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All over the world, children and adults are severely affected by acute gastroenteritis, caused by one of the emerging enteric pathogens, rotavirus C (RVC). At present, no extensive surveillance program is running for RVC in India, and its prevalence is largely unknown except cases of local outbreaks. Here, we intended to detect the presence of RVC in diarrheic children visiting or admitted to hospitals in Haldwani (state of Uttarakhand, India), a city located in the foothills of the Himalayas. During 2010–2013, we screened 119 samples for RVC by an RVC VP6 gene-specific RT-PCR. Of these, 38 (31.93%) were found positive, which is higher than the incidence rates reported so far from India. The phylogenetic analysis of the derived nucleotide sequences from one of the human RVC (HuRVC) isolates, designated as HuRVC/H28/2013/India, showed that the study isolate belongs to genotype I2, P2 and E2 for RVC structural genes 6 and 4 (VP6, and VP4) and non-structural gene 4 (NSP4), respectively. Furthermore, the VP6 gene of HuRVC/H28/2013/India shows the highest similarity to a recently-reported human-like porcine RVC (PoRVC/ASM140/2013/India, KT932963) from India suggesting zoonotic transmission. We also report a full-length NSP4 gene sequence of human RVC from India. Under the One-health platforms there is a need to launch combined human and animal RVC surveillance programs for a better understanding of the epidemiology of RVC infections and for implementing control strategies.Reoviridae, possess 11 double-stranded segments of RNA that encode six structural viral proteins (VP1, VP2, VP3, VP4, VP6, VP7) and five/six non-structural proteins (NSP1–NSP5/6) [7]. Based on the antigenic properties of the major inner capsid protein (VP6), RVs are subdivided into eight well-characterized species (A–H) and two putative species viz. I and J [8–10]. Humans and other mammalian species are affected by species A, B, C and H rotaviruses and birds by species D, F and G, and species E has been reported exclusively in pigs [7,8,11–17]. The newly-proposed species I is reported in dogs [18] and cats [19], whereas species J is found in bats [10].

Post rotavirus vaccine introduction in Mozambique (September 2015), we documented a decline in rotavirus-associated diarrhoea and genotypes changes in our diarrhoeal surveillance spanning 2008–2021. This study aimed to perform whole-genome sequencing of rotavirus strains from 2009 to 2012 (pre-vaccine) and 2017–2018 (post-vaccine). Rotavirus strains previously detected by conventional PCR as G2P[4], G2P[6], G3P[4], G8P[4], G8P[6], and G9P[6] from children with moderate-to-severe and less-severe diarrhoea and without diarrhoea (healthy community controls) were sequenced using Illumina MiSeq ® platform and analysed using bioinformatics tools. All these G and P-type combinations exhibited DS-1-like constellation in the rest of the genome segments as, I2-R2-C2-M2-A2-N2-T2-E2-H2. Phylogenetic analysis revealed that strains from children with and without diarrhoea clustered together with other Mozambican and global strains. Notably, the NSP4 gene of strains G3P[4] and G8P[4] in children with diarrhoea clustered with animal strains, such as bovine and caprine, with similarity identities ranging from 89.1 to 97.0% nucleotide and 89.5-97.0% amino acids. Our findings revealed genetic similarities among rotavirus strains from children with and without diarrhoea, as well as with animal strains, reinforcing the need of implementing studies with One Health approach in our setting, to elucidate the genetic diversity of this important pathogen.

Porcine reproductive and respiratory syndrome virus (PRRSV) has continuously mutated since its first isolation in China in 1996, leading to difficulties in infection prevention and control. Infections caused by PRRSV-2 strains are the main epidemic strains in China, as determined by phylogenetic analysis. In this study, we focused on the prevalence and genetic variations of the non-structural protein 4 (NSP4) from PRRSV-2 over the past 20 years in China. The fundamental biological properties of the NSP4 were predicted, and an analysis and comparison of NSP4 homology at the nucleotide and amino acid levels was conducted using 123 PRRSV-2 strains. The predicted molecular weight of the NSP4 protein was determined to be 21.1 kDa, and it was predicted to be a stable hydrophobic protein that lacks a signal peptide. NSP4 from different strains exhibited a high degree of amino acid (85.8–100%) and nucleotide sequence homology (81.0–100%). Multiple amino acid substitutions were identified in NSP4 among 15 representative PRRSV-2 strains. Phylogenetic analysis showed that the lineage 8 and 1 strains, the most prevalent strains in China, were indifferent clades with a long genetic distance. This analysis will help fully elucidate the parameters of the PRRSV NSP4 epidemic in China to lay a foundation for adequate understanding of the function of NSP4. Genetic information results from the accumulation of conserved and non-conserved sequences. The high conservation of the NSP4 gene determines the most basic life traits and functions of PRRSV. Analyzing the spatial structure of NSP4 protein and studying the genetic evolution of NSP4 not only provide the theoretical basis for how NSP4 participates in the regulation of the innate response of the host but also provide a target for genetic manipulation and a reasonable target molecule and structure for new drug molecules.

Human astroviruses (HAstVs) are considered important causative pathogens of acute gastroenteritis (AGE) in children under 5 years of age worldwide, along with group A rotavirus (RVA), norovirus (NoV), and enteric adenovirus (EAdV). The present study was aimed to both detect HAstV and its co-infections and investigate genetic analysis of circulating HAstV and co-infected virus in hospitalized children under 5 years of age with AGE in Iran. Accordingly, a sum of 200 stool specimens were screened by PCR for HAstV during 2021–2022. The HAstV was found in 0.5% of 200 specimens (n = 1) while was co-infected with RVA. The genetic and phylogenetic analysis indicated HAstV1 genotype, which clustered with viruses from lineage 1b, which has not been previously reported in Iran. The detected RVA strain belonged to G1 lineage II/P[8]-lineage III, which has been reported previously in Iran as the most common strain. The further genetic analysis of RVA VP6 and NSP4 demonstrated an atypical genotype pattern G1P[8]-I1-E2, as a mono-reassortant of a Wa-like genogroup, which appeared to be reassorted with the NSP4 gene of E2 genotype of the G2P[4] DS-1 genogroup. Although the clinical outcomes of the AGE-causing viruses co-infection is not yet entirely clear, it seems that future studies will be helpful to merge clinical and epidemiological data of co-infecting viruses for a more accurate medical and clinical relevance in symptomatic children.

In Brazil, molecular surveillance expanded after Rotarix™ vaccine introduction, alongside G2P[4] dominance. The G2P[6] genotype, despite sharing the same DS-1-like constellation as G2P[4] strains, remains rare. This retrospective study analyzed eight Brazilian G2P[6] strains (2012–2014) through RT-PCR and 11-segments sequencing, followed by phylogenetic analysis. Two distinct groups were identified: 2012–2013 strains (six) carried a DS-1-like backbone with the rare NSP4 E6 genotype, while 2014 strains (two) exhibited the classical DS-1-like constellation with E2. Phylogenetic analysis confirmed the two main clusters: 2012–2013 strains related to classical G2P[4] and uncommon global genotypes, and 2014 strains resembling emerging DS-1-like G1/G3/G8P[8] reassortants. The 2012–2013 strains clustered within G2-VP7 Lineage IVa, while the 2014 strains belonged to Lineage V, reflecting the global distribution of these variants. All VP4 genes were classified within the P[6]-Ia lineage, with phylogenetic analyses suggesting separate introductions from Asia and Africa. The E6 NSP4 gene segment identified in these strains has an undetermined origin and was not previously associated with G2P[6] strains in Brazil. Despite similarities to G2P[4], G2P[6] strains remain rare, with no genomic features explaining their limited spread. Phylogenetic data indicate multiple reassortment events and international viral exchange, highlighting Brazil’s role in RVA diversity. Ongoing full-genome surveillance is crucial to track rare variants and assess their public health relevance.

Objective To isolate a prevalent G9P[8] group A rotavirus (RVA) (N4006) in China and investigate its genomic and evolutionary characteristics, with the goal of facilitating the development of a new rotavirus vaccine. Methods The RVA G9P[8] genotype from a diarrhea sample was passaged in MA104 cells. The virus was evaluated by TEM, polyacrylamide gel electrophoresis, and indirect immunofluorescence assay. The complete genome of virus was obtained by RT-PCR and sequencing. The genomic and evolutionary characteristics of the virus were evaluated by nucleic acid sequence analysis with MEGA ver. 5.0.5 and DNASTAR software. The neutralizing epitopes of VP7 and VP4 (VP5* and VP8*) were analyzed using BioEdit ver. 7.0.9.0 and PyMOL ver. 2.5.2. Results The RVA N4006 (G9P[8] genotype) was adapted in MA104 cells with a high titer (10 5.5 PFU/mL). Whole-genome sequence analysis showed N4006 to be a reassortant rotavirus of Wa-like G9P[8] RVA and the NSP4 gene of DS-1-like G2P[4] RVA, with the genotype constellation G9-P[8]-I1-R1-C1-M1-A1-N1-T1-E2-H1 (G9P[8]-E2). Phylogenetic analysis indicated that N4006 had a common ancestor with Japanese G9P[8]-E2 rotavirus. Neutralizing epitope analysis showed that VP7, VP5*, and VP8* of N4006 had low homology with vaccine viruses of the same genotype and marked differences with vaccine viruses of other genotypes. Conclusion The RVA G9P[8] genotype with the G9-P[8]-I1-R1-C1-M1-A1-N1-T1-E2-H1 (G9P[8]-E2) constellation predominates in China and may originate from reassortment between Japanese G9P[8] with Japanese DS-1-like G2P[4] rotaviruses. The antigenic variation of N4006 with the vaccine virus necessitates an evaluation of the effect of the rotavirus vaccine on G9P[8]-E2 genotype rotavirus.

Non-structural protein 4 (NSP4) is a critical protein for rotavirus (RV) replication and assembly. This protein has multiple domains and motifs that predispose its function and activity. NSP4 has a sequence divergence in human and animal RVs. Recently, 14 genotypes (E1-E14) of NSP4 have been identified, and E1 and E2 have been shown to be the most common genotypes in human. The gene and protein sequence of NSP4 in RV-positive samples were inspected with the aim of NSP4 genotyping and variation analysis in viroporin and other domains. P and G typings of RV samples were carried out by WHO primers using a semi-multiplex PCR method. Non-typeable RV samples were amplified by conserved primers and sequenced. In viroporin and enterotoxin, conserved sequence was detected, and amino acids substitution with the same biochemical properties was found. Association of NSP4 genotype with P or G genotyping G1/G9 correlates with E1 genogroups. In electrophoretyping of RV, E2 genotype had a short pattern when compared to E1.

The present study reports the detection and molecular characterisation of rotavirus C (RVC) in sloth bears (Melursus ursinus) rescued from urban areas in India. Based on an RVC VP6 gene-targeted diagnostic RT-PCR assay, 48.3% (42/87) of sloth bears tested positive for RVC infection. The VP6, VP7, and NSP4 genes of three sloth bear RVC isolates (UP-SB19, 21, and 37) were further analysed. The VP6 genes of RVC UP-SB21 and 37 isolates were only 37% identical. The sequence identity, TM-score from structure alignment, and selection pressure (dN/dS) of VP6 UP-SB37 with pig and human RVCs isolates were (99.67%, 0.97, and 1.718) and (99.01%, 0.93, and 0.0340), respectively. However, VP6 UP-SB21 has an identity, TM-score, and dN/dS of (84.38%, 1.0, and 0.0648) and (99.63%, 1.0, and 3.7696) with human and pig RVC isolates, respectively. The VP7 genes from UP-SB19 and 37 RVC isolates were 79.98% identical and shared identity, TM-score, and dN/dS of 88.4%, 0.76, and 5.3210, along with 77.98%, 0.77, and 4.7483 with pig and human RVC isolates, respectively. The NSP4 gene of UP-SB37 RVC isolates has an identity, TM-score, and dN/dS of 98.95%, 0.76, and 0.2907, along with 83.12%, 0.34, and 0.2133 with pig and human RVC isolates, respectively. Phylogenetic analysis of the nucleotide sequences of the sloth bear RVC isolates assigned the isolate UP-SB37 to genotype G12, I2 for RVC structural genes VP7 and VP6, and E1 for NSP4 genes, respectively, while isolates UP-SB19 and UP-SB21 were classified as genotype G13 and GI7 based on the structural gene VP7, respectively. The study suggests that the RVCs circulating in the Indian sloth bear population are highly divergent and might have originated from pigs or humans, and further investigation focusing on the whole genome sequencing of the sloth bear RVC isolate may shed light on the virus origin and evolution.

Introduction of animal group A rotavirus (RVA) gene segments into the human RVA population is a major factor shaping the genetic landscape of human RVA strains. The VP6 and NSP4 genes of 74 G/P-genotyped RVA isolates collected in Rawalpindi during 2010 were analyzed, revealing the presence of VP6 genotypes I1 (60.8%) and I2 (39.2%) and NSP4 genotypes E1 (60.8%), E2 (28.3%) and E-untypable (10.8%) among the circulating human RVA strains. The typical human RVA combinations I1E1 and I2E2 were found in 59.4% and 24.3% of the cases, respectively, whereas 5.4% of the RVA strains were reassortants, i.e., either I1E2 or I2E1. The phylogeny of the NSP4 gene showed that one G2P[4] and two G1P[6] RVA strains clustered with porcine E1 RVA strains or RVA strains that were considered to be (partially) of porcine origin. In addition, the NSP4 gene segment of the unusual human G6P[1] RVA strains clustered closely with bovine E2 RVA strains, further strengthening the hypothesis of an interspecies transmission event. The study further demonstrates the role of genomic re-assortment and the involvement of interspecies transmission in the evolution of human RVA strains. The VP6 and NSP4 nucleotide sequences analyzed in the study received the GenBank accession numbers KC846908- KC846971 and KC846972-KC847037, respectively.

Rotavirus A (RVA) possesses a genome of 11 double-stranded (ds) RNA segments, and each segment encodes one protein, with the exception of segment 11. NSP4 is a non-structural multifunctional protein encoded by segment 10 that defines the E-genotype. From the 31 E-genotypes described, genotype E12 has been described in Argentina, Uruguay, Paraguay, and Brazil in RVA strains infecting different animal species and humans. In this work, we studied the evolutionary relationships of RVA strains carrying the E12 genotype in South America using phylogenetic and phylodynamic approaches. We found that the E12 genotype has a South American origin, with a guanaco (Lama guanicoe) strain as natural host. Interestingly, all the other reported RVA strains carrying the E12 genotype in equine, bovine, caprine, and human strains are related to RVA strains of camelid origin. The evolutionary path and genetic footprint of the E12 genotype were reconstructed starting with the introduction of non-native livestock species into the American continent with the Spanish conquest in the 16th century. The imported animal species were in close contact with South American camelids, and the offspring were exposed to the native RVA strains brought from Europe and the new RVA circulating in guanacos, resulting in the emergence of new RVA strains in the current lineages’ strongly species-specific adaption. In conclusion, we proposed the NSP4 E12 genotype as a genetic geographic marker in the RVA strains circulating in different animal species in South America.