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.

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.

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.

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.

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 sequence of an avian-origin H3N2 canine influenza virus isolated from dogs in South Korea.

An avian-origin Korean H3N2 canine influenza virus (CIV) strain, designated A/canine/Korea/01/2007 (H3N2), was isolated from nasal swabs of pet dogs exhibiting severe respiratory syndrome in 2007. In the present study, we report the first complete genome sequence containing 3' and 5' noncoding regions (NCRs) of H3N2 CIV, which will provide important insights into the molecular basis of pathogenesis, transmission, and evolution of CIV.

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).

A novel reassortant canine H3N1 influenza virus between pandemic H1N1 and canine H3N2 influenza viruses in Korea.

During recent canine influenza surveillance in South Korea, a novel H3N1 canine influenza virus (CIV) that is a putative reassortant between pandemic H1N1 2009 and H3N2 CIVs was isolated. Genetic analysis of eight genes of the influenza virus revealed that the novel H3N1 isolate presented high similarities (99.1-99.9 %) to pandemic influenza H1N1, except for in the haemagglutinin (HA) gene. The HA gene nucleotide sequence of the novel CIV H3N1 was similar (99.6 %) to that of CIV H3N2 isolated in Korea and China. Dogs infected with the novel H3N1 CIV did not show any notable symptoms, in contrast to dogs infected with H3N2 CIV. Despite no visible clinical signs of disease, nasal shedding of virus was detected and the infected dogs presented mild histopathological changes.

Association between nasal shedding and fever that influenza A (H3N2) induces in dogs.

BACKGROUND: Avian origin canine influenza virus was reported in Korea. The dog to dog contact transmission of the avian origin canine influenza virus (CIV) H3N2 and CIV H3N8 was shown by experimental contact transmission. This study was focused on viral excretion and fever in order to elucidate the epidemiological associations which might be helpful to control the disease transmissions in CIV outbreak in dogs. METHODS: An influenza seronegative 10-week-old Beagle dog was experimentally inoculated with the canine influenza virus A/canine/01/2007, subtype H3N2. Eight hours after inoculation, the infected dog was cohoused with seven uninfected Beagle dogs. Clinical signs including fever were recorded for 14 days post inoculation. RESULTS: The infected dog and four of seven contact dogs in the study showed clinical signs (sneezing, nasal discharge and coughing) during the study. Viral shedding occurred in all of the animals tested and began on 1 to 6 DPI in dogs with clinical signs. Elevated body temperatures above 39.5 °C (geometric mean temperature of 39.86 °C ± 0.49) were observed in all symptomatic dogs. The mean viral titer during fever was 2.99 log EID50/ml, which was significantly higher than the viral titer detected in the non fever. CONCLUSIONS: The data show that contact dogs with a canine influenza infected dog shed different levels of virus in their nasal excretions and demonstrate that clinical signs, including fever, significantly correlate with the viral shedding.

Molecular characterization and phylogenetic analysis of porcine epidemic diarrhea virus (PEDV) field isolates in Korea.

Porcine epidemic diarrhea virus (PEDV) has caused enteric disease with devastating impact since the first identification of PEDV in 1992 in Korea. In this study, we investigated molecular epidemiology, showed genetic diversity, and analyzed phylogenetic relationships of Korean PEDV field isolates with other PEDV reference strains. Genetic analysis of the complete M and ORF3 genes showed that each PEDV group had several unique characteristics, and this indicated that specific groups of PEDVs may be differentiated from the other PEDVs by specific nucleotide differences. Especially, ORF3 gene analysis can be used for discrimination between vaccine and wild-type PEDVs. Sequence and phylogenetic analysis showed that recent, prevalent Korean PEDV field isolates have close relationships to Chinese field strains and differ genetically from European strains and vaccine strains used in Korea. These results raise questions as to whether a new type of PEDV vaccine may be necessary for preventing PEDV infection more effectively in Korea.

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.

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.

Molecular detection of porcine kobuviruses in pigs in Korea and their association with diarrhea.

Kobuviruses are small, non-enveloped viruses with a single-stranded, positive-sense genomic RNA, belonging to the family Picornaviridae, a highly diverse family of important pathogens of human and other animals. Porcine kobuvirus has been found recently, and consequently, information about the virus is lacking. In this study, we identified porcine kobuviruses from pigs in Korea by RT-PCR, cloning and sequencing, and we showed the existence of genetic diversity among geographically separated porcine kobuviruses through genetic and phylogenetic analysis. Epidemiological studies of porcine kobuvirus linked to diarrhea indicated that porcine kobuvirus infections are endemic in diarrheic pigs in Korea. Statistical analysis of the porcine kobuvirus positive rate between diarrheic and healthy pigs as well as a survey for other enteric pathogens in diarrheic pigs suggests that porcine kobuvirus may play a role as a causative agent of gastroenteritis in pigs.

Prevalence and phylogenetic analysis of the isolated type I porcine reproductive and respiratory syndrome virus from 2007 to 2008 in Korea.

The first Korean strain of porcine reproductive and respiratory syndrome virus (PRRSV) was isolated in 1997, and it exhibited high similarity to strain VR-2332 (type II PRRSV; North American type). Recently, however, infection with type I PRRSV (European type) has also been reported in Korea. To date, preliminary data about type I PRRSV prevalence in Korea have not been reported. Here, using reverse transcriptase (RT)-PCR, we analyzed 383 archived field samples from 101 pig farms in Korea that were collected from 2007 to 2008. We identified 155 samples from 68 farms that were positive for PRRSV. Fifty-one samples (51/155; 32.9%) and 20 farms (20/68; 29.4%) were type I PRRSV-positive/type II PRRSV-negative. Furthermore, we tried to isolate the type I PRRSV from positive samples and seven type I PRRSV were isolated using PAM. The phylogenetic analysis using the type I PRRSV isolates (7 isolates) was performed based on open reading frame (ORF)5 (accession numbers GU325642 to GU325648) and ORF7 (accession numbers GU325635 to GU325641). In the phylogenetic study, seven type I PRRSV isolates were closely related with panEuropean based on ORF7, while they were genetically distinct from Lelystad virus and made a unique clade based on ORF5. The results of this study demonstrate that infection with type I PRRSV is not uncommon in Korean pig farms, which suggests that diagnosis and control of type I PRRSV should be considered in Korea. A new approach to vaccination against, and epidemiological analysis of, Korean PRRSV is urgently needed.

Porcine noroviruses and sapoviruses on Korean swine farms.

Porcine noroviruses (NoVs) and sapoviruses (SaVs), which belong to the family Caliciviridae, have been considered potential zoonotic agents for human infection, and several cases have been reported in Asian countries. In this study, a total of 537 porcine fecal samples collected from 64 swine farms in Korea were tested. Among 537 samples, porcine NoVs were detected by semi-nested RT-PCR in ten samples (1.9%), and porcine SaVs were detected by RT-PCR in 60 samples (11.2%), showing their circulation in Korea. The porcine NoVs were genetically related to strains of genotypes 11 and 18, of genogroup II (GII) of the genus Norovirus. The porcine SaV strains were genetically related to the porcine enteric calicivirus Cowden strain and to the previously identified Korean porcine strains in genogroup III (GIII) of the genus Sapovirus. In no case was co-infection with both NoV and SaV observed in one pig. This is the first report describing porcine NoVs identified in Korea.

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).

Multiplex PCR for the simultaneous detection of pseudorabies virus, porcine cytomegalovirus, and porcine circovirus in pigs.

The use of porcine organs is being developed as a means to alleviate the shortage of human organs for transplantation. Recommendations have been published for the microbiological specifications of organ-source pigs to reduce the possibility of a microorganism from pigs being inadvertently transferred to the recipient of the xenograft. The pseudorabies virus (PRV), porcine cytomegalovirus (PCMV), and porcine circovirus (PCV) are infectious agents in pigs that are considered to be of significance for the microbiological safety of xenotransplantation. A multiplex polymerase chain reaction (mPCR) was developed to detect and differentiate among PRV, PCMV, and PCV. The sensitivities of the multiplex PCR were 10(2.5) TCID(50)/ml for PRV, 10(1.8) TCID(50)/ml for PCMV, and 10(1.8) TCID(50)/ml for PCV. The lowest viral concentrations detected by single PCR were 10(1.5) TCID(50)/ml for PRV, 10(1.0) TCID(50)/ml for PCMV, and 10(1.4) TCID(50)/ml for PCV2. Non-specific reactions were not observed when other viruses, bacteria, and Vero cells were used to assess the multiplex PCR. The multiplex PCR was effective in detecting various combinations of one or more of these viruses in pig specimens collected for xenotransplantation.

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.