Ovine adenoviruses infecting sheep and goats in Türkiye: detection and molecular characterization of three different types.

Adenoviruses (AdVs) have been detected in a wide variety of animals. To date, eight types of AdVs in sheep and two types in goats have been identified, which belong to two distinct genera, Mastadenovirus and Atadenovirus. Typically, the term pneumo-enteritis is used to describe adenovirus-induced disease in small ruminants, which has been associated with both enteric and respiratory symptoms of varying severity. The aim of this study was to detect and identify AdVs of small ruminants belonging to the genera Mastadenovirus and Atadenovirus. For this purpose, diagnostic samples (47 lung, 27 intestine, and two pooled tissue samples including intestine and lung) from 49 small ruminants (39 sheep and 10 goats) were used. Following the viral DNA extraction, PCR was carried out by using the primers targeting the hexon gene in order to detect both mast- and atadenoviruses. Sequencing the amplified fragments revealed the presence of three types of ovine adenovirus (OAdV): OAdV-3, OAdV-4, and OAdV-8. Specifically, OAdV-3 was detected in two sheep and a goat while OAdV-4 and OAdV-8 were found in only one sheep each. There is still limited data on the interaction between the viruses in different adenovirus genera and the detected disease, as well as the genetic diversity of adenoviruses, especially in small ruminants. In conclusion, the detection of AdVs in lung and intestinal tissues of small ruminants in this study suggests that these viruses may have contributed to the disease and/or predisposed to other agents.

Molecular analysis of the env, LTR, and pX regions of bovine leukemia virus in dairy cattle of Türkiye.

Bovine leukemia virus is a retrovirus that causes enzootic bovine leukosis and is associated with global economic losses in the livestock industry. The aim of this study was to investigate the genotype determination of BLVs from cattle housed in 6 different farms in Türkiye and the characterization of their LTR and pX (tax, rex, R3, and G4 gene) regions. For this purpose, blood samples from 48 cattle infected with BLV were used. The phylogenetic analysis based on the env gene sequences revealed that all BLVs were clustered in genotype 1 (G1), and the sequences of the LTR (n = 48) and the pX region (n = 33) of BLVs were obtained. Also, analysis of these nucleic acid and amino acid sequences allowed assessments similar to those reported in earlier studies to be relevant to transactivation and pathogenesis. This study reports the molecular analysis of the LTR and pX region of BLVs in Türkiye for the first time.

Molecular characterization of picobirnaviruses in small ruminants with diarrhea in Turkey.

Picobirnaviruses (PBVs), detected in a wide range of host species, are viruses of which limited information is available about their pathogenic potential, ecology, or evolutionary characteristics. In this study, a molecular analysis of segment 2 encoding the PBV RNA-dependent RNA-polymerase (RdRp) in small ruminants with diarrhea in Turkey was undertaken. A total of 66 fecal samples or gut contents from diarrheic small ruminants including 55 sheep and 11 goats were screened. Four samples (6.06%), obtained from sheep in different farms, yielded the expected amplicon size for the genogroup I RdRp gene fragment, whereas no positivity was detected for genogroup II PBVs. Phylogenetic analysis revealed high levels of genetic diversity among the genogroup I PBVs. Additionally, all PBV infected sheep were also positive for rotavirus A. This study, reporting the presence of the PBVs in sheep Turkey for the first time, contributes to the molecular characterization and epidemiology of PBVs.

Whole-genome analysis of a rare G15P[21] group A rotavirus detected at a dairy cattle farm.

Group A rotaviruses (RVAs) are a major cause of severe enteritis in humans and animals. RVAs have been identified in several animal species and their genetic diversity, the segmented nature of their RNA genome and the ability to spill over from one species to another can generate new RVA strains. In this study, we investigated the genome constellations of an unusual, rare, bovine RVA strain, G15P[21], identified from a farm with neonatal diarrhoea of calves in 2006. In parallel, the genome constellations of other RVA strains with different G/P types identified from the same farm in the same time span (2006-2008) were analysed. The genome constellation of strain K53 was G15-P[21]-I2-R2-C2-M2-A13-N2-T9-E2-H3 and was similar, overall, to that of the other bovine RVA strains (G6/10-P[11]-I2-R2-C2-M2-A13-N2-T6-E2-H3) with the exception of the NSP3 segment (T9 vs T6). This study describes RVA genomes with different genotype combinations isolated at a farm and also contributes to the understanding of the diversity and evaluation of rotavirus in a global context.

Bovine coronavirus infections in Turkey: molecular analysis of the full-length spike gene sequences of viruses from digestive and respiratory infections.

Bovine coronavirus (BCoV) can be spread by animal activity. Although cattle farming is widespread in Turkey, there are few studies of BCoV. The aim of this study was to evaluate the current situation regarding BCoV in Turkey. This is the first study reporting the full-length nucleotide sequences of BCoV spike (S) genes in Turkey. Samples were collected from 119 cattle with clinical signs of respiratory (n = 78) or digestive tract (n = 41) infection on different farms located across widely separated provinces in Turkey. The samples were screened for BCoV using RT-nested PCR targeting the N gene, which identified BCoV in 35 samples (9 faeces and 26 nasal discharge). RT-PCR analysis of the S gene produced partial/full-length S gene sequences from 11 samples (8 faeces and 3 nasal discharge samples). A phylogenetic tree of the S gene sequences was made to analyze the genetic relationships among BCoVs from Turkey and other countries. The results showed that the local strains present in faeces and nasal discharge samples had many different amino acid changes. Some of these changes were shown in previous studies to be critical for tropism. This study provides new data on BCoV in Turkey that will be valuable in designing effective vaccine approaches and control strategies.

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.

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.

Isolation and genetic characterization of bovine ephemeral fever virus from epidemic-2020 in Turkey.

Bovine ephemeral fever virus (BEFV) infection occurs seasonally in many tropical and subtropical regions of Africa, Asia (including the Middle East), and Australia while it is exotic in Europe. In this study, the epidemiology of BEFV infection in Turkey that bridges southeastern Europe and Asia, geographically, was investigated according to the comparison of the nucleotide sequences of the virus caused the last epidemic in 2020 with those of the strains previously detected in Turkey as well as BEFV strains from other countries. In the phylogenetic analysis, based on an alignment of full-length G gene sequences, BEFVs from epidemic-2020 were located in Middle Eastern lineage and appear to represent most closely related BEFVs from India-2018 and 2019. The findings will contribute to a better understanding of BEFV epidemiology in Turkey.

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.

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.

An assessment of bovine herpes virus 4 as a causative agent in abortions and neonatal death.

Numerous viruses, including bovine viral diarrhoea virus (BVDV), bovine herpes virus 1 (BoHV-1) and bovine herpes virus 4 (BoHV-4), and other pathogens are the most common causes of reproductive disorders and are responsible for huge economic losses in livestock production. This study investigates the aetiological role of BoHV-4 in fertility problems such as abortions, stillbirth and birth with unviable calves. Retrospective samples from 38 animals, including 17 aborting cows, 17 aborted foetuses, three stillborn calves and one unviable newborn calf were analysed. The BoHV-4 genome was detected in 25 (65.7%) animals by polymerase chain reaction. In 14 of these infected animals, we detected co-infection with BVDV, while the co-presence of BoHV-1 was also detected in one animal. In addition to the high prevalence of BoHV-4 genome in materials related to fertility problems, isolation of BoHV-4 from the brain of one stillborn calf indicated a causal link between BoHV-4 and fertility problems, such as abortion, stillbirths or birth with unviable calves.

Identification of rotavirus A strains in small ruminants: first detection of G8P[1] genotypes in sheep in Turkey.

Although members of rotavirus group A (RVA) are major enteric pathogens of humans and animals of many species, their impact on the health of small ruminants is not well documented. In this study, we conducted a molecular analysis of VP4, VP7, VP6 and NSP4 genes of RVAs detected using a commercial antigen ELISA in small ruminants with or without diarrhea in Turkey. Of the RVAs detected in sheep, one strain (Kutahya) was characterized as genotype G8P[1]-I2-E2. Two others (Ankara-1 and Ankara-2) were identified as NSP4 E2 and VP6 I2 genotypes, although they were untyped for the VP4 and VP7 genes. The RVAs from two goats were characterized as genotype G6P [1]-I2-E2. This is the first detection of in goats RVA genotypes G6P [1], which had previously only been found in cattle in Turkey, and of RVA in sheep. The study extends our current knowledge about the circulation of two RVA G genotypes, G6 and G8, in goat herds, and the detection of the G8 genotype in sheep in Turkey. This provides further information about the molecular epidemiology of RVAs in different animal species and indicates that additional surveillance programs are needed to determine the epidemiology of RVA in small ruminants and other species.

Molecular and antigenic characterization of bovine herpesvirus type 1 (BoHV-1) strains from cattle with diverse clinical cases in Turkey.

The present study reports the molecular and antigenic characterization of 13 bovine herpesvirus type 1 (BoHV-1) field viruses obtained from cattle with different clinical cases in Turkey between 1992 and 2017. We selected glycoprotein C (gC) of BoHV-1 as a target to detect and/or verify presence of the virus in suspect materials followed by virus isolation (VI) in MDBK cells. In seven out of 13 BoHV-1 positive samples, cytophatic effects (CPEs) were observed in MDBK cell cultures, although only four virus samples reached a sufficient titer to use in phylogenetic assay, restriction endonuclease analysis (REA), and virus neutralization test (VNT). According to the results of sequence analysis of the 13 BoHV-1 positive samples, nine BoHV-1 field viruses were determined as BoHV-1.1 and four as BoHV-1.2. Using REA, we demonstrated that two of our isolated viruses could be categorized as BoHV-1.1 while the other two isolates were BoHV-1.2 subtypes. Differences between the BoHV-1.1 and BoHV-1.2 isolates were also detected in the VNT results by assaying 125 suspected serum samples after testing with isolated (KY748023, KY748022, KY748020, and KY748021) and reference viruses (BoHV-1 Cooper and BoHV-5 Texas 89). These results are indicating the need to correctly identify BoHV-1 field isolates to better understand the epidemiology and pathogenesis of infection. In addition, it would be useful to identify the subtypes circulating in the specific geographical area while determining vaccination preferences.

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.

Molecular characterization of bovine noroviruses and neboviruses in Turkey: detection of recombinant strains.

To investigate the molecular epidemiology and genetic diversity of bovine enteric caliciviruses, a total of 167 fecal samples from diarrheic calves were screened. Bovine noroviruses (BoNoVs) and neboviruses were detected in 56 (33.5%) and 37 (22.1%) fecal samples, respectively. Sequences of the RdRp and capsid gene of selected BoNoVs showed that the GIII.1 and GIII.2 genotypes were in circulation in Turkey. Two of the BoNoV strains were identified as recombinant strains (GIII.P1/GIII.2). All examined neboviruses possessed a Nebraska-like RdRp gene. The two nebovirus strains were classified into lineage 4 based on phylogenetic analysis of VP1 amino acid sequences. One of them showed evidence of a recombination event within the S domain. This study is thus the first to reveal the presence of the BoNoV GIII.1 genotype and recombinant strains of BoNoV and neboviruses in Turkey.

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.

Long-term study (2005-2010) on the vaccination with BoHV-1 glycoprotein E-deleted marker vaccine in selected two dairy herds in Turkey.

A follow-up study from 2005 to 2010 was carried out in two herds where eradication programme for the bovine herpes virus-1 (BoHV-1) infection depends on the vaccination with inactivated glycoprotein E-deleted vaccine that was started in 2001 following the vaccination with inactivated conventional vaccine between 1999 and 2001. For serological screening, a total of 12,976 sera sampled over several sampling times approximately 6 months of interval during 5 years (2005-2010) were tested for glycoprotein E (gE)- and glycoprotein B-specific antibodies using ELISA. According to the serological evidence, the long-term persistence of BoHV-1 antibodies, success of marker vaccine, first vaccination time of the calves in herds regularly vaccinated, etc. were discussed in this paper. In conclusion, the vaccination programme using gE (-) marker vaccines, with making efforts to prevent the other factors about transmission of infection, was suggested for the eradication of BoHV-1 infection in Turkey as many EU countries. This is the first report on the BoHV-1 eradication programme in some dairy cattle in Turkey.

Comparison of genetic characteristics of canine papillomaviruses in Turkey.

Papillomavirus (PV) infections often cause benign and malignant skin neoplasia in dogs. To date, twenty types of canine papillomaviruses (CPVs) have been described worldwide. A detailed molecular characterization of CPVs in Turkey is lacking. In the present study, oral and mucosal lesions from 13 dogs with suspected CPV infection from the Mediterranean and central Anatolian regions of Turkey were analyzed. The partial gene sequences of the L1, E6, and E7 regions were compared with those of CPV types in the GenBank database. The results showed that CPV-1 infection was the dominant type of canine papillomatosis in Turkey. In addition, there was no statistically significant association between the frequency of the disease and the age or gender of the dog (p>0.05). However, all the dogs were pedigree breeds, suggesting that the disease may be more prevalent among pure-bred dogs than mixed breeds.

Putative vaccine breakthrough event associated with heterotypic rotavirus infection in newborn calves, Turkey, 2015.

Group A rotaviruses (RVA) are regarded as major enteric pathogens of large ruminants, including cattle. Rotavirus vaccines administered to pregnant cows are commonly used to provide passive immunity that protects newborn calves from the clinical disease. In this study we report the detection of RVA from calves with severe diarrhea in a herd regularly vaccinated to prevent enteric infections including RVA. Diarrheic disease was observed in newborn calves aged 4-15days, with high morbidity and mortality rates, but no diarrhea was seen in adult animals. Rotavirus antigen was detected by enzyme-immunoassay in the intestinal content or the fecal samples of all examined animals. Besides RVA, bovine coronavirus and bovine enteric calicivirus were detected in some samples. Selected RVA strains were characterized by whole genome sequencing. Two strains, RVA/Cow-wt/TUR/Amasya-1/2015/G8P[5] and RVA/Cow-wt/TUR/Amasya-2/2015/G8P[5] were genotyped as G8-P[5]-I2-R2-C2-M2-A3-N2-T6-E2-H3 and showed >99% nucleotide sequence identity among themselves. This genomic constellation is fairly common among bovine RVA strains; however, phylogenetic analysis of the G8 VP7 gene showed close genetic relationship to some European human RVA strains (up to 98.4% nt identity). Our findings is the first indication regarding the circulation of G8 RVA strains in Turkey. Given that the administered RVA vaccines contained type G6 and G10 VP7 antigens some concerns raised with regard to the level of heterotypic protection elicited by the vaccine strains against circulating bovine G8 RVA strains. Enhancement of surveillance of circulating RVA strains in calves across Turkey is needed to support ongoing vaccination programs.

Identification of a Bovine Enteric Calicivirus, Kirklareli Virus, Distantly Related to Neboviruses, in Calves with Enteritis in Turkey.

A calicivirus was detected in neonatal calves with enteritis in Kirklareli, Thrace, Turkey. In the full-length genome, Kirklareli virus was related (48% nucleotide identity) to bovine enteric caliciviruses (Nebovirus genus). The virus was also detected in a herd in Ankara, Central Anatolia, but not in other Turkish prefectures.