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.

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 sequences of a Korean virulent porcine epidemic diarrhea virus and its attenuated counterpart.

A virulent porcine epidemic diarrhea virus (PEDV) strain, DR13, was obtained from suckling pigs suspected of having porcine epidemic diarrhea in 1999 in Korea, and its attenuated counterpart was derived from virulent strain DR13 by serial propagation in Vero cells. This report describes the first complete genome sequences of virulent PEDV and its attenuated counterpart, which will provide important insights into the molecular basis of the attenuation of PEDV.

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).

Complete genome sequences of porcine reproductive and respiratory syndrome viruses: perspectives on their temporal and spatial dynamics.

Porcine reproductive and respiratory syndrome virus (PRRSV) has been investigated extensively at the molecular level. Nevertheless, genome wide study on the temporal and spatial dynamics of the virus is non-existed. To explore this topic, we determined complete coding genome sequences for four PRRSV isolates and analyzed them together with 122 global published ones using the Bayesian coalescent approach as well as Bayesian inferences and maximum likelihood methods. All EU-type viruses belonged to one of two groups or were unclassified (5 isolates), and all NA-type isolates were divided into one of three major groups or were unclassified (1 isolate). Here, there was no apparent association between temporal or geographic origin and heterogeneity of global PRRSVs. Of the eight ORFs, ORF1a showed the most powerful evolutionary signal. Our findings also indicated that the PRRS virus evolved at a rate of 1.98 × 10-3 substitutions/site/year, and the most recent common ancestor of the virus existed 786.4 years ago. Here, EU-type viruses segregated 115.7 years ago, while NA-type isolates diverged 179.8 years before the present. In addition, our reconstruction of the effective population size depicted five phases of epidemic growth: an initial constant, followed sequentially by slow decrease, slight increase, sharp decline, and then a rapid expansion approaching the present.

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.

Tracing the genetic history of porcine reproductive and respiratory syndrome viruses derived from the complete ORF 5-7 sequences: a Bayesian coalescent approach.

To trace the genetic history of porcine reproductive and respiratory syndrome virus (PRRSV), we determined the complete sequences of ORFs 5 to 7 of four PRRSV isolates. These sequences were analyzed together with published sequences from 146 isolates from various parts of the world using a Bayesian coalescent approach as well as Bayesian inference and maximum-likelihood methods. All of the European-type (EU-type) viruses were classified into one of two groups or unclassified (4 isolates), while all North American-type (NA-type) viruses belonged to one of three major groups or were unclassified (5 isolates). Within each genotype, no apparent periodic and/or geographic influence on the evolution of PRRSVs was observed. The evolutionary rate of PRRSV isolates was estimated to be 1.55 × 10(-3) substitutions/site/year, and the time of the most recent common ancestor (TMRCA) was 491.2 years ago. Here, the TMRCA for the EU- and NA-type viruses was 58.7 and 62.6 years ago, respectively. A Bayesian skyline plot revealed that the viruses evolved at an almost constant population size until the late 1970s, when they experienced a population expansion that continued until the late 1980s. The population size then remained constant again until the early 2000s, when a rapid, sharp decline in the effective number of infections occurred.

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.

Sputum Processing Method for Lateral Flow Immunochromatographic Assays to Detect Coronaviruses.

Coronavirus causes an infectious disease in various species and crosses the species barriers leading to the outbreak of zoonotic diseases. Due to the respiratory diseases are mainly caused in humans and viruses are replicated and excreted through the respiratory tract, the nasal fluid and sputum are mainly used for diagnosis. Early diagnosis of coronavirus plays an important role in preventing its spread and is essential for quarantine policies. For rapid decision and prompt triage of infected host, the immunochromatographic assay (ICA) has been widely used for point of care testing. However, when the ICA is applied to an expectorated sputum in which antigens are present, the viscosity of sputum interferes with the migration of the antigens on the test strip. To overcome this limitation, it is necessary to use a mucolytic agent without affecting the antigens. In this study, we combined known mucolytic agents to lower the viscosity of sputum and applied that to alpha and beta coronavirus, porcine epidemic diarrhea virus (PEDV) and Middle East respiratory syndrome coronavirus (MERS-CoV), respectively, spiked in sputum to find optimal pretreatment conditions. The pretreatment method using tris(2-carboxyethyl)phosphine (TCEP) and BSA was suitable for ICA diagnosis of sputum samples spiked with PEDV and MERS-CoV. This sensitive assay for the detection of coronavirus in sputum provides an useful information for the diagnosis of pathogen in low respiratory tract.

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.

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.

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).

Complete Genome Sequences of Porcine Deltacoronavirus Strains DH1/2016 and DH2/2016 Isolated in South Korea.

Two porcine deltacoronavirus (PDCoV) strains, named DH1/2016 and DH2/2016, were isolated from feces of piglets which had severe watery diarrhea symptoms. A comparison of the complete genome sequences suggested that the DH1/2016 and DH2/2016 strains are highly homologous to each other and to PDCoVs isolated in early 2014 from the United States.

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.

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.

Inter- and intraspecies transmission of canine influenza virus (H3N2) in dogs, cats, and ferrets.

BACKGROUND: The emergence of zoonotic viruses in domestic animals is a significant public health concern. Canine influenza virus (CIV) H3N2 is a virus that can infect companion animals and is, therefore, a potential public health concern. OBJECTIVE: This study investigated the inter- and intraspecies transmission of CIV among dogs, cats, and ferrets, under laboratory conditions, to determine whether transmission of the virus was possible between as well as within these domestic animal species. METHOD: The transmission routes for inter- and intraspecies transmission were airborne and direct contact, respectively. Transmission was conducted through intranasal infection of dogs followed by exposure to either cats or ferrets and by comingling infected and naïve animals of the same species. RESULTS: The interspecies transmission of CIV H3N2 via airborne was only observed from dogs to cats and not from dogs to ferrets. However, direct intranasal infection of either cats or ferrets with CIV could induce influenza-like clinical signs, viral shedding, and serological responses. Additionally, naïve cats and ferrets could be infected by CIV via direct contact with infected animals of the same species. CONCLUSION: Cats appear to be another susceptible host of CIV H3N2, whereas ferrets are not likely natural hosts. The molecular-based mechanism of interspecies and intraspecies transmission of CIV H3N2 should be further studied.