Detection of bat-associated circoviruses in Korean bats.

In recent years, several novel circular single-stranded DNA viruses have been detected in various mammals, birds, insects, and environmental samples using metagenomic and high-throughput sequencing approaches. In this study, we tested for the presence of circoviruses in 243 bat fecal samples collected between 2018 and 2019 from 48 sampling sites across Korea. To detect circoviruses, nested PCR was performed with degenerate primers targeting a conserved replication-associated protein (rep) gene of circovirus/cyclovirus. Among 243 samples tested, a total of 37 fecal samples from 14 sampling sites were PCR-positive for circoviruses at a frequency rate of 15.23%. We obtained 36 partial rep gene sequences of circoviruses and one complete genome sequence of bat-associated circovirus 12, encompassing a genome size of 2097 nt containing two inversely arranged open reading frames and a conserved nonamer sequence in the apex of a stem-loop structure. In addition, we found four bat species that were harboring circoviruses in Korea based on species identification PCR of circovirus-positive bat fecal samples. Detailed sequence analysis indicated that the bat-associated circovirus sequences identified in this study were related to those of known bat and avian groups of circoviruses. Herein, we report evidence for the presence of bat-associated circoviruses in Korean bats.

Genomic Comparisons of Alphacoronaviruses and Betacoronaviruses from Korean Bats.

Coronaviruses are well known as a diverse family of viruses that affect a wide range of hosts. Since the outbreak of severe acute respiratory syndrome, a variety of bat-associated coronaviruses have been identified in many countries. However, they do not represent all the specific geographic locations of their hosts. In this study, full-length genomes representing newly identified bat coronaviruses in South Korea were obtained using an RNA sequencing approach. The analysis, based on genome structure, conserved replicase domains, spike gene, and nucleocapsid genes revealed that bat Alphacoronaviruses are from three different viral species. Among them, the newly identified B20-97 strain may represent a new putative species, closely related to PEDV. In addition, the newly-identified MERS-related coronavirus exhibited shared genomic nucleotide identities of less than 76.4% with other Merbecoviruses. Recombination analysis and multiple alignments of spike and RBD amino acid sequences suggested that this strain underwent recombination events and could possibly use hDPP4 molecules as its receptor. The bat SARS-related CoV B20-50 is unlikely to be able to use hACE2 as its receptor and lack of an open reading frame in ORF8 gene region. Our results illustrate the diversity of coronaviruses in Korean bats and their evolutionary relationships. The evolution of the bat coronaviruses related ORF8 accessory gene is also discussed.

The Epidemiological Characteristics of the Korean Bat Paramyxovirus between 2016 and 2019.

Bats are considered reservoirs of severe emerging human pathogens. Notably, bats host major mammalian paramyxoviruses from the family Paramyxoviridae, order Mononegavirales. In this study, paramyxoviruses were investigated by reverse transcription semi-nested polymerase chain reaction (RT-semi-nested PCR) and reverse transcription polymerase chain reaction (RT-PCR), based on the RT-semi-nested PCR using the consensus paramyxovirus primers targeting the RNA dependent-RNA-polymerase (RdRp) region. In addition, RT-PCR was performed using newly designed primers targeting regions of the fusion protein (F) and hemagglutinin-neuraminidase (HN). The dominant bat species in the collection site of paramyxoviruses were Miniopterus schreibersii, Myotis macrodactylus, Myotis petax, and Rhinolophus ferrumequinum. Paramyxoviruses were detected in four samples in 2016 and six in 2019. Meanwhile, in samples collected in 2017 and 2018, no paramyxoviruses were detected. Phylogenetic analysis based on the partial nucleotide sequences of RdRp, F, and HN proteins suggested that the viruses belonged to the proposed genus Shaanvirus. In conclusion, this study revealed that bat paramyxoviruses in Korea belonged to a single genus and circulated sporadically in several provinces, including Chungbuk, Gangwon, Jeju, and Jeonnam.

Long-term surveillance of bat coronaviruses in Korea: Diversity and distribution pattern.

Bats harbour diverse coronaviruses (CoVs), some of which are associated with zoonotic infections, as well as inter-species transmission. In this study, a total of 512 bat faecal samples from the bat habitats at different geographical locations in South Korea were investigated between 2016 and 2019. Seventy-eight samples were positive for coronaviruses (15.2%), comprising 68 alphacoronaviruses (13.3%) and 10 betacoronaviruses (2.0%). The positive rates tended to increase during the awakening (April) period. Notably, betacoronaviruses were only found in the site where Rhinolophus ferrumequinum was the major species of bats, and were related to SARS- and MERS-related CoVs identified in China and South Korea, respectively. No betacoronaviruses were closely related to SARS-CoV-2 in this study. Alphacoronaviruses were detected in the sites where Hypsugo alaschanicus, Miniopterus fuliginosus, Miniopterus schreibersii, Rhinolophus ferrumequinum, Myotis bombinus, Myotis macrodactylus and Myotis petax were found to be the major bat species. Furthermore, alphacoronaviruses had higher genetic diversity than betacoronaviruses and had a wider distribution in Korea. Considering that different bat species are co-roosting in crowded conditions in the same habitat, the diverse coronaviruses in Korean bats are likely to undergo cross-species transmission events due to the richness in host species. Therefore, continuous monitoring should be performed, especially at the awakening time of the hibernating bats in the habitats where diverse bat species co-roost, to better understand the evolution of coronaviruses in bats.

Isolation and characterization of novel bat paramyxovirus B16-40 potentially belonging to the proposed genus Shaanvirus.

The bat paramyxovirus B16-40 was first isolated in Korea in this study. Using the isolated virus, we could obtain not only genomic information, but also several biological characteristics of the virus. In the phylogenetic analysis, the virus was found to belong to the recently proposed genus Shaanvirus. Through sequence analyses and in vitro testing, the isolated virus was also found to have haemagglutinin-neuraminidase (HN) protein as one of the structural proteins. When mouse antiserum was generated against the isolated virus and tested, it was cross-reactive to human parainfluenza virus 1 in an indirect immunofluorescence assay but could not cross-neutralize human parainfluenza virus 1. In addition, the bat paramyxovirus B16-40 was not infectious in the mouse model. Collectively, this study provided basic information on further classification of the bat paramyxovirus B16-40 and related viruses in the proposed genus Shaanvirus.

Complete mitochondrial genome of a troglobite millipede Antrokoreana gracilipes (Diplopoda, Juliformia, Julida), and juliformian phylogeny.

The complete mitochondrial genome of a troglobite millipede Antrokoreana gracilipes (Verhoeff, 1938) (Dipolopoda, Juliformia, Julida) was sequenced and characterized. The genome (14,747 bp) contains 37 genes (2 ribosomal RNA genes, 22 transfer RNA genes and 13 protein-encoding genes) and two large non-coding regions (225 bp and 31 bp), as previously reported for two diplopods, Narceus annularus (order Spirobolida) and Thyropygus sp. (order Spirostreptida). The A + T content of the genome is 62.1% and four tRNAs (tRNA(Ser(AGN)), tRNA(Cys), tRNA(Ile) and tRNA(Met)) have unusual and unstable secondary structures. Whereas Narceus and Thyropygus have identical gene arrangements, the tRNA(Thr) and tRNA(Trp) of Antrokoreana differ from them in their orientations and/or positions. This suggests that the Spirobolida and Spirostreptida are more closely related to each other than to the Dipolopoda. Three scenarios are proposed to account for the unique gene arrangement of Antrokoreana. The data also imply that the Duplication and Nonrandom Loss (DNL) model is applicable to the order Julida. Bayesian inference (BI) and maximum likelihood (ML) analyses using amino acid sequences deduced from the 12 mitochondrial protein-encoding genes (excluding ATP8) support the view that the three juliformian members are monophyletic (BI 100%; ML 100%), that Thyropygus (Spirostreptida) and Narceus (Spirobolida) are clustered together (BI 100%; ML 83%), and that Antrokoreana (Julida) is a sister of the two. However, due to conflict with previous reports using cladistic approaches based on morphological characteristics, further studies are needed to confirm the close relationship between Spirostreptida and Spirobolida.

A new species of Cavernocypris Hartmann, 1964 (Crustacea: Ostracoda) from caves in South Korea.

Surveys of caves in South Korea yielded a new species of the ostracod genus Cavernocypris Hartmann, 1964, herein described as Cavernocypris danielopoli Smith & Kamiya n. sp. This is the fifth described species in this widely distributed genus, and can be discriminated from its congeners by the highly triangular shape of the carapace in lateral view, caused by a large dorsal protrusion on the left valve. A second triangular-shaped morphotype was also collected, differing from the first by a lower carapace and less pronounced dorsal protrusion. As it is not clear at present if this represents a separate species or intraspecific variation, it is left in open nomenclature. The lack of a pigmented eye, a highly triangular carapace and no carapace pigmentation suggests that Cavernocypris danielopoli Smith & Kamiya n. sp. is the most adapted species in the genus to a stygobiotic lifestyle. Another Korean species, Cavernocypris coreana (McKenzie, 1972), found in caves and spring discharges, has a reduced number of antennule segments, common for stygobiotic species, but this feature is lacking in the new species. This suggests that within the genus, species have followed different evolutionary pathways to adapt to the subterranean realm.

Geographical distribution of bat flies (Diptera: Nycteribiidae and Streblidae), including two new records, Nycteribia allotopa and N. formosana, collected from bats (Chiroptera: Rhinolophidae and Vespertilionidae) in the Republic of Korea.

As part of the 65(th) Medical Brigade, U.S. Army, arthropod-borne disease surveillance program and in collaboration with the Korea National Institute of Biological Resources (NIBR), bats were captured from caves and abandoned mines in the Republic of Korea. A total of 39 adult bat flies including five species of Nycteribiidae [Penicillidia jenynsii, Nycteribia parvula, N. formosana, N. allotopa mikado, and an unidentified species of Nycteribia (N. cf. formosana)], and one species of Streblidae, Brachytarsina kanoi, were collected from bats belonging to two families, Rhinolophidae and Vespertilionidae. This is the first report of N. allotopa mikado and N. formosana from the Republic of Korea.