NSP4 Genes Research Articles

Insertion/deletion hotspots in the Nsp2, Nsp3, S1, and ORF8 genes of SARS-related coronaviruses

The genome of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) contains many insertions/deletions (indels) from the genomes of other SARS-related coronaviruses. Some of the identified indels have recently reported to involve relatively long segments of 10–300 consecutive bases and with diverse RNA sequences around gaps between virus species, both of which are different characteristics from the classical shorter in-frame indels. These non-classical complex indels have been identified in non-structural protein 3 (Nsp3), the S1 domain of the spike (S), and open reading frame 8 (ORF8). To determine whether the occurrence of these non-classical indels in specific genomic regions is ubiquitous among broad species of SARS-related coronaviruses in different animal hosts, the present study compared SARS-related coronaviruses from humans (SARS-CoV and SARS-CoV-2), bats (RaTG13 and Rc-o319), and pangolins (GX-P4L), by performing multiple sequence alignment. As a result, indel hotspots with diverse RNA sequences of different lengths between the viruses were confirmed in the Nsp2 gene (approximately 2500–2600 base positions in the overall 29,900 bases), Nsp3 gene (approximately 3000–3300 and 3800–3900 base positions), N-terminal domain of the spike protein (21,500–22,500 base positions), and ORF8 gene (27,800–28,200 base positions). Abnormally high rate of point mutations and complex indels in these regions suggest that the occurrence of mutations in these hotspots may be selectively neutral or even benefit the survival of the viruses. The presence of such indel hotspots has not been reported in different human SARS-CoV-2 strains in the last 2 years, suggesting a lower rate of indels in human SARS-CoV-2. Future studies to elucidate the mechanisms enabling the frequent development of long and complex indels in specific genomic regions of SARS-related coronaviruses would offer deeper insights into the process of viral evolution.

Genetic characterization of porcine reproductive and respiratory syndrome virus from Eastern China during 2017-2022.

Porcine reproductive and respiratory syndrome (PRRS) is an immunosuppressive disease caused by PRSS virus (PRRSV). PRRSV mainly causes reproductive disorders in pregnant sows and respiratory diseases in piglets. Recently, it has emerged as one of the most important diseases of the pig industry across the globe. In this study, we have collected 231 samples from differently sized pig farms in Eastern China from 2017 to 2022 to investigate the epidemic characteristics of the disease. All samples were screened by RT-PCR and analyzed further using Nsp2 and ORF5 genes. The result showed that the positive rate of PRRSV was 24% (54/231). Phylogenetic analysis (13 positive samples) revealed that all isolates belonged to genotype 2, and they were mainly distributed in four lineages (i.e., lineage 1, 3, 5, and 8). Nsp2 is the most variable protein among all PRRSV NSPs, several isolates from this study had amino acid deletions within Nsp2 compared to that of strain VR-2332. The major structural protein glycoprotein (GP5) protein is encoded by ORF5. Epitope analysis of the 13 isolated strains and additional reference strains revealed that all 13 strains had some mutations on the decoy epitope, the primary neutralizing epitope, T cell epitopes, and B cell epitopes. This study showed that the prevalent PRRSV strain in Eastern China was still HP-PRRSV, while the proportion of NADC30-like and NADC34-like strains have increased. This study further enriches the epidemiological data of PRRS in Eastern China and provides a theoretical basis for vaccine development and prevention and control of the disease across the region.

Full genome characterization of a human-porcine reassortment G12P[7] rotavirus and its pathogenicity in piglets.

In recent years, increasing numbers of cases of acute gastroenteritis caused by Group A rotavirus (RVA) G12 strains have been reported in humans from many countries around the world, but G12 RVA detection in animals is currently less reported. Pigs are an important animal reservoir of zoonotic RVs and a mixing vessel for RVs. In 2020, RVA infection cases in piglets increased in China, which attracted more attention. During an epidemiological survey, a new type of porcine G12P[7] strain (CN127) was detected in pig farms across several provinces. Complete genome analyses revealed that strain CN127 possessed a Wa-like backbone with a genotype constellation of G12-P[7]-I1-C1-M1-R1-A8-N1-T1-E1-H1. The A8 genotype is indicative of its porcine rotavirus origin. Sequence identities and phylogenetic analyses showed that the VP2, VP4, NSP1, NSP4 and NSP5 genes were most closely related to those of porcine rotaviruses, but the VP1, VP6, VP7 and NSP2-3 genes were most closely related to those of human rotaviruses. CN127 likely emerged due to genetic reassortment between porcine and human rotavirus. In vivo experiments showed that CN127 infection caused gastrointestinal tract lesions in piglets and histopathological changes in the lung, liver and mesenteric lymph nodes (MLNs). In the small intestine, RVA antigen was detected in the duodenum and jejunum but not in the ileum. In the extra-intestinal tissues, RVA antigen was detected in the lung but not in the MLNs. Viral RNA was detected in the intestinal and extra-intestinal tissues as well as blood. This study reveals that RVA G12P[7] may become an epidemicstrain in China and also provides further evidence that cocirculating human and porcine strains could produce new genotype rotaviruses with high virulence in piglets.

Quantitative Assay of SARS-CoV-2 RNA and Level of Proinflammatory Protein Gene Transcripts in Peripheral Blood Leukocytes after a Novel Coronavirus Infection.

The possibility of finding persistent SARS-CoV-2 viral particles in human peripheral blood leukocytes after a novel coronavirus infection was shown. The results of droplet digital PCR showed that 19 of 24 examined subjects had from 4 to 555 copies of the Nsp4 SARS-CoV-2 gene in 5-6 months after infection. The presence of this transcript in peripheral blood leukocytes was associated with reduced expression of FOXP3 gene and increased level of RORγ gene mRNA. The copy number of the Nsp4 gene negatively correlated with the level of FOXP3 gene mRNA (r=-0.45; p=0.028), but showed a positive correlation with the DANCR long non-coding RNA (r=0.94; p<0.001). In SARS-CoV-2-positive healthy individuals, the level of TLR2, NLRP3, and IL1B gene transcripts was higher than in SARS-CoV-2-negative donors. The presence of SARS-CoV-2 in a persistent form is probably associated with impaired immunosuppression and the development of chronic inflammation in apparently healthy volunteers after a new coronavirus infection.

Rapid Spread in Japan of Unusual G9P[8] Human Rotavirus Strains Possessing NSP4 Genes of E2 Genotype.

The emergence of unusual G9P[8]-E2 human rotaviruses in the Tokyo metropolitan area, Japan, in 2018 has been reported. During rotavirus strain surveillance in different regions of Japan (Mie, Okayama, and Chiba prefectures), G9P[8]-E2 strains were detected in children with diarrhea from all three prefectures. Here, we characterized the whole genome of seven representative G9P[8]-E2 strains. In the full-genome-based analysis, the seven study strains exhibited a unique genotype configuration with the NSP4 gene of genogroup 2 in a genogroup 1 genomic backbone: G9-P[8]-I1-R1-C1-M1-A1-N1-T1-E2-H1. This genotype constellation was shared by the Tokyo G9P[8]-E2 strains. Phylogenetic analysis showed that all 11 genes, except NSP4, of the seven study strains appeared to have originated from co-circulating Wa-like G9P[8]-E1 strains. In contrast, NSP4 appeared to have originated from the co-circulating DS-1-like G2P[4]-E2 strains. Thus, G9P[8]-E2 strains appear to be derived through reassortment between G9P[8]-E1 and G2P[4]-E2 strains in Japan. Notably, the seven study G9P[8]-E2 strains and Tokyo G9P[8]-E2 strains were revealed to have 11-segment genomes almost indistinguishable from one another in their sequences (99.3-100%), indicating all these G9P[8]-E2 strains had a common origin. To our knowledge, this is the first description of the rapid spread of G9P[8]-E2 strains across a country.

Rotavirus C infections in asymptomatic piglets in India, 2009-2013: genotyping and phylogenetic analysis of all genomic segments.

Asymptomatic infection with rotavirus C (RVC) was observed in pigs in India, with a detection rate of 20%. Sequencing of the VP6, VP7, and NSP4 genes of RVC strains identified the genotypes I7/I10, G1, and E5, respectively. Full genome sequencing of one of these strains revealed that the genotypes of the VP4, VP1, VP2, VP3, NSP1, NSP2, NSP3, and NSP5 genes were P1, R1, C1, M3, A1, N5, T5, and H1, respectively. The detection of porcine RVC strains at two different locations in India at different time points strongly suggests that they are circulating continuously in the pig population through asymptomatic infections.

Establishment of a reverse transcription real-time quantitative PCR method for Getah virus detection and its application for epidemiological investigation in Shandong, China.

Getah virus (GETV) is a mosquito-borne, single-stranded, positive-sense RNA virus belonging to the genus Alphavirus of the family Togaviridae. Natural infections of GETV have been identified in a variety of vertebrate species, with pathogenicity mainly in swine, horses, bovines, and foxes. The increasing spectrum of infection and the characteristic causing abortions in pregnant animals pose a serious threat to public health and the livestock economy. Therefore, there is an urgent need to establish a method that can be used for epidemiological investigation in multiple animals. In this study, a real-time reverse transcription fluorescent quantitative PCR (RT-qPCR) method combined with plaque assay was established for GETV with specific primers designed for the highly conserved region of GETV Nsp1 gene. The results showed that after optimizing the condition of RT-qPCR reaction, the minimum detection limit of the assay established in this study was 7.73 PFU/mL, and there was a good linear relationship between viral load and Cq value with a correlation coefficient (R2) of 0.998. Moreover, the method has good specificity, sensitivity, and repeatability. The established RT-qPCR is 100-fold more sensitive than the conventional RT-PCR. The best cutoff value for the method was determined to be 37.59 by receiver operating characteristic (ROC) curve analysis. The area under the curve (AUC) was 0.956. Meanwhile, we collected 2,847 serum specimens from swine, horses, bovines, sheep, and 17,080 mosquito specimens in Shandong Province in 2022. The positive detection rates by RT-qPCR were 1%, 1%, 0.2%, 0%, and 3%, respectively. In conclusion, the method was used for epidemiological investigation, which has extensive application prospects.

Genomic and evolutionary characteristics of G9P[8], the dominant group a rotavirus in China (2016-2018).

G9P[8] became the predominant rotavirus A (RVA) genotype in China in 2012. To evaluate its genetic composition at the whole-genome level, 115 G9P[8] RVA strains isolated from children under 5 years old were sequenced and characterized. All 13 strains in 2016 and 2017 and an additional 54 strains in 2018 were genotyped as G9-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1. The other 48 strains in 2018 were all genotyped as G9-P[8]-I1-R1-C1-M1-A1-N1-T1-E2-H1, with the NSP4 gene characterized as a DS-1-like genotype. The time of the most recent common ancestor (tMRCA) and evolution rates of the VP7, VP4, and NSP4 (E1 and E2) genes of these strains were estimated by Bayesian evolutionary dynamics analysis. We estimated the evolution rates (nt substitutions per site per year) as 1.38 × 10–3 [the 95% highest posterior density (HPD) was 1.09–1.72 × 10–3] for VP7, 0.87 × 10–3 (95% HPD: 0.75–1.00 × 10–3) for VP4, 0.56 × 10–3 (95% HPD: 0.41–0.73 × 10–3) for NSP4-E1, and 1.35 × 10–3 (95% HPD: 0.92–1.86 × 10–3) for NSP4-E2. The tMRCA was estimated to be 1935.4 (95% HPD: 1892.4–1961.3) for VP7, 1894.3 (95% HPD: 1850.5–1937.8) for VP4, 1929.4 (95% HPD: 1892.4–1961.3) for NSP4-E1, and 1969.2 (95% HPD: 1942.2–1985.3) for NSP4-E2. The baseline genetic information in this study is expected to improve our understanding of the genomic and evolutionary characteristics of the rotavirus genome. Furthermore, it will provide a basis for the development of next-generation rotavirus vaccines for humans.

Epidemiological and Genomic Characterisation of Middelburg and Sindbis Alphaviruses Identified in Horses with Febrile and Neurological Infections, South Africa (2014-2018).

Although Old World alphaviruses, Middelburg- (MIDV) and Sindbis virus (SINV), have previously been detected in horses and wildlife with neurologic disease in South Africa, the pathogenesis and clinical presentation of MIDV and SINV infections in animals are not well documented. Clinical samples from horses across South Africa with acute or fatal neurologic and febrile infections submitted between 2014–2018 were investigated. In total, 69/1084 (6.36%) and 11/1084 (1.01%) horses tested positive for MIDV and SINV, respectively, by real-time reverse transcription (RT) PCR. Main signs/outcomes for MIDV (n = 69): 73.91% neurological, 75.36% fever, 28.99% icterus and anorexia, respectively, 8.70% fatalities; SINV (n = 11): 54.54% neurological, 72.73% fever, 36.36% anorexia and 18.18% fatalities. MIDV cases peaked in the late summer/autumn across most South African provinces while SINV cases did not show a clear seasonality and were detected in fewer South African provinces. MIDV could still be detected in blood samples via RT-PCR for up to 71,417 and 21 days after onset of signs in 4 horses respectively, suggesting prolonged replication relative to SINV which could only be detected in the initial sample. Phylogenetic analyses based on partial sequences of the nsP4 (MIDV n = 59 and SINV n = 7) and E1 (MIDV n = 45) genes, as well as full genome sequences (MIDV n = 6), clustered the MIDV and SINV strains from the present study with previously detected strains. MIDV infection appears to be more prevalent in horses than SINV infection based on RT-PCR results, however, prevalence estimates might be different when also considering serological surveillance data.

Emergence of a novel PRRSV-1 strain in mainland China: A recombinant strain derived from the two commercial modified live viruses Amervac and DV.

Porcine reproductive and respiratory syndrome virus 1 (PRRSV-1) is one of the main pathogens causing porcine reproductive and respiratory syndrome (PRRS). In recent years, the rate of PRRSV-1 detection in China has gradually increased, and the PRRSV-1 strains reported in China belong to subtype I (Global; Clade A-L). In the present study, a novel PRRSV-1 strain, TZJ2134, was found during epidemiological surveillance of PRRSV-1 in Shandong Province in China. We obtained two fragments of the TZJ2134 genome: TZJ2134-L12 (located at nt 1672-nt 2112 in the partial Nsp2 gene) and TZJ2134-(A+B) (located at nt 7463-nt 11272 in the partial Nsp9, complete Nsp10 and partial Nsp11 genes). Phylogenetic and recombination analyses based on the two sequences showed that TZJ2134 is a recombinant strain derived from two commercial PRRSV-1 modified live vaccine (MLV) strains (the Amervac vaccine and DV vaccine strains) that formed a new recombinant subgroup of DV+Amervac-like isolates with other strains. However, PRRSV-1 MLV is not currently allowed for use in China. This study is the first to detected recombinant PRRSV-1 MLV strain in China and provides new data for the epidemiological study of PRRSV-1 in China. The existence of the TZJ2134 strain is a reminder that the swine surveillance at the Chinese customs should be strengthened.

Synergistic interactions of repurposed drugs that inhibit Nsp1, a major virulence factor for COVID-19

Nsp1 is one of the first proteins expressed from the SARS-CoV-2 genome and is a major virulence factor for COVID-19. A rapid multiplexed assay for detecting the action of Nsp1 was developed in cultured lung cells. The assay is based on the acute cytopathic effects induced by Nsp1. Virtual screening was used to stratify compounds that interact with two functional Nsp1 sites: the RNA-binding groove and C-terminal helix-loop-helix region. Experimental screening focused on compounds that could be readily repurposed to treat COVID-19. Multiple synergistic combinations of compounds that significantly inhibited Nsp1 action were identified. Among the most promising combinations are Ponatinib, Rilpivirine, and Montelukast, which together, reversed the toxic effects of Nsp1 to the same extent as null mutations in the Nsp1 gene.

Identification of conserved regions from 230,163 SARS-CoV-2 genomes and their use in diagnostic PCR primer design.

BackgroundAs the rapidly evolving characteristic of SARS-CoV-2 could result in false negative diagnosis, the use of as much sequence data as possible is key to the identification of conserved viral sequences. However, multiple alignment of massive genome sequences is computationally intensive.ObjectiveTo extract conserved sequences from SARS-CoV-2 genomes for the design of diagnostic PCR primers using a bioinformatics approach that can handle massive genomic sequences efficiently.MethodsA total of 230,163 full-length viral genomes were retrieved from the NCBI SARS-CoV-2 Resources and GISAID EpiCoV database. This number was reduced to 14.11% following removal of 5′-/3′-untranslated regions and sequence dereplication. Fast, reference-based, multiple sequence alignments identified conserved sequences and specific primer sets were designed against these regions using a conventional tool. Primer sets chosen among the candidates were evaluated by in silico PCR and RT-qPCR.ResultsOut of 17 conserved sequences (totaling 4.3 kb), two primer sets targeting the nsp2 and ORF3a genes were picked that exhibited > 99.9% in silico amplification coverage against the original dataset (230,163 genomes) when a 5% mismatch between the primers and target was allowed. In addition, the primer sets successfully detected nine SARS-CoV-2 variant RNA samples (Alpha, Beta, Gamma, Delta, Epsilon, Zeta, Eta, Iota, and Kappa) in experimental RT-qPCR validations.ConclusionIn addition to the RdRp, E, N, and S genes that are targeted commonly, our approach can be used to identify novel primer targets in SARS-CoV-2 and should be a priority strategy in the event of novel SARS-CoV-2 variants or other pandemic outbreaks.Supplementary InformationThe online version contains supplementary material available at 10.1007/s13258-022-01264-7.

Phylogenetic and Mutation Analysis of the Venezuelan Equine Encephalitis Virus Sequence Isolated in Costa Rica from a Mare with Encephalitis.

Venezuelan Equine Encephalitis virus (VEEV) is an arboviral pathogen in tropical America that causes lethal encephalitis in horses and humans. VEEV is classified into six subtypes (I to VI). Subtype I viruses are divided into epizootic (IAB and IC) and endemic strains (ID and IE) that can produce outbreaks or sporadic diseases, respectively. The objective of this study was to reconstruct the phylogeny and the molecular clock of sequences of VEEV subtype I complex and identify mutations within sequences belonging to epizootic or enzootic subtypes focusing on a sequence isolated from a mare in Costa Rica. Bayesian phylogeny of the VEEV subtype I complex tree with 110 VEEV complete genomes was analyzed. Evidence of positive selection was evaluated with Datamonkey server algorithms. The putative effects of mutations on the 3D protein structure in the Costa Rica sequence were evaluated. The phylogenetic analysis showed that Subtype IE-VEEV diverged earlier than other subtypes, Costa Rican VEEV-IE ancestors came from Nicaragua in 1963 and Guatemala in 1907. Among the observed non-synonymous mutations, only 17 amino acids changed lateral chain groups. Fourteen mutations located in the NSP3, E1, and E2 genes are unique in this sequence, highlighting the importance of E1-E2 genes in VEEV evolution.

Rotational diffusometric sensor with isothermal amplification for ultra-sensitive and rapid detection of SARS-CoV-2 nsp2 cDNA

In the wake of a pandemic, the development of rapid, simple, and accurate molecular diagnostic tests can significantly aid in reducing the spread of infections. By combining particle imaging with molecular assays, a quick and highly sensitive biosensor can readily identify a pathogen at low concentrations. Here, we implement functionalized particle-enabled rotational diffusometry in combination with loop-mediated isothermal amplification for the rapid detection of the SARS-CoV-2 nsp2 gene in the recombinant plasmid as a proof of concept for COVID-19 diagnostics. By analyzing the images of blinking signals generated by these modified particles, the change in micro-level viscosity due to nucleic acid amplification was measured. The high sensitivity of rotational diffusometry enabled facile detection within 10 min, with a limit of detection of 70 ag/μL and a sample volume of 2 μL. Tenfold higher detection sensitivity was observed for rotational diffusometry in comparison with real-time PCR. In addition, the system stability and the effect of temperature on rotational diffusometric measurements were studied and reported. These results demonstrated the utility of a rotational diffusometric platform for the rapid and sensitive detection of SARS-CoV-2 cDNA fragments.

Studies on Detection, Prevalence and Molecular Characterization of Avian Rotavirus

Rotaviruses are the foremost cause of acute gastroenteritis. Outbreaks of rotavirus infection can lead to significant economic losses in livestock and poultry industry. Group A and D rotavirus are the predominant enteric viruses groups in birds. Limited prevalence studies are available to date in India. Poultry samples consisting of 20 fecal samples and 80 intestinal content samples were screened by VP6 gene based diagnostic PCR for AvRVA and AvRVD. All samples were negative for group A rotavirus. Five fecal samples and 22 intestinal samples were positive for AvRVD-VP6 gene giving an amplicon of 185 bp giving a prevalence of 27%. Positive samples screened for VP4, VP7, NSP4 and NSP5 genes of AvRVD were negative for VP4, VP7 and NSP4 but six samples were positive for NSP5 gene giving an amplicon of 652 bp size. Cloning and sequencing showed 99.4 to 100% sequence similarity among all six isolates at nucleotide level. The percent nucleotide similarity with chicken RVD isolates was in the range 78.2-85.1%. All isolates showed highest similarity of 85% with chicken isolate from South Korea followed by Germany (81.5%) and Australia (78.2%). The phylogenetic analysis exhibited all six isolates clustered together in a separate clade with isolates from Australia, Germany and South Korea.

Epidemiology of Group A rotavirus in rodents and shrews in Bangladesh

Rodents and shrews live in close proximity to humans and have been identified as important hosts of zoonotic pathogens. This study aimed to detect Group A rotavirus (RVA) and its potential risk factors in rodents and shrews in Bangladesh. We captured 417 small mammals from 10 districts with a high degree of contact between people and domestic animals and collected rectal swab samples between June 2011 and October 2013. We tested the swab samples for RVA RNA, targeting the NSP3 gene segment using real-time reverse transcription-polymerase chain reaction (rRT-PCR). Overall, RVA prevalence was the same (6.7%) in both rodents and shrews. We detected RVA RNA in 5.3% of Bandicota bengalensis (4/76; 95% CI: 1.4–12.9), 5.1% of B. indica (4/79; 95% CI: 1.4–12.4), 18.2% of Mus musculus (4/22; 95% CI: 5.2–40.3), 6.7% of Rattus rattus (6/90; 95% CI: 2.5–13.9), and 6.7% of Suncus murinus (10/150; 95% CI: 3.2–11.9). We found significantly more RVA in males (10.4%; OR: 3.4; P = 0.007), animals with a poor body condition score (13.9%; OR: 2.7; P = 0.05), during wet season (8.3%; OR: 4.1; P = 0.032), and in urban land gradients (10.04%; OR: 2.9; P = 0.056). These findings form a basis for understanding the prevalence of rotaviruses circulating among rodents and shrews in this region. We recommend additional molecular studies to ascertain the genotype and zoonotic potential of RVA circulating in rodents and shrews in Bangladesh.

Genetic Surveillance of Five SARS-CoV-2 Clinical Samples in Henan Province Using Nanopore Sequencing.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has rapidly spread and poses a major threat to public health worldwide. The whole genome sequencing plays a crucial role in virus surveillance and evolutionary analysis. In this study, five genome sequences of SARS-CoV-2 were obtained from nasopharyngeal swab samples from Zhengzhou, China. Following RNA extraction and cDNA synthesis, multiplex PCR was performed with two primer pools to produce the overlapped amplicons of ~1,200 bp. The viral genomes were obtained with 96% coverage using nanopore sequencing. Forty-five missense nucleotide mutations were identified; out of these, 5 mutations located at Nsp2, Nsp3, Nsp14, and ORF10 genes occurred with a <0.1% frequency in the global dataset. On the basis of mutation profiles, five genomes were clustered into two sublineages (B.1.617.2 and AY.31) or subclades (21A and 21I). The phylogenetic analysis of viral genomes from several regions of China and Myanmar revealed that five patients had different viral transmission chains. Taken together, we established a nanopore sequencing platform for genetic surveillance of SARS-CoV-2 and identified the variants circulating in Zhengzhou during August 2021. Our study provided crucial support for government policymaking and prevention and control of COVID-19.

VP7, VP4, and NSP4 genes of species a rotaviruses isolated from sewage in Nigeria, 2014/2015: partial sequence characterization and biophysical analysis of NSP4 (enterotoxin).

Species A rotavirus are an important cause of childhood gastroenteritis, and the main contributor to its pathogenicity is the enterotoxin (NSP4) protein. Some biophysical properties of partial NSP4 genes of RVAs isolated from sewage in Nigeria during 2014/2015 were investigated. Samples were typed by RT-PCR and Sanger sequencing of partial VP4, VP7 and NSP4 genes. Phylogeny identified lineages within genotypes, predicted glycosylation sites; hydrophobicity profiles and amino acid alignments were employed to determine some biophysical properties of the NSP4 protein. The VP7 sequences of our isolates were the most diversified, the majority of the isolates carried NSP4 genes of the E1 genotype. Genotype specific variations both in hydrophobicity and potential glycosylation were identified, mutations were highest within the H3 hydrophobic domain and VP4 binding domain. The study of RVA NSP4 genes from non-clinical samples revealed that there were structural consistencies with those of reference genes.

Whole genome analysis of rotavirus strains circulating in Benin before vaccine introduction, 2016–2018

Species A rotaviruses (RVA) still play a major role in causing acute diarrhea in children under five years old worldwide. Currently, an 11-gene classification system is used to designate the full genotypic constellations of circulating strains. Viral proteins and non-structural proteins in the order VP7-VP4-VP6-VP1-VP2-VP3-NSP1-NSP2-NSP3-NSP4-NSP5/6 are represented by the genotypes Gx-P[x]-Ix-Rx-Cx-Mx-Ax-Nx-Tx-Ex-Hx, respectively. In Benin, ROTAVAC® vaccine was introduced into the Expanded Programme on Immunization in December 2019. To monitor circulating RVA strains for changes that may affect vaccine performance, in-depth analysis of strains prior to vaccine introduction are needed. Here we report, the whole-gene characterization (11 ORFs) for 72 randomly selected RVA strains of common and unusual genotypes collected in Benin from the 2016 to 2018 seasons. The sequenced strains were 15 G1P[8], 20 G2P[4], 5 G9P[8], 14 G12P[8], 9 G3P[6], 2 G1P[6], 3 G2P[6], 2 G9P[4], 1 G12P[6], and 1 G1G9P[8]/P[4]. The study strains exhibited two genetic constellations designed as Wa-like G1/G9/G12-P[6]/P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1 and DS-1-like G2/G3/G12-P[4]/P[6]-I2-R2-C2-M2-A2-N2-T2-E2-H2. Genotype G9P[4] strains possessed a DS-1-like genetic constellation with an E6 NSP4 gene, G9-P[4]-I2-R2-C2-M2-A2-N2-T2-E6-H2. The mixed genotype showed both Wa-like and DS-1-like profiles with a T6 NSP3 gene G1/G9P[8]/[4]-I1/I2-R1/R2-C1/C2-M1/M2-A1/A2-N1/N2-T1/T6-E1/E6-H1/H2. At the allelic level, the analysis of the Benin strains, reference strains (with known alleles), vaccine strains (with known alleles) identified 2–13 and 1–17 alleles for DS-1-like and Wa-like strains, respectively. Most of the study strains clustered into previously defined alleles, but we defined 3 new alleles for the VP7 (G3 = 1 new allele and G12 = 2 new alleles) and VP4 (P[4] = 1 new allele and P[6] = 2 new alleles) genes which formed the basis of the VP7 and VP4 gene clusters, respectively. For the remaining 9 genes, 0-6 new alleles were identified for both Wa-like and DS-1-like strains. This analysis of whole genome sequences of RVA strains circulating in Benin described genetic point mutations and reassortment events as well as novel alleles. Further detailed studies on these new alleles are needed and these data can also provide a baseline for studies on RVA in the post-vaccination period.

Genomic diversity and intragenic recombination of species C rotaviruses.

Rotavirus C (RVC) is a major cause of diarrhoea in swine, cattle, and humans worldwide. RVC exhibits sequence diversity in all 11 genes, especially in VP4 and VP7, and all segment-based genotyping has been performed similar to rotavirus A. To date, recombination events have been reported in rotavirus A and B. However, there are no reports describing gene recombination of RVC, except for recombination in NSP3 between RVC and rotavirus H. In this study, nine porcine RVC strains identified in Japanese pigs were completely sequenced and analysed together with RVC sequences from the GenBank database. The analyses showed that sequences of the VP4, VP2, and NSP1 of several porcine RVC strains did not branch with any of those of the RVC strains in the GenBank database, suggesting new genotypes. Several homologous recombination events, between or within genotypes, were identified in the VP4, VP7, VP2, NSP1, and NSP3 genes. Of these, nine, one, and one intergenotypic recombination events in the VP4, VP2, and NSP3 genes, respectively, were supported with sufficient statistical values. Although these findings suggest occurrences of the intragenic recombination events in the RVC genome, potential sequence errors and poor sequence assemblies in the databases should be watched with care. The results in this study present data about the important recombination events of the RVCs, which influence evolution of the virus by aiding them to gain genetic diversity and plasticity, although further sequence data will be necessary to obtain more comprehensive understanding of such mechanisms.