Magnetic-bead-based DNA-capture-assisted real-time polymerase chain reaction and recombinase polymerase amplification for the detection of African swine fever virus.

African swine fever (ASF) is a deadly disease in swine caused by African swine fever virus (ASFV). The global spread of ASFV has resulted in significant economic losses worldwide. Improved early detection has been the most important first line of defense for preventing ASF outbreaks and for activating control measures. Despite the availability of rapid amplification methods, nucleic acid extraction from specimens still needs to be performed in a laboratory. To facilitate this step, we exploited the strong affinity of biotin-streptavidin binding by functionalizing streptavidin-coated magnetic beads with biotinylated oligonucleotide capture probes to efficiently capture genotype II ASFV DNA directly from crude clinical samples. The captured DNA is suitable for detection using real-time quantitative PCR (qPCR) and recombinase polymerase amplification (RPA). In this study, ASFV DNA was efficiently captured from swine feces, serum, and tissue samples. Both DNA-capture-assisted qPCR and RPA-based detection methods have a limit of detection (LOD) of 102 copies/µl, which is comparable to those of commercially available kits. In addition, an RPA-SYBR Green I method was developed for the immediate visual detection of ASFV DNA, which is time-saving and efficient for resource-limited field settings. In summary, a rapid, versatile, sequence-specific DNA capture method was developed to efficiently capture ASFV DNA from swine clinical samples and subsequent detection by qPCR and RPA, which has the potential to be used for robust screening and surveillance of ASFV and in point-of-care (POC) diagnostics.

Evidence for the circulation of genotype 4 Eurasian avian-like lineage swine H1N1 influenza A viruses on Korean swine farms, obtained using a newly developed one-step multiplex RT-qPCR assay.

Genotype 4 (G4) Eurasian avian-like lineage swine H1N1 influenza A viruses, which are reassortants containing sequences from the pandemic 2009 H1N1 virus lineage, triple-reassortant-lineage internal genes, and EA-lineage external genes, have been reported in China since 2013. These have been predominant in pig populations since 2016 and have exhibited pandemic potential. In this study, we developed a one-step multiplex RT-qPCR assay targeting the M, HA1, and PB2 genes to detect G4 and related EA H1N1 viruses, with detection limits of 1.5 × 101 copies/µL and 1.15 × 10-2 ng/µL for the purified PCR products and RNA templates, respectively. The specificity of the detection method was confirmed using various influenza virus subtypes. When the one-step multiplex RT-qPCR assay was applied to swine respiratory samples collected between 2020 and 2022 in Korea, a virus related to G4 EA H1N1 strains was detected. Phylogenetic analysis based on portions of all eight genome segments showed that the positive sample contained HA, NA, PB2, NS, and NP genes closely related to those of G4 EA H1N1 viruses, confirming the ability of our assay to accurately detect G4 EA H1N1 viruses in the field.

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.

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.

Correction to: Simultaneous detection of severe acute respiratory syndrome, Middle East respiratory syndrome, and related bat coronaviruses by real-time reverse transcription PCR.

Unfortunately, the concentration unit of plasmids was published incorrectly in the original publication of the article. The concentration unit, 'copies/ml' should be corrected to 'copies/µl'. This changes do not affect to the analytic sensitivity of the method because the detection limits of 50-100 copies/µL and 5-100 copies/µL using pUC57-SARS-pS2 (a template for SARS-CoV) and pGEM-MERS-S2 (a template for MERS-CoV), respectively, were as sensitive as other real-time PCR methods [1].

Serological evidence of H5-subtype influenza A virus infection in indigenous avian and mammalian species in Korea.

In Korea, H5-subtype highly pathogenic avian influenza (HPAI) has caused huge economic losses in poultry farms through outbreaks of H5N1 since 2003, H5N8 since 2013 and H5N6 since 2016. Although it was reported that long-distance migratory birds may play a major role in the global spread of avian influenza viruses (AIVs), transmission from such birds to poultry has not been confirmed. Intermediate hosts in the wild also may be a potential factor in viral transmission. Therefore, a total of 367 serum samples from wild animals were collected near major migratory bird habitats from 2011 to 2016 and tested by AIV-specific blocking ELISA and hemagglutination inhibition (HI) test. Two mammalian and eight avian species were seropositive according to the ELISA test. Among these, two mammalian (Hydropotes inermis and Prionailurus bengalensis) and three avian (Aegypius monachus, Cygnus cygnus, and Bubo bubo) species showed high HI titres (> 1,280) against one or two H5-subtype AIVs. As H. inermis (water deer), P. bengalensis (leopard cat), and B. bubo (Eurasian eagle owl) are indigenous animals in Korea, evidence of H5-subtype AIV in these animals implies that continuous monitoring of indigenous animals should be followed to understand interspecies transmission ecology of H5-subtype influenza viruses.

Virological and pathological characterization of an avian H1N1 influenza A virus.

Gene segments from avian H1N1 influenza A viruses have reassorted with other influenza viruses to generate pandemic strains over the past century. Nevertheless, little effort has been invested in understanding the characteristics of avian H1N1 influenza viruses. Here, we present the genome sequence and a molecular and virological characterization of an avian influenza A virus, A/wild bird/Korea/SK14/2014 (A/SK14, H1N1), isolated from migratory birds in South Korea during the winter season of 2014-2015. Full-genome sequencing and phylogenetic analysis revealed that the virus belongs to the Eurasian avian lineage. Although it retained avian-receptor binding preference, A/SK14 virus also exhibited detectable human-like receptor binding and was able to replicate in differentiated primary normal human bronchial epithelial cells. In animal models, A/SK14 virus was moderately pathogenic in mice, and virus was detected in nasal washes from inoculated guinea pigs, but not in direct-contact guinea pigs. Although A/SK14 showed moderate pathogenicity and no evidence of transmission in a mammalian model, our results suggest that the dual receptor specificity of A/SK14-like virus might allow for a more rapid adaptation to mammals, emphasizing the importance of further continuous surveillance and risk-assessment activities.

Simultaneous detection of severe acute respiratory syndrome, Middle East respiratory syndrome, and related bat coronaviruses by real-time reverse transcription PCR.

Since severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) coronaviruses (CoVs) share similar characteristics with respect to clinical signs, etiology, and transmission, methods for a rapid and accurate differential diagnosis are important. Therefore, the aim of this study was to develop a duplex real-time reverse transcription (RT)-PCR method for the simultaneous detection of these viruses. Primers and probes that target the conserved spike S2 region of human SARS-CoV, MERS-CoV, and their related bat CoVs were designed. The results of real-time RT-PCR showed specific reactions for each virus with adequate detection limits of 50-100 copies/mL and 5-100 copies/mL using pUC57-SARS-pS2 (a template for SARS-CoV) and pGEM-MERS-S2 (a template for MERS-CoV), respectively. In addition, this real-time RT-PCR system was able to detect the target viruses SARS-like bat CoV and MERS-CoV in bat fecal samples and sputum of MERS patients, respectively. Therefore, this newly developed real-time RT-PCR method is expected to detect not only SARS-CoV and MERS-CoV in humans but also several bat CoVs that are closely related to these viruses in bats.

Innate antiviral responses against Shaan virus infection in HEK293, A549 and MARC-145 cells and limited role of viperin against Shaan virus replication.

The Shaan virus is a new paramyxovirus species recently isolated from an insectivorous bat. Therefore, its replication characteristics remain unclear. We used transcriptome analysis and molecular experiments to examine host cell responses in human A549, HEK293, and monkey MARC-145 cell lines infected with the Shaan virus (ShaV/B16-40). Transcriptome data showed that Shaan virus infection induced innate immune responses associated with defense mechanisms against viral infection in all infected host cells. In real-time RT-PCR, IFN-α, -ß and -λ1 were significantly upregulated in response to infection with Shaan virus in A549 and HEK-293 cells. However, the expression of IFN-α and -λ1 did not change in MARC-145 infected cells, while IFN-ß significantly increased compared to the control in all the infected cell lines. In DEG analysis, the viperin expression pattern by Shaan virus infection varied depending on the host cell types or their origins. Viperin was highly induced at the RNA level by Shaan virus infection, and viperin protein expression was detected by western blotting. Although viperin, an ISG, has broad inhibitory effects on a range of viral pathogens, viperin knockdown or knock-in in the infected cells indicated that this protein did not markedly affect Shaan virus replication. Interestingly, these effects were independent of CMPK2 expression, which is beneficial for the antiviral effects of viperin. Therefore, the present results suggest that Shaan virus might have a strategy to evade the antiviral effect of viperin or not be significantly affected by viperin.

SARS-CoV-2 mutations acquired during serial passage in human cell lines are consistent with several of those found in recent natural SARS-CoV-2 variants.

Since the outbreak of coronavirus disease (COVID-19) in 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has evolved into diverse variants. Here, an early isolate of SARS-CoV-2 was serially passaged in multiple cell lines of human origin in triplicate, and selected mutations were compared to those found in natural SARS-CoV-2 variants. In the spike protein, Q493R and Q498R substitutions from passaged viruses were consistent with those in the B.1.1.529 (Omicron) variant. Y144del and H655Y substitutions from passaged viruses were also reported in B.1.1.7 (Alpha), P.1 (Gamma), and B.1.1.529 (Omicron) variants. Several single nucleotide polymorphisms (SNPs) found in first-passaged viruses have also been identified as selected mutation sites in serially passaged viruses. Considering the consistent mutations found between serially passaged SARS-CoV-2 and natural variants, there may be host-specific selective mutation patterns of viral evolution in humans. Additional studies on the selective mutations in SARS-CoV-2 experiencing diverse host environments will help elucidate the direction of SARS-CoV-2 evolution. Importance: SARS-CoV-2 isolate (SARS-CoV-2/human/KOR/KCDC03-NCCP43326/2020) was serially passaged in A549, CaCO2, and HRT-18 cells in triplicate. After 12 times of serial passages in each cell lines, several consistent selected mutations were found on spike protein between the serially passaged SARS-CoV-2 in human cell lines and recent natural variants of SARS-CoV-2 like omicron. On the non-spike protein genes, selected mutations were more frequent in viruses passaged in Caco-2 and HRT-18 cells (Colon epithelial-like) than in those passaged in A549 cells (Lung epithelial-like). In addition, several SNPs identified after one round of passaging were consistently identified as the selected mutation sites in serially passaged viruses. Thus, mutation patterns of SARS-CoV-2 in certain host environments may provide researchers information to understand and predict future SARS-CoV-2 variants.

α2,3-Linked Sialic Acids Are the Potential Attachment Receptor for Shaan Virus Infection in MARC-145 Cells.

Shaan virus (ShaV), a novel species of the genus Jeilongvirus, family Paramyxoviridae, was isolated from an insectivore bat (Miniopterus schreibersii) in Korea in 2016. ShaV particles contain a hemagglutinin-neuraminidase (HN) glycoprotein in their envelope that allows the virus to target cells. Typically, diverse paramyxoviruses with HN glycoprotein are reported to interact predominantly with sialic acids, but there are no studies of receptors for ShaV. In this study, the identification of potential receptors for ShaV was demonstrated using sialidase treatments, glycan microarray, magnetic bead-based virus binding assay, and neuraminidase inhibitor treatments. Pretreatment of MARC-145 cells with sialidase, which cleaves α2,3-linked sialic acids, showed higher inhibition of viral infection than α2,6-linked-specific sialidase. These data were supported by the binding of ShaV to predominantly α2,3-linked sialylated glycans in the screening of sialyl linkage patterns through glycan microarray. To further confirm the direct interaction between ShaV and α2,3-linked sialic acids, ShaV was incubated with α2,3- or α2,6-linked sialylated glycans conjugated to magnetic beads, and binding signals were detected only for α2,3-linked sialylated glycans. In addition, the potential of sialic acids as a receptor was demonstrated by the viral replication inhibitory effect of the neuraminidase inhibitor 2,3-dehydro-2-deoxy-N-acetylneuraminicacid (DANA) in the mature virion release steps. Collectively, these results support that α2,3-linked sialic acids are the potential receptor for ShaV infection in MARC-145 cells. IMPORTANCE Bats host major mammalian paramyxoviruses, and novel paramyxoviruses are increasingly being reported around the world. Shaan virus (ShaV), from the genus Jeilongvirus, family Paramyxoviridae, has a potential attachment glycoprotein, HN. Here, we identify that ShaV binds to sialic acid and demonstrate that α2,3-linked sialic acids are the potential receptor for ShaV infection. The presented data regarding ShaV receptor specificity will enable studies into the viral tropism for the host and contribute to the development of new antiviral strategies targeting viral receptors.

Detection of Porcine circovirus 3 from captured wild boars in Korea.

Porcine circovirus 3 (PCV3) is a newly discovered ssDNA virus. The virus was first reported in pigs suffering from several clinical syndromes, including porcine dermatitis and nephropathy syndrome, reproductive disorders, respiratory disease and myocarditis. PCV3 was recently reported in wild boars with high prevalence as well. In this study, 266 wild boar anal swab, feces, nasal swab and whole blood samples were collected from three mainland provinces and one island province (Chungbuk, Gangwon, Gyeonggi, Jeju) of South Korea between 2019 and 2020 including 119 from male, 142 from female and 5 undetermined. PCV3 was diagnosed targeting conserved rep (replication associated protein) gene region using Direct PCR and sequencing. Out of 266 tested samples, 15 were positive for PCV3 with detection frequency at 5.6%. Among 266 samples tested, we obtained 14 partial rep gene sequences and one complete genome sequence of PCV3 with a genome size of 2000nt. Here we present the evidence of PCV3 circulation in Korean wild boars.

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.

Pipetting-based immunoassay for point-of-care testing: Application for detection of the influenza A virus.

Point-of-care tests (POCT) for pathogens are considered important for low-resource countries and facilities. Although lateral flow immunoassays (LFIA) have many advantages including speed and ease of use, their sensitivity is limited without specific equipment. Furthermore, their response cannot be enhanced through enzymatic reactions. Owing to these limitations, LFIAs have not yet been generally adopted as the standard protocol for in vitro analysis of infectious pathogens. We aimed to develop a novel pipetting-based immunoassay using a removable magnetic ring-coupled pipette tip. The "magnetic bead-capture antibody-targeted protein complex" was simply purified by pipetting and quantified by enzymatic colour development or using a lateral flow system. This pipetting-based immunoassay was applied to detect the nucleoprotein (NP) of the influenza A virus. Using an HRP-conjugated monoclonal antibody as a probe, the assay allowed for specific and sensitive detection. Furthermore, when this assay was applied exclusively for antigen capture in the lateral flow system, the limit of detection improved 100-fold and displayed greater sensitivity than the lateral flow system alone. Therefore, the pipetting-based immunoassay may be potentially used as a sensitive POCT to clinically detect a target antigen.

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.