Characterization of replication and variations in genome segments of a bat reovirus, BatMRV/B19-02, by RNA-seq in infected Vero-E6 cells.

Mammalian orthoreoviruses (MEVs) that can cause enteric, respiratory, and encephalitic infections have been identified in a wide variety of mammalian species. Here, we report a novel MRV type 1 strain detected in Miniopterus schreibersii that may have resulted from reassortment events. Using next-generation RNA sequencing (RNA-seq), we found that the ratios of the RNA levels of the 10 reovirus segments in infected cells were constant during the late stages of infection. We also discovered that the relative abundance of each segment differed. Notably, the relative abundance of M2 (encoding the µ1 protein) and S4 (encoding the σ3 protein) RNAs was higher than that of the others throughout the infection. Additionally, massive junctions were identified. These results support the hypothesis that defective genome segments are generated and that cross-family recombination occurs. These data may further the study of gene function, viral replication, and virus evolution.

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.

Diagnostic performance and clinical feasibility of a novel one-step RT-qPCR assay for simultaneous detection of multiple severe acute respiratory syndrome coronaviruses.

Coronavirus disease 2019 (COVID-19) is an acute respiratory infection caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Other coronaviruses (CoVs) can also infect humans, although the majority cause only mild respiratory symptoms. Because early diagnosis of SARS-CoV-2 is critical for preventing further transmission events and improving clinical outcomes, it is important to be able to distinguish SARS-CoV-2 from other SARS-related CoVs in respiratory samples. Therefore, we developed and evaluated a novel reverse transcription quantitative polymerase chain reaction (RT-qPCR) assay targeting the genes encoding the spike (S) and membrane (M) proteins to enable the rapid identification of SARS-CoV-2, including several new circulating variants and other emerging SARS-like CoVs. By analysis of in vitro-transcribed mRNA, we established multiplex RT-qPCR assays capable of detecting 5 × 10° copies/reaction. Using RNA extracted from cell culture supernatants, our multiple simultaneous SARS-CoV-2 assays had a limit of detection of 1 × 10° TCID50/mL and showed no cross-reaction with human CoVs or other respiratory viruses. We also validated our method using human clinical samples from patients with COVID-19 and healthy individuals, including nasal swab and sputum samples. This novel one-step multiplex RT-qPCR assay can be used to improve the laboratory diagnosis of human-pathogenic CoVs, including SARS-CoV-2, and may be useful for the identification of other SARS-like CoVs of zoonotic origin.

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.

Complete Coding Sequence of a Swine Influenza A Variant (H3N2) Virus Isolated in the Republic of Korea in 2017.

Cases of human infection with a swine influenza A virus variant have been reported in the United States, and since 2011, H3N2 variant viruses have also been regularly isolated from swine in the Republic of Korea. Here, we genetically characterized an influenza A H3N2 isolate (A/swine/P17-4/2017). BLASTN analysis of the 8 gene sequences revealed a high degree of nucleotide similarity (97.0 to 99.0%) to porcine strains circulating in the Republic of Korea and the United States. Specifically, we found a high degree of similarity in the nucleotide matrix gene to those of recent isolates from North Carolina. Therefore, continuous epidemiological surveillance is necessary to monitor the variation and evolution of influenza A viruses.