A Robust Quadruple Protein-Based Indirect ELISA for Detection of Antibodies to African Swine Fever Virus in Pigs.

African swine fever (ASF) emerged in domestic pigs and wild boars in China in 2018 and rapidly spread to neighboring Asian countries. Currently, no effective vaccine or diagnostic tests are available to prevent its spread. We developed a robust quadruple recombinant-protein-based indirect enzyme-linked immunosorbent assay (QrP-iELISA) using four antigenic proteins (CD2v, CAP80, p54, and p22) to detect ASF virus (ASFV) antibodies and compared it with a commercial kit (IDvet) using ASFV-positive and -negative serum samples. The maximum positive/negative value was 24.033 at a single antigen concentration of 0.25 µg/mL and quadruple ASFV antigen combination of 1 µg/mL at a 1:100 serum dilution. Among 70 ASFV-positive samples, 65, 67, 65, 70, 70, and 14 were positive above the cut-offs of 0.121, 0.121, 0.183, 0.065, 0.201, and 0.122, for CD2v, CAP80, p54, p22-iELISA, QrP-iELISA, and IDvet, respectively, with sensitivities of 92.9%, 95.7%, 92.9%, 100%, 100%, and 20%, respectively, all with 100% specificity. The antibody responses in QrP-iELISA and IDvet were similar in pigs infected with ASFV I. QrP-iELISA was more sensitive than IDvet for early antibody detection in pigs infected with ASFV II. These data provide a foundation for developing advanced ASF antibody detection kits critical for ASF surveillance and control.

Potential for transmission of naturally mutated H10N1 avian influenza virus to mammalian hosts and causing severe pulmonary disease.

Subtype H10 avian influenza viruses (AIV) are distributed worldwide in wild aquatic birds, and can infect humans and several other mammalian species. In the present study, we investigated the naturally mutated PB2 gene in A/aquatic bird/South Korea/SW1/2018 (A/SW1/18, H10N1), isolated from wild birds during the 2018-2019 winter season. This virus was originally found in South Korea, and is similar to isolates from mainland China and Mongolia. It had low pathogenicity, lacked a multi-basic cleavage site, and showed a binding preference for α2,3-linked sialic acids. However, it can infect mice, causing severe disease and lung pathology. SW1 was also transmitted by direct contact in ferrets, and replicated in the respiratory tract tissue, with no evidence of extrapulmonary spread. The pathogenicity and transmissibility of SW1 in mouse and ferret models were similar to those of the pandemic strain A/California/04/2009 (A/CA/04, H1N1). These factors suggest that subtype H10 AIVs have zoonotic potential and may transmit from human to human, thereby posing a potential threat to public health. Therefore, the study highlights the urgent need for closer monitoring of subtype H10 AIVs through continued surveillance of wild aquatic birds.

Kinetics of adaptive immune responses after administering mRNA-Based COVID-19 vaccination in individuals with and without prior SARS-CoV-2 infections.

OBJECTIVE: We aimed to compare the adaptive immune response in individuals with or without prior SARS-CoV-2 infections following the administration of mRNA-based COVID-19 vaccines. METHODS: A total of 54 participants with ages ranging from 37 to 56 years old, consisting of 23 individuals without a history of SARS-CoV-2 infection (uninfected group) and 31 individuals with prior infection of SARS-CoV-2 (infected group) who have received two doses of mRNA SARS-CoV-2 vaccines were enrolled in this study. We measured the IFN-γ level upon administration of BNT162b2 (PF) or mRNA-1273 (MO) by QuantiFERON SARS-CoV-2. The production of neutralizing antibodies was evaluated by a surrogate virus neutralization assay, and the neutralizing capacity was assessed by a plaque reduction neutralization test (PRNT50). The immune response was compared between the two groups. RESULTS: A significantly higher level of IFN-γ (p < 0.001) and neutralization antibodies (p < 0.001) were observed in the infected group than those in the uninfected group following the first administration of vaccines. The infected group demonstrated a significantly higher PRNT50 titer than the uninfected group against the Wuhan strain (p < 0.0001). Still, the two groups were not significantly different against Delta (p = 0.07) and Omicron (p = 0.14) variants. Following the second vaccine dose, T- and B-cell levels were not significantly increased in the infected group. CONCLUSION: A single dose of mRNA-based COVID-19 vaccines would boost immune responses in individuals who had previously contracted SARS-CoV-2.

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.

A Whole-Genome Analysis of the African Swine Fever Virus That Circulated during the First Outbreak in Vietnam in 2019 and Subsequently in 2022.

Since its initial report in Vietnam in early 2019, the African swine fever (ASF), a highly lethal and severe viral swine disease worldwide, continues to cause outbreaks in other Southeast Asian countries. This study analyzed and compared the genomic sequences of ASF viruses (ASFVs) during the first outbreak in Hung Yen (VN/HY/2019-ASFV1) and Quynh Phu provinces (VN/QP/2019-ASFV1) in Vietnam in 2019, and the subsequent outbreak in Hung Yen (VN/HY/2022-ASFV2) in 2022, to those of other ASFV strains. VN/HY/2019-ASFV1, VN/QP/2019-ASFV1, and VN/HY/2022-ASFV2 genomes were 189,113, 189,081, and 189,607 bp in length, encoding 196, 196, and 203 open reading frames (ORFs), respectively. VN/HY/2019-ASFV1 and VN/QP/2019-ASFV1 shared a 99.91-99.99% average nucleotide identity with genotype II strains. Variations were identified in 28 ORFs in VN/HY/2019-ASFV1 and VN/QP/2019-ASFV1 compared to 20 ASFV strains, and 16 ORFs in VN/HY/2022-ASFV2 compared to VN/HY/2019-ASFV1 and VN/QP/2019-ASFV1. Vietnamese ASFV genomes were classified as IGR II variants between the I73R and I329L genes, with two copy tandem repeats between the A179L and A137R genes. A phylogenetic analysis based on the whole genomes of 27 ASFV strains indicated that the Vietnamese ASFV strains are genetically related to Estonia 2014, ASFV-SY18, and Russia/Odintsovo_02/14. These results reveal the complete genome sequences of ASFV circulating during the first outbreak in 2019, providing important insights into understanding the evolution, transmission, and genetic variation of ASFV in Vietnam.

Conductive Thread-Based Immunosensor for Pandemic Influenza A (H1N1) Virus Detection.

Infectious agents such as viruses pose significant threats to human health, being transmitted via direct contact as well as airborne transmission without direct contact, thus requiring rapid detection to prevent the spread of infectious diseases. In this study, we developed a conductive thread-based immunosensor (CT-IS), a biosensor to easily detect the presence of airborne viruses. CT-IS utilizes an antibody that specifically recognizes the HA protein of the pandemic influenza A (pH1N1) virus, which is incorporated into the conductive thread. The antigen-antibody interaction results in increased strain on the conductive thread in the presence of the pH1N1 virus, resulting in increased electrical resistance of the CT-IS. We evaluated the performance of this sensor using the HA protein and the pH1N1 virus, in addition to samples from patients infected with the pH1N1 virus. We observed a significant change in resistance in the pH1N1-infected patient samples (positive: n = 11, negative: n = 9), whereas negligible change was observed in the control samples (patients not infected with the pH1N1 virus; negative). Hence, the CT-IS is a lightweight fiber-type sensor that can be used as a wearable biosensor by combining it with textiles, to detect the pH1N1 virus in a person's vicinity.

Establishment of multicenter COVID-19 therapeutics preclinical test system in Republic of Korea.

Throughout the recent COVID-19 pandemic, South Korea led national efforts to develop vaccines and therapeutics for SARS-CoV-2. The project proceeded as follows: 1) evaluation system setup (including Animal Biosafety Level 3 (ABSL3) facility alliance, standardized nonclinical evaluation protocol, and laboratory information management system), 2) application (including committee review and selection), and 3) evaluation (including expert judgment and reporting). After receiving 101 applications, the selection committee reviewed pharmacokinetics, toxicity, and efficacy data and selected 32 final candidates. In the nonclinical efficacy test, we used golden Syrian hamsters and human angiotensin-converting enzyme 2 transgenic mice under a cytokeratin 18 promoter to evaluate mortality, clinical signs, body weight, viral titer, neutralizing antibody presence, and histopathology. These data indicated eight new drugs and one repositioned drug having significant efficacy for COVID-19. Three vaccine and four antiviral drugs exerted significant protective activities against SARS-CoV-2 pathogenesis. Additionally, two anti-inflammatory drugs showed therapeutic effects on lung lesions and weight loss through their mechanism of action but did not affect viral replication. Along with systematic verification of COVID-19 animal models through large-scale studies, our findings suggest that ABSL3 multicenter alliance and nonclinical evaluation protocol standardization can promote reliable efficacy testing against COVID-19, thus expediting medical product development.

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.

Multiple HA substitutions in highly pathogenic avian influenza H5Nx viruses contributed to the change in the NA subtype preference.

Novel highly pathogenic avian influenza (HPAI) H5Nx viruses are predominantly circulating worldwide, with an increasing potential threat of an outbreak in humans. It remains largely unknown how the stably maintained HPAI H5N1 suddenly altered its neuraminidase (NA) to other NA subtypes, which resulted in the emergence and evolution of H5Nx viruses. Here, we found that a combination of four specific amino acid (AA) substitutions (S123P-T156A-D183N- S223 R) in the hemagglutinin (HA) protein consistently observed in the H5Nx markedly altered the NA preference of H5N1 viruses. These molecular changes in H5N1 impaired its fitness, particularly viral growth and the functional activities of the HA and NA proteins. Among the AA substitutions identified, the T156A substitution, which contributed to the NA shift, also dramatically altered the antigenicity of H5N1 viruses, suggesting an occurrence of antigenic drift triggered by selective pressure. Our study shows the importance of how HA and NA complement each other and that antigenic drift in HA can potentially cause a shift in the NA protein in influenza A virus evolution.

CRISPR/Cas-Assisted Colorimetric Biosensor for Point-of-Use Testing for African Swine Fever Virus.

African swine fever virus (ASFV) causes a highly contagious and fatal disease affecting both domesticated and wild pigs. Substandard therapies and inadequate vaccinations cause severe economic damages from pig culling and removal of infected carcasses. Therefore, there is an urgent need to develop a rapid point-of-use approach that assists in avoiding the spread of ASFV and reducing economic loss. In this study, we developed a colorimetric sensing platform based on dual enzymatic amplification that combined the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 12a (Cas12a) system and the enzyme urease for accurate and sensitive detection of ASFV. The mechanism of the sensing platform involves a magnetic bead-anchored urease-conjugated single-stranded oligodeoxynucleotide (MB@urODN), which in the presence of ASFV dsDNA is cleaved by activated CRISPR/Cas12a. After magnetically separating the free urease, the presence of virus can be confirmed by measuring the colorimetric change in the solution. The advantage of this method is that it can detect the presence of virus without undergoing a complex target gene duplication process. The established method detected ASFV from three clinical specimens collected from porcine clinical tissue samples. The proposed platform is designed to provide an adequate, simple, robust, highly sensitive and selective analytical technique for rapid zoonotic disease diagnosis while eliminating the need for vast or specialized tools.

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.

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.

Severe acute respiratory syndrome coronavirus 2 and influenza A virus co-infection alters viral tropism and haematological composition in Syrian hamsters.

The ongoing coronavirus disease 2019 pandemic and its overlap with the influenza season lead to concerns over severe disease caused by the influenza virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) co-infections. Using a Syrian hamster co-infection model with SARS-CoV-2 and the pandemic influenza virus A/California/04/2009 (H1N1), we found (a) more severe disease in co-infected animals, compared to those infected with influenza virus alone but not SARS-CoV-2 infection alone; (b) altered haematological changes in only co-infected animals and (c) altered influenza virus tropism in the respiratory tracts of co-infected animals. Overall, our study revealed that co-infection with SARS-CoV-2 and influenza virus is associated with altered disease severity and tissue tropism, as well as haematological changes, compared to infection with either virus alone.

Laboratory information management system for COVID-19 non-clinical efficacy trial data.

BACKGROUND: As the number of large-scale studies involving multiple organizations producing data has steadily increased, an integrated system for a common interoperable format is needed. In response to the coronavirus disease 2019 (COVID-19) pandemic, a number of global efforts are underway to develop vaccines and therapeutics. We are therefore observing an explosion in the proliferation of COVID-19 data, and interoperability is highly requested in multiple institutions participating simultaneously in COVID-19 pandemic research. RESULTS: In this study, a laboratory information management system (LIMS) approach has been adopted to systemically manage various COVID-19 non-clinical trial data, including mortality, clinical signs, body weight, body temperature, organ weights, viral titer (viral replication and viral RNA), and multiorgan histopathology, from multiple institutions based on a web interface. The main aim of the implemented system is to integrate, standardize, and organize data collected from laboratories in multiple institutes for COVID-19 non-clinical efficacy testings. Six animal biosafety level 3 institutions proved the feasibility of our system. Substantial benefits were shown by maximizing collaborative high-quality non-clinical research. CONCLUSIONS: This LIMS platform can be used for future outbreaks, leading to accelerated medical product development through the systematic management of extensive data from non-clinical animal studies.

Relationship between SARS-CoV-2 antibody titer and the severity of COVID-19.

BACKGROUND: It remains unclear whether high titers of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies aggravate clinical manifestations in patients or whether severe clinical manifestations result in high antibody titers. Thus, we investigated the cause-effect relationship between SARS-CoV-2 antibody titers and disease severity. METHODS: We prospectively enrolled patients admitted with the diagnosis of coronavirus disease-19 (COVID-19) from February 2020 to August 2020. We measured SARS-CoV-2 antibody titers, namely anti-receptor-binding domain (RBD) antibody and neutralizing antibody (NAb), from blood samples and calculated the chest radiograph (CXR) scores of the patients to evaluate the severity of COVID-19. RESULTS: Overall, 40 patients with COVID-19 were enrolled. Pneumonia was observed in more than half of the patients (25/40, 60%). SARS-CoV-2 antibody titers were higher in patients who were aged >60 years (anti-RBD antibodies, P = 0.003 and NAb, P = 0.009), presented with pneumonia (P = 0.006 and 0.007, respectively), and required oxygen therapy (P = 0.003 and 0.004, respectively) than in those who were not. CXR scores peaked (at 15-21 days after the onset of symptoms) statistically significantly earlier than SARS-CoV-2 antibody titers (at 22-30 days for NAb and at 31-70 days for anti-RBD antibody). There was a close correlation between the maximum CXR score and the maximum SAR-CoV-2 antibody titer. CONCLUSIONS: Based on the comparison of the peak time of SARS-CoV-2 antibody titers with the CXR score after symptom onset, we suggest that severe clinical manifestations result in high titers of SARS-CoV-2 antibodies.

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.

Photothermal inactivation of universal viral particles by localized surface plasmon resonance mediated heating filter membrane.

This study introduces localized surface plasmon resonance (L-SPR) mediated heating filter membrane (HFM) for inactivating universal viral particles by using the photothermal effect of plasmonic metal nanoparticles (NPs). Plasmonic metal NPs were coated onto filter membrane via a conventional spray-coating method. The surface temperature of the HFM could be controlled to approximately 40-60 °C at room temperature, owing to the photothermal effect of the gold (Au) NPs coated on them, under irradiation by visible light-emitting diodes. Due to the photothermal effect of the HFMs, the virus titer of H1Npdm09 was reduced by > 99.9%, the full inactivation time being < 10 min, confirming the 50% tissue culture infective dose (TCID50) assay. Crystal violet staining showed that the infectious samples with photothermal inactivation lost their infectivity against Mardin-Darby Canine Kidney cells. Moreover, photothermal inactivation could also be applied to reduce the infectivity of SARS-CoV-2, showing reduction rate of 99%. We used quantitative reverse transcription polymerase chain reaction (qRT-PCR) techniques to confirm the existence of viral genes on the surface of the HFM. The results of the TCID50 assay, crystal violet staining method, and qRT-PCR showed that the effective and immediate reduction in viral infectivity possibly originated from the denaturation or deformation of membrane proteins and components. This study provides a new, simple, and effective method to inactivate viral infectivity, leading to its potential application in various fields of indoor air quality control and medical science.

Surface-enhanced Raman scattering-based immunoassay for severe acute respiratory syndrome coronavirus 2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has affected humans worldwide for over a year now. Although various tests have been developed for the detection of SARS-CoV-2, advanced sensing methods are required for the diagnosis, screening, and surveillance of coronavirus disease 2019 (COVID-19). Here, we report a surface-enhanced Raman scattering (SERS)-based immunoassay involving an antibody pair, SERS-active hollow Au nanoparticles (NPs), and magnetic beads for the detection of SARS-CoV-2. The selected antibody pair against the SARS-CoV-2 antigen, along with the magnetic beads, facilitates the accurate direct detection of the virus. The hollow Au NPs exhibit strong, reproducible SERS signals, allowing sensitive quantitative detection of SARS-CoV-2. This assay had detection limits of 2.56 fg/mL for the SARS-CoV-2 antigen and 3.4 plaque-forming units/mL for the SARS-CoV-2 lysates. Furthermore, it facilitated the identification of SARS-CoV-2 in human nasopharyngeal aspirates and diagnosis of COVID-19 within 30 min using a portable Raman device. Thus, this assay can be potentially used for the diagnosis and prevention of COVID-19.

Reassortant Highly Pathogenic H5N6 Avian Influenza Virus Containing Low Pathogenic Viral Genes in a Local Live Poultry Market, Vietnam.

Sites of live poultry trade and marketing are hot spots for avian influenza virus (AIV) transmission. We conducted active surveillance at a local live poultry market (LPM) in northern Vietnamese provinces in December 2016. Feces samples from the market were collected and tested for AIV. A new reassorted AIV strain was isolated from female chickens, named A/chicken/Vietnam/AI-1606/2016 (H5N6), and was found to belong to group C of clade 2.3.4.4 H5N6 highly pathogenic (HP) AIVs. The neuraminidase gene belongs to the reassortant B type. The viral genome also contained polymerase basic 2 and polymerase acidic, which were most closely related to domestic-duck-origin low pathogenic AIVs in Japan (H3N8) and Mongolia (H4N6). The other six genes were most closely related to poultry-origin H5N6 HP AIVs in Vietnam and had over 97% sequence identity with human AIV isolate A/Guangzhou/39715/2014 (H5N6). The new reassorted AIV isolate A/chicken/Vietnam/AI-1606/2016 (H5N6) identified in this study exemplifies AIVs reassortment and evolution through contact among wild birds, poultry farms, and LPMs. Therefore, active surveillance of AIVs is necessary to prevent potential threats to human and animal health.

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.