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.

IntroductionBovine 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 MethodsA 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.ResultsAccording 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.ConclusionAlthough 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.

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.

Torque teno virus (TTV) was first detected in humans, and since then it has been reported in many host species, such as monkeys, cats, pigs, seagulls and dogs. The aim of this study was to investigate the presence of Torque teno canis virus (TTCaV) in shelter dogs housed in Sivas Municipal Animal Shelter, Turkey. Faecal specimens, including diarrheic and non-diarrheic (n=202), were collected from dogs of various age groups. In total, 32.18% (65/202) of samples were found positive for TTCaV. Out of the 65 positive samples, 34.64% (44/127) samples were from adult diarrheic dogs and 26.09% (6/23) belonged to diarrheic puppies. On the other hand, 28.84% (15/52) positive samples were detected from clinically healthy dogs. Eight sequences showed close homology among themselves, however, the sequences of two samples (CANEL130 and CANEL140) were genetically distinct from other published sequences. This is the first report on the detection of TTCaV in adult dogs and puppies in Turkey, and provides evidence that TTCaV cannot be considered as the sole cause of diarrhea.

The emergence of the Omicron variant has reinforced the importance of continued SARS-CoV-2 evolution and its possible impact on vaccine effectiveness. Specifically, mutations in the receptor-binding domain (RBD) are critical to comprehend the flexibility and dynamicity of the viral interaction with the human agniotensin-converting enzyme 2 (hACE2) receptor. To this end, we have applied a string of deep structural and genetic analysis tools to map the substitution patterns in the S protein of major Omicron sub-variants (n = 51) with a primary focus on the RBD mutations. This head-to-head comparison of Omicron sub-variants revealed multiple simultaneous mutations that are attributed to antibody escape, and increased affinity and binding to hACE2. Our deep mapping of the substitution matrix indicated a high level of diversity at the N-terminal and RBD domains compared with other regions of the S protein, highlighting the importance of these two domains in a matched vaccination approach. Structural mapping identified highly variable mutations in the up confirmation of the S protein and at sites that critically define the function of the S protein in the virus pathobiology. These substitutional trends offer support in tracking mutations along the evolutionary trajectories of SAR-CoV-2. Collectively, the findings highlight critical areas of mutations across the major Omicron sub-variants and propose several hotspots in the S proteins of SARS-CoV-2 sub-variants to train the future design and development of COVID-19 vaccines.