Neonatal calf diarrhea (NCD) is one of the major health issues for cattle industry and it is still a challenge for many countries including Turkiye. Rotaviruses are the main causative agents of NCD and due to their genomic structure, rotaviruses are tended to mutate rapidly and evolve by reassortment which lead to novel genotypes. Reassortment of rotaviruses can culminate with not only novel genotypes but also strains that cause the generation of genotypes with interspecies and zoonotic transmission. According to the current data of Rotavirus Classification Working Group, there are 28 R (VP1), 24 C (VP2), 24 M (VP3), 58 P (VP4), 32 I (VP6), 42 G (VP7), 39 A (NSP1), 28 N (NSP2), 28 T (NSP3), 32 E (NSP4), and 28 H (NSP5/6) genotypes of rotaviruses which means incredible increase of novel genotypes and discovery of these by advanced sequence or metagenomics technologies.
Because of the high mutation rate of rotaviruses, in the present study it is aimed to isolate the virus and reveal the genotype of the field isolate that cause NCD in Turkiye in order to find out if there is any novel genotype that were not circulate before our country. According to the results of virus isolation, seven BRV isolates were obtained and one of them which was from Kirsehir province of Turkiye was investigated with shotgun metagenomics. According to shotgun metagenomics 9 out of 11 viral genomic segments could be sequenced; 5 segments were in full-length and 4 segments were in partial, and the genotype constellation of KIRSEHIR strain was determined as G10-P[X]-I2-R2-C2-M2-AX-N2-T6-E2-H3, indicating that the field isolate from Kirsehir has a common genotype constellation with other Turkish BRV strains.
In routine screening and/or epidemiological studies of NCD, antigen ELISA, RT-PCR and real-time RT-PCR were commonly used techniques [25]. Virus isolation is a classical method for viral diagnosis; however, it is time consuming, labor intensive, and sometimes it cannot be possible to isolate the virus. Besides the routine diagnosis of the NCD using RT-PCR, virus isolation was performed in this study and BRV was successfully isolated. This study is the first to use shotgun metagenomics for BRV field isolate in Turkiye. Alkan et al., (2010) had investigated BRV isolates from Turkiye in order to find out only the G and P genotypes, and the rest of the viral genome segments were missing in their study. In the present study, nine segments of a Turkish BRV field isolate were sequenced and deposited in GenBank (OQ082570-OQ082578).
Sanger and next-generation sequencing technologies were commonly used techniques for sequencing of viral genomes and these techniques had been carried out for RVA genomes in Turkiye [27–29]. Another way for sequencing viruses is using forward and reverse primers that based on overlapping strategy [30]. Recent technology of metagenomics provides partial or full-length sequences of viruses and assists to discover unidentified microorganisms in a sample. Metagenomics has been used for many viral agents including rotaviruses [31–33]. This study includes the first shotgun metagenomics for genotype determination of a Turkish BRV field isolate. Oxford nanopore technologies has been shown that able to give near-complete or complete sequences of rotavirus genome segments [31]. In the present study 9 out of 11 segments of KIRSEHIR strain were able to be sequenced. One can speculate that if other primer-based sequencing technologies would use for KIRSEHIR strain, it may be possible to obtain whole genome sequence of the isolate.
There are few studies that performed partial or full-length genome sequencing of BRV in Turkiye. The sequence data were obtained different provinces of Turkiye such as Ankara, Igdir, Samsun, Eskisehir, Erzurum, Tekirdag, Amasya [26, 29, 34]; however, there was no sequence data from Kirsehir. This study provides the BRV field isolate sequences from Kirsehir for the first time.
In the GenBank there are six Turkish BRV strains that have whole genotype constellation: K53, K56, K060, K063, Amasya-1 and Amasya-2, and their genotypes are listed in Table 3. The genotype of KIRSEHIR strain was determined as G10-P[X]-I2-R2-C2-M2-AX-N2-T6-E2-H3, indicating that its genomic constellation is similar to K060 and K063 strains (Table 3), except the VP4 and NSP1 genes that cannot be sequenced and genotypes are unknown. All Turkish BRV strains have N2 genotype (NSP2), however, KIRSEHIR strain has the highest nucleotide identity (96.45%) with Amasya-1 and Amasya-2. On the other hand, although they have same genotype constellation, KIRSEHIR strain has lower nucleotide identity (up to 88.34%) with K060 and K063 strains. Another remarkable result was that although all Turkish BRV strains including KIRSEHIR strain have the same C2 genotype (VP2) (Table 3), the VP2 nucleotide identity was only close to K060, K063, Amasya-1 and Amasya-2. These results indicate that even though the strains are classified within the same genotype, bovine rotaviruses also have a high mutation rate and exhibit large amounts of nucleotide changes in Turkiye. These results once again have demonstrated the importance of screening and surveillance for bovine rotavirus genotypes.
Rotaviruses can be transmitted as interspecies as a result of genetic reassortment and this situation may result in genetic similarity between rotaviruses that infect different species. There are some examples for interspecies transmission of RVA from bovine to heterologous species, such as from bovine to rabbit (K1130027 strain), from bovine to horse (OH-4 strain) [35, 36], and with some recent data, from bovine to feline transmission was shown in other studies [37, 38]. It was previously shown that the NSP5 gene of feline RVA strain shares high nucleotide identity with BRV (97%) and it was suggested to have been acquired from a bovine rotavirus through reassortment [38]. A feline RVA strain (FRV537) from Japan which is thought to transmitted from bovine species to a cat [39], showed close relationship with the NSP4 (94.94% nt, 97.71% aa identity and similarity) and the VP3 (84.18% nt, 83.27% aa identity, 85.45% aa similarity) genes of the present bovine RVA field isolate (Table 4 and Table 5). On the other hand, the VP2 and NSP5/6 genes of KIRSEHIR strain were genetically close to feline and canine RVA strains from Italy, Germany, Japan, Australia and USA which exhibited few aa substitutions. In a recent study, the R11-035 strain of a rotavirus, which was transferred from a cat to a human, acquired NSP3 through intergenotype reassortment with strains found in pigs before being transmitted [17]. Phylogenetic analysis of a recent study revealed that some of the genes of the rotavirus (VP4, VP7, NSP1, NSP3, NSP4, and NSP5) were similar to those found in human feline-like rotaviruses, while other genes (VP1, VP2, VP3, VP6, and NSP2) were more similar to those found in human bovine-like rotaviruses [37] The results of this study are consistent with previously published findings in the area. These results demonstrated that rotaviruses have international, intercontinental and interspecies transmission and Turkish bovine RVA field isolate can be the source of reassortment that culminates with interspecies transmission.
The VP6, VP7, and NSP4 were found to be more conserved among Turkish bovine RVA strains but still have nt/aa identities with some animal (i.e., horse, buffalo, cat, dog) and human RVA strains. The VP1, VP2 and VP3 of KIRSEHIR strain had high identity variation covering different RVA strains from other species and human rotavirus vaccine strains, indicating that these genes are hypervariable and may have interspecies transmission possibility. All non-structural genes of KIRSEHIR strain have interspecies variability. Interestingly, in the phylogeny of the NSP4, numerous Japanese equine RVA strains and a Japanese feline RVA strain were genetically closer to KIRSEHIR strain which may be indicative for both interspecies and international/intercontinental transmission that culminate in genetic variability of rotaviruses.