Phylodynamic characteristics of the Russian population of rotavirus А (Reoviridae: Sedoreovirinae: Rotavirus) based on the VP6 gene

Introduction. Rotavirus A is one of the leading causes of acute gastroenteritis in children in the first years of life. Rotavirus infection is currently classified as a preventable infection. The most abundant rotavirion protein is VP6.Material and methods. Phylogenetic analysis and calculation of phylodynamic characteristics were carried out for 262 nucleotide sequences of the VP6 gene of rotavirus species A, isolated in Russia, using the BEAST v.1.10.4 software package. The derivation and analysis of amino acid sequences was performed using the MEGAX program.Results. This study provides phylodynamic characteristics of the rotaviruses in Russia based on the sequences coding VP6 protein. Bayesian analysis showed the circulation of rotaviruses of three sublineages of genotype I1 and three sublineages of genotype I2 in Russia. The level of accumulation of mutations was established, which turned out to be similar for genotypes I1 and I2 and amounted to 7.732E-4 and 1.008E-3 nucleotides/site/year, respectively. The effective population sizes based on nucleotide sequences of the VP6 I1 and I2 genotypes are relatively stable while after the 2000s there is a tendency of its decreasing. Comparative analysis of the amino acid sequences in the region of the intracellular neutralization sites A (231–260 aa) and B (265–292 aa) made it possible to reveal a mutation in position V252I in a proportion of Russian strains of genotype I1 some strains of genotypes I1 and I2 had mutation I281V. These substitutions were not associated with any sublineages to which the strains belong. The analysis of three T-cell epitopes revealed four amino acid differences (in aa positions 305, 315, 342, 348) that were associated with the first or second genogroup.Conclusion. Based on the phylodynamic characteristics and amino acid composition of antigenic determinants, it was concluded that the VP6 protein is highly stable and could potentially be a good model for development of a rotavirus vaccine.

Phylogenetic Study of VP6 Gene of Bovine Rotavirus A and Molecular Survey of Bovine Rotaviruses B and C, and Human G and P Genotypes of Rotavirus A in Calves in Iran

Background: Rotavirus (RV) is one of the most important causes of diarrhea in the calf and human neonates. Rotaviruses are divided into nine different serogroups, of which group A is more important compared to other groups. Objective: This study was performed because of the lack of information about the importance and prevalence of bovine rotaviruses B (RVB) and C (RVC) and human genotypes of rotavirus A (RVA) in the bovine population in Iran. Phylogenetic analysis of VP6 of bovine RVA was the second aim of the present study. Materials and Methods: A total of 581 stool specimens were collected from diarrheic calves of 14 provinces and were analyzed by reverse transcription polymerase chain reaction (RT-PCR) and 485 of them were investigated by PAGE electrophoresis to determine the frequency of three rotaviruses A (RVA), B (RVB), and C (RVC). The presence of human G and P genotypes in Iranian bovine population was also evaluated using semi-nested multiplex RT-PCR. Results: RVA was detected by RT-PCR (VP6 gene detection) in 16.2% (94/581) and by PAGE in 22.16% (108/485) and no positive cases of RVB and RVC were confirmed by polyacrylamide gel electrophoresis (PAGE). This study showed that non-A RV groups (B and C) have little role in calf diarrhea in Iran. The results of the phylogenetic study of VP6 sequences of rotaviruses A identified in this study showed that they all belonged to genotype I2 and were classified into three different branches. Specimen isolated in Zanjan showed the highest difference (maximum identity of 94%) with other sequences and clustered along with the Japanese strain, R22. Human G and P genotypes were not found in the studied samples. Conclusion: The results showed that non-A rotaviruses and human genotypes of RVA are of little importance in calf rotavirus diarrhea in Iran. Also, there is the first phylogenetic study of rotavirus A VP6 protein in Iran.

Molecular characterization of unusual G10P[33], G6P[14] genomic constellations and evidence of zooanthroponosis in bovines

Group A rotaviruses (RVA) are a major cause of diarrhea in neonatal calves and children. The present study examined G/P combinations and genetic characteristics of RVAs in diarrheic bovine calves in Western India. RVAs were detected in 27 samples (17.64%) with predominance of G10P[11] (51.85%), followed by previously unreported genomic constellations, G6P[14] (14.81%), and, G6P[4] (7.40%) and G10P[33] (3.70%). Sequencing and phylogenetic analysis revealed circulation of G10 (Lineage-5), G6 (Lineage-2), P[11] (Lineage-3), P[14] (proposed Lineage-8) and P[4] (Lineage-3) genotypes. The predominant G10P[11] strains were typical bovine strains and exhibited genotypic homogeneity. The rare, G10P[33] strain, had VP7 and VP4 genes of bovine origin but resemblance of VP6 gene with simian strain indicated possible reassortment between bovine and simian (SA11-like) strains. The VP6 and VP7 genes of other two rare strains, G6P[14] and G6P[4], were similar to those of bovine stains, but the VP4 was closely related to those of the human-bovine like and human strains, respectively. Additionally, in VP4 gene phylogenetic tree Indian P[14] strains constituted a closely related genetic cluster distinct from the other P[14] strains, hence Lineage-8 was proposed for them. These findings indicated that bovines could serve as source for anthropozoonotic transmission of G6P[14] strains while zooanthroponotic transmission followed by reassortment with human strain gave rise to G6P[4] strains. The observations of present study reinforce the potential of rotaviruses to cross the host-species barrier and undergo reassortant to increase genetic diversity which necessitates their continuous surveillance for development and optimization of prevention strategies against zoonotic RVAs.

IDENTIFICATION OF ROTAVIRUS I- AND E-GENOTYPES BY MULTIPLEX PCR METHOD

Introduction. In recent years the presence of reassortant rotavirus strains is increasingly mentioned in the world due to the application of the full-genome based classification system. Information on the circulation of such strains in the territory of Russia is limited. The aim of this work was the development of the approach for determination of genotypes of segments encoding VP6 (I) and NSP4 (E) to reveal reassortant strains. Material and Methods. Rotavirus-positive samples were studied by means of nucleotide sequencing and multiplex PCR. Phylogenetic analysis was conducted using the Bayesian approach. Results. Three alleles of the VP6 gene (I1-1, I2-IV, I2-VII) and seven alleles of the NSP4 gene (E1-I, E1-III, E2-VI, E2-VII, E2-X, E2-XII, E3) were detected on the base of nucleotide sequences of Nizhny Novgorod rotaviruses. Taking into account these results, the oligonucleotide primers specific to genotypes I1, I2, I3 and E1, E2, E3 were designed. Optimal conditions for multiplex PCR were chosen. The method was tested using the strains collected in Nizhny Novgorod in 2018. The diversity of I and E genotypes was determined and various combinations with G and P genotypes were identified. Discussion. G9-P[8]-I1-E1 rotaviruses were predominant (32.7 %) and G2-P[4]-I2-E2 rotaviruses were in second place (29.1 %). Strains with genotypes G4-P[8]-I1-E2, G3-P[8]-I2-E2 and G2-P[4]-I2-E1 were detected sporadically. They had genes of two rotavirus genogroups, so can be considered to be reassortant. Conclusion. The proposed approach is a useful tool for the characterization of rotaviruses in the conditions of the beginning of vaccination against rotavirus infection in Russia.

Rotavirus A associated pathology of intestine and mesenteric lymph nodes and occurrence in bovine calves of Gwalior and Bareilly regions

To understand the pathology of natural cases of rotavirus (RVA) in bovine calves, a total of 40 cases below 6 months died due to diarrhoea were studied, out of which 7 cases (17.5%) turned positive for RVA by RT-PCR. Histopathology of small intestine showed loss of villous enterocytes, blunting and fusion of villi, elongation of crypts and mononuclear cells infiltration in the lamina-propria. The mesenteric lymph nodes were severely depleted of lymphocytes. These changes were corroborated with presence of RVA antigen in sections by dFAT and nucleic acid by RT-PCR. The fluorescent signals were more in mesenteric lymph nodes than in intestine. Besides, 115 rectal fecal samples were also collected from calves for RVA detection by RT-PCR using VP6 gene specific sets of primers. Dead carcasses of calves (n= 40) belonged to organized dairy farm of Bareilly, while rectal fecal samples belonged to both organized (n= 38) and unorganized farms (n= 77) of Bareilly and Gwalior. The overall occurrence of RVA was 19.3% (30/155), comprising 5/37 cases (13.5%) from Gwalior (MP) and 25/118 cases (21.1%) from Bareilly (UP). These findings suggest the infection of RVA widely prevalent in calves and have potential to escape from the intestinal site to mesenteric lymph nodes.

Is Porcine Kobuvirus 1 a Typical Diarrhoeic Pathogen of Piglets?

The objective of this study was to show if porcine kobuvirus 1 (PKV-1) participates in the development of diarrhoea in piglets. The experiments were focused on comparing the occurrence of PKV-1 with the occurrence of rotavirus A (RVA) infection in suckling pigs on Slovak pig farms. A total of 91 rectal swabs of piglets (age < 28 days) were collected from 8 pig farms. RT-PCR was employed to detect PKV-1 through amplification of the 495 bp fragment of the 3D gene using primers KoVF/ KoVR, and RVA was detected through amplification of the 309 bp fragment of the VP6 gene using primers rot3 and rot5. As expected, the detection of RVA in diarrhoeic piglets was 56.8 % (P < 0.01), while only 14.8 % in healthy animals. These results confirm that RVA is one of the main causes of diarrhoea in young piglets. Comparatively, PKV-1 was detected in approximately equal numbers in the same group of both healthy and diarrhoeic pigs, with 74.1 % in healthy animals and 81.1 % in diarrhoeic animals, which was not statistically significant (P < 0.05). The level of co-infection of both viruses was 11.1 % in healthy animals. A portion of 48.6 % (P < 0.01) of diarrhoeic animals were found with RVA and PKV-1 coinfections. The results of this study indicate that while RVA is an enteric virus, PKV-1 cannot confidently be confirmed as an enteric pathogen.