Jobin Jose Kattoor

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 hosts 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.

Molecular evidence of group D rotavirus in commercial broiler chicks in India

Group D rotavirus (RVD) is one of the evolving viral causes of acute gastroenteritis in avian species all over the world but its frequency in Indian poultry is not known. We report here the first sequence-confirmed RVD infection in 1–2 week old broiler chicks from northern India. Initial confirmation of type D rotavirus was done using polyacrylamide gel electrophoresis and VP6 gene based reverse-transcriptase-PCR (RT-PCR) for group D rotavirus, which produced a specific 742 bp amplicon in positive cases. The intestinal contents which showed presence of group D rotavirus strain were designated as RVD/Avian/India/PTN-14/2012/ GXP[X] and RVD/Avian/India/UKD-48/2012/GXP[X]. The comparative sequence analysis based on partial VP6 gene of type D rotavirus Indian strains revealed higher homology with the VP6 genes of the avian group D rotaviruses from Brazil, Germany, Netherlands, Bangladesh and UK, both at the nucleotide (87.2–89.6%) and amino acid (98.7–99.5%) levels. These two Indian avian RVD isolates clustered together, away from other Asian group D isolates from Bangladesh. The analysis of group specific VP6 protein showed amino acid changes at only two positions i.e. 228 and 384 when compared to the reference group D rotavirus strain (GenBank accession number: GU733448) confirming conserved nature of this protein. This seems to be the first sequence-confirmed detection of group D avian rotavirus in broiler chicks from India. The findings emphasise the importance of this virus in enteric infections of Indian poultry flocks. The study also emphasises the need for intensifying the epidemiological surveillance for group D rotaviruses in the near future.

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

ABSTRACT 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.

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

ABSTRACT 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.

Development of VP6 Gene Specific Reverse Transcription(RT)-PCR Assay for Detectionof Avian Group D Rotavirus in Diarrheic Chickens

Development of VP6 Gene Specific Reverse Transcription(RT)-PCR Assay for Detection of Avian Group D Rotavirus in Diarrheic Chickens The group D rotavirus (RVD) is an evolving viral cause of acute gastroenteritis in avian species world over. Despite the increase in the frequency of avian RVD infections world over, isolates from Indian poultry have yet to be characterised. Paucity of epidemiological studies with unavailability of sensitive and rapid detection methods for RVD in poultry led to design of the present study, aiming at development and validation of a RT-PCR assay, targeting the conserved group specific region of VP6 gene of RVD, for virus detection purpose.

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.

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.

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.

Analysis of structure-function relationship in porcine rotavirus A enterotoxin gene

Rotavirus (RV)-infected piglets are presumed to be latent sources of heterologous RV infection in humans and other animals. In RVs, non-structural protein 4 (NSP4) is the major virulence factor with pleiotropic properties. In this study, we analyzed the nsp4 gene from porcine RVs isolated from diarrheic and non-diarrheic cases at different levels of protein folding to explore correlations to diarrhea-inducing capabilities and evolution of nsp4 in the porcine population. Full-length nsp4 genes were amplified, cloned, sequenced, and then analyzed for antigenic epitopes, RotaC classification, homology, genetic relationship, modeling of NSP4 protein, and prediction of post-translational modification. RV presence was observed in both diarrheic and non-diarrheic piglets. All nsp4 genes possessed the E1 genotype. Comparison of primary, secondary, and tertiary structure and the prediction of post-translational modifications of NSP4 from diarrheic and non-diarrheic piglets revealed no apparent differences. Sequence analysis indicated that nsp4 genes have a multi-phyletic evolutionary origin and exhibit species independent genetic diversity. The results emphasize the evolution of the E9 nsp4 genotype from the E1 genotype and suggest that the diarrhea-inducing capability of porcine RVs may not be exclusively linked to its enterotoxin gene.