Development of a BoHV-4 viral vector expressing tgD of BoHV-1 and evaluation of its immunogenicity in mouse model.

In recent years, Bovine herpesvirus 4 (BoHV-4) has emerged as an attractive gene delivery viral vector, mainly for vaccination purposes in the veterinary field. In the present study, a new infectious clone of the BoHV-4 genome carrying a bacterial artificial chromosome vector (BoHV-4-BAC) was developed by homologous recombination in mammalian cell culture and bacterial systems, and exploited to express a truncated form of glycoprotein D (tgD) of Bovine herpesvirus 1 (BoHV-1) (BoHV-4-tgD∆TK) as a vaccine candidate. This construct's immunogenicity was compared to a DNA vector expressing the same antigen (pC-tgD) in a BALB/c mouse model. After the mice were immunized, total and specific antibody responses, cytokine responses, total splenocyte cells proliferation/cytotoxicity, and virus neutralization assays were conducted to analyze the immune response elicited by both constructs. Mice from both vaccine groups developed significant humoral and cellular immune responses after a booster dose regime was conducted on day 28 post-injection. In almost all immunological assays, BoHV-4-tgDΔTK induced as high an immune response as pC-tgD. In both vaccine constructs, neutralizing antibodies were a significant determining factor in protection against BoHV-1, even after the first injection. We conclude that a BoHV-4-based viral vector offers an effective immunization strategy as an alternative to DNA-based immunization platforms, at least to combat BoHV-1.

New members to Arctic-like lineage of canine distemper virus from Turkey.

Canine distemper virus (CDV) causes a multisystemic fatal disease, briefly named as distemper, in domestic and wild animals. Molecular characterization studies serve to identify local strains, accordingly, helps to determine the scope of vaccination in prevention of distemper. We aimed with this study to update the molecular status of CDV in domestic dogs in Turkey. Sequence analysis of the H gene revealed that novel Turkish sequences formed a separated clade in Arctic-like lineage. Italian clade which mainly included strains originated from wild canid or non-canid localized nearly to novel Turkish clade. Codons 530th and 549th determining the affinity of domestic or wild animals to distemper were Asparagine and Tyrosine, respectively. This report presented the presence of CDV strains belonging to Arctic-like lineage for the first time in domestic dogs in Turkey. The findings pave the way for the reassessment of the circulation and geographical shifting of Arctic-like lineages of CDV.

An indirect enzyme-linked immunosorbent assay for the identification of antibodies to Senecavirus A in swine.

BACKGROUND: Senecavirus A (SVA), a member of the family Picornaviridae, genus Senecavirus, is a recently identified single-stranded RNA virus closely related to members of the Cardiovirus genus. SVA was originally identified as a cell culture contaminant and was not associated with disease until 2007 when it was first observed in pigs with Idiopathic Vesicular Disease (IVD). Vesicular disease is sporadically observed in swine, is not debilitating, but is significant due to its resemblance to foreign animal diseases, such as foot-and-mouth disease (FMD), whose presence would be economically devastating to the United States. IVD disrupts swine production until foreign animal diseases can be ruled out. Identification and characterization of SVA as a cause of IVD will help to quickly rule out infection by foreign animal diseases. METHODS: We have developed and characterized an indirect ELISA assay to specifically identify serum antibodies to SVA. Viral protein 1, 2 and 3 (VP1, VP2, VP3) were expressed, isolated, and purified from E. coli and used to coat plates for an indirect ELISA. Sera from pigs with and without IVD symptoms as well as a time course following animals from an infected farm, were analyzed to determine the antibody responses to VP1, VP2, and VP3. RESULTS: Antibody responses to VP2 were higher than VP1 and VP3 and showed high affinity binding on an avidity ELISA. ROC analysis of the SVA VP2 ELISA showed a sensitivity of 94.2% and a specificity of 89.7%. Compared to IFA, the quantitative ELISA showed an 89% agreement in negative samples and positive samples from 4-60 days after appearance of clinical signs. Immune sera positive for FMDV, encephalomyocarditis virus, and porcine epidemic diarrhea virus antibodies did not cross-react. CONCLUSIONS: A simple ELISA based on detection of antibodies to SVA VP2 will help to differentially diagnose IVD due to SVA and rule out the presence of economically devastating foreign animal diseases.

Urinary detection of toscana virus nucleic acids in neuroinvasive infections.

BACKGROUND: Toscana virus (TOSV) is a sandfly-borne pathogen causing febrile diseases and neuroinvasive infections in humans. Definitive diagnosis of TOSV infections frequently requires the detection of viral RNA in cerebrospinal fluid (CSF) or in circulation, which can be achieved prior to seroconversion. OBJECTIVES: To evaluate TOSV excretion in urine and impact of urine as a diagnostic specimen. STUDY DESIGN: A total of 82 plasma, CSF and urine samples were collected from 24 individuals with a preliminary diagnosis of atypical viral encephalitis, where frequent bacterial fungal and viral causes were ruled out. Phlebovirus and WNV nucleic acids were investigated via real-time and nested polymerase chain reaction (PCR) assays. Commercial immunofluorescence assays were employed for viral IgM detection. Amplicons were characterized via cloning and sequencing. RESULTS: Phlebovirus PCR yielded positive results in 7 out of 14 samples that comprise 4 plasma and 3 urine specimens from 3 individuals. Amplicons were characterized as TOSV genotype A. Investigation of the follow-up samples suggested that virus shedding in urine coincides or follows viremia. Despite conserved sequences observed in paired or sequential plasma-urine specimens, L693S substitution in the viral polymerase was characterized in a urine sample. CONCLUSIONS: These preliminary findings indicate that urine can be employed as a additional clinical sample for TOSV RNA detection in suspected cases, especially in individuals where specimens for viral diagnostics during the early stages of the infection are not available.