation with bat species or the spatiotemporal distribution of Korean bat astroviruses.

Minimizing an outbreak of avian botulism (Clostridium botulinum type C) in Incheon, South Korea.

An outbreak of botulism occurred over a two-month period beginning July 20, 2016. In all, 697 wild birds were found paralyzed or dead at the Namdong reservoir and 11 Gong-gu. Using a mouse bioassay, type C botulinum toxin was identified in the bird serum, liquid cultures of soil samples, and maggot extracts. To minimize further infection of wild birds, we opened the floodgates of the Namdong reservoir adjacent to the Yellow Sea; this decreased the water temperature and the nutrient load such as nitrogen and phosphorus. The outbreak stopped shortly after taking these actions. It is not known if these efforts decreased the number of dead and diseased wild birds. Our study demonstrates one potential approach to minimize future botulism outbreaks among wild birds and their habitats.