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.

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.

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].

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.

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.

Identification of canine norovirus in dogs in South Korea.

BACKGROUND: Canine noroviruses (CaNoVs) are classified into genogroups GIV, GVI, and GVII and have been detected in fecal samples from dogs since their first appearance in a dog with enteritis in Italy in 2007. CaNoVs may be a public health concern because pet animals are an integral part of the family and could be a potential reservoir of zoonotic agents. Nonetheless, there was no previous information concerning the epidemiology of CaNoV in South Korea. In the present study, we aimed to detect CaNoV antigens and to investigate serological response against CaNoV in dogs. RESULTS: In total, 459 fecal samples and 427 sera were collected from small animal clinics and animal shelters housing free-roaming dogs in geographically distinct areas in South Korea. For the detection of CaNoV, RT-PCR was performed using target specific primers, and nucleotide sequences of CaNoV isolates were phylogenetically analyzed. Seroprevalence was performed by ELISA based on P domain protein. CaNoVs were detected in dog fecal samples (14/459, 3.1%) and were phylogenetically classified into the same cluster as previously reported genogroup GIV CaNoVs. Seroprevalence was performed, and 68 (15.9%) of 427 total dog serum samples tested positive for CaNoV IgG antibodies. CONCLUSION: This is the first study identifying CaNoV in the South Korean dog population.

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.

Attenuation of the virulence of a recombinant influenza virus expressing the naturally truncated NS gene from an H3N8 equine influenza virus in mice.

Equine influenza virus (EIV) causes a highly contagious disease in horses and other equids. Recently, we isolated an H3N8 EIV (A/equine/Kyonggi/SA1/2011) from a domestic horse in South Korea that exhibited symptoms of respiratory disease, and found that the EIV strain contained a naturally mutated NS gene segment encoding a truncated NS1 protein. In order to determine whether there was an association between the NS gene truncation and viral virulence, a reverse genetics system was applied to generate various NS gene recombinant viruses using the backbone of the H1N1 A/Puerto Rico/8/1934 (PR/8) virus. In a mouse model, the recombinant PR/8 virus containing the mutated NS gene of the Korean H3N8 EIV strain showed a dramatically reduced virulence: it induced no weight loss, no clinical signs and no histopathological lesions. However, the mice infected with the recombinant viruses with NS genes of PR/8 and H3N8 A/equine/2/Miami/1963 showed severe clinical signs including significant weight loss and 100% mortality. In addition, the levels of the pro-inflammatory cytokines; IL-6, CCL5, and IFN-γ, in the lungs of mice infected with the recombinant viruses expressing a full-length NS1 were significantly higher than those of mice infected with the virus with the NS gene from the Korean H3N8 EIV strain. In this study, our results suggest that the C-terminal moiety of NS1 contains a number of virulence determinants and might be a suitable target for the development of a vaccine candidate against equine influenza.

Gouleako and Herbert viruses in pigs, Republic of Korea, 2013.

Several viruses in the family Bunyaviridae are pathogenic to animals and cause vector-borne zoonoses. In 2013, investigation of cause of death of 9 pigs on 1 farm in the Republic of Korea found infection with Gouleako and Herbert viruses. Subsequent investigation revealed high prevalence of these viruses among pigs throughout the country.

Characterization of a complete genome of a circular single-stranded DNA virus from porcine stools in Korea.

Porcine circular single-stranded DNA viruses have been just identified from swine feces in Korea. This virus was mentioned as bovine stool-associated circular DNA virus (BoSCV)-like virus discovered from porcine stools. However, the thorough characteristics of the virus were not identified. Therefore, this research focuses on finding a full genome sequence and analyzing the genetic features of the virus. The virus, now called porcine stool-associated circular DNA virus in Korea (PoSCV Kor), consists of 2,589 bases forming circular structure. It has two major ORFs inversely encoding replicase and capsid protein, with each stem-loop structure between 5' ends and 3' ends of the two putative ORFs. This characteristics is the same as PoSCV in New Zealand, but different from chimpanzee stool-associated circular virus (ChiSCVs) and BoSCV, which have one stem-loop structure. Therefore, it would be sure that PoSCV Kor is very similar to PoSCV in respect to the genetic aspect; the same number of nucleotide bases and the amino acid identity of replicase and capsid protein (96 and 93 %, respectively). This fact could be certified through the finding that PoSCV Kor and PoSCV are in the same cluster by phylogenetic analysis based on the comparison with full-sequences of other circular ssDNA viruses.

Inhibition of porcine endogenous retrovirus in PK15 cell line by efficient multitargeting RNA interference.

To effectively suppress porcine endogenous retroviruses (PERV)s, RNAi technique was utilized. RNAi is the up-to-date skill for gene knockdown which simultaneously multitargets both gag and pol genes critical for replication of PERVs. Previously, two of the most effective siRNAs (gag2, pol2) were found to reduce the expression of PERVs. Concurrent treatment of these two siRNAs (gag2+pol2) showed knockdown efficiency of up to 88% compared to negative control. However, despite the high initial knockdown efficiency 48 h after transfection caused by siRNA, it may only be a transient effect of suppressing PERVs. The multitargeting vector was designed, containing both gag and pol genes and making use of POL II miR Expression Vector, which allowed for persistent and multiple targeting. This is the latest shRNA system technique expressing and targeting like miRNA. Through antibiotics resistance characteristics utilizing this vector, miRNA-transfected PK15 cells (gag2-pol2) were selected during 10 days. An 88.1% reduction in the level of mRNA expression was found. In addition, we performed RT-activity analysis and fluorescence in situ hybridization assay, and it demonstrated the highest knockdown efficiency in multitargeting (gag2+pol2) miRNA group. Therefore, according to the results above, gene knockdown system (siRNA and shRNA) through multitargeting strategy could effectively inhibit PERVs.

Complete genome sequences of a Korean virulent porcine epidemic diarrhea virus and its attenuated counterpart.

A virulent porcine epidemic diarrhea virus (PEDV) strain, DR13, was obtained from suckling pigs suspected of having porcine epidemic diarrhea in 1999 in Korea, and its attenuated counterpart was derived from virulent strain DR13 by serial propagation in Vero cells. This report describes the first complete genome sequences of virulent PEDV and its attenuated counterpart, which will provide important insights into the molecular basis of the attenuation of PEDV.

Complete genome analysis of porcine enterovirus B isolated in Korea.

The complete genome sequence of porcine enterovirus B (PEV-B) from a Korean isolate was analyzed. The genome size was 7,393 bp. Previously, full genome sequences of PEV-B had been reported from the United Kingdom, Hungary, and China. The Korean PEV-B isolate presented polyprotein gene nucleotide sequence similarities of 77.9, 73.7, 78.9, and 80.3%, respectively, to PEV-B UKG/410/73, LP54, PEV15, and Chinese strains (Ch-ah-f1).

Vacuoles isolated from Saccharomyces cerevisiae inhibit differentiation of 3T3-L1 adipocyte.

Yeast vacuoles contain various hydrolytic enzymes including lipase. They play important roles in intracellular signaling and metabolism. Using these characteristics, the aim of this study is to determine effects of yeast vacuoles on the triglyceride accumulation and differentiation of pre-adipocytes to adipocytes using 3T3-L1 cells. The accumulation of lipid droplets and triglyceride were reduced after treatment with vacuoles. As a result of not maintaining the expression of C/EBPß and C/EBPδ in vacuole-treated adipocytes, expression levels of C/EBPα and PPARγ in vacuole-treated adipocytes were significantly reduced. The expression of adiponectin in the late differentiation stage was increased compared to that in the control. By confirming that vacuolar enzymes also inhibit differentiation of adipocytes same as vacuoles, it can be concluded that the adipogenesis inhibitory effect of vacuoles is by lipase of vacuolar enzymes. Yeast-derived vacuoles could be an important source for inhibiting accumulation of lipids and obesity-related inflammation by suppressing adipogenesis.

Distinction of Male and Female Trees of Ginkgo biloba Using LAMP.

Ginkgo biloba is utilized as food, medicine, wood, and street trees among other things. The objective of this study was to develop a loop-mediated isothermal amplification (LAMP) assay for gender distinction of G. biloba. Male-specific SCAR gene can be utilized to identify G. biloba gender using LAMP. The optimized LAMP conditions, temperature 60 °C, 2-mM MgSO4, and [F3/B3]:[FIP/BIP] primer ratio of 1:4 were selected as final conditions. The G. biloba SCAR LAMP displayed a sensitivity of 10 ng when amplified by concentration under the optimum conditions. Additionally, it demonstrated a particular response in male with SYBR Green I in LAMP analysis that can be a more powerful tool for field and scale-up applications. Our work represents a first attempt to identify G. biloba gender using LAMP and offers an efficient and reliable tool for roadside landscaping.

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.

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.