CD8+ lymphocytes but not B lymphocytes are required for protection against chronic hepatitis E virus infection in chickens.

Hepatitis E is an important global disease, causing outbreaks of acute hepatitis in many developing countries and sporadic cases in industrialized countries. Hepatitis E virus (HEV) infection typically causes self-limiting acute hepatitis but can also progress to chronic disease in immunocompromised individuals. The immune response necessary for the prevention of chronic infection is T cell-dependent; however, the arm of cellular immunity responsible for this protection is not currently known. To investigate the contribution of humoral immunity in control of HEV infection and prevention of chronicity, we experimentally infected 20 wild-type (WT) and 18 immunoglobulin knockout (JH-KO) chickens with a chicken strain of HEV (avian HEV). Four weeks postinfection (wpi) with avian HEV, JH-KO chickens were unable to elicit anti-HEV antibody but had statistically significantly lower liver lesion scores than the WT chickens. At 16 wpi, viral RNA in fecal material and liver, and severe liver lesions were undetectable in both groups. To determine the role of cytotoxic lymphocytes in the prevention of chronicity, we infected 20 WT and 20 cyclosporine and CD8+ antibody-treated chickens with the same strain of avian HEV. The CD8 + lymphocyte-depleted, HEV-infected chickens had higher incidences of prolonged fecal viral shedding and statistically significantly higher liver lesion scores than the untreated, HEV-infected birds at 16 wpi. The results indicate that CD8 + lymphocytes are required for viral clearance and reduction of liver lesions in HEV infection while antibodies are not necessary for viral clearance but may contribute to the development of liver lesions in acute HEV infection.

B-Cell-Deficient and CD8 T-Cell-Depleted Gnotobiotic Pigs for the Study of Human Rotavirus Vaccine-Induced Protective Immune Responses.

Genetically modified pigs have become available recently. In this study, we established the gnotobiotic pig model of human rotavirus (HRV) infection using cloned pigs with homozygous disruption in the gene encoding immunoglobulin heavy chain (HCKO), which totally impairs B-cell development. To clarify importance of B cells and cytotoxic T cells in rotavirus immunity, CD8 cells in a subset of the pigs were depleted by injecting antipig CD8 antibodies and the immune phenotypes of all pigs were examined. HCKO pigs, CD8 cell-depleted HCKO pigs, and wild-type (WT) pigs were vaccinated with an attenuated HRV vaccine and challenged with virulent HRV. Protection against HRV infection and diarrhea was assessed postchallenge and detailed T-cell subset responses were determined pre- and postchallenge. Significantly longer duration of virus shedding was seen in vaccinated HCKO pigs than in WT pigs, indicating the importance of B cells in vaccine-induced protective immunity. Vaccinated HCKO/CD8(-) pigs shed significantly higher number of infectious virus than WT pigs and non-CD8-depleted HCKO pigs, indicating the importance of CD8 T cells in controlling virus replication. Therefore, both B cells and CD8 T cells play an important role in the protection against rotavirus infection. HCKO and HCKO/CD8(-) pigs did not differ significantly in diarrhea and virus shedding postchallenge; increased CD4 and CD8(-) γδ T-cell responses probably compensated partially for the lack of CD8 T cells. This study demonstrated that HCKO pigs can serve as a valuable model for dissection of protective immune responses against viral infections and diseases.

A novel pathogenic Mammalian orthoreovirus from diarrheic pigs and Swine blood meal in the United States.

UNLABELLED: Since May 2013, outbreaks of porcine epidemic diarrhea have devastated the U.S. swine industry, causing immense economic losses. Two different swine enteric coronaviruses (porcine epidemic diarrhea virus and Delta coronavirus) have been isolated from the affected swine population. The disease has been reported from at least 32 states of the United States and other countries, including Mexico, Peru, Dominican Republic, Canada, Columbia, Ecuador, and Ukraine, with repeated outbreaks in previously infected herds. Here we report the isolation and characterization of a novel mammalian orthoreovirus 3 (MRV3) from diarrheic feces of piglets from these outbreaks in three states and ring-dried swine blood meal from multiple sources. MRV3 could not be isolated from healthy or pigs that had recovered from epidemic diarrhea from four states. Several MRV3 isolates were obtained from chloroform-extracted pig feces or blood meal in cell cultures or developing chicken embryos. Biological characterization of two representative isolates revealed trypsin resistance and thermostability at 90°C. NextGen sequencing of ultrapurified viruses indicated a strong homology of the S1 segment to mammalian and bat MRV3. Neonatal piglets experimentally infected with these viruses or a chloroform extract of swine blood meal developed severe diarrhea and acute gastroenteritis with 100% mortality within 3 days postinfection. Therefore, the novel porcine MRV3 may contribute to enteric disease along with other swine enteric viruses. The role of MRV3 in the current outbreaks of porcine epidemic diarrhea in the United States remains to be determined, but the pathogenic nature of the virus warrants further investigations on its epidemiology and prevalence. IMPORTANCE: Porcine orthoreoviruses causing diarrhea have been reported in China and Korea but not in the United States. We have isolated and characterized two pathogenic reassortant MRV3 isolates from swine fecal samples from porcine epidemic diarrhea outbreaks and ring-dried swine blood meal in the United States. These fecal and blood meal isolates or a chloroform extract of blood meal induced severe diarrhea and mortality in experimentally infected neonatal pigs. Genetic and phylogenetic analyses of two MRV3 isolates revealed that they are identical but differed significantly from nonpathogenic mammalian orthoreoviruses circulating in the United States. The present study provides a platform for immediate development of suitable vaccines and diagnostics to prevent and control porcine orthoreovirus diarrhea.

Production of transgenic pigs that express porcine endogenous retrovirus small interfering RNAs.

BACKGROUND: The presence of multiple copies of porcine endogenous retrovirus (PERV) within the pig genome, and the demonstration that replication competent PERV, that infect human cells in culture, can be isolated from pig cells, directly impacts the drive towards the development of pigs for xenotransplantation. The development of technology to produce pigs that do not propagate PERV has the potential to facilitate the development of xenotransplantation products for human use, and as such, is the focus of this investigation. The shear number of PERV loci, most of which are defective or pseudogenes, renders conventional gene targeting impractical, if not impossible, to inactivate all PERV provirus within the pig genome, including potential replication competent PERV arising from spontaneous recombination. The recently developed RNA interference (RNAi) technology to knockdown/silence post-transcriptional gene expression, offers a promising alternative to achieving this goal. METHODS: Here, the combination of nuclear transfer cloning and RNAi technology was used to produce pigs that may not propagate PERV. Small interfering RNAs (siRNA) were expressed as short hairpin RNAs (shRNA) against the gag and pol PERV genes, respectively, under the control of a RNA polymerase III (pol III), or a pol II promoter. PERV gag and pol model-genes, in combination with a Green Fluorescent Protein (GFP) reporter system, were developed to assess in vitro PERV target knockdown. Two shRNAs were selected, and transgenic pigs were produced that expressed the anti-gag and -pol shRNAs, in tandem, under the control of a ubiquitous pol II promoter. RESULTS: The anti-gag and -pol shRNAs, effectively knocked down expression of the PERV model-genes, and also endogenous PERV within cells in vitro. PERV knockdown was achieved whether the shRNA was expressed under the control of a RNA pol III, or a pol II promoter. Three litters of cloned pigs were produced. The shRNA construct was expressed by all the transgenic cloned animals, and within all the tissues of transgenic animals tested. PERV expression at the mRNA and PERV particulate levels in the pigs was virtually undetectable, compared with the infectious levels expressed by the positive control PK15 cell line in vitro. Immunofluorescence and Western blotting, with an anti-PERV-envelope antibody, did not detect PERV in pig tissues or cells whether activated or not, as compared to the positive control on PK15 cells. CONCLUSIONS: The stable long-term expression of anti-PERV siRNAs was shown to be effective in knocking down PERV expression in cells. However, the very low (sometimes undetectable), and variable levels of expression of PERV in normal pigs make it difficult to obtain suitable control animals for comparison, to assess knockdown of PERV in vivo. This was demonstrated by the observation that even cloned non-transgenic littermates, express levels of PERV as low as that of some of their siRNA transgenic littermates. Further analysis is required to conclusively quantitate in vivo effects in the shRNA transgenic pigs.

Efficient production of transgenic cloned calves using preimplantation screening.

The genetic manipulation of donor cells before nuclear transfer (NT) enables prior selection for transgene integration. However, selection for genetically modified cells using antibiotic drugs often results in mixed populations, resulting in a mixture of transgenic and nontransgenic donor cells for NT. In this study, we attempted to develop efficient strategies for the generation of human bile salt-stimulated lipase (BSSL) transgenic cows. Preimplantation screening by either biopsy or green fluorescent protein (GFP) expression was used to detect NT-derived BSSL transgenic embryos to ensure that the calf born would be transgenic. We compared the development rates of NT-derived embryos from G418- and GFP-selected donor cells. There were no significant differences (P < 0.001) in cleavage rate (67.2% vs. 60.0%) and blastocyst formation rate (44.9% vs. 41.2%). We also compared the pregnancy rates of the G418/biopsy and GFP preimplantation screened NT-derived blastocysts. The Day 40 pregnancy rate of the G418/biopsy group (40%) was lower than that of the GFP group (57%), but the calf birth rate of the G418/biopsy group (40%) was higher than that of the GFP group (21%). Healthy BSSL transgenic calves were born after both screening processes. This is the first report of biopsy-screened cloned transgenic animals. The results suggest that both selection methods are useful for detecting transgenic NT embryos without negatively affecting their development into viable transgenic offspring.

Targeted disruption of the alpha1,3-galactosyltransferase gene in cloned pigs.

Galactose-alpha1,3-galactose (alpha1,3Gal) is the major xenoantigen causing hyperacute rejection in pig-to-human xenotransplantation. Disruption of the gene encoding pig alpha1,3-galactosyltransferase (alpha1,3GT) by homologous recombination is a means to completely remove the alpha1,3Gal epitopes from xenografts. Here we report the disruption of one allele of the pig alpha1,3GT gene in both male and female porcine primary fetal fibroblasts. Targeting was confirmed in 17 colonies by Southern blot analysis, and 7 of them were used for nuclear transfer. Using cells from one colony, we produced six cloned female piglets, of which five were of normal weight and apparently healthy. Southern blot analysis confirmed that these five piglets contain one disrupted pig alpha1,3GT allele.