Prevalence of viral agents causing swine reproductive failure in Korea and the development of multiplex real-time PCR and RT-PCR assays.

This study aimed to investigate the prevalence of viral agents causing reproductive failure in pigs in Korea. In addition, two types of multiplex real-time PCR (mqPCR) were developed for the simultaneous detection of Aujeszky's disease virus (ADV) and porcine parvovirus (PPV) in mqPCR and encephalomyocarditis virus (EMCV) and Japanese encephalitis virus (JEV) in reverse transcription mqPCR (mRT-qPCR). A total of 150 aborted fetus samples collected from 2020 to 2022 were analyzed. Porcine reproductive and respiratory syndrome virus was the most prevalent (49/150 32.7%), followed by porcine circovirus type 2 (31/150, 20.7%), and PPV1 (7/150, 4.7%), whereas ADV, EMCV, and JEV were not detected. The newly developed mqPCR and mRT-qPCR could simultaneously detect and differentiate with high sensitivities and specificities. When applied to aborted fetuses, the newly developed mqPCR for PPV was 33.3% more sensitivities than the previously established diagnostic method. Amino acid analysis of the VP2 sequences of PPV isolates revealed considerable similarity to the highly pathogenic Kresse strain. This study successfully evaluated the prevalence of viral agents causing reproductive failure among swine in Korea, the developed mqPCR and mRT-qPCR methods could be utilized as effective and accurate diagnostic methods for the epidemiological surveillance of ADV, PPV, EMCV, and JEV.

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.

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.

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).

Establishment of a p30-based lateral flow assay for African swine fever virus detection.

African swine fever virus (ASFV) has continuously devastated the global pig industry. Viral persistence causes problems in large pig farms and kills small farms. Timely diagnostic tools play an important role in controlling outbreaks and minimizing losses. In this study, we developed a lateral flow assay to detect ASFV on-site. The VDRG® ASFV Ag Rapid Kit was established using two monoclonal antibodies (mAbs) against the p30 protein. The conjunction pad of the kit was coated with a mixture of the mAb and colloidal gold. This rapid kit was capable of detecting 11.5 ng of antigen and 0.16 HAD50 of virus from samples, in 20 min for the entire procedure. It passed cross-specific tests using common viruses that cause infectious diseases in pigs. ASFV was detected after 4 days in experimental infection in pigs by the kit. The specificity and sensitivity of the kit for clinical samples were 99.88% and 84.52% (93.8% for samples with a Ct value below 30), respectively. Finally, the kit can detect 100% positive herd outbreaks. The VDRG® ASFV Ag Rapid Kit presents a useful point-of-care tool for ASFV detection.

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.

Enhanced detection and serotyping of foot-and-mouth disease virus serotype O, A, and Asia1 using a novel multiplex real-time RT-PCR.

Rapid and accurate detection and serotyping of foot-and-mouth disease (FMD) virus (FMDV) is essential for implementing control policies against emergent FMD outbreaks. Current serotyping assays, such as VP1 reverse transcription-polymerase chain reaction (RT-PCR)/sequencing (VP1 RT-PCR/sequencing) and antigen detection enzyme-linked immunosorbent assay (ELISA), have problems with increasing serotyping failure of FMDVs from FMD outbreaks. This study was conducted to develop a multiplex real-time RT-PCR for specific detection and differential serotyping of FMDV serotype O, A, and Asia 1 directly from field clinical samples. Primers and probes were designed based on 571 VP1 coding region sequences originated from seven pools. Multiplex real-time RT-PCR using these primers and probes demonstrated serotype-specific detection with enhanced sensitivity compared to VP1 RT-PCR/sequencing for reference FMDV (n = 24). Complete serotyping conformity between the developed multiplex real-time RT-PCR and previous VP1 RT-PCR/sequencing was demonstrated using FMDV field viruses (n = 113) prepared in cell culture. For FMDV field clinical samples (n = 55), the serotyping rates of multiplex real-time RT-PCR and VP1 RT-PCR/sequencing were 92.7% (51/55) and 72.7% (40/55), respectively. Moreover, the developed multiplex real-time RT-PCR demonstrated improved FMDV detection (up to 33.3%) and serotyping (up to 67.7%) capabilities for saliva samples when compared with 3D real-time RT-PCR and VP1 RT-PCR/sequencing, during 10 days of challenge infection with FMDV serotype O, A, and Asia 1. Collectively, this study suggests that the newly developed multiplex real-time RT-PCR assay may be useful for the detection and differential serotyping of FMDV serotype O, A, and Asia 1 in the field.

Development of an indirect ELISA against African swine fever virus using two recombinant antigens, partial p22 and p30.

African swine fever (ASF) is a highly fatal viral disease affecting pigs. It is caused by the ASF virus (ASFV), and causes serious economic losses to the swine industry worldwide, including in Korea. Commercially available enzyme-linked immunosorbent assay (ELISA) kits for detecting anti-ASFV antibodies are used for the diagnosis and surveillance of ASF. In this study, an ELISA was developed to detect anti-ASFV antibodies using two recombinant proteins, p22 and p30, from genotype II ASFV. Recombinant transmembrane domain-deleted p22 (p22∆TM) and p30 were expressed in E.coli vector system pET32a and mixed for use as antigens in indirect ELISA. The p22∆TM/p30-based indirect ELISA was validated using 31 sera from genotype I ASFV-infected pigs and 1133 sera from uninfected pigs. Area under the curve of this test was 0.999 [95 % concentration interval 0.992-1.000], and sensitivity and specificity were 93.5 % and 99.8 %, respectively. The between run coefficient of variation for internal quality control serum was 6.61 %. In the seroconversion analysis, the p22∆TM/p30-based indirect ELISA showed equal or better ability to detect antibodies in pigs experimentally challenged with ASFV p72 genotypes I and II (p < 0.05). In conclusion, the p22∆TM/p30-based indirect ELISA is a reliable diagnostic method for detecting anti-ASFV antibodies.

Antimicrobial susceptibility testing of two Lawsonia intracellularis isolates associated with proliferative hemorrhagic enteropathy and porcine intestinal adenomatosis in South Korea.

This study represents the first published data on antimicrobial susceptibility of Asian isolates of Lawsonia intracellularis. We assessed MICs of 16 antimicrobials for two isolates of L. intracellularis recovered from diseased pigs in South Korea, one from a finisher pig with acute proliferative hemorrhagic enteropathy in 2002 and the other from a grower pig with porcine intestinal adenomatosis in 2010. Tylosin and tilmicosin were found to be the most active against L. intracellularis both intracellularly (MICs, 0.25 to 0.5 µg/ml and 0.125 µg/ml, respectively) and extracellularly (MICs, 0.25 to 0.5 µg/ml and 1 µg/ml, respectively).

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.

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.

Experimental infection of dogs with avian-origin canine influenza A virus (H3N2).

Susceptible dogs were brought into contact with dogs experimentally infected with an avian-origin influenza A virus (H3N2) that had been isolated from a pet dog with severe respiratory syndrome. All the experimentally infected and contact-exposed dogs showed elevated rectal temperatures, virus shedding, seroconversion, and severe necrotizing tracheobronchitis and bronchioalveolitis.

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).

Transmission of avian influenza virus (H3N2) to dogs.

In South Korea, where avian influenza virus subtypes H3N2, H5N1, H6N1, and H9N2 circulate or have been detected, 3 genetically similar canine influenza virus (H3N2) strains of avian origin (A/canine/Korea/01/2007, A/canine/Korea/02/2007, and A/canine/Korea/03/2007) were isolated from dogs exhibiting severe respiratory disease. To determine whether the novel canine influenza virus of avian origin was transmitted among dogs, we experimentally infected beagles with this influenza virus (H3N2) isolate. The beagles shed virus through nasal excretion, seroconverted, and became ill with severe necrotizing tracheobronchitis and bronchioalveolitis with accompanying clinical signs (e.g., high fever). Consistent with histologic observation of lung lesions, large amounts of avian influenza virus binding receptor (SAalpha 2,3-gal) were identified in canine tracheal, bronchial, and bronchiolar epithelial cells, which suggests potential for direct transmission of avian influenza virus (H3N2) from poultry to dogs. Our data provide evidence that dogs may play a role in interspecies transmission and spread of influenza virus.

An immunochromatography assay for rapid antemortem diagnosis of dogs suspected to have canine distemper.

A new assay was developed for rapid and antemortem diagnosis of canine distemper (CD). This immunochromatography (IC)-based assay, which employs two monoclonal anti-CDV antibodies, was compared with nested PCR. When serial dilutions of purified CDV were tested, the CDV detection limits of the nested PCR and IC assays were 2 x 10(2)TCID(50)/ml and 5 x 10(2)TCID(50)/ml, respectively. Nasal irrigation fluid, conjunctival swabs, and blood lymphocytes from 66 dogs suspected to have CD were tested. Preliminary IC experiments revealed that the optimal sample volume and reaction time were 100 microl and 5 min, respectively. Relative to nested PCR, the sensitivity and specificity of the IC assay was maximal (100% and 100%, respectively) when conjunctival swabs were tested. This is significant because conjunctival swab specimens are easy to obtain in the early phase of CD infection. However, with blood lymphocytes and nasal samples, the IC assay was slightly less sensitive (89.7% and 85.7%, respectively) and specific (94.6% and 100%, respectively) than nested PCR. Since this novel IC assay does not require special instruments, it is a simple enough for dog owners to use. Since early detection of CD would allow appropriate treatment and quarantine to be instituted quickly, such a test would help reduce the morbidity and mortality associated with CD help to prevent its spread to other animals.

Impact of porcine group A rotavirus co-infection on porcine epidemic diarrhea virus pathogenicity in piglets.

Porcine epidemic diarrhea virus (PEDV) and porcine group A rotavirus (PGAR) are the main causative agents of acute diarrhea in piglets. In South Korea, PGAR is prevalent in piglets naturally infected with PEDV. Piglets naturally co-infected with PEDV and PGAR appeared to have severe and prolonged diarrhea that was distinct from that commonly observed. The aim of this study was to determine the impact of PGAR co-infection on PEDV pathogenicity in piglets. Thirty-six colostrum-deprived, one-day old, Large White-Duroc crossbred pigs were randomly divided into four equal groups: PEDV, PEDV/PGAR, PGAR, and control groups. The piglets were euthanized at 1, 2, or 3 days post-inoculation (DPI) to measure the villous height:crypt depth (VH:CD) ratio and to collect fecal samples for RT-PCR and virus isolation. No significant differences in mean VH:CD ratio and clinical symptoms (diarrhea, vomiting, dehydration, and anorexia) were observed between the PEDV/PGAR-infected and PEDV-infected groups of piglets at 1, 2 and 3 DPI; however, at 2 and 3 DPI, PGAR was detected in all fecal samples by RT-PCR and virus isolation. These findings failed to detect any interaction between PEDV and porcine rotavirus in the small intestines of piglets, suggesting that concurrent infection of PGAR may not synergistically enhance intestinal villous atrophy of piglets with PEDV disease. We propose that the severe diarrhea exhibited in PEDV and PGAR co-infected piglets may be more associated with the immunity level of the host rather than to any synergistic effect of PGAR on PEDV enteritis.

Effects of epidermal growth factor on atrophic enteritis in piglets induced by experimental porcine epidemic diarrhoea virus.

Epidermal growth factor (EGF) promotes gastrointestinal mucosal recovery by stimulating the mitogenic activity of intestinal crypt epithelial cells. The aim of this study was to determine the effects of EGF on atrophic enteritis induced in piglets by experimental infection with porcine epidemic diarrhoea virus (PEDV) strain Dr13. Two groups of 12 conventional, colostrum-deprived, 1-day-old, large White-Duroc cross breed piglets were inoculated orally with PEDV (3 x 10(5) 50% tissue culture infective doses), with or without EGF (10 microg/kg/day, intraperitoneally once daily for 4 days after infection) and compared to 12 uninfected, untreated control piglets. PEDV+EGF piglets had less severe clinical signs than PEDV only piglets at 48 and 60 h post-infection (hpi). Histologically, the ratio of villous height:crypt depth of PEDV+EGF piglets was significantly higher than PEDV only piglets at 36 and 48 hpi. Immunohistochemistry for Ki67 demonstrated increased proliferation in intestinal crypt epithelial cells of PEDV+EGF piglets compared to PEDV only piglets at 36, 48 and 60 hpi. EGF stimulates proliferation of intestinal crypt epithelial cells and promotes recovery from atrophic enteritis in PEDV-infected piglets.