Emerging porcine Enterovirus G infections, epidemiological, complete genome sequencing, evolutionary and risk factor analysis in India.

The current study reports the in-depth analysis of the epidemiology, risk factors, and molecular characterization of a complete genome of Enterovirus G (EV-G) isolated from Indian pigs. We analysed several genes of EV-G isolates collected from various provinces in India, using phylogenetic analysis, recombination detection, SimPlot, and selection pressure analyses. Our analysis of 534 porcine faecal samples revealed that 11.61% (62/534) of the samples were positive for EV-G. While the G6 genotype was the most predominant, our findings showed that Indian EV-G strains also clustered with EV-G types G1, G6, G8, and G9. Furthermore, Indian EV-G strains exhibited the highest nucleotide similarity with Vietnamese (81.3%) and Chinese EV-G isolates (80.3%). Moreover, we identified a recombinant Indian EV-G strain with a putative origin from a Japanese isolate and South Korean EV-G isolate. In summary, our findings provide significant insights into the epidemiology, genetic diversity, and evolution of EV-G in India.

Migratory Gaddi sheep and goats as potential carriers of Theileria infection: a molecular survey.

Theileriosis caused by parasites of the genus Theileria, is a vector-borne haemoprotozoan parasitic disease of critical concern in small ruminants. This study aimed to explore the infection status of migratory Gaddi sheep and goats with parasites from the Theileria genus in concurrence with ectoparasite infestations using molecular methods. Seventy three apparently healthy animals were randomly sampled from different flocks of migratory Gaddi sheep and goats and were systematically screened for ectoparasitic infestations. Molecular investigation for theileriosis was conducted using the genus wide polymerase chain reaction (PCR) technique. Out of 56 (76.71%) animals positive for the genus Theileria, 2 randomly selected amplicons were sequenced and subjected to BLAST analysis and were showing 99.71% identity with Theileria luwenshuni, a pathogenic Theileria species of small ruminants. To confirm the presence of T. luwenshuni, species-specific PCR was attempted to identify that 38 (52.05%) animals were infected by T. luwenshuni. On analysing the molecular prevalence data of Theileria to the ectoparasitism, it was evident that the infection existed in the animals irrespective of the type of ectoparasitic infestation and even T. luwenshuni was found in non-infested animals also. This is the first report of subclinical infections of T. luwenshuni in sheep and goats of Northern India and its potential carrier status. The asymptomatic carrier status of these nomadic animals is a matter possessing serious implications on the disease transmission rates and the production economics of small ruminant production in this region.

Evolutionary and codon usage preference insights into spike glycoprotein of SARS-CoV-2.

Interaction of SARS-CoV-2 spike glycoprotein with the ACE2 cell receptor is very crucial for virus attachment to human cells. Selected mutations in SARS-CoV-2 S-protein are reported to strengthen its binding affinity to mammalian ACE2. The N501T mutation in SARS-CoV-2-CTD furnishes better support to hotspot 353 in comparison with SARS-CoV and shows higher affinity for receptor binding. Recombination analysis exhibited higher recombination events in SARS-CoV-2 strains, irrespective of their geographical origin or hosts. Investigation further supports a common origin among SARS-CoV-2 and its predecessors, SARS-CoV and bat-SARS-like-CoV. The recombination events suggest a constant exchange of genetic material among the co-infecting viruses in possible reservoirs and human hosts before SARS-CoV-2 emerged. Furthermore, a comprehensive analysis of codon usage bias (CUB) in SARS-CoV-2 revealed significant CUB among the S-genes of different beta-coronaviruses governed majorly by natural selection and mutation pressure. Various indices of codon usage of S-genes helped in quantifying its adaptability in other animal hosts. These findings might help in identifying potential experimental animal models for investigating pathogenicity for drugs and vaccine development experiments.

Genomic Analysis of an Indian G8P[1] Caprine Rotavirus-A Strain Revealing Artiodactyl and DS-1-Like Human Multispecies Reassortment.

The surveillance studies for the presence of caprine rotavirus A (RVA) are limited in India, and the data for the whole-genome analysis of the caprine RVA is not available. This study describes the whole-genome-based analysis of a caprine rotavirus A strain, RVA/Goat-wt/IND/K-98/2015, from a goat kid in India. The genomic analysis revealed that the caprine RVA strain K-98, possess artiodactyl-like and DS-1 human-like genome constellation G8P[1]-I2-R2-C2-M2-A3-N2-T6-E2-H3. The three structural genes (VP2, VP4, and VP7) were close to caprine host having nucleotide-based identity range between 97.5 and 98.9%. Apart from them, other gene segments showed similarity with either bovine or human like genes, ultimately pointing toward a common evolutionary origin having an artiodactyl-type backbone of strain K-98. Phylogenetically, the various genes of the current study isolate also clustered inside clades comprising Human-Bovine-Caprine isolates from worldwide. The current findings add to the knowledge on caprine rotaviruses and might play a substantial role in designing future vaccines or different alternative strategies combating such infections having public health significance. To the best of our knowledge, this is the first report on the whole-genome characterization of a caprine RVA G8P[1] strain from India. Concerning the complex nature of the K-98 genome, whole-genome analyses of more numbers of RVA strains from different parts of the country are needed to comprehend the genomic nature and genetic diversity among caprine RVA.

First report and genetic characterization of porcine astroviruses of lineage 4 and 2 in diarrhoeic pigs in India.

Porcine astroviruses (PAstVs) have extended their distribution globally and have a high prevalence; however, their clinical significance is still under investigation. Thus far, information about their prevalence and diversity in the Indian pig population is unknown. This study is the first report on the prevalence and genetic characterization of PAstVs in diarrhoeic piglets in India. From January 2013 to December 2017, 757 samples were screened using an RT-PCR assay and PAstV infection was detected in 17.6% (133/757) pigs. Of the 133 positive samples, 79 (59.4%) were positive for PAstV alone, whereas 54 (40.6%) were found to be co-infected with porcine rotavirus A (PoRVA). Phylogenetic analysis of RdRp/capsid gene region revealed high genetic heterogeneity among PAstV sequences, with a predominance of PAstV lineage 4 and detection of lineage 2. The lineage 4 PAstVs exhibited 61.2%-94.5% sequence similarity at the nucleotide level to other reported sequences, whereas lineage 2 strain shared 66.0%-71.6% sequence identity with cognate sequences of the same lineage. This is the first report on PAstV and circulation of lineages 4 and 2 in India. Further, phylogenetic analysis indicates a multiphyletic origin of PAstV strains and suggests cross-border circulation of PAstVs with a similar genetic configuration in Asian countries.

Epidemiologic Status of Picobirnavirus in India, A Less Explored Viral Disease.

Since the unexpected discovery of picobirnaviruses (PBV) in 1988, they have been reported in many animals including mammals and birds, which comprises both terrestrial and marine species. Due to their divergent characteristics to other viral taxa they are classified into a new family Picobirnaviridae. Although their pathogenicity and role in causing diarrhea still remains a question since they have been discovered in symptomatic and asymptomatic cases both. Recent studies employing state-of-art molecular tools have described their presence in various clinical samples, like stool samples from different mammals and birds, respiratory tracts of pigs and humans, sewage water, different foods, etc. Furthermore, their epidemiological status from different parts of the world in different hosts has also increased. Due to their diverse host and irregular host pattern their role in causing diarrhea remains alien. The heterogeneity nature can be ascribed to segmented genome of PBV, which renders them prone to continuous reassortment. Studies have been hampered on PBVs due to their non-adaptability to cell culture system. Here, we describe the molecular epidemiological data on PBVs in India and discusses the overall status of surveillance studies carried out till date in India.

Frequency distribution of porcine rotavirus-A and capsid protein gene based sequence and phylogenetic analysis indicating marked heterogeneity among prevailing strains, India.

Rotavirus (RV), is an etiological agent of acute infantile diarrhea in both humans and animals including poultry. Among the eight accepted species/types of RVs, RV-A is genetically and antigenically the most diverse. RV-A associated enteritis is a major problem in the weaning and post-weaning piglets. Due to high genetic variability in the antigenic regions, RV-A is thought to have high interspecies jumping probability. In this study, comparatively a large sized sample (n = 757) was screened, where the samples were collected from diarrheic porcine population of north (Uttar Pradesh), North eastern (Assam, Nagaland, Meghalaya, Tripura, Manipur, Mizoram and Arunachal Pradesh) and Southern states of India (Kerala, Karnataka and Tamil Nadu). The VP6 gene based reverse-transcription (RT)-PCR based screening of the samples for RV-A identified 42.4% (321/757) positivity, where highest identification was from Uttar Pradesh 119 (37.07%), followed by 74 (23.05%), 34 (10.6%), 31 (9.65%), 21 (6.54%), 15 (4.67%), 11 (3.43%), 8 (2.49%), 3 (0.93%), 3 (0.93%) and 2 (0.62%) from Assam, Nagaland, Meghalaya, Tripura, Kerala, Manipur, Mizoram, Arunachal Pradesh, Karnataka and Tamil Nadu, respectively. Percentage identity calculation of the VP6 gene sequences from different porcine RV-A revealed 77.1-97.3% identity within the Indian porcine RV-A strains of the current study. Phylodendrogram and percent identity based analysis of the amplified and sequenced full length VP6 gene confirmed the presence of new VP6 genotypes (I1 and I5). Although, there are reports of detection of porcine RV-A based on VP6 gene from India, no lineage/genotype based characterization is available for the target gene. Till date, only a single VP6 type (I2) has been confirmed from pig population of India. Here, the findings confirm the circulation of diverse RV-A strains in porcine population in India.

Identification and In Silico Characterization of a Genetically Distinct Avian Rotavirus D Capsid Gene, VP7.

Rotavirus D (RV-D) is gaining importance as a cause of gastroenteritis and runting and stunting syndrome (RSS) in poultry. To date, information is scarce on the molecular analysis of RV-D isolates worldwide. In this study, the VP7 gene, a major constituent of outer capsid structural protein, from a RV-D isolate (UKD48) obtained from Uttarakhand state was analyzed. Phylogenetically, the RV-D isolate was found to be closely related to a South Korean strain, and the nucleotide percent identity varied from 80.4–84.2% with other RV-D strains available globally. Furthermore, domain investigation within 21 aligned amino acid sequences of the VP7 gene affirmed that this gene has several domains: a conserved glycosylation site (N–I–T) having an important role in protein folding; a N-terminal signal peptide (“ITG”) for endoplasmic reticulum retention; and two hydrophobic sites for elucidating transmembrane portions, antigenic structures, and so forth. The findings suggest that the VP7 gene of the Indian RV-D isolate is genetically distinct from those of other avian RV-Ds. Although biological evidence is still needed to prove the functional characteristics of these domains in outer capsid structural proteins, the present study adds new knowledge and derives the need for further investigation.

Species C Rotaviruses in Children with Diarrhea in India, 2010-2013: A Potentially Neglected Cause of Acute Gastroenteritis.

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].

Avian Group D Rotaviruses: Structure, Epidemiology, Diagnosis, and Perspectives on Future Research Challenges.

In 1981, a new virus (virus 132) was described for the first time with morphological and biochemical similarities to rotaviruses (RVs), but without antigenic similarity to any of the previously known rotavirus groups. Subsequently, it was re-designated as D/132, and formed a new serogroup among rotaviruses, the group D rotavirus (RVD). Since their identification, RVs are the leading cause of enteritis and diarrhea in humans and various animal species, and are also associated with abridged growth, particularly in avian species. Recently, RVD has been suggested to play a role in the pathogenesis of runting and stunting syndrome (RSS), alongside other viruses such as reovirus, astrovirus, coronavirus, and others, all of which cause colossal economic losses to the poultry industry. RVD has been reported from several countries worldwide, and to date, only one complete genome sequence for RVD is available. Neither an immunodiagnostic nor a vaccine is available for the detection and prevention of RVD infection. Despite our growing understanding about this particular group, questions remain regarding its exact prevalence and pathogenecity, and the disease-associated annual losses for the poultry industry. Here, we describe the current knowledge about the identification, epidemiology, diagnosis, and prevention of RVD in poultry.

Unexpected detection of porcine rotavirus C strains carrying human origin VP6 gene.

BACKGROUND: Rotavirus C (RVC), a known etiological agent of diarrheal outbreaks, mainly inflicts swine population globally with sporadic incidence in human, cattle, ferret, mink and dog. OBJECTIVE: To demonstrate the presence of RVC in Indian swine population and characterization of its selected structural (VP6) and non-structural (NSP4 and NSP5) genes. METHODS: A total of 108 diarrheic samples from different regions of India were used. Isolated RNA was loaded onto polyacrylamide gel to screen for the presence of RVs through the identification of specific electrophoretic genomic migration pattern. To characterize the RVC strains, VP6 gene and NSP4 and NSP5 genes were amplified, sequenced and analyzed. RESULTS: Based on VP6 gene specific diagnostic RT-PCR, the presence of RVC was confirmed in 12.0% (13/108) piglet fecal specimens. The nucleotide sequence analysis of VP6 gene, encoding inner capsid protein, from selected porcine RVC (PoRVC) strains revealed more than 93% homologies to human RVC strains (HuRVC) of Eurasian origin. These strains were distant from hitherto reported PoRVCs and clustered with HuRVCs, owning I2 genotype. However, the two non-structural genes, i.e. NSP4 and NSP5, of these strains were found to be of swine type, signifying a re-assortment event that has occurred in the Indian swine population. CONCLUSION: The findings indicate the presence of human-like RVC in Indian pigs and division of RVC clade with I2 genotype into further sub-clades. To the best of our knowledge, this appears to be the first report of RVC in Indian swine population. Incidence of human-like RVC VP6 gene in swine supports its subsequent zoonotic prospective.

Ebola virus - epidemiology, diagnosis, and control: threat to humans, lessons learnt, and preparedness plans - an update on its 40 year's journey.

Ebola virus (EBOV) is an extremely contagious pathogen and causes lethal hemorrhagic fever disease in man and animals. The recently occurred Ebola virus disease (EVD) outbreaks in the West African countries have categorized it as an international health concern. For the virus maintenance and transmission, the non-human primates and reservoir hosts like fruit bats have played a vital role. For curbing the disease timely, we need effective therapeutics/prophylactics, however, in the absence of any approved vaccine, timely diagnosis and monitoring of EBOV remains of utmost importance. The technologically advanced vaccines like a viral-vectored vaccine, DNA vaccine and virus-like particles are underway for testing against EBOV. In the absence of any effective control measure, the adaptation of high standards of biosecurity measures, strict sanitary and hygienic practices, strengthening of surveillance and monitoring systems, imposing appropriate quarantine checks and vigilance on trade, transport, and movement of visitors from EVD endemic countries remains the answer of choice for tackling the EBOV spread. Herein, we converse with the current scenario of EBOV giving due emphasis on animal and veterinary perspectives along with advances in diagnosis and control strategies to be adopted, lessons learned from the recent outbreaks and the global preparedness plans. To retrieve the evolutionary information, we have analyzed a total of 56 genome sequences of various EBOV species submitted between 1976 and 2016 in public databases.

Analysis of codon usage pattern evolution in avian rotaviruses and their preferred host.

Rotavirus infection is a worldwide problem, with occurrence of highly divergent viruses classified in 8 species (A-H). We report here the evolution assessment of codon usage patterns in virus-host system in avian rotavirus (AvRV) of species RVA, RVD, RVF and RVG (preferentially affecting birds). The nucleotide contents, codon usage bias (CUB), relative synonymous codon usage (RSCU), and effective number of codons (ENCs) values were investigated targeting overexpressing major inner capsid viral protein (VP6) of these AvRV species. The results confirm that the evolutionary characteristics influences the rotavirus (RV) genetic diversity and impact of host's natural selection on the AvRVs codons. Synonymous codon usage patterns were evaluated following multivariate statistical procedures on all available AvRV coding gene sequences. RSCU trees accommodated all AvRV species and preferred host sequences in one topology confirming greater imminence of AvRVs with the host chicken cell genes. Similarly, the codon adaptation index (CAI) results also displayed a higher adaptation of AvRVs to its chicken host. The codon preference analysis of RVs revealed that VP6 gene express more proficiently in the yeast system, whereas, codon optimization might be required for the effectual expression in Escherichia coli and Homo sapiens. The findings provide basic evidence on the dynamics of AvRV evolution and its host adaptation, which could be exploited for additional research on avian species in future.