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).

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.

Development of recombinant Yarrowia lipolytica producing virus-like particles of a fish nervous necrosis virus.

Nervous necrosis virus (NNV) causes viral encephalopathy and retinopathy, a devastating disease of many species of cultured marine fish worldwide. In this study, we used the dimorphic non-pathogenic yeast Yarrowia lipolytica as a host to express the capsid protein of red-spotted grouper nervous necrosis virus (RGNNV-CP) and evaluated its potential as a platform for vaccine production. An initial attempt was made to express the codon-optimized synthetic genes encoding intact and N-terminal truncated forms of RGNNV-CP under the strong constitutive TEF1 promoter using autonomously replicating sequence (ARS)-based vectors. The full-length recombinant capsid proteins expressed in Y. lipolytica were detected not only as monomers and but also as trimers, which is a basic unit for formation of NNV virus-like particles (VLPs). Oral immunization of mice with whole recombinant Y. lipolytica harboring the ARS-based plasmids was shown to efficiently induce the formation of IgG against RGNNV-CP. To increase the number of integrated copies of the RGNNV-CP expression cassette, a set of 26S ribosomal DNA-based multiple integrative vectors was constructed in combination with a series of defective Ylura3 with truncated promoters as selection markers, resulting in integrants harboring up to eight copies of the RGNNV-CP cassette. Sucrose gradient centrifugation and transmission electron microscopy of this high-copy integrant were carried out to confirm the expression of RGNNV-CPs as VLPs. This is the first report on efficient expression of viral capsid proteins as VLPs in Y. lipolytica, demonstrating high potential for the Y. lipolytica expression system as a platform for recombinant vaccine production based on VLPs.

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.

Genetic Characteristics and Immunogenicity of Pandemic H1N1 Influenza Virus Isolate from Pig in Korea.

A pandemic influenza A (H1N1) virus strain was isolated from a pig farm in Korea in December 2009. The strain was propagated in and isolated from both the Madin-Darby canine kidney cell line and embryonated eggs. The partial and complete sequences of the strain were identical to those of A/California/04/2009, with >99% sequence similarity in the HA, NA, M, NS, NP, PA, PB1, and PB2 genes. The isolated strain was inactivated and used to prepare a swine influenza vaccine. This trial vaccine, containing the new isolate that has high sequence similarity with the pandemic influenza A (H1N1) virus, resulted in seroconversion in Guinea pigs and piglets. This strain could therefore be a potential vaccine candidate for swine influenza control in commercial farms.

A new set of rDNA-NTS-based multiple integrative cassettes for the development of antibiotic-marker-free recombinant yeasts.

The traditional yeast Saccharomyces cerevisiae has been widely used as a host system to produce recombinant proteins and metabolites of great commercial value. To engineer recombinant yeast that stably maintains expression cassettes without an antibiotic resistance gene, we developed new multiple integration cassettes by exploiting the non-transcribed spacer (NTS) of ribosomal DNA (rDNA) in combination with defective selection markers. The 5' and 3'-fragments of rDNA-NTS2 were used as flanking sequences for the expression cassettes carrying a set of URA3, LEU2, HIS3, and TRP1 selection markers with truncated promoters of different lengths. The integration numbers of NTS-based expression cassettes, ranging from one to ∼30 copies, showed a proportional increase with the extent of decreased expression of the auxotrophic markers. The NTS-based cassettes were used to construct yeast strains expressing the capsid protein of red-spotted grouper necrosis virus (RG-NNVCP) in a copy number-dependent manner. Oral administration of the recombinant yeast, harboring ∼30 copies of the integrated RG-NNVCP cassettes, provoked efficient immune responses in mice. In contrast, for the NTS cassettes expressing a truncated 3-hydroxyl-3-methylglutaryl-CoA reductase, the integrant carrying only 4 copies was screened as the highest producer of squalene, showing a 150-fold increase compared to that of the wild-type strain. The multiple integrated cassettes were stably retained under prolonged nonselective conditions. Altogether, our results strongly support that rDNA-NTS integrative cassettes are useful tools to construct recombinant yeasts carrying optimal copies of a desired expression cassette without an antibiotic marker gene, which are suitable as oral vaccines or feed additives for animal and human consumption.

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.

Efficacy of a combined inactivated porcine reproductive and respiratory syndrome virus vaccine using North American and European strains in specific pathogen free pigs.

In Korea, porcine reproductive and respiratory syndrome (PRRS) is caused by European (type 1) and North American (type 2) strains of PRRS virus (PRRSV). In the present study, the efficacy of a multi-strain PRRSV vaccine inactivated with binary ethylenimine (BEI) was evaluated in pigs. The vaccine contained one type 1 strain (GCEU0907) and two type 2 strains (GC4019 and GC6262). Three vaccinated groups (four pigs per group) and three mock vaccinated groups (four pigs per group) were challenged with infectious PRRSV (strains GC4019, GC6262 or GCEU0907), then euthanased at 28 days post-infection. Mean anti-PRRSV neutralising antibody titres were significantly higher in the vaccinated groups than in the mock vaccinated groups. Mean blood virus titres in the mock vaccinated groups were significantly higher than those in the vaccinated groups from 5 to 28 days post-infection. On pathological examination, there were less severe macroscopic and microscopic lesions in vaccinated pigs compared with mock vaccinated pigs.

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.

Comparative pathology of pigs infected with Korean H1N1, H1N2, or H3N2 swine influenza A viruses.

BACKGROUND: The predominant subtypes of swine influenza A virus (SIV) in Korea swine population are H1N1, H1N2, and H3N2. The viruses are genetically close to the classical U.S. H1N1 and triple-reassortant H1N2 and H3N2 viruses, respectively. Comparative pathogenesis caused by Korean H1N1, H1N2, and H3N2 SIV was evaluated in this study. FINDINGS: The H3N2 infected pigs had severe scores of gross and histopathological lesions at post-inoculation days (PID) 2, and this then progressively decreased. Both the H1N1 and H1N2 infected pigs lacked gross lesions at PID 2, but they showed moderate to severe pneumonia on PID 4, 7 and 14. The pigs infected with H1N1 had significant scores of gross and histopathological lesions when compared with the other pigs infected with H1N2, H3N2, and mock at PID 14. Mean SIV antigen-positive scores were rarely detected for pigs infected with H1N2 and H3N2 from PID 7, whereas a significantly increased amount of viral antigens were found in the bronchioles and alveolar epithelium of the H1N1infected pigs at PID 14. CONCLUSIONS: We demonstrated that Korean SIV subtypes had different pulmonary pathologic patterns. The Korean H3N2 rapidly induced acute lung lesions such as broncho-interstitial pneumonia, while the Korean H1N1 showed longer course of infection as compared to other strains.

A novel canine influenza H3N2 virus isolated from cats in an animal shelter.

The interspecies transmission of avian-origin H3N2 canine influenza virus (CIV) to dogs was first reported in 2007. The present study characterized a novel CIV H3N2 isolated from cats in an animal shelter. A comparative analysis of the deduced amino acid sequences of the A/Canine/Korea/CY009/2010(H3N2) (CY009) and A/Feline/Korea/FY028/2010 (H3N2) (FY028) strains isolated from dogs and cats, respectively, in the animal shelter identified point mutations in 18 amino acid positions within eight viral genes. Interestingly, CY009 and FY028 replicated well in specific pathogen-free embryonated chicken eggs and in mice, respectively. Mice infected with the FY028 strain exhibited significant over expression of IL-10, TNF-α, and IFN-γ (p<0.001) at 3 days postinfection. Thus, an emergency monitoring system should be developed to identify influenza mutations that occur during interspecies transmission in companion animals and for continuous public health surveillance.

Complete Genome Sequence of a Canine-Origin H3N2 Feline Influenza Virus Isolated from Domestic Cats in South Korea.

A canine-origin Korean H3N2 feline influenza virus (FIV), A/feline/Korea/01/2010 (H3N2), was isolated in 2010 from a dead cat with severe respiratory disease. Here, we report the first complete genome sequence of this virus, containing 3' and 5' noncoding regions, which will help elucidate the molecular basis of the pathogenesis, transmission, and evolution of FIV.

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).

Identification of reassortant pandemic H1N1 influenza virus in Korean pigs.

Since the 2009 pandemic human H1N1 influenza A virus emerged in April 2009, novel reassortant strains have been identified throughout the world. This paper describes the detection and isolation of reassortant strains associated with human pandemic influenza H1N1 and swine influenza H1N2 (SIV) viruses in swine populations in South Korea. Two influenza H1N2 reassortants were detected, and subtyped by PCR. The strains were isolated using Madin- Darby canine kidney (MDCK) cells, and genetically characterized by phylogenetic analysis for genetic diversity. They consisted of human, avian, and swine virus genes that were originated from the 2009 pandemic H1N1 virus and a neuraminidase (NA) gene from H1N2 SIV previously isolated in North America. This identification of reassortment events in swine farms raises concern that reassortant strains may continuously circulate within swine populations, calling for the further study and surveillance of pandemic H1N1 among swine.

Identification of a novel single-stranded, circular DNA virus from bovine stool.

We report the identification of a novel single-stranded, circular DNA virus isolated from bovine stool. The virus, named bovine stool-associated circular DNA virus (BoSCV), has a genome comprising 2600 bases of circular ssDNA, with two putative ORFs encoding replicase and capsid proteins, arranged inversely. The stem-loop structure was located between the 3' ends of the two putative ORFs, as in chimpanzee stool-associated circular virus (ChimpSCV) and unlike other circular DNA viruses, including members of the families Circoviridae, Nanoviridae and Geminiviridae. BoSCV was also genetically similar to ChimpSCV, with approximately 30 % identity in the replicase and capsid proteins. A phylogenetic analysis based on the replicase protein showed that BoSCV and ChimpSCV are in the same clade. A field survey using BoSCV-specific PCRs targeting ORF1 detected BoSCV and BoSCV-like sequences in bovine and porcine stool samples. BoSCV appears to belong to a new genus of circular DNA viruses.

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.

The assessment of efficacy of porcine reproductive respiratory syndrome virus inactivated vaccine based on the viral quantity and inactivation methods.

BACKGROUND: There have been many efforts to develop efficient vaccines for the control of porcine reproductive and respiratory syndrome virus (PRRSV). Although inactivated PRRSV vaccines are preferred for their safety, they are weak at inducing humoral immune responses and controlling field PRRSV infection, especially when heterologous viruses are involved. RESULTS: In all groups, the sample to positive (S/P) ratio of IDEXX ELISA and the virus neutralization (VN) titer remained negative until challenge. While viremia did not reduce in the vaccinated groups, the IDEXX-ELISA-specific immunoglobulin G increased more rapidly and to significantly greater levels 7 days after the challenge in all the vaccinated groups compared to the non-vaccinated groups (p < 0.05). VN titer was significantly different in the 106 PFU/mL PRRSV vaccine-inoculated and binary ethylenimine (BEI)-inactivated groups 22 days after challenge (p < 0.05). Consequently, the inactivated vaccines tested in this study provided weak memory responses with sequential challenge without any obvious active immune responses in the vaccinated pigs. CONCLUSIONS: The inactivated vaccine failed to show the humoral immunity, but it showed different immune response after the challenge compared to mock group. Although the 106 PFU/mL-vaccinated and BEI-inactivated groups showed significantly greater VN titers 22 days after challenge, all the groups were already negative for viremia.