CA-CAS-01-A: A Permissive Cell Line for Isolation and Live Attenuated Vaccine Development Against African Swine Fever Virus.

African swine fever virus (ASFV) is the causative agent of the highly lethal African swine fever disease that affects domestic pigs and wild boars. In spite of the rapid spread of the virus worldwide, there is no licensed vaccine available. The lack of a suitable cell line for ASFV propagation hinders the development of a safe and effective vaccine. For ASFV propagation, primary swine macrophages and monocytes have been widely studied. However, obtaining these cells can be time-consuming and expensive, making them unsuitable for mass vaccine production. The goal of this study was to validate the suitability of novel CA-CAS-01-A (CAS-01) cells, which was identified as a highly permissive cell clone for ASFV replication in the MA-104 parental cell line for live attenuated vaccine development. Through a screening experiment, maximum ASFV replication was observed in the CAS-01 cell compared to other sub-clones of MA-104 with 14.89 and log10 7.5 ± 0.15 Ct value and TCID50/ml value respectively. When CAS-01 cells are inoculated with ASFV, replication of ASFV was confirmed by Ct value for ASFV DNA, HAD50/ml assay, TCID50/ml assay, and cytopathic effects and hemadsoption were observed similar to those in primary porcine alveolar macrophages after 5th passage. Additionally, we demonstrated stable replication and adaptation of ASFV over the serial passage. These results suggest that CAS-01 cells will be a valuable and promising cell line for ASFV isolation, replication, and development of live attenuated vaccines.

Development of optimized protocol for culturing African swine fever virus field isolates in MA104 cells.

The goal of this study was to identify a candidate commercial cell line for the replication of African swine fever virus (ASFV) by comparing several available cell lines with various medium factors. In the sensitivity test of cells, MA104 and MARC-145 had strong potential for ASFV replication. Next, MA104 cells were used to compare the adaptation of ASFV obtained from tissue homogenates and blood samples in various infectious media. At the 10th passage, the ASFV obtained from the blood sample had a significantly higher viral load than that obtained from the tissue sample (P = 0.000), exhibiting a mean cycle threshold (Ct) value = 20.39 ± 1.99 compared with 25.36 ± 2.11. For blood samples, ASFV grew on infectious medium B more robustly than on infectious medium A (P = 0.006), corresponding to a Ct value = 19.58 ± 2.10 versus 21.20 ± 1.47. African swine fever virus originating from blood specimens continued to multiply gradually and peaked in the 15th passage, exhibiting a Ct value = 14.36 ± 0.22 in infectious medium B and a Ct value = 15.42 ± 0.14 in infectious medium A. When ASFV was cultured from tissue homogenates, however, there was no difference (P = 0.062) in ASFV growth between infectious media A and B. A model was developed to enhance ASFV replication through adaptation to MA104 cells. The lack of mutation at the genetic segments encoding p72, p54, p30, and the central hypervariable region (CVR) in serial culture passages is important in increasing the probability of maintaining immunogenicity when developing a vaccine candidate.

L'objectif de cette étude était d'identifier une lignée cellulaire commerciale candidate pour la réplication du virus de la peste porcine africaine (ASFV) en comparant plusieurs lignées cellulaires disponibles et différents milieux. Lors du test de sensibilité des cellules, MA104 et MARC-145 présentaient un fort potentiel pour la réplication d'AFSV. Par la suite, les cellules MA104 ont été utilisées pour comparer l'adaptation d'ASFV obtenu d'homogénats de tissus et d'échantillons de sang dans différents milieux. Au dixième passage, l'ASFV obtenu de l'échantillon de sang avait une charge virale significativement plus élevée que celle obtenue de l'échantillon de tissu (P = 0,000), avec une valeur seuil moyenne de cycles (Ct) de 20,39 ± 1,99 comparativement à 25,36 ± 2,11. Pour les échantillons sanguins, l'ASFV a poussé sur le milieu B de manière plus robuste que sur le milieu A (P = 0,006), ce qui correspond à une valeur Ct de 19,58 ± 2,10 versus 21,20 ± 1,47. L'ASFV provenant des échantillons sanguins continua de se multiplier graduellement et atteignit un pic au 15e passage, avec une valeur Ct de 14,36 ± 0,22 dans le milieu B et une valeur Ct de 15,42 ± 0,14 dans le milieu A. Toutefois, lorsque l'ASFV fut cultivé à partir des homogénats de tissus, il n'y avait pas de différence (P = 0,062) dans la croissance d'ASFV entre les milieux A et B. Un modèle a été développé pour augmenter la réplication d'ASFV par adaptation aux cellules MA104. L'absence de mutation au segment génétique codant pour p72, p54, p30, et la région hypervariable centrale (CVR) dans des passages en série en culture est importante en augmentant la probabilité de maintenir une immunogénicité lors du développement d'un vaccin candidat.(Traduit par Docteur Serge Messier).

The Isolation and Replication of African Swine Fever Virus in Primary Renal-Derived Swine Macrophages.

African swine fever virus (ASFV) causes hemorrhagic disease in domestic pigs by replicating mainly in monocyte/macrophage lineages. Various primary cells including pulmonary alveolar macrophages have been used for the propagation of ASFV on this account. However, ethical constraints and consistency problems exist as it is necessary to harvest same phenotype of primary cells in order to continue a study. We suggested renal-derived swine macrophages as a novel primary cell candidate to address these issues. These primary cells proved to be permissive to both cell adapted ASFV and a wild-type ASFV. Compared to the commercial cell line MA-104, the renal-derived macrophages were more suitable to isolate the field virus. The consistent molecular characteristics of the renal-derived macrophages were demonstrated by immunocytochemistry with antibodies against macrophage cell surface markers including CD163, CD172a, and Iba-1. Viral protein p30 and p72 expression in ASFV infected macrophages was confirmed by immunocytochemistry by use of specific monoclonal antibodies. We observed increase of cell-free viral DNA and infectious virus titer in infected cell supernatant in successive days-post-infection. These results demonstrated that primary renal-derived swine macrophages are useful for ASFV isolation and propagation in terms of cell phenotypes, susceptibility to the virus, and virus production.

Genetic and pathogenic diversity of severe fever with thrombocytopenia syndrome virus (SFTSV) in South Korea.

To investigate nationwide severe fever with thrombocytopenia syndrome virus (SFTSV) infection status, we isolated SFTSVs from patients with suspected severe fever with thrombocytopenia syndrome (SFTS) in 207 hospitals throughout South Korea between 2013 and April 2017. A total of 116 SFTSVs were isolated from 3137 SFTS-suspected patients, with an overall 21.6% case fatality rate. Genetic characterization revealed that at least 6 genotypes of SFTSVs were co-circulating in South Korea, with multiple reassortments among them. Of these, the genotype B-2 strains were the most prevalent, followed by the A and F genotypes. Clinical and epidemiologic investigations revealed that genotype B strains were associated with the highest case fatality rate, while genotype A caused only one fatality among 10 patients. Further, ferret infection studies demonstrated varying clinical manifestations and case mortality rates with different strains of SFTSV, which suggests this virus could exhibit genotype-dependent pathogenicity.

A Novel Neuraminidase-Dependent Hemagglutinin Cleavage Mechanism Enables the Systemic Spread of an H7N6 Avian Influenza Virus.

In this study, we demonstrate a novel mechanism for hemagglutinin (HA) activation in a naturally occurring H7N6 avian influenza A virus strain, A/mallard duck/Korea/6L/2007 (A/Mdk/6L/07). This novel mechanism allows for systemic infection of chickens, ducks, and mice, and A/Mdk/6L/07 can replicate in vitro without exogenous trypsin and exhibits broad tissue tropism in animals despite the presence of a monobasic HA cleavage motif (PEIPKGR/G). The trypsin-independent growth phenotype requires the N6 neuraminidase and the specific recognition of glycine at the P2 position of the HA cleavage motif by a thrombin-like protease. Correspondingly, viral growth is significantly attenuated by the addition of a thrombin-like protease inhibitor (argatroban). These data provide evidence for a previously unrecognized virus replication mechanism and support the hypothesis that thrombin-mediated HA cleavage is an important virulence marker and potential therapeutic target for H7 influenza viruses.IMPORTANCE The identification of virulence markers in influenza viruses underpins risk assessment programs and the development of novel therapeutics. The cleavage of the influenza virus HA is a required step in the viral life cycle, and phenotypic differences in viruses can be caused by changes in this process. Here, we describe a novel mechanism for HA cleavage in an H7N6 influenza virus isolated from a mallard duck. The mechanism requires the N6 protein and full activity of thrombin-like proteases and allows the virus to cause systemic infection in chickens, ducks, and mice. The thrombin-mediated cleavage of HA is thus a novel virulence determinant of avian influenza viruses.

Rapid and simple colorimetric detection of multiple influenza viruses infecting humans using a reverse transcriptional loop-mediated isothermal amplification (RT-LAMP) diagnostic platform.

BACKGROUND: In addition to seasonal influenza viruses recently circulating in humans, avian influenza viruses (AIVs) of H5N1, H5N6 and H7N9 subtypes have also emerged and demonstrated human infection abilities with high mortality rates. Although influenza viral infections are usually diagnosed using viral isolation and serological/molecular analyses, the cost, accessibility, and availability of these methods may limit their utility in various settings. The objective of this study was to develop and optimized a multiplex detection system for most influenza viruses currently infecting humans. METHODS: We developed and optimized a multiplex detection system for most influenza viruses currently infecting humans including two type B (both Victoria lineages and Yamagata lineages), H1N1, H3N2, H5N1, H5N6, and H7N9 using Reverse Transcriptional Loop-mediated Isothermal Amplification (RT-LAMP) technology coupled with a one-pot colorimetric visualization system to facilitate direct determination of results without additional steps. We also evaluated this multiplex RT-LAMP for clinical use using a total of 135 clinical and spiked samples (91 influenza viruses and 44 other human infectious viruses). RESULTS: We achieved rapid detection of seasonal influenza viruses (H1N1, H3N2, and Type B) and avian influenza viruses (H5N1, H5N6, H5N8 and H7N9) within an hour. The assay could detect influenza viruses with high sensitivity (i.e., from 100 to 0.1 viral genome copies), comparable to conventional RT-PCR-based approaches which would typically take several hours and require expensive equipment. This assay was capable of specifically detecting each influenza virus (Type B, H1N1, H3N2, H5N1, H5N6, H5N8 and H7N9) without cross-reactivity with other subtypes of AIVs or other human infectious viruses. Furthermore, 91 clinical and spiked samples confirmed by qRT-PCR were also detected by this multiplex RT-LAMP with 98.9% agreement. It was more sensitive than one-step RT-PCR approach (92.3%). CONCLUSIONS: Results of this study suggest that our multiplex RT-LAMP assay may provide a rapid, sensitive, cost-effective, and reliable diagnostic method for identifying recent influenza viruses infecting humans, especially in locations without access to large platforms or sophisticated equipment.

Shedding and Transmission Modes of Severe Fever With Thrombocytopenia Syndrome Phlebovirus in a Ferret Model.

BACKGROUND: Although human-to-human transmission of severe fever with thrombocytopenia syndrome phlebovirus (SFTSV) via direct contact with body fluids has been reported, the role of specific body fluids from SFTSV-infected hosts has not been investigated in detail. METHODS: To demonstrate the virus transmission kinetics in SFTSV-infected hosts, we adapted the ferret infection model and evaluated the virus shedding periods, virus titers, and transmission modes from various specimens of infected ferrets. RESULTS: Large amounts of infectious SFTSV are shed through nasal discharge, saliva, and urine from SFTSV-infected ferrets. Virus could be detected from 2 dpi and persisted until 12 dpi in these specimens, compared with the relatively short virus-shedding period in sera. Further, transmission studies revealed that SFTSV can be transmitted to close direct and indirect contact naïve animals through various mediums, especially through contact with serum and urine. Further, ferrets contacted with human urine specimens from SFTSV-positive patients were successfully infected with SFTSV, suggesting that urine specimens could be a source of SFTSV infection in humans. CONCLUSIONS: Our results demonstrate that the SFTSV can be shed in various body fluids for more than 12 days and that these specimens could be a source for direct or indirect transmission through close personal contact.

Development of a SFTSV DNA vaccine that confers complete protection against lethal infection in ferrets.

Although the incidence of severe fever with thrombocytopenia syndrome virus (SFTSV) infection has increased from its discovery with a mortality rate of 10-20%, no effective vaccines are currently available. Here we describe the development of a SFTSV DNA vaccine, its immunogenicity, and its protective efficacy. Vaccine candidates induce both a neutralizing antibody response and multifunctional SFTSV-specific T cell response in mice and ferrets. When the vaccine efficacy is investigated in aged-ferrets that recapitulate fatal clinical symptoms, vaccinated ferrets are completely protected from lethal SFTSV challenge without developing any clinical signs. A serum transfer study reveals that anti-envelope antibodies play an important role in protective immunity. Our results suggest that Gn/Gc may be the most effective antigens for inducing protective immunity and non-envelope-specific T cell responses also can contribute to protection against SFTSV infection. This study provides important insights into the development of an effective vaccine, as well as corresponding immune parameters, to control SFTSV infection.

Seroprevalence of Severe Fever with Thrombocytopenia Syndrome Phlebovirus in Domesticated Deer in South Korea.

Severe fever with thrombocytopenia syndrome phlebovirus (SFTSV) has a wide host range. Not only has it been found in humans, but also in many wild and domesticated animals. The infection of breeding deer on farms is a particularly worrisome public health concern due to the large amount of human contact and the diverse use of deer products, including raw blood. To investigate the prevalence of breeding domesticated deer, we examined the SFTSV infection rate on deer farms in South Korea from 2015 to 2017. Of the 215 collected blood samples, 0.9% (2/215) were found to be positive for viral RNA by PCR, and sequence analysis showed the highest homology with the KADGH human isolate. Both SFTSV-specific recombinant N and Gn protein-based ELISAs revealed that 14.0% (30/215) and 7.9% (17/215) of collected blood specimens were positive for SFTSV antibody. These results demonstrate that the breeding farm deer are exposed to SFTSV and could be a potential infection source for humans through direct contact or consumption of byproducts.

Preclinical evaluation of the efficacy of an H5N8 vaccine candidate (IDCDC-RG43A) in mouse and ferret models for pandemic preparedness.

Because H5N1 influenza viruses continuously threaten the public health, the WHO has prepared various clades of H5N1 mock-up vaccines as one of the measures for pandemic preparedness. The recent worldwide outbreak of H5Nx virus which belongs to clade 2.3.4.4 and of which H5N6 subtype belongs and already caused human infection also increases the need of pandemic vaccine for such novel emerging viruses. In this study, we evaluated the protective efficacy and immunogenicity of an egg-based and inactivated whole-virus H5N8 (IDCDC-RG43A) developed by CDC containing HA and NA gene of the parent virus A/gyrfalcon/Washington/41088-6/2014. Mice vaccinated two times elicited low to moderate antibody titer in varying amount of antigen doses against the homologous H5N8 vaccine virus and heterologous intra-clade 2.3.4.4 H5N6 (A/Sichuan/26221/2014) virus. Mice immunized with at least 3.0 µg/dose of IDCDC-RG43A with aluminum hydroxide adjuvant were completely protected from lethal challenge with the mouse-adapted H5N8 (A/Environment/Korea/ma468/2015, maH5N8) as well as cleared the viral replication in tissues including lung, brain, spleen, and kidney. Vaccinated ferrets induced high antibody titers against clade 2.3.4.4 H5N8/H5N6 viruses and the antibody showed high cross-reactivity to clade 2.2 H5N1 but not to clade 1 and 2.3.4 viruses as measured by hemagglutinin inhibition and serum neutralization assays. Furthermore, administration of the vaccine in ferrets resulted in attenuation of clinical disease signs and virus spread to peripheral organs including lung, spleen, and kidney from high dose challenge with maH5N8 virus. The protective and immunogenic characteristic of the candidate vaccine are essential attributes to be considered for further clinical trials as a pre-pandemic vaccine for a potential pandemic virus.

Ferret animal model of severe fever with thrombocytopenia syndrome phlebovirus for human lethal infection and pathogenesis.

Severe fever with thrombocytopenia syndrome phlebovirus (SFTSV), listed in the most dangerous pathogens by the World Health Organization, has 12-30% fatality rates with a characteristic thrombocytopenia syndrome. With a majority of clinically diagnosed SFTSV patients older than ~50 years of age, age is a critical risk factor for SFTSV morbidity and mortality. Here, we report an age-dependent ferret model of SFTSV infection and pathogenesis that fully recapitulates the clinical manifestations of human infections. Whereas young adult ferrets (≤2 years of age) did not show any clinical symptoms and mortality, SFTSV-infected aged ferrets (≥4 years of age) demonstrated severe thrombocytopenia, reduced white blood cell counts and high fever with 93% mortality rate. Moreover, a significantly higher viral load was observed in aged ferrets. Transcriptome analysis of SFTSV-infected young ferrets revealed strong interferon-mediated anti-viral signalling, whereas inflammatory immune responses were markedly upregulated and persisted in aged ferrets. Thus, this immunocompetent age-dependent ferret model should be useful for anti-SFTSV therapy and vaccine development.

Simple, Rapid and Sensitive Portable Molecular Diagnosis of SFTS Virus Using Reverse Transcriptional Loop-Mediated Isothermal Amplification (RT-LAMP).

Recently, human infections caused by severe fever with thrombocytopenia syndrome virus (SFTSV), which can lead to fatality, have dramatically increased in East Asia. With the unavailability of vaccines or antiviral drugs to prevent and/or treat SFTSV infection, early rapid diagnosis is critical for prevention and control of the disease. Here, we report the development of a simple, rapid and sensitive portable detection method for SFTSV infection applying reverse transcription-loop mediated isothermal amplification (RT-LAMP) combined with one-pot colorimetric visualization and electro-free reaction platform. This method utilizes a pocket warmer to facilitate diagnosis in a resource-limited setting. Specific primers were designed to target the highly-conserved region of L gene of SFTSV. The detection limit of the RT-LAMP assay was approximately 100 viral genome copies from three different SFTSV strains. This assay exhibited comparable sensitivity to qRT-PCR and 10-fold more sensitivity than conventional RT-PCR, with a rapid detection time of 30 to 60 minutes. The RT-LAMP assay using SFTSV clinical specimens has demonstrated a similar detection rate to qRT-PCR and a higher detection rate compared to conventional RT-PCR. Moreover, there was no observed cross-reactive amplification of other human infectious viruses including Japanese Encephalitis Virus (JEV), Dengue, Enterovirus, Zika, Influenza and Middle East Respiratory Syndrome Coronavirus (MERS-CoV). This highly sensitive, electro- and equipment-free rapid colorimetric visualization method is feasible for resource-limited SFTSV field diagnosis.

Seroprevalence and genetic characterization of severe fever with thrombocytopenia syndrome virus in domestic goats in South Korea.

Severe fever with thrombocytopenia syndrome (SFTS) is a newly emerging tick-borne infectious disease caused by the SFTS virus (SFTSV). To investigate the prevalence of SFTSV in domestic goats in South Korea, we collected blood samples in commercial slaughterhouses in Chungbuk Province in 2017. Of the 207 samples tested, 4 (2%) were found to be positive for viral RNA by RT-PCR and 30 (14.4%) were positive for SFTSV antibody as detected by a nucleocapsid (NP) protein-based ELISA. Phylogenetic analysis of the non-structural protein (NS) sequences showed that all viruses belonged to the genotype B, although they were clustered into two different sublineages that showed the highest homology with the KR612076-JP01 and KY789441-CB3 human isolate from South Korea. Further, we confirmed the specificity of seropositive goat sera by FRNT50 and western blotting analysis and found differential cross-reactivity of the sera with genotype A and B SFTSV strains. Collectively, this study suggests that relatively high numbers of goats are infected by antigenically different SFTSV strains, which might have a potential for zoonotic infection.

Generation of a High-Growth Influenza Vaccine Strain in MDCK Cells for Vaccine Preparedness.

As shown during the 2009 pandemic H1N1 (A(H1N1)pdm09) outbreak, egg-based influenza vaccine production technology is insufficient to meet global demands during an influenza pandemic. Therefore, there is a need to adapt cell culture-derived vaccine technology using suspended cell lines for more rapid and larger-scale vaccine production. In this study, we attempted to generate a high-growth influenza vaccine strain in MDCK cells using an A/Puerto/8/1934 (H1N1) vaccine seed strain. Following 48 serial passages with four rounds of virus plaque purification in MDCK cells, we were able to select several MDCK-adapted plaques that could grow over 108 PFU/ml. Genetic characterization revealed that these viruses mainly had amino acid substitutions in internal genes and exhibited higher polymerase activities. By using a series of Rg viruses, we demonstrated the essential residues of each gene and identified a set of high-growth strains in MDCK cells (PB1D153N, M1A137T, and NS1N176S). In addition, we confirmed that in the context of the high-growth A/PR/8/34 backbone, A/California/7/2009 (H1N1), A/Perth/16/2009 (H3N2), and A/environment/Korea/deltaW150/2006 (H5N1) also showed significantly enhanced growth properties (more than 107 PFU/ml) in both attached- and suspended-MDCK cells compared with each representative virus and the original PR8 vaccine strain. Taken together, this study demonstrates the feasibility of a cell culture-derived approach to produce seed viruses for influenza vaccines that are cheap and can be grown promptly and vigorously as a substitute for egg-based vaccines. Thus, our results suggest that MDCK cell-based vaccine production is a feasible option for producing large-scale vaccines in case of pandemic outbreaks.

Comparison of the pathogenic potential of highly pathogenic avian influenza (HPAI) H5N6, and H5N8 viruses isolated in South Korea during the 2016-2017 winter season.

Highly pathogenic avian influenza (HPAI) A(H5N6) and A(H5N8) virus infections resulted in the culling of more than 37 million poultry in the Republic of Korea during the 2016/17 winter season. Here we characterize two representative viruses, A/Environment/Korea/W541/2016 [Em/W541(H5N6)] and A/Common Teal/Korea/W555/2017 [CT/W555(H5N8)], and evaluate their zoonotic potential in various animal models. Both Em/W541(H5N6) and CT /W555(H5N8) are novel reassortants derived from various gene pools of wild bird viruses present in migratory waterfowl arising from eastern China. Despite strong preferential binding to avian virus-type receptors, the viruses were able to grow in human respiratory tract tissues. Em/W541(H5N6) was found to be highly pathogenic in both chickens and ducks, while CT/W555(H5N8) caused lethal infections in chickens but did not induce remarkable clinical illness in ducks. In mice, both viruses appeared to be moderately pathogenic and displayed limited tissue tropism relative to HPAI H5N1 viruses. Em/W541(H5N6) replicated to moderate levels in the upper respiratory tract of ferrets and was detected in the lungs, brain, spleen, liver, and colon. Unexpectedly, two of three ferrets in direct contact with Em/W541(H5N6)-infected animals shed virus and seroconverted at 14 dpi. CT/W555(H5N8) was less pathogenic than the H5N6 virus in ferrets and no transmission was detected. Given the co-circulation of different, phenotypically distinct, subtypes of HPAI H5Nx viruses for the first time in South Korea, detailed virologic investigations are imperative given the capacity of these viruses to evolve and cause human infections.

Comparison of the virulence and transmissibility of canine H3N2 influenza viruses and characterization of their canine adaptation factors.

Recent canine influenza outbreaks have raised concerns about the generation of pathogenic variants that may pose a threat to public health. Here, we examine avian-like H3N2 canine influenza viruses (CIVs) isolated from 2009 to 2013 in South Korea from dogs. Phylogenetic analysis revealed that these viruses are closely related to strains previously isolated from dogs in Korea and China. However, molecular characterization demonstrated non-synonymous mutations between the canine viruses, particularly in the putative H3 antigenic sites, NA stalk regions, and in the internal genes of the 2012-2013 isolates compared with the 2009 isolate. Animal experiments showed that three representative isolates (A/canine/Korea/AS-01/2009(AS-01/09), A/canine/Korea/AS-05/2012(AS-05/12) and A/canine/Korea/AS-11/2013(AS-11/13), were readily droplet transmitted between dogs, whereas AS-05/12 induced more severe clinical disease and was lethal in dogs compared with AS-01/09. Although all viruses were able to infect ferrets, AS-05/12 consistently yielded higher nasal wash titers and was transmissible to ferrets via airborne droplets. Using reverse genetics, we show that the NA, NP, and M genes of CIV are critical for the adaptation of avian H3N2 viruses, and the resulting reassortant genotypes promote viral growth in dogs in a manner similar to that of the wild-type AS-01/09 virus. Taken together, these results demonstrate that CIVs continuously evolve in dogs thereby allowing them to gain a foothold in mammalian hosts. Importantly, we elucidated the genetic contributions of the NA, NP, and M genes to the adaptability of CIVs derived from the avian H3N2 virus.

Evaluation of two different enzyme-linked immunosorbent assay for severe fever with thrombocytopenia syndrome virus diagnosis.

To develop the large scale serological assay for severe fever with thrombocytopenia syndrome virus (SFTSV) infection, we evaluated two different enzyme-linked immunosorbent assay (ELISA) methods using nucleocapsid protein (NP) and Gn proteins of CB1 (genotype B) SFTSV strains. The NP-based ELISA tests showed more sensitive with broad cross-reactivity between two different genotype A and B strains compared with those of Gn-based ELISA tests. However, Gn-based ELISA showed more genotype specificity and specificity. These result suggested that NP-based ELISA test could be applicable for general sero-prevalence studies of SFTSV infections, while Gn-based ELISA could be applicable for a certain specific genotype sero-prevalence study.

Pathogenicity and genetic characterisation of a novel reassortant, highly pathogenic avian influenza (HPAI) H5N6 virus isolated in Korea, 2017.

We investigated influenza A(H5N6) viruses from migratory birds in Chungnam and Gyeonggi Provinces, South Korea following a reported die-off of poultry in nearby provinces in November 2017. Genetic analysis and virulence studies in chickens and ducks identified our isolate from December 2017 as a novel highly pathogenic avian influenza virus. It resulted from reassortment between the highly virulent H5N8 strain from Korea with the N6 gene from a low-pathogenic H3N6 virus from the Netherlands.

Altered virulence of Highly Pathogenic Avian Influenza (HPAI) H5N8 reassortant viruses in mammalian models.

Recently identified highly pathogenic avian influenza (HPAI) H5N8 viruses (clade 2.3.4.4) are relatively low to moderately pathogenic in mammalian hosts compared with HPAI H5N1 viruses. In this study, we generated reassortant viruses comprised of A/MD/Korea/W452/2014(H5N8) with substitution of individual genes from A/EM/Korea/W149/2006(H5N1) to understand the contribution of each viral gene to virulence in mammals. Substituting the PB2 gene segment or the NA gene segment of the H5N8 virus by that from the H5N1 virus resulted in significantly enhanced pathogenicity compared with the parental H5N8 virus in mice. Of note, substitution of the PB2 gene segment of the H5N8 virus by that from the H5N1 virus resulted in a 1000-fold increase in virulence for mice compared with the parental virus (MLD50 decreased from 105.8 to 102.5 EID50). Further, the W452W149PB2 virus also induced the highest virus titers in lungs at all time points and the highest levels of inflammatory cytokine responses among all viruses tested. This high virulence phenotype was also confirmed by high viral titers in the respiratory tracts of infected ferrets. Further, a mini-genome assay revealed that W452W149PB2 has significantly increased polymerase activity (p < 0.001). Taken together, our study demonstrates that a single gene substitution from other avian influenza viruses can alter the pathogenicity of recent H5N8 viruses, and therefore emphasizes the need for intensive monitoring of reassortment events among co-circulating avian and mammalian viruses.