Genetic diversity of group A rotaviruses associated with repeated outbreaks of diarrhea in a farrow-to-finish farm: identification of a porcine rotavirus strain bearing a novel VP7 genotype, G26

Group A rotaviruses (GARs) are one of the most common causes of diarrhea in suckling pigs. Although a number of G and P genotypes have been identified in porcine GARs, few attempts have been made to study the molecular epidemiology of these viruses associated with diarrhea outbreaks within a farm over an extended period of time. Here, we investigated the molecular characteristics of GARs that caused four outbreaks of diarrhea among suckling pigs in a farrow-to-finish farm over the course of a year. G and P genotyping of GARs detected at each outbreak demonstrated genetic diversity in this farm as follows: G9P[23] was detected at the first outbreak, G9P[13]/[22] and G9P[23] at the second, G3P[7] at the third, and G9P[23], G5P[13]/[22], and P[7] combined with an untypeable G genotype at the fourth. Sequence analysis of the detected GARs revealed that such genetic diversity could have resulted not only from the introduction of new GAR strains, but also from gene reassortment between GAR strains within the farm. Further, the GAR strain carrying the untypeable G genotype was shown to be a novel porcine GAR bearing a new G26 genotype, as confirmed by the Rotavirus Classification Working Group

Novel Human Rotavirus Genotype G5P[7] from Child with Diarrhea, Cameroon

We report characterization of a genotype G5P[7] human rotavirus (HRV) from a child in Cameroon who had diarrhea. Sequencing of all 11 gene segments showed similarities to >5 genes each from porcine and human rotaviruses. This G5P[7] strain exemplifies the importance of heterologous animal rotaviruses in generating HRV genetic diversity through reassortment

Identification of a G2-like porcine rotavirus bearing a novel VP4 type, P[32]

A porcine group A rotavirus (GARV) strain, 61/07/Ire, was isolated from a 4–5 week asymptomatic piglet, during an epidemiological survey of porcine herds in Southern Ireland, in 2007. The nucleotide (nt) and amino acid (aa) sequence of the full-length VP4 protein of the PoRV strain 61/07/Ire was determined. Based on the entire VP4 open reading frame (nt), strain 61/07/Ire displayed ≤ 76.5% identity to representatives of the established 31 P-types, a value far lower than the percentage identity cutoff value (80%) established by the Rotavirus Classification Working Group (RCWG) to define a novel P genotype. Strain 61/07/Ire revealed low aa identity, ranging from 57.1% to 83.6%, to the cognate sequences of representatives of the various P genotypes. The aa identity was lower in the VP8* trypsin-cleavage fragment of the VP4, which encompasses the VP4 hypervariable region, ranging from 36.9% to 75.3%. Sequence analyses of the VP7, VP6, and NSP4 genes revealed that the GARV strain 61/07/Ire possessed a G2-like VP7, an E9 NSP4 genotype and an I5 VP6 genotype. Altogether, these results indicate that the GARV strain 61/07/Ire should be considered as a prototype of a new VP4 genotype, P[32], and provide further evidence for the vast heterogeneity of group A rotaviruses

Molecular determinants of rotavirus virulence

Rotaviruses are the single most important etiological agent of severe diarrhoea in infants and young children in both developed and developing countries. The World Health Organisation has identified the development of a rotavirus vaccine as a priority area for routine childhood immunisation to control rotavirus infections. However, the candidate vaccine strains have not been very successful. The main aim of this project was to map rotavirus virulence to its gene segments. Such studies can help in developing better vaccines for the control of rotavirus induced diarrhoea. A three step approach was undertaken (i) development of an animal model, (ii) construction and characterisation of reassortants between rotavirus strains of different virulence, (iii) mapping virulence to rotavirus gene segments. The mouse model developed revealed that the outcome of rotavirus infection was influenced by viral dose and viral strain as well as by host age and host strain. Homologous murine rotavirus strain was found to be most virulent. Among the heterologous strains studied, the OSU strain was found to be most virulent and UKtc strain the least virulent. The CD- 1 strain of mouse was found to be the most susceptible to virus infection and C57/BL the least susceptible. A very simple and rapid nucleic acid extraction method has been developed that requires only one centrifugation step and circumvents the use of any hazardous organic chemicals, which can be applied to very large numbers of samples saving time and labour. Rotavirus reassortants were constructed in a variety of ways and their genotype determined from relative mobility of their gene segments on polyacrylamide gels and restriction enzyme digestion of PCR amplified products. Twenty two reassortants (2%) were identified out of more than 1100 progeny clones examined and these reassortants belonged to 15 different genotypes. Possible reasons for obtaining this low number of reassortants are discussed. No reassortant could be identified between a murine rotavirus and other heterologous rotavirus strains. Preliminary sequence of VP7 gene of murine rotavirus strains, EDIM and EBR, was found to be different to the published rotavirus sequences including the recently published five murine rotavirus strains. The virulence mapping studies conducted in mice with some of the 22 reassortants obtained in the present study showed that gene 4 of the OSU and UKtc strains was involved in virulence. Segment 5 of OSU strain and segments 5, and 8 of UKtc strain may also be involved in virulence

Molecular characterization of a new porcine rotavirus P genotype found in an asymptomatic pig in Slovenia

AbstractRotaviral RNA was detected in the stool sample of an asymptomatic fattening pig at a Slovenian pig farm. To characterize the rotavirus, RT-PCR was used, employing primers specific for the VP7, VP4 and NSP4 genes. Specific products were purified and the sequencing reaction was performed for the molecular analysis of amplified genes. Nucleotide and amino acid sequences of the VP7 gene were found highly identical (85.3–88.1% and 90.7–91.6%) to G1 genotype strains. Phylogenetic and molecular analyses of the VP7 antigen regions revealed the sample to be from a new lineage of G1 genotype. In the molecular analysis of the VP4 gene, only 70.9% nucleotide (76.2% amino acid) identity was found with the most related rotavirus VP4 gene from GenBank. Following this, the NSP4 gene was also analyzed. After the phylogenetic analysis, it clustered with the NSP4 B genotype, but also seemed to represent a new lineage of this genotype. This new rotavirus strain, named P21-5, differed greatly from all rotaviruses characterized so far in all three genes analyzed. The virulence of this strain is not clear yet and has to be investigated

Occurrence of rotavirus a genotypes and other enteric pathogens in diarrheic suckling piglets from Spanish swine farms

Species A rotavirus (RVA) is a major viral pathogen causing diarrhea in suckling piglets. Studies on its genetic heterogeneity have implications for vaccine efficacy in the field. In this study, fecal samples (n = 866) from diarrheic piglets younger than 28 days were analyzed over a two-year period (2018–2019). Samples were submitted from 426 farms located in 36 provinces throughout Spain and were tested using real-time PCR (qPCR) and reverse transcription real-time PCR (RT-qPCR) for five enteric pathogens. The individual prevalence was 89.4%, 64.4%, 44.9%, 33.7% and 4.4% for Clostridiumperfringens, Clostridioides (formerly Clostridium) difficile, species A rotavirus, species C rotavirus and porcine epidemic diarrhea virus, respectively. Most specimens (96.9%) were positive for at least one of the target pathogens, and more than 80% of samples harbored mixed infections. Nucleotide sequencing of 70 specimens positive for RVA revealed the presence of the VP7 genotypes G4, G9, G3, G5, G11 and the VP4 genotypes P7, P23, P6 and P13, with the combinations G4P7 and G9P23 being the most prevalent, and especially in the areas with the highest pig population. The study shows the extensive genetic diversity of RVA strains as well as discrepancies with the genotypes contained in the vaccine available in Spain, and multiple amino acid differences in antigenic epitopes of different G- and P- genotypes with the vaccine strains. Further investigations are needed to determine the efficacy of the vaccine to confer clinical protection against heterologous strains

Molecular Characterization of the Porcine Group A Rotavirus NSP2 and NSP5/6 Genes from São Paulo State, Brazil, in 2011/12

Rotaviruses are responsible for the acute diarrhea in various mammalian and avian species. The nonstructural proteins NSP2 and NSP5 are involved in the rotavirus replication and the formation of viroplasm, cytoplasmic inclusion bodies within which new viral particles morphogenesis and viral RNA replication occur. There are few studies on the genetic diversity of those proteins; thus this study aims at characterizing the diversity of rotavirus based on NSP2 and NSP5 genes in rotaviruses circulating in Brazilian pig farms. For this purpose, 63 fecal samples from pig farms located in six different cities in the São Paulo State, Brazil, were screened by nested RT-PCR. Seven strains had the partial nucleotide sequencing for NSP2, whereas in six, the total sequencing for NSP5. All were characterized as genotype H1 and N1. The nucleotide identity of NSP2 genes ranged from 100% to 86.4% and the amino acid identity from 100% to 91.5%. For NSP5, the nucleotide identity was from 100% to 95.1% and the amino acid identity from 100% to 97.4%. It is concluded that the genotypes of the strains circulating in the region of study are in agreement with those reported in the literature for swine and that there is the possibility of interaction between human and animal rotaviruses

The role of NSP1 in rotavirus pathogenesis

NSP1, a non-structural protein encoded by rotavirus gene segment 5, has been suggested as a virulence determinant for rotavirus and to function as an antagonist of the interferon signalling pathway. Although non-essential for rotavirus replication in cell culture, and is the least conserved in all rotavirus proteins, NSP1 from different rotavirus strains of different species has been demonstrated to interact with several cellular proteins involved in the IFNβ induction pathway. NSP1 from a bovine rotavirus strain (UKtc) has been shown to interact with and to degrade IRF3 in a proteasome dependent manner whereas NSP1 from a porcine rotavirus strain (OSU) fails to target IRF3 but is able to interfere with IFNβ production via similar targeting of β-TrCP. The research presented in this thesis sought to gain a better understanding of the molecular determinants of NSP1 specificity for targeting the IFNβ pathway by mapping the regions in NSP1 sequences responsible for targeting specific cellular proteins. NSP1 hybrid constructs with sequences from both UKtcNSP1 and OSUNSP1 were generated and their interactions with both IRF3 and β-TrCP were tested in a series of assays. The initial attempts to map interaction sites using the mammalian two-hybrid assay were not successful. No reporter plasmid signal was generated indicating the expected interaction. The failure of this assay might be due to the insufficient expression of the NSP1 proteins as subsequent modification of the expression vector\ud was shown to improve the expression level of NSP1 proteins in subsequent reporter assay analysis. Using IFNβ promoter reporter assays to demonstrate the functional consequence of NSP1 action in IRF3, it was found that the constructs containing the entire Cterminal part of UKtcNSP1 were able to reduce IRF3-induced IFNβ promoter activity. Such constructs also caused IRF3 degradation in a proteasome dependent manner in agreement with previous studies. However, the sequence containing the last 135 amino acids from UKtcNSP1 was not sufficient for these activities. Collectively, these data suggested that the sequence between amino acid position 165 and 135 from the C-terminus are required for this interaction and subsequent degradation of IRF3. Similar experiments focused on determining the interaction site for β-TrCP on NSP1 were more difficult to interpret according the data presented. Unexpectedly in the light of published data, not only OSUNSP1 was able to degrade β-TrCP but UKtcNSP1 appeared to have the similar effect, as well as two reciprocal pairs of NSP1 hybrid constructs. In summary, it appears that sequences from the C-terminal part of UKtcNSP1 can function in a heterogeneous NSP1 context to target IRF3 from human cells. Further analysis is clearly required to fulfil the understanding of the role of NSP1 in rotavirus pathogenesis, including its interaction with β-TrCP