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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly transmissible and potentially fatal virus. So far, most comprehensive analyses encompassing clinical and transcriptional manifestation have concentrated on the lungs. Here, we confirmed evident signs of viral infection in the lungs and spleen of SARS-CoV-2-infected K18-hACE2 mice, which replicate the phenotype and infection symptoms in hospitalized humans. Seven days post viral detection in organs, infected mice showed decreased vital signs, leading to death. Bronchopneumonia due to infiltration of leukocytes in the lungs and reduction in the spleen lymphocyte region were observed. Transcriptome profiling implicated the meticulous regulation of distress and recovery from cytokine-mediated immunity by distinct immune cell types in a time-dependent manner. In lungs, the chemokine-driven response to viral invasion was highly elevated at 2 days post infection (dpi). In late infection, diseased lungs, post the innate immune process, showed recovery signs. The spleen established an even more immediate line of defense than the lungs, and the cytokine expression profile dropped at 7 dpi. At 5 dpi, spleen samples diverged into two distinct groups with different transcriptome profile and pathophysiology. Inhibition of consecutive host cell viral entry and massive immunoglobulin production and proteolysis inhibition seemed that one group endeavored to survive, while the other group struggled with developmental regeneration against consistent viral intrusion through the replication cycle. Our results may contribute to improved understanding of the longitudinal response to viral infection and development of potential therapeutics for hospitalized patients affected by SARS-CoV-2.

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 10

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.

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. Supplementary Information The online version contains supplementary material available at 10.1007/s00284-021-02661-z.

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.

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., This article reports the epidemiological data of Porcine circovirus 3 in captured wild boar populations of Korea.

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.

Mammalian Orthoreoviruses have been isolated from a wide variety of mammalian species that can cause enteric, respiratory and encephalitis infections. We report a novel MRV type 1 detected in Miniopterus schreibersii may have resulted from reassortment events. Using next-generation sequencing RNA-seq, we found that the ratios of relative abundances in RNA levels of the ten reovirus segments are constant during the late stages of infection in infected cells. We also discovered that the related RNA abundance level of each gene differed. Remarkably, the relative abundances of total RNA of M2 (coding µ1 protein) and S4 (coding σ3 protein) genes were higher than others throughout infection time. In addition, massive junctions were defined. These are evidence to support the hypothesis of generating defective genome segments and cross-family recombination mechanisms. These findings may support further the study of gene function, viral replication and virus evolution in the host.

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.

African swine fever (ASF) is a highly infectious disease of pigs caused by African swine fever virus (ASFV). In order to identify potential genetic variations among ASFV strains circulating in Vietnam, 26 ASFV isolates from organs and blood samples collected from domestic pigs from 23 different provinces of northern, central and southern Vietnam during 2019-2020 ASF outbreaks were genetically characterized. Nucleotide sequences were determined for a portion of the B646L (p72) gene, the complete E183L (p54) gene, the variable region of EP402R (CD2v), the central variable region (CVR) of pB602L, and a tandem repeat sequence (TRS) between the I73R and I329L genes. Analysis of the partial B646L (p72) and EP402R (CD2v) gene sequences and the full-length E183L (p54) gene sequence showed that all 26 ASFV isolates belonged to genotype II and serotype VIII and that they were identical to the strain Georgia/2007/1 and all ASFV strains sequenced in China. The TRS between the I73R and I329L genes contained a 10-nucleotide insertion that was observed in the Chinese ASFV strain CN201801 isolated from domestic pigs in 2018, but not in the Georgia/2007/1 and China/Jilin/2018/boar strains isolated from wild boar in China. This is the first intra-epidemic genome analysis reported for the ASFV strains circulating in Vietnam.

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.

The canine influenza virus (CIV) has spread globally from East Asia to the United States and mutated and evolved to generate various CIVs. Since 2010, the mutant CIVs found in China and Korea have presented increased virulence in mice, guinea pigs and ferrets, which has raised concerns about public health and outbreak of a severe canine flu. We analysed and compared the morphology, cellular uptake and pathogenicity of CIV variants in host animals, to determine their characteristics. The Chinese mutant, A/canine/Jiangsu/06/2010[H3N2](JS10), has two amino acid insertions at the distal end of the NA stalk, and A/canine/Korea/01/2007[H3N2](KR07) presented comparable efficiency of cell uptake and a similar morphology to spherical or small ovoid particles. However, KR07M generated from swapping of M segment of the pandemic isolate, A/California/04/2009 [H1N1] (CA04) into KR07 alone accounted for morphologic change and higher efficiency of cell uptake to the wild-type CIV. This study will provide an insight into the pathogenesis, transmission and evolution of CIVs and help determine future countermeasures.

Dengue virus (DENV) is a major infectious viral pathogen that affects millions of individuals worldwide every year, causing a potentially fatal syndrome, while no commercial antiviral drugs are yet available. To develop an antiviral against dengue fever, it is necessary to understand the relationship between DENV and host cells, which could provide a basis for viral dynamics and identification of inhibitory drug targets. In this study, we designed DiD-loaded and BODIPY-ceramide-encapsulated DENV-polymersome hybrid nanovesicles (DENVSomes) prepared by an extrusion method, which trigger red fluorescence in the endosome and green in the Golgi. DENVSome monitors the dynamics of host cell-virus interaction and tracking in living cells with novel state-of-the-art imaging technologies that show images at high resolution. Also, DENVSome can be exploited to screen whether candidate antiviral drugs interact with DENVs. Consequently, we successfully demonstrated that DENVSome is an efficient tool for tracking and unraveling the mechanisms of replication and drug screening for antiviral drugs of DENV.

The pandemic coronavirus disease 2019 (COVID-19) is characterized as an acute respiratory infection in the majority of cases and is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, other coronaviruses (CoVs) can infect humans, although the majority only cause mild respiratory symptoms. As early diagnosis of SARS-CoV-2 is critical to prevent further transmission events and to improve clinical outcomes, it is important to be able to distinguish SARS-CoV-2 from other CoVs in respiratory samples. Therefore, we developed and evaluated a novel quantitative reverse transcription PCR (RT-qPCR) assay, targeting the spike (S) and membrane (M) genes, to enable the rapid identification of SARS-CoV-2 including several new circulating variants, and other emerging pan-SARS-like CoVs. Using RNA extracted from cell culture supernatants, our multiple simultaneous SARS-CoV-2 assays confirmed a limit of detection of 1 × 100 TCID50/ml and no cross-reaction with human coronaviruses or other respiratory viruses. We also validated our method using human clinical samples from COVID-19 patients 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 coronaviruses, including SARS-CoV-2, and may be useful for the identification of other pan-SARS-like CoVs of zoonotic origin.

Aquatic birds are known to be a reservoir for the most common influenza A viruses (IAVs). In the annual surveillance program, we collected the feces of migratory birds for the detection of IAVs in South Korea in November 2016. A novel reassorted H3N3 avian influenza virus (AIV) containing genes from viruses of wild and domestic birds was identified and named A/aquatic bird/South Korea/sw006/2016(H3N3). The polymerase basic 2 (PB2) and non-structural (NS) genes of this isolate are most closely related to those of wild-bird-origin AIV, while the polymerase basic 1 (PB1), polymerase acidic (PA), hemagglutinin (HA), nucleoprotein (NP), neuraminidase (NA), and matrix (M) genes are most closely related to those of domestic-bird-origin AIV. A/aquatic bird/South Korea/sw006/2016 contains PA, NP, M, and NS genes were most closely related to those of AIV subtype H4 and PB2, PB1, and HA genes that are most closely related to those of AIV subtype H3N8, while the NA gene was most closely related to those of subtype H10, which was recently detected in humans in China. These results suggest that novel reassortment of AIV strains occurred due to interaction between wild and domestic birds. Hence, we emphasize the need for continued surveillance of avian influenza virus in bird populations.

Highlights • RNA adjuvant was developed from the CrPV intergenic region IRES. • The RNA adjuvant functioned as an adjuvant with protein-based vaccines. • The RNA adjuvant increased vaccine efficacy and induced balanced Th1/Th2 response. • The RNA adjuvant enhanced APC chemotaxis., An ideal adjuvant should increase vaccine efficacy through balanced Th1/Th2 responses and be safe to use. Recombinant protein-based vaccines are usually formulated with aluminum (alum)-based adjuvants to ensure an adequate immune response. However, use of alum triggers a Th2-biased immune induction, and hence is not optimal. Although the adjuvanticity of RNA has been reported, a systematic and overall investigation on its efficacy is lacking. We found that single strand RNA (termed RNA adjuvant) derived from cricket paralysis virus intergenic region internal ribosome entry site induced the expression of various adjuvant-function-related genes, such as type 1 and 2 interferon (IFN) and toll-like receptor (TLR), T cell activation, and leukocyte chemotaxis in human peripheral blood mononuclear cells; furthermore, its innate and IFN transcriptome profile patterns were similar to those of a live-attenuated yellow fever vaccine. This suggests that protein-based vaccines formulated using RNA adjuvant function as live-attenuated vaccines. Application of the RNA adjuvant in mouse enhanced the efficacy of Middle East respiratory syndrome spike protein, a protein-subunit vaccine and human papillomavirus L1 protein, a virus-like particle vaccine, by activating innate immune response through TLR7 and enhancing pAPC chemotaxis, leading to a balanced Th1/Th2 responses. Moreover, the combination of alum and the RNA adjuvant synergistically induced humoral and cellular immune responses and endowed long-term immunity. Therefore, RNA adjuvants have broad applicability and can be used with all conventional vaccines to improve vaccine efficacy qualitatively and quantitively.

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.

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.

We report the complete mitogenome of the common dolphin, Delphinus delphis. Overall structure of the 16,387 bp mitogenome was very similar to those of other delphinid species, including the ancient D. delphis individuals. Multigene phylogeny revealed that D. delphis was most closely related to Stenella coeruleoalba, and clustered well with other species within the subfamily Delphininae.

As agonists of TLR7/8, single-stranded RNAs (ssRNAs) are safe and promising adjuvants that do not cause off-target effects or innate immune overactivation. However, low stability prevents them from mounting sufficient immune responses. This study evaluates the adjuvant effects of ssRNA derived from the cricket paralysis virus intergenic region internal ribosome entry site, formulated as nanoparticles with a coordinative amphiphile, containing a zinc/dipicolylamine complex moiety as a coordinative phosphate binder, as a stabilizer for RNA-based adjuvants. The nanoformulated ssRNA adjuvant was resistant to enzymatic degradation in vitro and in vivo, and that with a coordinative amphiphile bearing an oleyl group (CA-O) was approximately 100 nm, promoted effective recognition, and improved activation of antigen-presenting cells, leading to better induction of neutralizing antibodies following single immunization. Hence, CA-O may increase the efficacy of ssRNA-based adjuvants, proving useful to meet the urgent need for vaccines during pathogen outbreaks.

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., 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.

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.

Fraser’s dolphin (Lagenodelphis hosei Fraser, 1956) is one of the least known and most recently described species in the family Delphinidae, and its complete mitogenome has yet to be determined. Herein, we report the first complete mitogenome of L. hosei. The newly obtained 16,390 bp L. hosei mitogenome showed the highest average nucleotide identity value with the Burrunan dolphin (Tursiops australis, 97.3%), and its overall structure was similar to those from other delphinid species. Multigene phylogeny using the 13 protein coding genes in the mitogenome revealed that L. hosei was well clustered with other species within the subfamily Delphininae, and the overall tree topology was congruent with recent phylogenetic studies of this species. These results provide important insights for conservation genetics and further evolutionary studies of the oceanic dolphins.