Isolation and characterization of Korean porcine deltacoronavirus strain KNU16-07.

Porcine deltacoronavirus (PDCoV) has emerged in several pig-raising countries and has been a causative pathogen associated with diarrheal diseases in South Korea since 2014. In the present study, we were able to isolate and cultivate a Korean PDCoV strain (KNU16-07) in cell culture and investigate its pathogenicity. PDCoV-inoculated piglets showed watery diarrhea accompanied by acute enteritis in the natural host. Sequencing analysis demonstrated the genetic stability of KNU16-07 for at least thirty serial passages.

Efficacy of an inactivated genotype 2b porcine epidemic diarrhea virus vaccine in neonatal piglets.

Massive outbreaks of porcine epidemic diarrhea virus (PEDV) recurred in South Korea in 2013-2014 and affected approximately 40% of the swine breeding herds across the country, incurring a tremendous financial impact on producers and consumers. Despite the nationwide use of commercially available attenuated and inactivated vaccines in South Korea, PEDV has continued to plague the domestic pork industry, raising concerns regarding their protective efficacies and the need for new vaccine development. In a previous study, we isolated and serially cultivated a Korean PEDV epidemic strain, KOR/KNU-141112/2014, in Vero cells. With the availability of a cell culture-propagated PEDV strain, we are able to explore vaccination and challenge studies on pigs. Therefore, the aim of the present study was to produce an inactivated PEDV vaccine using the KNU-141112 strain and evaluate its effectiveness in neonatal piglets. Pregnant sows were immunized intramuscularly with the inactivated adjuvanted monovalent vaccine at six and three weeks prior to farrowing. Six-day-old piglets born to vaccinated or unvaccinated sows were challenged with the homogeneous KNU-141112 virus. The administration of the inactivated vaccine to sows greatly increased the survival rate of piglets challenged with the virulent strain, from 0% to approximately 92% (22/24), and significantly reduced diarrhea severity including viral shedding in feces. In addition, litters from unvaccinated sows continued to lose body weight throughout the experiment, whereas litters from vaccinated sows started recovering their daily weight gain at 7 days after the challenge. Furthermore, strong neutralizing antibody responses to PEDV were verified in immunized sows and their offspring, but were absent in the unvaccinated controls. Altogether, our data demonstrated that durable lactogenic immunity was present in dams administrated with the inactivated vaccine and subsequently conferred critical passive immune protection to their own litters against virulent PEDV infection.

Immunoprophylactic effect of chicken egg yolk antibody (IgY) against a recombinant S1 domain of the porcine epidemic diarrhea virus spike protein in piglets.

Porcine epidemic diarrhea virus (PEDV) is a highly contagious enteric pathogen of swine causing high mortality rates in piglets. PEDV outbreaks have occurred continuously in most swine-producing Asian countries and have recently emerged in the United States, leading to large economic losses for both the Asian and US pig industries. The spike (S) protein of PEDV consists of the S1 and S2 domains, responsible for virus binding and fusion, respectively. The involvement of the S1 domain in specific high-affinity interactions with the cellular receptor and induction of neutralizing antibodies in the natural host makes it a logical target for the development of effective vaccines and therapeutics against PEDV. Passive immunization by oral administration of egg yolk antibodies (IgY) obtained from immunized chickens provides an alternative source of specific antibodies for the prevention and treatment of PEDV in newborn piglets. In this study, we produced an IgY against the PEDV S1 protein and investigated its immunoprophylactic effect in neonatal piglets. A codon-optimized PEDV S1 gene consisting of amino acid residues 25-749 was synthesized and used to establish a stable porcine cell line constitutively expressing a recombinant PEDV S1 protein containing the chicken immunoglobulin Fc fragment at its C-terminus. The purified recombinant S1 protein was found to mediate potent immune responses in immunized hens. We next tested the ability of oral passive immunization with anti-PEDV S1 IgY to protect piglets against PEDV. Specific chicken IgY against the S1 protein was orally administered to neonatal piglets, and their responses subsequent to a virulent PEDV challenge were monitored. The results showed that oral administration of anti-PEDV S1 IgY efficiently protects neonatal piglets against PEDV, suggesting its potential as a prophylactic or therapeutic agent against acute PEDV infection.

Characterization in vitro and in vivo of a novel porcine parainfluenza virus 5 isolate in Korea.

A novel porcine parainfluenza 5 (pPIV5), KNU-11, in the genus Rubulavirus of the subfamily Paramyxovirinae, was isolated from the lung of a piglet in Korea in 2011. To understand the importance of this virus as an infectious agent, in vitro and in vivo characteristics of KNU-11 virus was investigated. KNU-11 was remarkably cytopathogenic, showing distinct cell rounding and clumping evident in porcine alveolar macrophage (PAM), porcine kidney (PK-15), and swine testicle (ST) cells within 12h postinfection and capable of hemagglutinating guinea pig red blood cells. Interestingly, this cytopathology was found to be absent in cell lines from other mammalian species. To evaluate the in vitro immunity of the pPIV5 isolate, we sought to explore alteration of inflammatory cytokine and chemokine expression in PAM cells infected with KNU-11 by using quantitative real-time RT-PCR. Most cytokine and chemokine genes including type 1 interferons (IFN-α/ß) and IFN-related antiviral genes were found to be significantly elevated in KNU-11 virus-infected PAM cells. A serum neutralization test-based serosurvey demonstrated that neutralizing antibodies against KNU-11 are readily detected in domestic swine populations, suggesting high prevalence of pPIV5 in Korean pig farms. Animal studies showed that KNU-11 fails to establish an acute respiratory illness, indicating that pPIV5 is non- or very mildly pathogenic to pigs.

Human telomerase reverse transcriptase-immortalized porcine monomyeloid cell lines for the production of porcine reproductive and respiratory syndrome virus.

Porcine reproductive and respiratory syndrome virus (PRRSV) shows highly restricted cell tropism and targets subpopulations of differentiated macrophages such as porcine alveolar macrophages (PAM) in the natural host. Although primary PAM cells would be ideal for in vitro virus production, they are not only difficult and expensive for establishment but cannot be frozen reliably for long-term storage and use. Apart from PAM cells, African green monkey kidney derived Marc-145 cells are used commonly for virus propagation. However, concerns have been raised regarding a possible modification of specific epitopes associated with virus neutralization because of distinct virus entry between PAM and Marc-145 cells. In order to overcome these problems, the present study was aimed to generate immortalized porcine monocyte/macrophage cell lines and to evaluate their potential for PRRSV production. Primary PAM cells were transfected stably with the human telomerase reverse transcriptase (hTERT) cDNA by a retrovirus vector so that constitutive expression of the hTERT protein allows cells to proliferate indefinitely. The newly immortalized PAM clones were shown to exert functional telomerase activity, indicating sustained expression of hTERT. In addition, telomerase-immortalization of PAMs did not affect expression levels of the native CD163 receptor on their surface. It was further demonstrated that these continuous PAM cell lines are fully permissive for the efficient growth of both type 1 and 2 PRRSV strains. The findings suggest that the hTERT-immortalized PAM cell lines can enable us to facilitate the continued use of PAMs for virus isolation and production and to provide a promising tool for viral pathogenesis and immune function studies.

Generation of a porcine alveolar macrophage cell line for the growth of porcine reproductive and respiratory syndrome virus.

Porcine reproductive and respiratory syndrome virus (PRRSV) has a marked tropism for cells of the monocyte-macrophage lineage and accordingly, replicates in fully differentiated alveolar macrophages in the natural host. Despite the identification of several putative receptors for PRRSV on porcine alveolar macrophages (PAM), only CD163 was found to be able to make non-permissive cells susceptible to PRRSV, indicating a requirement for CD163 in productive infection. Interestingly, the preliminary experiments revealed that the immortalized PAM cell line, which was previously shown to fail to support PRRSV replication, does not express detectable levels of CD163. These data suggest that there may be a correlation between the CD163 undetectable expression level and PRRSV non-susceptibility in the continuous PAM cell line. In this study, therefore, it was attempted to stably transfect non-permissive PAM cells with CD163 cDNA to generate cell lines constitutively expressing CD163 and to evaluate their permissivity to PRRSV. The newly established PAM cell lines were demonstrated to express robust levels of CD163 and to be fully permissive for both type 1 and 2 PRRSV strains. This PRRSV-permissive PAM cell line will be a valuable tool not only to facilitate virus propagation but also to advance in vitro studies on virus pathogenesis.

Genetic differentiation of the nucleocapsid protein of Korean isolates of porcine epidemic diarrhoea virus by RT-PCR based restriction fragment length polymorphism analysis.

A reverse transcriptase polymerase chain reaction (RT-PCR) based restriction fragment length polymorphism (RFLP) analysis based on the nucleocapsid (N) gene was developed to differentiate between field isolates of porcine epidemic diarrhoea virus (PEDV) and a vaccine strain, J-vac. Thirteen field isolates of PEDV from Korea were distinguishable from the vaccine strain and the prototype PEDV strain CV777 by RFLP using Tru9I. RFLP patterns in 11 of 13 field PEDV isolates were different from the vaccine strain using AspLEI, HgaI and MspR9I. Sequence analysis of the PEDV N gene revealed that Korean field PEDV isolates had 93.6% and 89.6% identity with the vaccine virus at nucleotide and amino acid sequence levels, respectively, suggesting progressive point mutations of the PEDV genome in the field. RFLP analysis of the PEDV N gene is a promising tool for distinguishing field strains from the vaccine-derived virus.

Effect of genotypic and biotypic differences among PRRS viruses on the serologic assessment of pigs for virus infection.

Genetic, antigenic, and pathogenic variability is known to exist among porcine reproductive and respiratory syndrome (PRRS) viruses and has garnered great attention for diagnostics and disease control/prevention. A comparative serologic study was conducted on five field and two cell-attenuated viruses to determine if serologic responses to PRRS virus infection could be influenced by biotypic and/or genotypic differences of the viruses. The isolates used for the study varied in their virulence to pigs and in genomic sequences. Ten pigs were inoculated with each isolate (1x10(3) TCID(50)) via the intranasal route. All inoculated animals became viremic during the study period. Some animals inoculated with the attenuated viruses remained seronegative until the end of the study (42 days post-inoculation (PI)), but all of the animals inoculated with field viruses developed enzyme-linked immunosorbent assay (ELISA)- and indirect fluorescent antibody (IFA)-detectable antibodies, regardless of the virus strain used in the IFA assay. In contrast, a great degree of variation in the onset and level of serum-virus neutralization (SVN) antibody was observed by individual pigs and by each virus. The reactivity of SVN antibody was highly specific for homologous viruses. Cross-neutralization titers were better correlated with sequence homology of open-reading frames (ORFs) 4 and 5 among the viruses than any other structural genes. We conclude that the biotypic difference among PRRS viruses may affect the kinetic of humoral immune response in infected pigs. The IFA test may be used as a confirmatory test when a false-positive ELISA result is suspected or vise-a-versa, but SVN antibody titers are highly affected by antigenic variability.

Molecular typing of VapA-positive Rhodococcus equi isolates from Jeju native horses, Korea.

We recently demonstrated the presence of virulence-associated protein antigen (VapA)-positive Rhodococcus equi in Jeju Island, Korea. These bacteria contained one of two distinct plasmid types, a 90-kb type II plasmid, which has been found in isolates from the native Kiso horses of Japan, and a new variant, a 90-kb type V plasmid. However, the genotypic characters of the VapA-positive R. equi from Jeju native horses and their environments are poorly understood. Ninety-eight isolates from soil samples and 89 isolates from fecal samples were collected from five farms that breed or have bred Jeju native horses, and were tested for the presence of VapA by immunoblotting and PCR. Of the 98 soil isolates and 89 fecal isolates, seven and 13 were VapA-positive R. equi, respectively. In 2003, two Jeju foals died suddenly and were brought to the Faculty of Veterinary Medicine, Cheju National University, for postmortem examination. Pure cultures of R. equi were isolated from the lung lesions of both foals. Of the 16 clinical isolates, 14 were VapA-positive R. equi. Of the 34 VapA-positive clinical and environmental isolates, 16 contained the 90-kb type II plasmid and 18 contained a 90-kb type V plasmid. Pulsed-field gel electrophoresis (PFGE) patterns of the VapA-positive isolates from Jeju horses and Kiso horses, containing the 90-kb type II plasmid, were compared and formed two distinct groups. Furthermore, 18 virulent isolates containing the 90-kb type V plasmid formed two distinct PFGE groups (of 16 and two isolates). These results demonstrate that two virulence plasmid types are widespread in R. equi in Jeju native horses. However, there is little diversity in the PFGE patterns of virulent isolates, suggesting the clonal spread of virulent R. equi. The PFGE pattern of the virulent R. equi isolates from Jeju native horses in Korea is not identical to those of Kiso native horses in Japan.

Rhodococcus equi virulence plasmids recovered from horses and their environment in Jeju, Korea: 90-kb type II and a new variant, 90-kb type V.

Rhodococcus equi was isolated from fecal and soil samples from four native Jeju horse farms and six Thoroughbred farms in Jeju, Korea. The isolates were examined for the presence of virulence-associated 15-17-kDa antigens (VapA) by colony blotting, using the monoclonal antibody 10G5, and for the gene encoding VapA by PCR. R. equi was isolated from all 36 soil samples collected from the 10 farms with between 5.0 x 10(2) and 7.5 x 10(4) colony-forming units (cfu) per gram of soil, and from 37 of 40 fecal samples with between 5.0 x 10(1) and 1.1 x 10 (5) cfu per gram of feces. Virulent R. equi was isolated from seven farms and appeared in 2.0% of isolates (10 of 508). Of the 10 virulent isolates, four contained a 90-kb type II plasmid, which has been found in isolates from the Kiso native horses of Japan, and the other six contained a new variant, which did not display the EcoRI and EcoT22I digestion patterns of the 10 representative plasmids already reported (85-kb types I, II, III, and IV; 87-kb types I and II; 90-kb types I, II, III, and IV). We designated the new variant as the "90-kb type V" plasmid, because its EcoRI digestion pattern is similar to that of the 90-kb type II plasmid. This is the first report of the prevalence of virulent R. equi in Jeju, Korea. The same virulence plasmid type is found in both Korean and Japanese isolates, providing insight into the origin, ancestry, and dispersal of native horses in Korea and Japan.