Bovine papular stomatitis virus as a vaccine vector for cattle.

Virus vectored vaccines are not available commercially for cattle even though compelling potential applications exist. Bovine papular stomatitis virus (BPSV), a highly prevalent parapoxvirus, causes self-limited oral lesions in cattle. Ability of virus to accommodate large amounts of foreign DNA, induce low level of antiviral immunity, and circulate and likely persist in cattle populations, make BPSV an attractive candidate viral vector. Here, recombinant BPSV were constructed expressing either Bovine herpesvirus 1 (BoHV-1) glycoprotein gD (BPSVgD), or gD and gB (BPSVgD/gB). Immunization of BPSV serologically-positive calves with BPSVgD or BPSVgD/gB induced BoHV-1 neutralization antibodies and provided protection for three of four animals following a high dose BoHV-1 challenge at day 70 pi. Results indicate BPSV suitability as a candidate virus vector for cattle vaccines.

African Swine Fever Virus CD2v Protein Induces ß-Interferon Expression and Apoptosis in Swine Peripheral Blood Mononuclear Cells.

African swine fever (ASF) is a hemorrhagic disease of swine characterized by massive lymphocyte depletion in lymphoid tissues due to the apoptosis of B and T cells, a process likely triggered by factors released or secreted by infected macrophages. ASFV CD2v (EP402R) has been implicated in viral virulence and immunomodulation in vitro; however, its actual function(s) remains unknown. We found that CD2v expression in swine PK15 cells induces NF-κB-dependent IFN-ß and ISGs transcription and an antiviral state. Similar results were observed for CD2v protein treated swine PBMCs and macrophages, the major ASFV target cell. Notably, treatment of swine PBMCs and macrophages with CD2v protein induced apoptosis. Immunoprecipitation and colocalization studies revealed that CD2v interacts with CD58, the natural host CD2 ligand. Additionally, CD58 knockdown in cells or treatment of cells with an NF-κB inhibitor significantly reduced CD2v-mediated NF-κB activation and IFN-ß induction. Further, antibodies directed against CD2v inhibited CD2v-induced NF-κB activation and IFN-ß transcription in cells. Overall, results indicate that ASFV CD2v activates NF-κB, which induces IFN signaling and apoptosis in swine lymphocytes/macrophages. We propose that CD2v released from infected macrophages may be a significant factor in lymphocyte apoptosis observed in lymphoid tissue during ASFV infection in pigs.

Attenuated strain of CVB3 with a mutation in the CAR-interacting region protects against both myocarditis and pancreatitis.

Coxsackievirus B3 (CVB3), is commonly implicated in myocarditis, which can lead to dilated cardiomyopathy, in addition to causing acute pancreatitis and meningitis. Yet, no vaccines are currently available to prevent this infection. Here, we describe the derivation of a live attenuated vaccine virus, termed mutant (Mt) 10, encoding a single amino acid substitution H790A within the viral protein 1, that prevents CVB3 infection in mice and protects from both myocarditis and pancreatitis in challenge studies. We noted that animals vaccinated with Mt 10 developed virus-neutralizing antibodies, predominantly containing IgG2a and IgG2b, and to a lesser extent IgG3 and IgG1. Furthermore, by using major histocompatibility complex class II dextramers and tetramers, we demonstrated that Mt 10 induces antigen-specific T cell responses that preferentially produce interferon-γ. Finally, neither vaccine recipients nor those challenged with the wild-type virus revealed evidence of autoimmunity or cardiac injury as determined by T cell response to cardiac myosin and measurement of circulating cardiac troponin I levels, respectively. Together, our data suggest that Mt 10 is a vaccine candidate that prevents CVB3 infection through the induction of neutralizing antibodies and antigen-specific T cell responses, the two critical components needed for complete protection against virus infections in vaccine studies.

A parapoxviral virion protein targets the retinoblastoma protein to inhibit NF-κB signaling.

Poxviruses have evolved multiple strategies to subvert signaling by Nuclear Factor κB (NF-κB), a crucial regulator of host innate immune responses. Here, we describe an orf virus (ORFV) virion-associated protein, ORFV119, which inhibits NF-κB signaling very early in infection (≤ 30 min post infection). ORFV119 NF-κB inhibitory activity was found unimpaired upon translation inhibition, suggesting that virion ORFV119 alone is responsible for early interference in signaling. A C-terminal LxCxE motif in ORFV119 enabled the protein to interact with the retinoblastoma protein (pRb) a multifunctional protein best known for its tumor suppressor activity. Notably, experiments using a recombinant virus containing an ORFV119 mutation which abrogates its interaction with pRb together with experiments performed in cells lacking or with reduced pRb levels indicate that ORFV119 mediated inhibition of NF-κB signaling is largely pRb dependent. ORFV119 was shown to inhibit IKK complex activation early in infection. Consistent with IKK inhibition, ORFV119 also interacted with TNF receptor associated factor 2 (TRAF2), an adaptor protein recruited to signaling complexes upstream of IKK in infected cells. ORFV119-TRAF2 interaction was enhanced in the presence of pRb, suggesting that ORFV119-pRb complex is required for efficient interaction with TRAF2. Additionally, transient expression of ORFV119 in uninfected cells was sufficient to inhibit TNFα-induced IKK activation and NF-κB signaling, indicating that no other viral proteins are required for the effect. Infection of sheep with ORFV lacking the ORFV119 gene led to attenuated disease phenotype, indicating that ORFV119 contributes to virulence in the natural host. ORFV119 represents the first poxviral protein to interfere with NF-κB signaling through interaction with pRb.

Zika Virus Encoding Nonglycosylated Envelope Protein Is Attenuated and Defective in Neuroinvasion.

Zika virus (ZIKV), a mosquito-transmitted flavivirus responsible for sporadic outbreaks of mild and febrile illness in Africa and Asia, reemerged in the last decade causing serious human diseases, including microcephaly, congenital malformations, and Guillain-Barré syndrome. Although genomic and phylogenetic analyses suggest that genetic evolution may have led to the enhanced virulence of ZIKV, experimental evidence supporting the role of specific genetic changes in virulence is currently lacking. One sequence motif, VNDT, containing an N-linked glycosylation site in the envelope (E) protein, is polymorphic; it is absent in many of the African isolates but present in all isolates from the recent outbreaks. In the present study, we investigated the roles of this sequence motif and glycosylation of the E protein in the pathogenicity of ZIKV. We first constructed a stable full-length cDNA clone of ZIKV in a novel linear vector from which infectious virus was recovered. The recombinant ZIKV generated from the infectious clone, which contains the VNDT motif, is highly pathogenic and causes lethality in a mouse model. In contrast, recombinant viruses from which the VNDT motif is deleted or in which the N-linked glycosylation site is mutated by single-amino-acid substitution are highly attenuated and nonlethal. The mutant viruses replicate poorly in the brains of infected mice when inoculated subcutaneously but replicate well following intracranial inoculation. Our findings provide the first evidence that N-linked glycosylation of the E protein is an important determinant of ZIKV virulence and neuroinvasion.IMPORTANCE The recent emergence of Zika virus (ZIKV) in the Americas has caused major worldwide public health concern. The virus appears to have gained significant pathogenicity, causing serious human diseases, including microcephaly and Guillain-Barré syndrome. The factors responsible for the emergence of pathogenic ZIKV are not understood at this time, although genetic changes have been shown to facilitate virus transmission. All isolates from the recent outbreaks contain an N-linked glycosylation site within the viral envelope (E) protein, whereas many isolates of the African lineage virus lack this site. To elucidate the functional significance of glycosylation in ZIKV pathogenicity, recombinant ZIKVs from infectious clones with or without the glycan on the E protein were generated. ZIKVs lacking the glycan were highly attenuated for the ability to cause mortality in a mouse model and were severely compromised for neuroinvasion. Our studies suggest glycosylation of the E protein is an important factor contributing to ZIKV pathogenicity.

Branched chain α-ketoacid dehydrogenase kinase 111-130, a T cell epitope that induces both autoimmune myocarditis and hepatitis in A/J mice.

INTRODUCTION: Organ-specific autoimmune diseases are believed to result from immune responses generated against self-antigens specific to each organ. However, when such responses target antigens expressed promiscuously in multiple tissues, then the immune-mediated damage may be wide spread. METHODS: In this report, we describe a mitochondrial protein, branched chain α-ketoacid dehydrogenase kinase (BCKDk ) that can act as a target autoantigen in the development of autoimmune inflammatory reactions in both heart and liver. RESULTS: We demonstrate that BCKDk protein contains at least nine immunodominant epitopes, three of which, BCKDk 71-90, BCKDk 111-130 and BCKDk 141-160, were found to induce varying degrees of myocarditis in immunized mice. One of these, BCKDk 111-130, could also induce hepatitis without affecting lungs, kidneys, skeletal muscles, and brain. In immunogenicity testing, all three peptides induced antigen-specific T cell responses, as verified by proliferation assay and/or major histocompatibility complex class II/IAk dextramer staining. Finally, the disease-inducing abilities of BCKDk peptides were correlated with the production of interferon-γ, and the activated T cells could transfer disease to naive recipients. CONCLUSIONS: The disease induced by BCKDk peptides could serve as a useful model to study the autoimmune events of inflammatory heart and liver diseases.

Fiber muscle types in adult female pigs as determined by combinating histochemical and immunohistochemical methods

Las fibras musculares esqueléticas del cerdo han sido tradicionalmente tipificadas como I, IIA y IIB, más sus formas híbridas, determinando la actividad de la enzima miosina ATPasa miofibrilar. Recientemente, la utilización de anticuerpos monoclonales contra isoformas de cadena pesada de miosina, electroforesis y estudios moleculares, han documentado la existencia, además, de la isoforma IIx. El objetivo de este estudio fue determinar la existencia de las isoformas de miosina, presentes en los distintos tipos fibrilares, utilizando técnics histoquímicas e inmunohistoquímicas combinadas, dentro de una unidad experimental compuesta por muestras musculares del M. longissimo del dorso, en cerdas de 150 kg promedio. Las muestras fueron obtenidas 45 minutos después de la faena, mediante escisión y congeladas en acetona enfriada con hielo seco. Cortes seriados de 10 µm de espesor fueron tratados por la técnicas de mATPasa modificada por Nwoye (1982), a pH 4,6, NADH-TR e inmunohistoquímica. Histoquímicamente fueron identificados cinco grupos de fibras: tipo I oscuras, IIA claras, y tres tipos intermedios. Los ensayos inmunohistoquímicos permitieron identificar las isoformas ß lenta I, IIa, IIx y IIb presentes en fibras de tipo I, IIA, IIX, IIB y un grupo híbrido IIAX