Genetic diversity, frequency and concurrent infections of picobirnaviruses in diarrhoeic calves in Turkey.

Calf diarrhoea is one of the major problems in cattle farming with high morbidity and mortality in herds. Two enteric viruses, bovine rotavirus (BRV) and bovine coronavirus (BCoV), are the leading cause of gastroenteritis in young calves, whereas picobirnaviruses (PBVs) are often associated with diarrhoea. In the present study, the faecal specimens of 127 diarrhoeic bovines (less than 1-month-old) were employed to investigate the infection frequencies of these three pathogens. Results indicated that frequencies of BRV and BCoV in diarrhoeic calves were 38.58% and 29.92%, respectively. The 7.08% of bovine calf samples (9 out of 127) were found to be positive for PBV genogroup I. Sequence analysis further revealed the high genetic heterogeneity within representative PBV sequences. Additionally, both PBV-BCoV (n = 2) and BCoV-BRV-PBV (n = 1) co-infections were detected in bovine calves for the first time. Consequently, our findings pointed out the highly divergent nature of PBVs without regard to exact host or territory and the occasional co-existence with other enteric agents.

Molecular analysis of goose parvovirus field strains from a Derzsy's disease outbreak reveals local European-associated variants.

Since its first recognition in the early 1960s, Derzsy's disease has caused significant economic losses in the goose meat industry through the world. Today, Derzsy's disease still maintains its importance for small-scale waterfowl farming, despite not having a significant impact on public health. In the present study, we investigated the distribution of goose parvovirus (GPV) and its potential variants from a 2019 outbreak in Turkey. Tissue samples were obtained from infected eggs and goslings that were raised in distinct farming areas of the various provinces. For this purpose, a novel primer set for amplification of a 630-bp region of VP3 was designed to confirm GPV infection by conventional PCR method. A 4709-base nucleotide sequence including the structural, non-structural, and 5' inverted terminal repeat regions was obtained from three samples from the Central Anatolian region. Multiple sequence comparisons and phylogenetic analysis demonstrated that the field strains clustered with European group 2 and contained a series of unique amino acid substitutions that might affect the virulence of the virus. These results confirmed that European-related field strains caused the outbreak in Asia Minor, and this might assist in understanding the circulation of GPV in Asia and Europe.

GENETIC DIVERSITY of OVINE HERPESVIRUS 2 STRAINS OBTAINED FROM MALIGNANT CATARRHAL FEVER CASES in EASTERN TURKEY.

Malignant Catarrhal Fever (MCF) is a generalized, definitive lethal disease affecting the epithelial and lymphoid tissues of the respiratory and digestive tract, mainly cattle and some wild ruminants such as deer, buffalo or antelope. The sheep-related form of MCF is known to be present in Turkey and is caused by ovine herpesvirus 2 (OvHV-2). The aim of this study was to reveal the genetic diversity of OvHV-2 strains obtained from MCF cases in Eastern Turkey where the livestock industry has an important impact on economic activities. For this purpose, RTA (Replication and transcription activator), FGARAT (formylglycineamide ribotide amidotransferase) and some of glycoprotein genes (Ov7, Ov8 ex2, ORF27 and Ov9.5) were investigated in blood samples from 24 cattles, clinically diagnosed with MCF. Genomic data of chosen samples were furthermore used to characterize and undergo combined phylogenetic analysis to determine possible alleles and subvariants. The results showed that high level of OvHV-2 diversity existed in selected genes and strains carrying allelic variants might circulate both in two geographically distinct regions and in a region itself. Moreover, three different OvHV-2 types and various subtypes were identified based on multi locus approach. This study provides important data to epidemiological research and thereby helps to determine the source of the virus and understand the spread of the disease.

Simultaneous Deletion of Virulence Factors and Insertion of Antigens into the Infectious Laryngotracheitis Virus Using NHEJ-CRISPR/Cas9 and Cre-Lox System for Construction of a Stable Vaccine Vector.

Infectious laryngotracheitis virus (ILTV) is a promising vaccine vector due to its heterologous gene accommodation capabilities, low pathogenicity, and potential to induce cellular and humoral arms of immunity. Owing to these characteristics, different gene-deletion versions of ILTVs have been successfully deployed as a vector platform for the development of recombinant vaccines against multiple avian viruses using conventional recombination methods, which are tedious, time-demanding, and error-prone. Here, we applied a versatile, and customisable clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 accompanied with Cre-Lox system to simultaneously delete virulence factors and to insert foreign genes in the ILTV genome. Using this pipeline, we successfully deleted thymidine kinase (TK) and unique short 4 (US4) genes and inserted fusion (F) gene of the Newcastle disease virus without adversely affecting ILTV replication and expression of the F protein. Taken together, the proposed approach offers novel tools to attenuate (by deletion of virulence factor) and to generate multivalent (by insertion of heterologous genes) vaccine vectors to protect chickens against pathogens of poultry and public health importance.

Detection and first molecular characterisation of three picornaviruses from diarrhoeic calves in Turkey.

The involvement of picornaviruses in calf diarrhoea was evaluated by the analysis of 127 faecal samples collected from diarrhoeic calves during 2014-2016. Virus detections were carried out by PCR using generic or specific primer pairs. One-third of the faecal samples (33.86%) were found to be positive for one or more of the studied viruses. Bovine kobuvirus was detected in 22.83%, bovine hungarovirus in 11.02%, while bovine enterovirus 1 in 5.51% of the samples. The sequences of the PCR products indicated the existence of novel variants in all the three virus species. When comparing the partial sequences, the nucleotide sequence identities between our newly detected viruses and those previously deposited to the GenBank ranged between 76 and 99%. Phylogenetic analyses revealed a novel lineage within the species Hunnivirus A. Our findings suggest that these viruses should be regarded as possible aetiological agents of calf diarrhoea. Based on the newly determined sequences, we designed and tested a new generic PCR primer set for the more reliable detection of bovine hungaroviruses. This is the first report on the molecular detection of the presence of bovine hungarovirus, bovine kobuvirus and bovine enterovirus 1 in the faecal samples of diarrhoeic calves in Turkey.

Detection and Molecular Analysis of Bovine Enteric Norovirus and Nebovirus in Turkey.

INTRODUCTION: Bovine Norovirus (BoNeV) which has been confirmed in Asia, America, and Europe, seems to be distributed worldwide, even though only reported from a number of countries. Bovine noroviruses are predominantly detected in diarrhoeic animals rather than neboviruses. The study reveals the importance of noro- and neboviruses in early age diarrhoea of calves. MATERIAL AND METHODS: A total of 127 stool samples were collected from three provinces located in the central region of Turkey. Samples were subjected to nucleic acid isolation and reverse transcription and polymerase chain reaction (PCR). Positive samples were sequenced and analysed. RESULTS: According to PCR, five samples (3.93%) were found to be positive for bovine norovirus while 32 (25.19%) samples were found to be positive for bovine nebovirus. Phylogenetic analysis indicated that the novel Turkish norovirus strains were found to be of genotype III.2 and all novel neboviruses were substituted under Nebraska-like strains. CONCLUSION: Although predominantly bovine noroviruses are detected worldwide, the study indicated that bovine neboviruses were more prevalent in the studied area. We suggest that bovine neboviruses are more frequently responsible for calf diarrhoea than supposed by virologists. This is also the first report of neboviruses other than Kirklareli virus which is distantly related to neboviruses detected in Turkey.