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.

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.

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.

Identification of African swine fever virus genomic DNAs in wild boar habitats within outbreak regions in South Korea.

An African swine fever (ASF) outbreak in wild boars was first reported on October 2, 2019, in South Korea. Since then, additional cases were reported in South Korea's border areas. We here report the identification of ASF virus (ASFV) DNAs from two out of eight environmental abiotic matter samples collected from areas where ASF-positive wild boar carcasses were found. Comparative genomic investigations suggested that the contaminating ASFV DNAs originated from the wild boar whose carcass had been found near the positive sample sites. This is the first report on the identification of ASF viral material in wild boar habitats.

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.

The difference of detection rate of avian influenza virus in the wild bird surveillance using various methods.

Korea is located within the East Asian-Australian flyway of wild migratory birds during the fall and winter seasons. Consequently, the likelihood of introduction of numerous subtypes and pathotypes of the Avian influenza (AI) virus to Korea has been thought to be very high. In the current study, we surveyed wild bird feces for the presence of AI virus that had been introduced to Korea between September 2017 and February 2018. To identify and characterize the AI virus, we employed commonly used methods, namely, virus isolation (VI) via egg inoculation, real-time reverse transcription-polymerase chain reaction (rRT-PCR), conventional RT-PCR (cRT-PCR) and a newly developed next generation sequencing (NGS) approach. In this study, 124 out of 11,145 fresh samples of wild migratory birds tested were rRT-PCR positive; only 52.0% of VI positive samples were determined as positive by rRT-PCR from fecal supernatant. Fifty AI virus specimens were isolated from fresh fecal samples and typed. The cRT-PCR subtyping results mostly coincided with the NGS results, although NGS detected the presence of 11 HA genes and four NA genes that were not detected by cRT-PCR. NGS analysis confirmed that 12% of the identified viruses were mixed-subtypes which were not detected by cRT-PCR. Prevention of the occurrence of AI virus requires a workflow for rapid and accurate virus detection and verification. However, conventional methods of detection have some limitations. Therefore, different methods should be combined for optimal surveillance, and further studies are needed in aspect of the introduction and application of new methods such as NGS.

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.

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.

Detection of waterborne norovirus genogroup I strains using an improved real time RT-PCR assay.

Noroviruses (NoVs) are the major global source of acute gastroenteritis (AGE) outbreaks. To detect NoVs, real-time reverse transcription-quantitative PCR (RT-qPCR) assays have been widely employed since the first decade of the 21st century. We developed a redesigned probe, JJV1PM, for RT-qPCR assay detection of NoV genogroup (G) I strains. The new RT-qPCR assay using the JJV1PM-probe showed broader strain reactivity for 10 NoV GI genotypes, while the old method, using the JJV1PT-probe assay, detected only 7 NoV GI genotypes in a validation panel using human fecal specimens. The improved RT-qPCR assay was also successfully applied to water samples. The JJV1PM-probe assay identified 7 NoV GI genotypes, whereas the JJV1PT-probe assay detected only 2 NoV GI genotypes from water samples. Notably, groundwater-borne NoV GI strains detected by the improved JJV1PM-probe assay were associated with groundwater-borne AGE outbreaks in South Korea. The results of this study underscore the importance of the evaluation of RT-qPCR assays using recently circulating NoV strains prior to field application.

First detection of West Nile virus in domestic pigeon in Korea.

West Nile virus (WNV) is a mosquito-borne zoonotic pathogen that has spread throughout Europe and the United States. Recently, WNV spread to East and Southeast Asia, and great efforts have been made in South Korea to prevent the spread of WNV from neighboring countries. In this study, we diagnosed the first case of WNV in pigeons (Columba livia domestica) residing in cities using a competitive enzyme-linked immunosorbent assay and confirmed it with nested reverse transcription polymerase chain reaction analysis and sequencing. This is the first report to provide convincing evidence that WNV is present within South Korea.

Complete nucleotide sequence analysis of the norovirus GII.4 Sydney variant in South Korea.

Norovirus is the primary cause of acute gastroenteritis in individuals of all ages. In Australia, a new strain of norovirus (GII.4) was identified in March 2012, and this strain has spread rapidly around the world. In August 2012, this new GII.4 strain was identified in patients in South Korea. Therefore, to examine the characteristics of the epidemic norovirus GII.4 2012 variant in South Korea, we conducted KM272334 full-length genomic analysis. The genome of the gg-12-08-04 strain consisted of 7,558 bp and contained three open reading frame (ORF) composites throughout the whole genome: ORF1 (5,100 bp), ORF2 (1,623 bp), and ORF3 (807 bp). Phylogenetic analyses showed that gg-12-08-04 belonged to the GII.4 Sydney 2012 variant, sharing 98.92% nucleotide similarity with this variant strain. According to SimPlot analysis, the gg-12-08-04 strain was a recombinant strain with breakpoint at the ORF1/2 junction between Osaka 2007 and Apeldoorn 2008 strains. This study is the first report of the complete sequence of the GII.4 Sydney 2012 strain in South Korea. Therefore, this may represent the standard sequence of the norovirus GII.4 2012 variant in South Korea and could therefore be useful for the development of norovirus vaccines.

Development of enhanced primer sets for detection of norovirus.

Norovirus (NV) is a major viral pathogen that causes nonbacterial acute gastroenteritis and outbreaks of food-borne disease. The genotype of NV most frequently responsible for NV outbreaks is GII.4, which accounts for 60-80% of cases. Moreover, original and new NV variant types have been continuously emerging, and their emergence is related to the recent global increase in NV infection. In this study, we developed advanced primer sets (NKI-F/R/F2, NKII-F/R/R2) for the detection of NV, including the variant types. The new primer sets were compared with conventional primer sets (GI-F1/R1/F2, SRI-1/2/3, GII-F1/R1/F2, and SRII-1/2/3) to evaluate their efficiency when using clinical and environmental samples. Using reverse transcription polymerase chain reaction (RT-PCR) and seminested PCR, NV GI and GII were detected in 91.7% (NKI-F/R/F2), 89.3% (NKII-F/R/R2), 54.2% (GI-F1/R1/F2), 52.5% (GII-F1/R1/F2), 25.0% (SRI-1/2/3), and 32.2% (SRII-1/2/3) of clinical and environmental specimens. Therefore, our primer sets perform better than conventional primer sets in the detection of emerged types of NV and could be used in the future for epidemiological diagnosis of infection with the virus.