Assessment of replication of bovine herpesvirus type 4 in human glioblastoma and breast cancer cells as a potential oncolytic virus.

Oncolytic viruses have been extensively used in cancer treatment due to their tropism, selective replication only in tumor cells, and possible synergic interaction with other therapeutics. Different researchers have demonstrated that bovine herpesvirus 4 (BoHV-4), a member of the gammaherpesviridae family, has oncolytic potential in some human-origin cancer cell lines like glioma through the selective replication strategy. Using four apoptosis detection methods, namely MTT, LDH, TUNEL, and Annexin V assays, we evaluated the apoptotic effect of BoHV-4 Movar33/63 reference strain along with a recombinant BoHV-4 expressing EGFP in U87 MG cells (human glioblastoma cell line), MDA MB-231 (human breast cancer cell line), and MCF10a (non-tumorigenic human mammary epithelial cell line). Our findings indicate that this virus can replicate and induce apoptosis in these cell lines and hinder in vitro proliferation in a dose-dependent manner. In conclusion, BoHV-4 has in vitro potential as a novel oncolytic virus in human cancer therapy. However, its replication potential in the MCF10a cells as a non-tumorigenic human mammary epithelial cell line is a concern in using this virus in cancer therapy, at least against human mammary tumors. Further studies must therefore be conducted to examine the specific apoptotic pathways induced by this virus to move on to further experiments.

Genomic and phylogenetic characterization of ChPV2, a novel goat PV closely related to the Xi-PV1 species infecting bovines.

BACKGROUND: Papillomaviruses (PVs) infecting artiodactyls are very diverse, and only second in number to PVs infecting primates. PVs associated to lesions in economically important ruminant species have been isolated from cattle and sheep. METHODS: Potential PV DNA from teat lesions of a Damascus goat was isolated, cloned and sequenced. The PV genome was analyzed using bioinformatics approaches to detect open reading frames and to predict potential features of encoded proteins as well as putative regulatory elements. Sequence comparison and phylogenetic analyses using the concatenated E1E2L2L1 nucleotide and amino acid alignments was used to reveal the relationship of the new PV to the known PV diversity and its closest relevants. RESULTS: We isolated and characterized the full-genome of novel Capra hircus papillomavirus. We identified the E6, E7, E1, E2, L2, L1 open reading frames with protein coding potential and putative active elements in the ChPV2 proteins and putative regulatory genome elements. Sequence similarities of L1 and phylogenetic analyses using concatenated E1E2L2L1 nucleotide and amino acid alignments suggest the classification as a new PV type designated ChPV2 with a phylogenetic position within the XiPV genus, basal to the XiPV1 species. ChPV2 is not closely related to ChPV1, the other known goat PV isolated from healthy skin, although both of them belong confidently into a clade composed of PVs infecting cervids and bovids. Interestingly, ChPV2 contains an E6 open reading frame whereas all closely related PVs do not CONCLUSION: ChPV2 is a novel goat PV closely related to the Xi-PV1 species infecting bovines. Phylogenetic relationships and genome architecture of ChPV2 and closely related PV types suggest at least two independent E6 losses within the XiPV clade.

A teat papillomatosis case in a Damascus goat (Shami goat) in Hatay province, Turkey: a new putative papillomavirus?

Papillomaviruses (PVs) are epitheliotropic viruses that cause benign proliferative lesions in the skin (warts or papillomas) and mucous membranes of their natural hosts. Recently, new PVs have been found in many animal species. The most common current approach for identifying novel PV types is based on PCR, using various consensus or degenerated primer (broad-range primers), designed on the basis of the multiple alignment of nucleotide or amino acid sequences of a large number of different human papillomaviruses (HPV). PVs have been classified according to the sequence similarity of one of their capsid proteins, L1, without taking into account other regions of the genome and without considering the phenotypic characteristics of the viral infection. In this study, we performed molecular detection and typing of a PV in a goat with teat papillomatosis. Firstly, PCR was performed using the FAP59/FAP64 and MY09/MY11 primer pairs for the L1 gene region. The PV DNA was found to be positive only with the FAP59/FAP64 primer pair. PV DNA was then tested with three primer sets in four different combinations (L2Bf/FAP64, L2Bf/L1Br, FAP59/FAP64, L1Bf/LCRBr) for the gene region encoding the L1, L2 and LCR proteins. The goat teat papilloma sample was amplified using FAP59/FAP64 primers and two primer pairs (L2Bf/FAP64 and L2Bf/L1Br). We obtained products matching approximately 604 bp of the L1 region of the virus. PV DNA was used for typing using sequence analysis/PCR with some type-specific primers for bovids, caprids and cervids. The results of the sequence analysis suggested one new putative PV type with sequence identity ranging from 46.45 to 80.09% to other known papillomaviruses, including Capra hircus papillomavirus (ChPV-2), bovine papillomavirus (BPV) 6, 7, 10, 11 and 12, Rangifer tarandus papillomavirus 3 (RtPV-3) and BPV-7Z (Alpine wild ruminant papillomavirus; Cervus elaphus papillomavirus). We therefore propose that this is the first identification of a new putative type, MG523274 (HTY-goat-TR2016), in a goat with teat papillomatosis. It is essential to identify PV types in different animal species and investigate their prevalence/distribution and clinical consequences in order to develop appropriate prophylactic and/or therapeutic procedures and to determine the interspecies transmission potential and evolution of PVs.

Identification and molecular characterization of two papillomavirus genotypes in teat papillomatosis cases in hair goats, in Türkiye.

Papillomaviruses can cause benign or malignant proliferations on the host's skin and mucous membranes. Recent genetic studies have identified many new papillomavirus types. In this study, molecular detection and typing was performed on papilloma samples from eight hair goats with teat papillomatosis. The papilloma samples were tested with degenerate (FAP59/FAP64,MY09/MY11) and type-specific primers. One sample was positive with degenerate (MY09/MY11) and two samples were positive with Caprahircus papillomavirus-1 type specific primers. The MY09/MY11 primer sequence indicated identity to the closest Ovine papillomavirus type-2 (77.9%). The ChPV-1 primer sequence was similar to the closest ChPV-1 (99.4%). Investigating papillomavirus types in different animal species is important for determining the evolution, prevalence, host range, and interspecies transmission potential of papillomaviruses, and to indicate suitable therapeutics for later development.

Molecular characterisation of ovine herpesvirus type 2 (OvHV-2) in Turkey.

In this study, the physical examination of 22 cattle revealed clinical signs of malignant catarrhal fever (MCF). Peripheral blood leukocyte (PBL) samples of the 22 cattle, and nasal (n = 7) and conjunctival (n = 9) swab samples from 16 sheep from two different farms, were taken for laboratory examination. The clinical diagnosis of MCF in cows was confirmed by the detection of ovine herpesvirus type 2 (OvHV-2) DNA by polymerase chain reaction (PCR). OvHV-2 DNA was detected by nested-PCR in PBL of one cow with clinical signs and nasal (1/7)-conjunctival(1/9) swab samples of two sheep housed in the same barn. According to the sequence analysis, three slightly divergent viruses were detected. The results indicate the need for additional research in different regions of Turkey to gain a better understanding of the incidence of MCF and its implications for the livestock industry.

Prevalance of Schmallenberg orthobunyavirus (SBV) infection in sampled ruminants in Turkey's Eastern Mediterranean region between 2015 and 2017.

Schmallenberg orthobunyavirus (SBV), first discovered in 2011, belongs to the Orthobunyavirus genus of the Peribunyaviridae family. SBV, which predominantly infects ruminants, can cause severe fetal malformations when pregnant animals are infected during a critical phase of gestation. In this study, 1590 blood serum samples from cattle, sheep, and goats were obtained for serological investigation and 1604 specimens for virological investigation (including 1414 whole blood with EDTA, 165 vaginal swab samples from aborting animals, and tissue samples from 25 dead and/or aborted fetuses) in private and family-type ruminant establishments in Turkey's Eastern Mediterranean region. All the blood serum samples were tested for the presence of antibodies using ELISA, which showed SBV antibodies in 29.11% (95% CI: 26.89%-31.35%). The virological samples were tested using real-time RT-PCR for SBV nucleic acid presence, which showed 3.17% (95% CI:2.32%-4.04%) were positive. Finally, 10 different Culicoides species (a total of 29,156 Culicoides, including 16,005 females and 13,151 males) were tested to identify the vectors thought to carry infections in the region. However, no SBV nucleic acid was detected in the Culicoides pools.

Genetic variability of bovine herpesvirus type 4 (BoHV-4) field strains from Turkish cattle herds.

Bovine herpesvirus type 4 (BoHV­4) is a common virus in the world that is detected in clinically ill or in apparently healthy cattle. This study provides a molecular characterization of BoHV­4 strains from 24 cattle some showing respiratory and/or reproductive problems and some without any apparent clinical sign. This study also reported the growth properties of five BoHV­4 field isolates. The 24 sampled cattle came from 13 different herds in 10 provinces collected between 2007 and 2018. Phylogenetic analysis using partially amplified nucleotide sequences of ORF8 genes coding glycoprotein B (n = 24) and ORF3 genes coding thymidine kinase (n = 9), demonstrated genetic variability among the BoHV­4 strains analysed. The partial gB gene sequences clustered in three different genotypes (genotype I, II and III) were located within the genotype I cluster, such as Movar strain. The analysis of the five BoHV­4 strains isolated from vaginal swabs (n = 2), nasal swab (n = 1), and brain samples (n = 2) revealed no significant differences in their growth properties in MDBK cell culture.

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.

Detection of genotype 1 bovine leukemia virus from a C.schultzei pool: Do Culicoides spp. have a role on the transmission of bovine leukemia virus?

Bovine leukemia virus (BLV) is known as the etiological agent of Enzootic bovine leukosis (EBL), which is the most common neoplastic disease of cattle. While the major route of virus transmission is believed to be iatrogenic, BLV proviral DNA has been identified in biological materials, including nasal secretions, saliva, milk, colostrum, and semen, and in several insect species, including horses flies. However, insects' role in the natural transmission of BLV has not been clearly demonstrated. This study assessed the possible role of midges - Culicoides spp. - in BLV transmission. BLVs were genetically characterized and BLV infection seroprevelance was determined in 224 cattle sampled from 27 different small family herds in five different districts in Hatay province, southern Turkey. Out of the 25 Culicoides spp. pools, one (4.0%; 1/25) was a C.schultzei pool while 2.67% (6/224) of the sampled cattle were positive for BLV nucleic acid. The seroprevalance rates for the sampled herds and all sampled cattle were 7.40% (2/27) and 1.33% (3/224), respectively. According to the phylogenetic analysis, the sequences of the BLVs from the cattle (n = 6) and the one BLV-positive C.schultzei pool clustered on genotype 1 (G1) BLVs. Although these results do not reveal the exact role of Culicoides spp. or other midges flies in BLV transmission, the simultaneous presence of same substitions in BLVs from both cattle and a C.schultzei pool is noteworthy. Further studies on the env gene and other BLV gene regions detected from cattle and C.schultzei pools are ongoing to understand the possible epidemiological relationship between cattle and flies.

Bovine Herpesvirus Type 4 (BoHV-4) Vector Delivering Nucleocapsid Protein of Crimean-Congo Hemorrhagic Fever Virus Induces Comparable Protective Immunity against Lethal Challenge in IFNα/ß/γR-/- Mice Models.

Crimean-Congo hemorrhagic fever virus (CCHFV) is the causative agent of a tick-borne infection with a significant mortality rate of up to 40% in endemic areas, with evidence of geographical expansion. Due to a lack of effective therapeutics and control measures, the development of a protective CCHFV vaccine remains a crucial public health task. This paper describes, for the first time, a Bovine herpesvirus type 4 (BoHV-4)-based viral vector (BoHV4-∆TK-CCHFV-N) and its immunogenicity in BALB/c and protection potential in IFNα/ß/γR-/- mice models in comparison with two routinely used vaccine platforms, namely, Adenovirus type 5 and a DNA vector (pCDNA3.1 myc/His A), expressing the same antigen. All vaccine constructs successfully elicited significantly elevated cytokine levels and specific antibody responses in immunized BALB/c and IFNα/ß/γR-/- mice. However, despite highly specific antibody responses in both animal models, the antibodies produced were unable to neutralize the virus in vitro. In the challenge experiment, only the BoHV4-∆TK-CCHFV-N and Ad5-N constructs produced 100% protection against lethal doses of the CCHFV Ank-2 strain in IFNα/ß/γR-/- mice. The delivery platforms could not be compared due to similar protection rates in IFNα/ß/γR-/- mice. However, during the challenge experiment in the T cell and passive antibody transfer assay, BoHV4-∆TK-CCHFV-N was dominant, with a protection rate of 75% compared to others. In conclusion, vector-based CCHFV N protein expression constitutes an effective approach for vaccine development and BoHV-4 emerged as a strong alternative to previously used viral vectors.

First genetic characterization of equine adenovirus type 1 (EAdV-1) in Turkey.

Equine adenovirus type 1 (EAdV-1) is a cause of repiratory tract infection in equids. In present study for the first time in Turkey, the prevalence of EAdV-1 in nasal swab samples obtained from horses showing respiratory symptoms was investigated by polymerase chain reaction (PCR), and molecular characterization of the hexon gene detected in the Turkish (TR) strain was performed. Overall, the prevalence of EAdV-1 was found low (1.4%) as indicated by a positive PCR reaction from the nasal swab extracts tested. Phylogenetic analysis based on the partial sequences of the hexon gene of a TR-EAdV-1 strain with those of previously isolated AdVs from different mammals and an EAdV-1 M1 strain showed that the EAdV-1 strains were placed into a unique cluster. Although the TR-EAdV-1 strain was closely related to CAV-1, CAV-2 and bat adenovirus reference strains, larger-scale studies are necessary to better understand the molecular epidemiology and population structure of EAdV-1 in Turkey.