Investigation of canine chaphamaparvovirus, canine bufavirus, and canine adenovirus in dogs with diarrhea: First report of novel canine bufavirus in Turkey.

Viral enteritis is a significant cause of death among dogs younger than 6 months. In this study, the presence of canine chaphamaparvovirus (CaChPV), canine bufavirus (CBuV), and canine adenovirus (CAdV) was investigated in 62 diarrheal dogs previously tested for other viral pathogens (canine parvovirus type 2, canine coronavirus, and canine circovirus). CBuV was detected in two dogs (3.22%) and CaChPV in one dog (1.61%). One dog tested positive for three parvoviruses (CPV-2b, CBuV, and CaChPV). All dogs tested negative to CAdV-1/CAdV-2. A long genome fragment of one of the two identified CBuVs and of the CaChPV was obtained and analyzed. New Turkish CBuVs had high identity rates (96%-98% nt; 97%-98% aa) with some Italian CBuV strains (CaBuV/9AS/2005/ITA and CaBuV/35/2016/ITA). The phylogenetic analysis powerfully demonstrated that these viruses belonged to a novel genotype (genotype 2). A part of the genome ChPV-TR-2021-19 revealed high identity rates (> 98% nt and > 99% aa) with some Canadian CaChPV strains (NWT-W88 and NWT-W171) and the Italian CaChPV strain Te/37OVUD/2019/IT. This study is the first report on the detection of CBuV-2 and the concomitant presence of three canine parvoviruses in Turkey. The obtained data will contribute to the molecular epidemiology and the role in the etiology of enteric disease of new parvoviruses.

Predominance and first complete genomic characterization of canine parvovirus 2b in Turkey.

Viral enteritis is a significant threat to domestic dogs. The two primary pathogens that cause viral enteritis in dogs are canine coronavirus (CCoV) and canine parvovirus (CPV). In this study, we investigated the occurrence of CPV-2, CCoV, and canine circovirus coinfection by characterizing circulating subtypes of CPV-2 in faecal samples from symptomatic dogs admitted to veterinary clinics located in Ankara, Elazig, Kayseri, and Kocaeli provinces of Turkey, between 2019 and 2022. Virus detection by PCR and RT-PCR revealed that CPV-2 was present in 48 (77.4%) samples, and no other agents were detected. Based on the occurrence of the codon GAT at positions 1276 to 1278 (coding for aspartate at residue 426) of VP2, all CPV-2 isolates were confirmed to be of the CPV-2b subtype. The complete genome sequences of two CPV-2b isolates showed a high degree of similarity to and phylogenetic clustering with Australian and East Asian strains/isolates. The predominant CPV strain circulating in the three different regions of Turkey was found to be a CPV-2b strain containing the amino acid substitutions at Y324I and T440A, which commonly contribute to immune escape. This is the first report of complete genomic analysis of CPV-2 isolates circulating in symptomatic domestic dogs in Turkey. The evolution of CPV-2 has raised questions about the efficacy of current vaccination regimes and highlights the importance of monitoring the emergence and spread of new CPV-2 variants.

First report of Serotype-1 Marek's disease virus (MDV-1) with oncogenic form in backyard turkeys in Turkey: a molecular analysis study.

BACKGROUND: Marek's disease (MD) is a lymphoproliferative disease caused by Gallid alphaherpesvirus 2 (GaHV-2, MDV-1), which primarily affects chickens. However, the virus is also able to induce tumors and polyneuritis in turkeys, albeit less frequently than in chickens. RESULTS: This is the first study in Turkey reporting the molecular characterization of a MDV-1 strain detected in a flock of backyard turkeys exhibiting visceral lymphoma. Here, MEQ, vIL-8, pp38 and 132-bp tandem repeat regions, which are frequently preferred in the pathotyping of MDV-1, were examined. It was determined that the MEQ gene of MDV-1/TR-21/turkey strain obtained in the present study encoded 339 amino acids (1020 nt) and had four proline-rich repeat regions (PPPP). Based on the nucleotide sequence of the MEQ gene of the MDV-1/TR-21/turkey strain, a phylogenetic tree was created using the MEGA-X software with the Maximum Likelihood Method (in 1000 replicates). Our strain was highly identical (> 99.8) to the Italian/Ck/625/16, Polish (Polen5) and some Turkish (Layer-GaHV-2-02-TR-2017, Tr/MDV-1/19) MDV-1 strains. Also, nt and aa sequences of the MEQ gene of our strain were 99.1 and 99.41% identical to another Turkish strain (MDV/Tur/2019) originated from chickens. Sequence analysis of pp38 and vIL-8 genes also supported the above finding. The identity ratios of nucleotide and amino acid sequences of vIL-8 and pp38 genes of MDV-1/TR-21/turkey strain were 99.64-100% and 99.79-100%, respectively, when compared with those of the Polish strain. According to 132-bp tandem repeat PCR results, the MDV-1/TR-21/turkey strain had five copies. CONCLUSIONS: These results suggested that the MDV-1/TR-21/turkey strain obtained from backyard turkeys can be either very virulent or very virulent plus pathotype, though experimental inoculation is required for precise pathotyping.

Development of a protective inactivated vaccine against Crimean-Congo hemorrhagic fever infection.

Crimean-Congo hemorrhagic fever (CCHF) is an emerging zoonotic infectious disease caused by Crimean-Congo hemorrhagic fever virus (CCHFV). The first clinical CCHF infection was described in 1944 in the Crimean Peninsula, exclusively in humans, with case-fatality rates exceeding 30%. The increasing number of cases, high mortality rate, and lack of effective therapy make CCHF a serious threat to public health and a potential bioterrorism agent. The present study evaluated the development, immunogenicity, and immune response durations for cell-culture-derived inactivated vaccine (CCVax) formulations in comparison with those of mouse-brain-derived vaccine (MBVax) formulations. In this study, the Kelkit06 CCHF virus strain was propagated in both suckling mice and Vero E6 cells, and purified with a sucrose gradient. Formalin-inactivated vaccine candidates were formulated at various doses [low dose (LD), 5 µg; medium dose (MD), 10 µg; high dose (HD), 20 µg)] and mixed with an alum adjuvant. BALB/c mice received the same doses of the vaccine formulations three times at 3-week intervals. The humoral endpoint IgG responses were evaluated and compared for the MBVax and CCVax treatments. The duration of the presence of IgG and neutralizing antibody (Ab) titers was evaluated and compared until up to 1 year after immunization. The humoral IgG responses indicated that the CCVax and MBVax candidates enhanced the IgG endpoint titers in a dose-dependent manner, which were induced more strongly in all the CCVax groups than in the MBVax mice. The fold changes in neutralizing Ab levels were also found to be higher in the CCVax groups: between 2- and 7.6-fold after the second week of the last immunization. The neutralization titers peaked 4 months after immunization in all the vaccine-receiving groups, but these were still comparable at the end of the first year. The CCVax formulations induced higher IgG and neutralizing Ab titers at all the measured time points. In this study, we showed that cell-culture-purified and formalin-inactivated vaccine candidates induced strong and robust immunity in vaccinated mice dose-dependently, more so than mouse-brain-derived vaccines.

Detection and molecular characterization of a highly oncogenic Marek's disease virus from vaccinated hens in Turkey.

Marek's disease (MD) is a highly contagious neoplastic disease of chickens associated with economic losses, often due to visceral lymphomas. The etiological agent is MD virus serotype 1 (MDV-1), also called Gallid alphaherpesvirus 2 (GaHV-2). Despite intensive vaccination, MDV is constantly evolving and maintaining its presence in the world. The aim of this study was to genetically analyze a highly oncogenic MDV/Tur/2019 strain obtained from a poultry farm in Turkey's Elazig province in 2019. Genes associated with viral pathogenicity and oncogenicity Marek's EcoRI-Q-encoded protein (MEQ), phosphoprotein-38 (pp38), and viral interleukin 8 (vIL-8) were selected for this purpose. The vIL-8 nucleotide sequence showed high similarity (100% identity) to some European (EU-1, Polen 5) and Asian (03 India, GADVASU-M2) MDV strains. The pp38 nucleotide sequence showed high similarity (100% identity) to some American (CU-2, JM/102W, RB1B) and European (MD70/13, ATE2539) MDV strains. There were no disrupted four-proline molecules (PPPP) within the transactivation domain of the MEQ. However, according to phylogenetic results, the MDV/Tur/2019 strain was included in cluster 2a alongside European MDV strains (Polish, Hungarian, Italian) with very virulent and very virulent plus pathotypes. In conclusion, we believe that the MDV/Tur/2019 strain obtained from turkey herpesvirus (HVT)-vaccinated chickens has a very virulent or very virulent plus pathotype. Although this result provides some clues regarding the virulence of this strain, in vivo studies are needed to achieve exact pathotyping. Further, combination of HVT with the CVI988 strain should be used for vaccination to provide the best protection, as highly pathogenic MDV strains can break sterile immunity against the HVT vaccine. Keywords: GaHV-2; Marek's disease; oncogenes; Turkey.

Detection and molecular characterisation of feline viruses from swab samples.

Feline calicivirus (FCV), feline alphaherpesvirus 1 (FHV-1) and feline panleukopenia virus (FPLV) as well as retroviral agents such as feline leukaemia virus (FeLV) and feline immunodeficiency virus (FIV) are important viral pathogens of cats. The aim of this study was to detect and characterise FHV-1, FPLV, FeLV, FIV and feline foamy virus (FFV) in oropharyngeal, nasal and conjunctival swabs from 93 cats that had been screened for FCV previously. We wanted to determine the possible risk factors for infection with these viruses. The prevalence was found to be 12.9% for FHV-1 and 9.7% for FPLV. FIV was detected only in two samples and FeLV in one sample, whereas the presence of FFV was not demonstrated in any of the clinical samples. The statistical analysis of the results showed that breed, age, health status, and lifestyle are important predisposing factors to FHV-1 (P < 0.05). For FPLV, only clinically unhealthy animals were found to be at risk (P < 0.001). Sequence analysis revealed that the two FIV-positive samples in this study contained different (A and B) subtypes of the virus. This is the first report on the occurrence of subtype A FIV in Turkey.

Interpretation of SARS-CoV-2 behaviour on different substrates and denaturation of virions using ethanol: an atomic force microscopy study.

Coronavirus (SARS-CoV-2) is a respiratory infection virus that was first detected in Wuhan, China. The virus causes COVID-19 disease and the outbreak was recognised as a pandemic by the World Health Organization (WHO) in March 2020. SARS-CoV-2 virion was first imaged using cryo-electron microscopy by the Chinese Center for Disease Control and Prevention (CDC). Atomic Force Microscopy is a unique technique that can allow imaging of biomolecules under different conditions. In this work, we used Atomic Force Microscopy to characterize SARS-CoV-2 on tissue culture polystyrene (TCPS) and glass coverslip surfaces. We isolated SARS-CoV-2 and drop casted it on coverslip glass and tissue culture polystyrene surfaces. We analyzed height profiles, density, and aggregation behavior of the virion on glass and polystyrene surfaces. We observed the coffee ring effect on the drop casted samples and close packing of virions near the coffee rings on both surfaces with relatively higher virion distribution on the tissue culture polystyrene (TCPS) substrates. We compare virion agglomeration on the two types of surfaces. Finally, we applied ethanol disinfectant to virions on the surface to visualize the effect of ethanol and image the ultrastructure of SARS-CoV-2.

Complete genome analysis of highly pathogenic bovine ephemeral fever virus isolated in Turkey in 2012.

Relatively high prevalence and mortality rates of bovine ephemeral fever (BEF) have been reported in recent epidemics in some countries, including Turkey, when compared with previous outbreaks. A limited number of complete genome sequences of BEF virus (BEFV) are available in the GenBank Database. In this study, the complete genome of highly pathogenic BEFV isolated during an outbreak in Turkey in 2012 was analyzed for genetic characterization. The complete genome of the Turkish BEFV isolate was amplified by reverse transcription-polymerase chain reaction (RT-PCR) and sequenced. It was found that the complete genome of the Turkish BEFV isolate was 14,901 nt in length. The complete genome sequence obtained from the study showed 91-92% identity at nucleotide level to Australian (BB7721) and Chinese (Bovine/China/Henan1/2012) BEFV isolates. Phylogenetic analysis of the glycoprotein gene of the Turkish BEFV isolate also showed that Turkish isolates were closely related to Israeli isolates. Because of the limited number of complete BEFV genome sequences, the results from this study will be useful for understanding the global molecular epidemiology and geodynamics of BEF.

Immunization of knock-out α/ß interferon receptor mice against high lethal dose of Crimean-Congo hemorrhagic fever virus with a cell culture based vaccine.

Crimean-Congo hemorrhagic fever (CCHF) is an acute tick-borne zoonotic disease. The disease has been reported in many countries of Africa, Asia, the Middle East, and in Eurasia. During the past decade, new foci of CCHF have emerged in the Balkan Peninsula, southwest Russia, the Middle East, western China, India, Africa, and Turkey. CCHF virus produces severe hemorrhagic manifestations in humans with fatality rates up to 30%. Vaccine development efforts have been significantly hampered by a lack of animal models and therefore, no protective vaccine has been achieved. Lately, IFN α/ß receptor deficient (IFNAR-/-) mice have been established as a novel small animal model of CCHF virus infection. In the present study, we found that IFNAR-/- mice highly susceptible to CCHF virus Turkey-Kelkit06 strain. Immunization with the cell culture based vaccine elicited a significant level of protection against high dose challenge (1,000 PPFU) with a homologous CCHF virus in IFNAR-/- mice.

A large-scale outbreak of bovine ephemeral fever in Turkey, 2012.

Regional cases of bovine ephemeral fever (BEF) were documented previously in Turkey. Previous cases were confirmed in South-East Turkey with low cow mortality. Recent BEF-suspected outbreaks with high mortality were documented in many regions of Turkey in 2012. The aim of study was the epidemiological examination of the outbreak and molecular characterization of the viruses detected from the outbreak. For this reason, blood samples were collected from BEF-suspected outbreak regions. From the results of RT-PCR, high rate of BEF-suspected samples (48/60 or 80%) was found positive for BEF virus (BEFV) RNA. The nucleotide sequences of the G1 region of G gene of BEFV in the current study during the 2012 outbreak were grouped into cluster II of BEFV. It was suggested that BEFV may be spread out to other neighbor countries in the future years.

Antibodies to Schmallenberg virus in domestic livestock in Turkey.

This, partly retrospective study, was designed to determine the seroprevalence of Schmallenberg virus (SBV), a new Orthobunyavirus first reported in Germany in late 2011, in domestic ruminants from the Middle Black Sea, West, and Southeast regions of Turkey. An indirect enzyme-linked immunosorbent assay was used to screen serum samples collected from slaughterhouse animals between 2006 and 2013. The overall seroprevalence was 335/1,362 (24.5 %) with 325/816 (39.8 %), 5/307 (1.6 %), 3/109 (2.8 %), and 2/130 (1.5 %) recorded in cattle, sheep, goats, and Anatolian water buffalo, respectively. This is the first study to demonstrate the presence of antibodies to SBV in Turkish ruminants; it indicates that cattle are more susceptible to infection than sheep, goats, or buffalo and that exposure of domestic ruminants to SBV in Turkey may have occurred up to 5 years prior to the first recorded outbreak of the disease in 2011.

Pseudo-plaque reduction neutralization test (PPRNT) for the measurement of neutralizing antibodies to Crimean-Congo hemorrhagic fever virus.

BACKGROUND: Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne virus of the genus Nairovirus family Bunyaviridae, which are enveloped viruses containing tripartite, negative polarity, single-stranded RNA. CCHF is characterized by high case mortality, occurring in Asia, Africa, the Middle East and Europe. Currently, there are no specific treatments or licensed vaccines available for CCHFV. Recently, two research groups have found adult mice with defective interferon responses allowed to lethal CCHFV infection. These mouse models could provide invaluable information for further studies. Efforts to develop a vaccine against CCHFV are being made. To determine the efficacy of vaccine candidates it is important to conduct serological studies that can accurately measure levels of protective antibodies. In the present study, a pseudo-plaque reduction neutralization test (PPRNT) based on enzyme-catalyzed color development of infected cells probed with anti-CCHFV antibodies was used to measure neutralization antibody of CCHFV. METHODS: Sixty-nine human serum samples (20 acute and 49 convalescent) were tested. The presence of CCHFV antibodies was determined and confirmed by a commercial ELISA kit. CCHFV RNA was determined by RT-PCR. All the samples were analyzed by PPRNT and fluorescent focus reduction neutralization test (FFRNT) to measure of CCHFV-neutralizing antibodies. RESULTS: Pseudo-plaque reduction neutralization test showed a high sensitivity (98%), specificity (100%) and agreement (96,6%) in qualitative comparison with those of the FFRNT. There was a high correlation between the titers obtained in PPRNT and FFRNT (R2 = 0.92). The inter- and intra-assay variation of PPRNT revealed good reproducibility and positive cut-off of PPRNT was defined as 1:4 by the geometric mean titers for the individual samples distributed. CONCLUSION: The pseudo-plaque reduction neutralization test described in this study is a fast, reproducible and sensitive method for the measurement of CCHF neutralizing antibodies. This novel assay could serve as useful tools for CCHF research in epidemiology, vaccine development and other studies of immunity. It also provides an alternative to PRNT when viruses with no or poor CPE in cell culture.

Application of the pseudo-plaque assay for detection and titration of Crimean-Congo hemorrhagic fever virus.

A pseudo-plaque assay was developed for detection and quantitation of Crimean-Congo hemorrhagic fever virus Turkey-Kelkit06. Enzyme-catalyzed color development of infected cells probed with anti-Crimean-Congo hemorrhagic fever virus antibodies was used for determining the titer of Crimean-Congo hemorrhagic fever Turkey-Kelkit06 and for its detection in samples from persons infected with the Crimean-Congo hemorrhagic fever virus. The pseudo-plaque assay accuracy was confirmed by comparing pseudo-plaque assay titers with fluorescent immunofocus assay and focus formation assay titers using three stocks of virus. No significant difference in virus titers of Crimean-Congo hemorrhagic fever Turkey-Kelkit06 among the three methods was observed. The pseudo-plaque assay is more sensitive than the fluorescent immunofocus assay for detecting the virus in primary isolates of Crimean-Congo hemorrhagic fever virus collected from humans, but no difference in sensitivity between the two methods was observed in the cell-adapted strain of Crimean-Congo hemorrhagic fever Turkey-Kelkit06. The pseudo-plaque assay is suitable for titration of Crimean-Congo hemorrhagic fever Turkey-Kelkit06, which does not develop plaques, suggesting it may also be suitable for the detection of other viruses.

The complete genome analysis of Crimean-Congo hemorrhagic fever virus isolated in Turkey.

Crimean-Congo hemorrhagic fever virus (CCHFV) is the causative agent of a tick-borne disease with high mortality rates in humans. The distribution of CCHFV includes over 30 countries in Asia, the Middle East, southeastern Europe, and Africa. It was first recognized in Turkey in 2002, with an increasing number of cases reported between 2002 and 2009. Recent analysis of complete genome sequences of CCHFV isolates has revealed that the genomic plasticity of the virus is surprisingly high for an arthropod-borne virus. We have determined the complete nucleotide and deduced amino acid sequences of strain CCHFV Turkey-Kelkit06 isolated from the blood of a patient in an endemic region of Turkey in 2006. The complete sequence length of the CCHFV Turkey-Kelkit06 strain is 19,186 nt, consisting of a 1673 nt S segment, a 5364 nt M segment, and a 12,149 nt L segment. Based on the analysis of S, M, and L segments, CCHFV Turkey-Kelkit06 clustered in Group V, which represents the Europe/Turkey geographic lineage. Although glycoproteins encoded by the M gene are the most variable part of the CCHFV Turkey-Kelkit06 strain, some functional domains of the glycoproteins are well conserved. Here, we report the complete sequence and genome organization of the CCHFV Turkey-Kelkit06 strain and its phylogenetic relationship to other strains of CCHFV. Collecting data on viral sequences among isolates from CCHF epidemics may provide valuable information regarding the molecular basis of the epidemic potential of the virus.

Crimean-Congo hemorrhagic fever virus: genetic analysis and tick survey in Turkey.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne virus in the family Bunyaviridae, genus Nairovirus. The virus is transmitted to humans through infected tick bites or from direct contact with viremic animals or humans. In the present study, a total of 1,015 adult ticks were collected from cattle (603 specimens), sheep (17 specimens), and goats (395 specimens) in the Kelkit Valley in Turkey. Four tick species were recognized on the animals in the surveyed region. The most abundant species were Rhipicephalus bursa and Hyalomma marginatum marginatum, at 47.68% (484/1,015) and 46.40% (471/1,015), respectively. Reverse transcriptase PCR was used to recover partial sequences of the CCHFV small (S) genome segment. The presence of CCHFV was determined in 3 of 33 (9.09%) R. bursa pools and in 1 of 31 (3.22%) H. m. marginatum pools. Virus sequences from R. bursa were extremely different from those of the Greek CCHFV strain (U04958) isolated from an R. bursa tick. Phylogenetic analysis indicated that the CCHFV isolates obtained in this study clustered in group 5, whose range encompasses southwestern Russian and Kosovo. This is the first evidence of CCHFV in ticks from Turkey. Even though Hyalomma is the main vector for CCHFV, R. bursa may play a role in CCHFV transmission.