Characteristics and Pathogenicity of the Cell-Adapted Attenuated Porcine Epidemic Diarrhea Virus of the Non-S INDEL Cluster.

The high antigenic diversity of porcine epidemic diarrhea virus (PEDV) means that porcine epidemic diarrhea (PED) is a challenge for the global pig industry. Understanding the circulation of the virus to determine an optimal vaccine strategy is important in controlling the disease. In this study, we describe the genetic diversity of circulating PEDV based on the full sequences of spike genes of eight positive samples collected in Vietnam since 2018. Additionally, we developed a live attenuated vaccine candidate from the cell-adapted PEDV2 strain, which was continuously passaged until level 103 in VERO-CCL81 cells. PEDV2-p103, which belongs to the emerging non-S INDEL cluster, exhibited low virus shedding, did not induce lesions in the small intestine of challenged piglets, and had a high titer in the VERO-CCL81 cell at 48 h post-infection. These results suggest that the PEDV2-p103 strain could be a potential oral attenuated vaccine, and its immunogenicity and efficacy should be further assessed through in vivo tests.

A comprehensive review of SARS-CoV-2 genetic mutations and lessons from animal coronavirus recombination in one health perspective.

SARS-CoV-2 was originated from zoonotic coronaviruses and confirmed as a novel beta-coronavirus, which causes serious respiratory illness such as pneumonia and lung failure, COVID-19. In this review, we describe the genetic characteristics of SARS-CoV-2, including types of mutation, and molecular epidemiology, highlighting its key difference from animal coronaviruses. We further summarized the current knowledge on clinical, genetic, and pathological features of several animal coronaviruses and compared them with SARS-CoV-2, as well as recent evidences of interspecies transmission and recombination of animal coronaviruses to provide a better understanding of SARS-CoV-2 infection in One Health perspectives. We also discuss the potential wildlife hosts and zoonotic origin of this emerging virus in detail, that may help mitigate the spread and damages caused by the disease.

Molecular characterization of a Korean porcine epidemic diarrhea virus strain NB1.

In Korea, for the past 30 years (1987-present), porcine epidemic diarrhea (PED) has been established as an endemic situation in which multiple genogroups of classical G1 and G2b, and the recently introduced pandemic G2a, coexisted. Because of the dynamic nature of the virus, continuous field monitoring for PEDV strains is required. This study is the first to reveal prevalence of PEDV in 9 sampling provinces, with an overall detection rate of 6.70%. Porcine endemic diarrhea virus (PEDV) was present in pigs of all ages, especially in the non-PED vaccinated groups. The highest detection rate was in the finisher group (2.34%), followed by that in the newborn group (1.56%). Secondly, using Sanger sequencing, this study recovered a complete genome (28 005 nucleotides long) of NB1 strain from a farm severely affected by PED. Analyses of nucleotide and deduced amino acid sequences showed that NB1 differed from 18 other Korean PEDV mostly in 4 protein coding genes: ORF1a, ORF1b, S, and N. Two amino acid substitutions (V635E and Y681Q) in the COE and S1D neutralizing epitopes of NB1 resulted in antigenic index alteration of the adjacent sites, one of which contributed to a mutation that escaped neutralizing antibodies.

En Corée, pour les 30 dernières années (1987 à ce jour), la diarrhée épidémique porcine (DEP) s'est établie comme une situation endémique dans laquelle de multiples génogroupes des classiques G1 et G2b, ainsi que le G2a pandémique récemment introduit, ont coexisté. Étant donné la nature dynamique du virus, un suivi continu sur le terrain des souches de DEP est requis. La présente étude est la première à révéler la prévalence de DEP dans neuf provinces échantillonnées, avec un taux de détection global de 6,70 %. Le virus de la DEP (VDEP) était présent chez les porcs de tout âge, spécialement dans les groupes d'animaux non-vaccinés contre la DEP. Les animaux dans le groupe en finition avaient taux de détection le plus élevé (2,34 %), suivi par ceux du groupe des nouveau-nés (1,56 %). Deuxièmement, en utilisant le séquençage de Sanger, nous avons récupéré un génome complet (28 005 nucléotides de long) de la souche NB1 sur une ferme sévèrement affectée par la DEP. L'analyse des nucléotides et des séquences d'acides aminés déduites a montré que NB1 différaient de 18 autres VDEP coréens principalement dans quatre gènes codant pour protéines: ORF1a, ORF1b, S, et N. Deux substitutions d'acides aminés (V635E et Y681Q) dans les épitopes neutralisants COE et S1D de NB1 ont résulté en une altération de l'index antigénique des sites adjacents, dont l'un contribuait à une mutation qui échappait aux anticorps neutralisants.(Traduit par Docteur Serge Messier).

Comparison of the virulence of three H3N2 canine influenza virus isolates from Korea and China in mouse and Guinea pig models.

BACKGROUND: Avian-origin H3N2 canine influenza virus (CIV) has been the most common subtype in Korea and China since 2007. Here, we compared the pathogenicity and transmissibility of three H3N2 CIV strains [Chinese CIV (JS/10), Korean CIV (KR/07), and Korean recombinant CIV between the classic H3N2 CIV and the pandemic H1N1 virus (MV/12)] in BALB/c mouse and guinea pig models. The pandemic H1N1 (CA/09) strain served as the control. RESULTS: BALB/c mice infected with H1N1 had high mortality and obvious body weight loss, whereas no overt disease symptoms were observed in mice inoculated with H3N2 CIV strains. The viral titers were higher in the group MV/12 than those in groups JS/10 and KR/07, while the mice infected with JS/10 showed higher viral titers in all tissues (except for the lung) than the mice infected with KR/07. The data obtained in guinea pigs also demonstrated that group MV/12 presented the highest loads in most of the tissues, followed by group JS/10 and KR/07. Also, direct contact transmissions of all the three CIV strains could be observed in guinea pigs, and for the inoculated and the contact groups, the viral titer of group MV/12 and KR/07 was higher than that of group JS/10 in nasal swabs. These findings indicated that the matrix (M) gene obtained from the pandemic H1N1 may enhance viral replication of classic H3N2 CIV; JS/10 has stronger viral replication ability in tissues as compared to KR/07, whereas KR/07 infected guinea pigs have more viral shedding than JS/10 infected guinea pigs. CONCLUSIONS: There exists a discrepancy in pathobiology among CIV isolates. Reverse genetics regarding the genomes of CIV isolates will be helpful to further explain the virus characteristics.

Stability of virus-like particles of red-spotted grouper nervous necrosis virus in the aqueous state, and the vaccine potential of lyophilized particles.

Virus-like particles (VLPs) are multi protein complexes mimicking the structural properties of the native virus. The development of freeze-dried formulations of such complex protein structures remains a challenge. Red-spotted grouper nervous necrosis virus (RGNNV) causes mass mortality in fish culture, and RGNNV VLPs have been suggested to be promising vaccine candidates. In the present study, the stability of RGNNV VLPs in the liquid state was investigated over a 4-week period, along with the influence of freeze-drying on VLP stability. RGNNV VLPs were completely degraded after one week at 37 °C followed by 3 weeks at ambient temperature, and they were partially degraded after 4 weeks at 4 °C. Therefore, the inherent stability of RGNNV VLP in an aqueous milieu is insufficient for long-term storage. When RGNNV VLPs were freeze-dried in the presence or absence of sugar stabilizers, sorbitol was found to improve VLP stability whereas mannitol reduced it. VLP preparations freeze-dried with sorbitol or without stabilizer were as immunogenic as control (non-freeze dried) VLPs, whereas VLPs freeze-dried in mannitol were less immunogenic. These results indicate that freeze-dried RGNNV VLPs have potential as vaccines.

Oral vaccination through voluntary consumption of the convict grouper Epinephelus septemfasciatus with yeast producing the capsid protein of red-spotted grouper nervous necrosis virus.

Nervous necrosis viruses (NNV) cause serious economic losses in marine fish cultivation. The red-spotted grouper NNV (RGNNV) is the most common species of NNV worldwide. There have been many efforts to develop prophylactic NNV vaccines, and various types of vaccine candidate have been suggested. However, most were designed as injectable vaccines, which are not suitable for large-scale vaccination and cause too much stress to the fish. Oral vaccination through voluntary feeding is an ideal way to provide protective immunity to fish. In the present study, recombinant Saccharomyces cerevisiae producing RGNNV capsid protein was used as oral vaccine. The recombinant yeast was prepared in freeze-dried form after disruption. Convict groupers were divided into three groups, control, and oral and parenteral vaccination groups, each consisting of 700 fishes. The control group received no treatment, the parenteral group received one intraperitoneal injection of RGNNV virus-like particles, and the oral vaccination group consumed feed containing the lysed recombinant yeast; voluntary intake was allowed four times at one-week intervals. Both vaccination groups produced serum RGNNV neutralizing antibody titers of >103 (log 2, 9.96), sustained for at least 95days post-immunization. In addition, in response to challenge with RGNNV both groups suffered significantly reduced mortality and had reduced brain RGNNV titers. These results indicate that recombinant yeast-based oral fish vaccines have great potential for large-scale vaccination.

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.

Influenza virus vaccine for neglected hosts: horses and dogs.

This study provides information regarding vaccine research and the epidemiology of influenza virus in neglected hosts (horses and dogs). Equine influenza virus (EIV) causes a highly contagious disease in horses and other equids, and outbreaks have occurred worldwide. EIV has resulted in costly damage to the horse industry and has the ability of cross the host species barrier from horses to dogs. Canine influenza is a virus of equine or avian origin and infects companion animals that live in close contact with humans; this results in possible exposure to the seasonal epizootic influenza virus. There have been case reports of genetic reassortment between human and canine influenza viruses, which results in high virulence and the ability of transmission to ferrets. This emphasizes the need for vaccine research on neglected hosts to update knowledge on current strains and to advance technology for controlling influenza outbreaks for public health.

New emergence pattern with variant porcine epidemic diarrhea viruses, South Korea, 2012-2015.

Since outbreaks of porcine epidemic diarrhea virus (PEDV) in the United States in 2013, explosive outbreaks of PED in South Korea have infected all age groups of pigs in 2014-2015year. This study analyzed a large collection of the Spike protein coding gene to infer the spatial-temporal diffusion history of PEDV. The studying results suggested that PEDVs in Korea belonged to different genogroups. While classical G1 was continuingly circulating between provinces of Korea, the pandemic G2a were recently introduced from China and USA. By the application of Bayesian phylogeographical analysis, this study demonstrated the spatial-temporal transmission of PEDVs within Korea. Of the recent emerged G2a viruses, J3142 strains showed potential recombination breakpoint (376-2,143nt) of S1 gene between KNU1303_Korea strain_G2a (KJ451046) and 45RWVCF0712_Thailand strain_G2b (KF724935). The pandemic G2a virus was partial neutralized by the antibodies invoked by the G1- based PED vaccine virus.

Genetic Characterization of an Ancestral Strain of the Avian-Origin H3N2 Canine Influenza Virus Currently Circulating in East Asia.

H3N2 canine influenza virus emerged in South Korea in 2007 and subsequently spread to China and Thailand, causing epidemic or endemic respiratory diseases in dogs. Through intermammalian species transmission, the virus has also infected cats. However, no direct evidence of significant genetic evolution has been reported since its first emergence. Here, we describe in depth the genetic and molecular characteristics of the ancestral strain (i.e., the first virus isolate from South Korea) of the H3N2 canine influenza virus currently circulating in East Asia.

Comparison of the antigenic relationship between Japanese encephalitis virus genotypes 1 and 3.

PURPOSE: The Japanese encephalitis virus (JEV) genotype circulating in Korea has changed from G3 to G1. Therefore, the purpose of this study was to compare the antigenic relationship between the two genotypes by using antibody tests. MATERIALS AND METHODS: Blood samples from 42 sows and 216 horses were collected, and their seroprevalence was monitored using the hemagglutination inhibition and virus neutralization tests. Antisera against JEV G1 and G3 were isolated and prepared from guinea pigs. The cross-reactivity of these two viruses was then compared using the neutralizing antibody test. RESULTS: We found that there was a difference in the seropositive ratios of JEV G1 and G3. However, the difference was dependent on the antibody test used. There was also an observed difference in the antigenicity between the two genotypes, as ascertained using the neutralizing antibody test. CONCLUSION: There is an evident difference in JEV antigenicity between the genotypes G1 and G3. Therefore, we propose monitoring of the seroprevalence of JEV, and reevaluating the antigenicity of the current vaccine by using the relevant tests.

Viral dominance of reassortants between canine influenza H3N2 and pandemic (2009) H1N1 viruses from a naturally co-infected dog.

BACKGROUND: Since avian-origin H3N2 canine influenza virus (CIV) was first identified in South Korea in 2008, the novel influenza virus has been reported in several countries in Asia. Reverse zoonotic transmission of pandemic H1N1 (2009) influenza virus (pH1N1) has been observed in a broad range of animal species. Viral dominance and characterization of the reassortants of both viruses was undertaken in the present study. FINDINGS: Here we describe the viral dominance of 23 CIV reassortants between pH1N1 and canine H3N2 influenza viruses from a naturally co-infected dog. These results indicate that the M gene of pandemic H1N1 and the HA gene of canine H3N2 are predominant in the reassortants. Furthermore, unlike the original canine H3N2 virus, some reassortants showed high pathogenicity in mice. CONCLUSIONS: This study suggests that continuous monitoring of influenza infection in companion animals may be necessary to investigate the potential of the emergence of novel influenza viruses.

Porcine epidemic diarrhea: a review of current epidemiology and available vaccines.

Porcine epidemic diarrhea virus (PEDV), an Alphacoronavirus in the family Coronaviridae, causes acute diarrhea, vomiting, dehydration, and high mortality rates in neonatal piglets. PEDV can also cause diarrhea, agalactia, and abnormal reproductive cycles in pregnant sows. Although PEDV was first identified in Europe, it has resulted in significant economic losses in many Asian swine-raising countries, including Korea, China, Japan, Vietnam, and the Philippines. However, from April 2013 to the present, major outbreaks of PEDV have been reported in the United States, Canada, and Mexico. Moreover, intercontinental transmission of PEDV has increased mortality rates in seronegative neonatal piglets, resulting in 10% loss of the US pig population. The emergence and re-emergence of PEDV indicates that the virus is able to evade current vaccine strategies. Continuous emergence of multiple mutant strains from several regions has aggravated porcine epidemic diarrhea endemic conditions and highlighted the need for new vaccines based on the current circulating PEDV. Epidemic PEDV strains tend to be more pathogenic and cause increased death in pigs, thereby causing substantial financial losses for swine producers. In this review, we described the epidemiology of PEDV in several countries and present molecular characterization of current strains. We also discuss PEDV vaccines and related issues.

Canine susceptibility to human influenza viruses (A/pdm 09H1N1, A/H3N2 and B).

We investigated the infectivity and transmissibility of the human seasonal H3N2, pandemic (pdm) H1N1 (2009) and B influenza viruses in dogs. Dogs inoculated with human seasonal H3N2 and pdm H1N1 influenza viruses exhibited nasal shedding and were seroconverted against the viruses; this did not occur in the influenza B virus-inoculated dogs. Transmission of human H3N2 virus between dogs was demonstrated by observing nasal shedding and seroconversion in naïve dogs after contact with inoculated dogs. The seroprevalence study offered evidence of human H3N2 infection occurring in dogs since 2008. Furthermore, serological evidence of pdm H1N1 influenza virus infection alone and in combination with canine H3N2 virus was found in the serum samples collected from field dogs during 2010 and 2011. Our results suggest that dogs may be hosts for human seasonal H3N2 and pdm H1N1 influenza viruses.

Inefficient transmissibility of NS-truncated H3N8 equine influenza virus in dogs.

H3N8 equine influenza virus (EIV) causes respiratory diseases in the horse population, and it has been demonstrated that EIV can transmit into dogs owing to its availability on receptors of canine respiratory epithelial cells. Recently, we isolated H3N8 EIV from an EIV-vaccinated horse that showed symptoms of respiratory disease, and which has a partially truncated nonstructural gene (NS). However, it is not clear that the NS-truncated EIV has an ability to cross the host species barrier from horses to dogs as well. Here, we experimentally infected the NS-truncated H3N8 EIV into dogs, and monitored their clinical signs and viral load in respiratory organs to determine the virus's transmissibility.

Comparative analysis of virulence of a novel, avian-origin H3N2 canine influenza virus in various host species.

A novel avian-origin H3N2 canine influenza A virus (CIV) that showed high sequence similarities in hemagglutinin and neuraminidase genes with those of non-pathogenic avian influenza viruses was isolated in our routine surveillance program in South Korea. We previously reported that the pathogenicity of this strain could be reproduced in dogs and cats. In the present study, the host tropism of H3N2 CIV was examined by experimental inoculation into several host species, including chickens, pigs, mice, guinea pigs, and ferrets. The CIV infection resulted in no overt symptoms of disease in these host species. However, sero-conversion, virus shedding, and gross and histopathologic lung lesions were observed in guinea pig and ferrets but not in pigs, or mice. Based on the genetic similarity of our H3N2 CIV with currently circulating avian influenza viruses and the presence of α-2,3-linked rather than α-2,6-linked sialic acid receptors in the respiratory tract of dogs, we believed that this strain of CIV would have avian virus-like receptor specificity, but that seems to be contrary to our findings in the present study. Further studies are needed to determine the co-receptors of hemagglutinin or post-attachment factors related to virus internalization or pathogenesis in other animals.

Complete Genome Sequences of H3N2 Canine Influenza Virus with the Matrix Gene from the Pandemic A/H1N1 Virus.

We analyzed the complete genome sequence containing the 3' and 5' noncoding regions (NCRs) of H3N2 canine influenza virus (CIV) with the matrix gene from the pandemic A/H1N1 virus, which will provide a better understanding of the pathogenesis, transmission, and evolution of variant CIV.

Preliminary study about sublingual administration of bacteria-expressed pandemic H1N1 influenza vaccine in miniature pigs.

Sublingual (SL) administration of influenza vaccine would be non-invasive and effective way to give human populations protective immunity against the virus, especially when pandemic influenza outbreaks. In this study, the efficacy of pandemic influenza virus-based subunit vaccines was tested after sublingual (SL) adjuvant administration in pigs. Eight specific pathogen-free Yucatan pigs were divided into 4 groups: nonvaccinated but challenged (A) and vaccinated and challenged (B, C, and D). The vaccinated groups were subdivided by vaccine type and inoculation route: SL subunit vaccine (hemagglutinin antigen 1 [HA1] + wild-type cholera toxin [wtCT], B); IM subunit vaccine (HA1 + aluminum hydroxide, C); and IM inactivated vaccine (+ aluminum hydroxide, D). The vaccines were administered twice at a 2-week interval. All pigs were challenged with pandemic influenza virus (A/swine/GCVP-KS01/2009 [H1N1]) and monitored for clinical signs, serology, viral shedding, and histopathology. After vaccination, hemagglutination inhibition titre was higher in group D (320) than in the other vaccinated groups (40-80) at the time of challenge. The mobility and feed intake were reduced in group C. Both viral shedding and histopathological lesions were reduced in groups B and D. Although this study has limitation due to the limited number of pigs (2 pigs per a group), the preliminary data in this study provided the protective potential of SL administration of bacteria-expressed pandemic H1N1 influenza vaccine in pigs. There should be additional animal studies about effective adjuvant system and vaccine types for the use of SL influenza vaccination.

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.