Bat influenza vectored NS1-truncated live vaccine protects pigs against heterologous virus challenge.

Swine influenza is an important disease for the swine industry. Currently used whole inactivated virus (WIV) vaccines can induce vaccine-associated enhanced respiratory disease (VAERD) in pigs when the vaccine strains mismatch with the infected viruses. Live attenuated influenza virus vaccine (LAIV) is effective to protect pigs against homologous and heterologous swine influenza virus infections without inducing VAERD but has safety concerns due to potential reassortment with circulating viruses. Herein, we used a chimeric bat influenza Bat09:mH3mN2 virus, which contains both surface HA and NA gene open reading frames of the A/swine/Texas/4199-2/1998 (H3N2) and six internal genes from the novel bat H17N10 virus, to develop modified live-attenuated viruses (MLVs) as vaccine candidates which cannot reassort with canonical influenza A viruses by co-infection. Two attenuated MLV vaccine candidates including the virus that expresses a truncated NS1 (Bat09:mH3mN2-NS1-128, MLV1) or expresses both a truncated NS1 and the swine IL-18 (Bat09:mH3mN2-NS1-128-IL-18, MLV2) were generated and evaluated in pigs against a heterologous H3N2 virus using the WIV vaccine as a control. Compared to the WIV vaccine, both MLV vaccines were able to reduce lesions and virus replication in lungs and limit nasal virus shedding without VAERD, also induced significantly higher levels of mucosal IgA response in lungs and significantly increased numbers of antigen-specific IFN-γ secreting cells against the challenge virus. However, no significant difference was observed in efficacy between the MLV1 and MLV2. These results indicate that bat influenza vectored MLV vaccines can be used as a safe live vaccine to prevent swine influenza.

Pseudocowpox virus infection in an American bison (Bison bison).

BACKGROUND: The present report describes a case of pseudocowpox virus (PCPV) infection in a seven-year-old female bison euthanized due to a history of declining condition and sores on the vulva and udder. CASE PRESENTATION: External examination revealed multifocal, raised, keratinized plaques (0.5-2 cm) covering the skin of the ventral surface of the tail, perineum, caudoventral abdomen, udder, both inguinal recesses, and the medial aspects of both thighs. No significant gross lesions were present in the reminder of the tissues examined. Histopathological examination of the affected skin showed moderate epidermal hyperplasia with rete pegs, marked parakeratotic hyperkeratosis with crusts of degenerate neutrophils and cell debris, and few epithelial cells undergoing ballooning degeneration with occasional eosinophilic intracytoplasmic inclusion bodies (3-5 µm Bollinger body). Negative staining electron microscopy from skin revealed typical Parapoxvirus (PPV) particles, which were also confirmed by real-time PCR (Ct =18.6). Metagenomic analysis of the skin samples revealed only poxviruses. The bison parapox B2L envelope gene clustered with other parapox sequences identified from ruminants. CONCLUSIONS: This is the first report of PCPV virus infection in an American bison. Identification of novel susceptible hosts of parapox viruses sheds light on the viral evolution and highlights the importance of potential economic impact of this disease to the bison industry.

CD3ε+ Cells in Pigs With Severe Combined Immunodeficiency Due to Defects in ARTEMIS.

Severe combined immunodeficiency (SCID) is described as the lack of functional T and B cells. In some cases, mutant genes encoding proteins involved in the process of VDJ recombination retain partial activity and are classified as hypomorphs. Hypomorphic activity in the products from these genes can function in the development of T and B cells and is referred to as a leaky phenotype in patients and animals diagnosed with SCID. We previously described two natural, single nucleotide variants in ARTEMIS (DCLR1EC) in a line of Yorkshire pigs that resulted in SCID. One allele contains a splice site mutation within intron 8 of the ARTEMIS gene (ART16), while the other mutation is within exon 10 that results in a premature stop codon (ART12). While initially characterized as SCID and lacking normal levels of circulating lymphoid cells, low levels of CD3ε+ cells can be detected in most SCID animals. Upon further assessment, we found that ART16/16, and ART12/12 SCID pigs had abnormally small populations of CD3ε+ cells, but not CD79α+ cells, in circulation and lymph nodes. Newborn pigs (0 days of age) had CD3ε+ cells within lymph nodes prior to any environmental exposure. CD3ε+ cells in SCID pigs appeared to have a skewed CD4α+CD8α+CD8ß- T helper memory phenotype. Additionally, in some pigs, rearranged VDJ joints were detected in lymph node cells as probed by PCR amplification of TCRδ V5 and J1 genomic loci, as well as TCRß V20 and J1.1, providing molecular evidence of residual Artemis activity. We additionally confirmed that TCRα and TCRδ constant region transcripts were expressed in the thymic and lymph node tissues of SCID pigs; although the expression pattern was abnormal compared to carrier animals. The leaky phenotype is important to characterize, as SCID pigs are an important tool for biomedical research and this additional phenotype may need to be considered. The pig model also provides a relevant model for hypomorphic human SCID patients.

Novel Engraftment and T Cell Differentiation of Human Hematopoietic Cells in ART-/-IL2RG-/Y SCID Pigs.

Pigs with severe combined immunodeficiency (SCID) are an emerging biomedical animal model. Swine are anatomically and physiologically more similar to humans than mice, making them an invaluable tool for preclinical regenerative medicine and cancer research. One essential step in further developing this model is the immunological humanization of SCID pigs. In this work we have generated T- B- NK- SCID pigs through site directed CRISPR/Cas9 mutagenesis of IL2RG within a naturally occurring DCLRE1C (ARTEMIS)-/- genetic background. We confirmed ART-/-IL2RG-/Y pigs lacked T, B, and NK cells in both peripheral blood and lymphoid tissues. Additionally, we successfully performed a bone marrow transplant on one ART-/-IL2RG-/Y male SCID pig with bone marrow from a complete swine leukocyte antigen (SLA) matched donor without conditioning to reconstitute porcine T and NK cells. Next, we performed in utero injections of cultured human CD34+ selected cord blood cells into the fetal ART-/-IL2RG-/Y SCID pigs. At birth, human CD45+ CD3ε+ cells were detected in cord and peripheral blood of in utero injected SCID piglets. Human leukocytes were also detected within the bone marrow, spleen, liver, thymus, and mesenteric lymph nodes of these animals. Taken together, we describe critical steps forwards the development of an immunologically humanized SCID pig model.