Comparison of Porcine Airway and Intestinal Epithelial Cell Lines for the Susceptibility and Expression of Pattern Recognition Receptors upon Influenza Virus Infection.

Influenza viruses infect the epithelial cells of the swine respiratory tract. Cell lines derived from the respiratory tract of pigs could serve as an excellent in vitro model for studying the pathogenesis of influenza viruses. In this study, we examined the replication of influenza viruses in the MK1-OSU cell line, which was clonally derived from pig airway epithelium. MK1-OSU cells expressed both cytokeratin and vimentin proteins and displayed several sugar moieties on the cell membrane. These cells also expressed both Sial2-3Gal and Sial2-6Gal receptors and were susceptible to swine influenza A, but not to human B and C viruses. Interestingly, these cells were also permissive to infection by influenza D virus that utilized 9-O-acetylated glycans. To study the differences in the expression of pattern recognition receptors (PRRs) upon influenza virus infection in the respiratory and digestive tract, we compared the protein expression of various PRRs in MK1-OSU cells with that in the SD-PJEC cell line, a clonally derived cell line from the porcine jejunal epithelium. Toll-like receptor 7 (TLR-7) and melanoma differentiation-associated protein 5 (MDA5) receptors showed decreased expression in influenza A infected MK1-OSU cells, while only TLR-7 expression decreased in SD-PJEC cells. Further research is warranted to study the mechanism behind the virus-mediated suppression of these proteins. Overall, this study shows that the porcine respiratory epithelial cell line, MK1-OSU, could serve as an in-vitro model for studying the pathogenesis and innate immune responses to porcine influenza viruses.

Domestic pigs are susceptible to infection with influenza B viruses.

UNLABELLED: Influenza B virus (IBV) causes seasonal epidemics in humans. Although IBV has been isolated from seals, humans are considered the primary host and reservoir of this important pathogen. It is unclear whether other animal species can support the replication of IBV and serve as a reservoir. Swine are naturally infected with both influenza A and C viruses. To determine the susceptibility of pigs to IBV infection, we conducted a serological survey for U.S. Midwest domestic swine herds from 2010 to 2012. Results of this study showed that antibodies to IBVs were detected in 38.5% (20/52) of sampled farms, and 7.3% (41/560) of tested swine serum samples were positive for IBV antibodies. Furthermore, swine herds infected with porcine reproductive and respiratory syndrome virus (PRRSV) showed a higher prevalence of IBV antibodies in our 2014 survey. In addition, IBV was detected in 3 nasal swabs collected from PRRSV-seropositive pigs by real-time RT-PCR and sequencing. Finally, an experimental infection in pigs, via intranasal and intratracheal routes, was performed using one representative virus from each of the two genetically and antigenically distinct lineages of IBVs: B/Brisbane/60/2008 (Victoria lineage) and B/Yamagata/16/1988 (Yamagata lineage). Pigs developed influenza-like symptoms and lung lesions, and they seroconverted after virus inoculation. Pigs infected with B/Brisbane/60/2008 virus successfully transmitted the virus to sentinel animals. Taken together, our data demonstrate that pigs are susceptible to IBV infection; therefore, they warrant further surveillance and investigation of swine as a potential host for human IBV. IMPORTANCE: IBV is an important human pathogen, but its ability to infect other species, for example, pigs, is not well understood. We showed serological evidence that antibodies to two genetically and antigenically distinct lineages of IBVs were present among domestic pigs, especially in swine herds previously infected with PRRSV, an immunosuppressive virus. IBV was detected in 3 nasal swabs from PRRSV-seropositive pigs by real-time reverse transcription-PCR and sequencing. Moreover, both lineages of IBV were able to infect pigs under experimental conditions, with transmissibility of influenza B/Victoria lineage virus among pigs being observed. Our results demonstrate that pigs are susceptible to IBV infections, indicating that IBV is a swine pathogen, and swine may serve as a natural reservoir of IBVs. In addition, pigs may serve as a model to study the mechanisms of transmission and pathogenesis of IBVs.

In vitro and in vivo replication of influenza A H1N1 WSN33 viruses with different M1 proteins.

The M1 protein is a major structural protein that has multiple functions in various steps within the life cycle of the influenza A virus (IAV). However, little is currently known about the role of M1 in IAV replication in vivo and the associated pathogenesis. In this study, six isogenic H1N1 WSN33 viruses, constructed to express unique M1 proteins derived from various strains, subtypes or WSN33 itself, were tested to determine in vitro and in vivo functional exchangeability of M1 proteins in the replication and pathogenesis of the WSN33 virus. Despite five chimeric M1 viruses replicating to levels similar to those of the parental WSN33 virus in cell cultures, all M1 chimeras exhibited improved replication and enhanced virulence in mice when compared with the WSN33 virus. Interestingly, M1 proteins derived from swine viruses caused more severe clinical diseases than those from human or quail. These data indicate that the M1 protein is an important determinant of viral replication and pathogenic properties in mice, although the functions of M1 observed in vivo are not adequately reflected in simple infections of cultured cells. Chimeric M1 viruses that are variable in their clinical manifestations described here will aid future understanding of the role of M1 in IAV pathogenesis.

In vitro reassortment between endemic H1N2 and 2009 H1N1 pandemic swine influenza viruses generates attenuated viruses.

The pandemic H1N1 (pH1N1) influenza virus was first reported in humans in the spring of 2009 and soon thereafter was identified in numerous species, including swine. Reassortant viruses, presumably arising from the co-infection of pH1N1 and endemic swine influenza virus (SIV), were subsequently identified from diagnostic samples collected from swine. In this study, co-infection of swine testicle (ST) cells with swine-derived endemic H1N2 (MN745) and pH1N1 (MN432) yielded two reassortant H1N2 viruses (R1 and R2), both possessing a matrix gene derived from pH1N1. In ST cells, the reassortant viruses had growth kinetics similar to the parental H1N2 virus and reached titers approximately 2 log(10) TCID(50)/mL higher than the pH1N1 virus, while in A549 cells these viruses had similar growth kinetics. Intranasal challenge of pigs with H1N2, pH1N1, R1 or R2 found that all viruses were capable of infecting and transmitting between direct contact pigs as measured by real time reverse transcription PCR of nasal swabs. Lung samples were also PCR-positive for all challenge groups and influenza-associated microscopic lesions were detected by histology. Interestingly, infectious virus was detected in lung samples for pigs challenged with the parental H1N2 and pH1N1 at levels significantly higher than either reassortant virus despite similar levels of viral RNA. Results of our experiment suggested that the reassortant viruses generated through in vitro cell culture system were attenuated without gaining any selective growth advantage in pigs over the parental lineages. Thus, reassortant influenza viruses described in this study may provide a good system to study genetic basis of the attenuation and its mechanism.

New role of antibody in bacterial isolation.

To eliminate the interference caused by Pseudomonas aeruginosa in the isolation of Salmonella, a rabbit polyclonal antibody against P. aeruginosa was prepared by inoculating four New Zealand rabbits with the pathogen. The antiserum was purified using saturated ammonium sulfate and added into Rappaport-Vassiliadis medium with soya (RVS) broth and Muller-Kauffmann tetrathionate novobiocin broth (MKTTn broth) to evaluate whether it could inhibit the growth of P. aeruginosa. Observations by scanning electron microscopy demonstrated that P. aeruginosa was attacked and destroyed by the antibody when incubated for 10 min at 37 degrees C. The activity of the antibody was also effective against 11 other strains of P. aeruginosa. Twenty-six strains of Salmonella were mixed with P. aeruginosa in RVS and MKTTn broth at 37 degrees C for 12 h, respectively, and the cultures were plated on Salmonella chromogenic medium (SCM; Oxoid, Basingstoke, UK). Only Salmonella grew on SCM; five colonies were randomly selected for identification by VITEK 2 (bioMérieux, Lyon, France). Additionally, when mixed with two strains of Enterobacter cloacae (ATCC 700323 and YG001), the prepared antibody did not affect the growth of E. cloacae. The results demonstrated that the microbicidal activity of the antibody did not affect the tested Salmonella sp. or E. cloacae strains. Therefore, the antibody generated could be used to increase the accuracy of Salmonella isolation.

Analysis of the bacterial diversity existing on animal hide and wool: development of a preliminary PCR-restriction fragment length polymorphism fingerprint database for identifying isolates.

Twenty-one bacterial strains were isolated from imported cattle hide and rabbit wool using two types of media, nutrient broth, and nutrient broth with serum. The bacteria identified were Brevibacillus laterosporus, Leclercia adecarboxylata, Peptococcus niger, Bacillus circulans, Raoultella ornithinolytica, Bacillus subtilis, Bacillus cereus, Bacillus thermobacillus, Bacillus choshinensis, Bacillus sphaericus, Acinetobacter haemolyticus, Sphingomonas paucimobilis, Bacillus thuringiensis, Staphylococcus intermedius, Mycobacteria, Moraxella, Klebsiella pneumoniae, Ralstonia pickettii, Staphylococcus chromogenes, Comamonas testosteroni, and Cupriavidus pauculus. The 16s rDNA gene of each bacterium was amplified using the universal primers 27f and 1492r. The amplicons were digested with AvaI, BamHI, BgII, DraI, EcoRI, EcoRV, HindIII, HinfI, HpaI, PstI, SmaI, TaqII, XbaI, XmaI, AluI, XhoI, and PvuI individually. A specific fingerprint from the PCR-restriction fragment length polymorphism method based on 16s rDNA was obtained for each bacterium. The results showed that the method developed was useful not only for bacterial identification but also for the etiological investigation of pathogens in imported animal hair and wool.

Development and evaluation of a sensitive and quantitative assay for hirame rhabdovirus based on quantitative RT-PCR.

The aim of the present work was to develop a quantitative reverse-transcription polymerase chain reaction (qRT-PCR) assay using a TaqMan probe to detect and quantify hirame rhabdovirus (HRV). The results demonstrated that the assay had a detection limit of 100 copies of RNA per reaction and a log-linear range up to 10(8) copies of HRV RNA. Regression analysis demonstrated a significant correlation with an R(2) value of 0.9963 and a slope of -3.18 between the mean C(t) values and HRV cRNA. This assay was 100 times more sensitive than the conventional one-step RT-PCR assay. The qRT-PCR assay was found to be highly reproducible with intra- and inter-assay coefficients of variation of 0.37-1.72% and 1.37-3.79%, respectively. The primers and TaqMan probe were specific for HRV and did not react with either the spring viraemia of carp virus (SVCV), infectious pancreatic necrosis virus (IPNV), infectious haematopoietic necrosis virus (IHNV), marine birnavirus (MABV), viral hemorrhagic septicemia virus (VHSV), or viral nervous necrosis virus (VNNV). This assay was evaluated using 40 fish samples, indicating that such method offers considerable advantages over the classical virus isolation method currently used for HRV surveillance. In conclusion, the developed qRT-PCR assay was a reliable, specific and sensitive tool for the quantitative diagnosis of HRV in fish samples.

Development of a sensitive and quantitative assay for spring viremia of carp virus based on real-time RT-PCR.

A one-step real-time reverse transcription-polymerase chain reaction (RT-PCR) using a TaqMan probe to quantitatively detect spring viremia of carp virus (SVCV) is described. In this assay, a pair of primers amplifying an 81-bp DNA fragment and a TaqMan probe was designed targeting the conserved region at the SVCV glycoprotein (G) gene. To avoid the disadvantages arising from plasmids, an extension adding a T7 phage polymerase promoter to the 5' end of the antisense primer was carried out to obtain viral cRNA. Standardized cycle threshold (Ct) values for 10-fold serial dilutions of SVCV cRNA were achieved by real-time RT-PCR and used to create standard curves. A regression line between the mean Ct values and viral template concentrations over a 1:10(7) dilution range with an r(2) value (0.9916) and a slope (-3.36) and the coefficient of variation (intra- or inter-assay is <2% and <4%, respectively) indicated that the assay was highly reproducible. The assay was specific to SVCV and there was no cross-reactivity with other fish viruses (viral hemorrhagic septicemia virus, VHSV; infectious pancreatic necrosis virus, IPNV; grass carp reovirus, GCRV; epizootic haematopoietic necrosis virus, EHNV). The standard curve allows precise absolute quantitation and shows that the detection limit of the assay is 40 copies of the viral RNA. This one-step RT-PCR assay was evaluated using 60 clinical common carp samples collected during the year 2006, indicating such technology offers considerable advantages over conventional RT-PCR methods in current routine use for SVCV surveillance. This is the first report of the development of a one-step TaqMan RT-PCR for SVCV detection.

[Prokaryotic expression of the major antigenic domain of equine arteritis virus GL protein and the establishment of putative indirect ELISA assay].

According to the antigenic analysis of equine arteritis virus (EAV) GL protein, one pair of primers were designed, with which the gene fragment coding the high antigenic domain of EAV GL protein was amplified from the EAV genome. The cloned gene was digested with BamH I and Xho I and then inserted into pET-32a and resulted pET-GL1. The pET-GL1 was transformed into the host cell BL21(DE3) and the expression was optimized including cultivation temperature and concentration of IPTG. The aim protein was highly expressed and the obtained recombinant protein manifested well reactiongenicity as was confirmed by Western blot. The recombinant GL1 protein was purified by the means of His * Bind resin protein purification procedure. Then an indirect ELISA was established to detect antibody against EAV with the purified GL1 protein as the coating antigen. The result showed that the optimal concentration of coated antigen was 9.65 microg/mL and the optimal dilution of serum was 1:80. The positive criterion of this ELISA assay is OD (the tested serum) > 0.4 and OD (the tested serum) /OD (the negative serum) > 2.0. The iGL-ELISA was evaluated versus micro-virus neutralization test. The ELISA was performed on 900 sera from which were preserved by this lab during horse entry/exit inspection, the agreement (94.1%) of these test were considered suitable for individual serological detection. In another test which 180 sera samples were detected by iGL-ELISA and INGEZIM ELISA kit respectively. The agreement ratio between the two methods is 95.6%.

Polymerase chain reaction method to detect canis materials by amplification of species-specific DNA fragment.

Rapid identification of mammal materials in feeding stuffs and food is essential for effective control of a potential source of pathogens, such as those that cause bovine spongiform encephalopathy. A convenient polymerase chain reaction (PCR)-based assay was developed for detection and identification of a canis-specific mitochondrial DNA sequence in foodstuffs and food. The amplified canis-specific PCR product was a 213 base pair band from the D-loop DNA fragment of mitochondria, a high copy gene which should improve the possibility of amplifying template molecules of adequate size among the degraded DNA fragments brought about by heat denaturation. The specificity of this method was confirmed by 8 canis blood DNA samples (from different breeds of dog) and 9 noncanis animal blood DNA samples (bovine, sheep, porcine, chicken, fish, donkey, rabbit, deer, horse). This method was able to detect the presence of canis material in foodstuffs and in food mixtures even when the concentration of canis-derived meat was reduced to 0.05%. Furthermore, it did not appear to be affected by prolonged heat treatment. This method was developed for detection of canis materials in feeding stuffs, and occasionally for medical jurisprudence detection of canis-derived materials.