Background and objectives Zika virus cases were confirmed from Pune, Western India, during an outbreak in June 2024. We conducted an entomological investigation in the Pune municipal corporation area and aimed to characterise the Zika virus from Aedes aegypti mosquitoes at the genomic level. Methods An entomological survey was conducted covering 16 locations in the Pune Municipal Corporation, between June and August 2024. Adult mosquitoes were collected using aspirators, while larvae were collected with the standard dipping and pipetting method. Aedes vector density was assessed, and samples were pooled. Zika virus positivity was assessed using real-time RT-qPCR. Genomic characterisation of the strains was achieved by whole-genome sequencing using the Oxford Nanopore technology platform. Results A high density of Aedes mosquitoes was revealed in the Pune area. Zika virus positivity was observed in 34/128 (26.5%) of Aedes aegypti mosquito pools. Full genome sequences of two ZIKV isolates were recovered from adult mosquito pools. Their time-scaled phylogenetic analysis showed grouping within the Asian lineage, having a recent ancestral time of evolution around 2014, indicating the introduction of newer virus strains in the recent past in India. Mutations E-V473M and motif E-VNDT, indicative of faster viral replication and higher mosquito infectivity, were noted in only one of the two isolates. Interpretation and conclusions With a high positivity of the Zika virus in Aedes aegypti, this study highlights the ongoing risk prevailing in the Pune municipal corporation area. Distinct characteristics of the circulating strains of the virus, and their evolutionary links to different global strains, underlines the importance of continuous genomic surveillance in vectors and hosts.

Background Varicella, commonly known as chickenpox is a neglected disease in India with numerous outbreaks reported in the last decade. Unfortunately, varicella vaccine is not included in India’s Universal Immunization Program (UIP), and active surveillance is not in place. Investigation of suspected varicella cases from various Indian regions was done to understand disease burden and molecular epidemiology. Methods Between 2016 and 2025, 195 clinical cases from suspected chickenpox cases were referred for virological investigation from five States and one Union Territory of India. Samples were analyzed for Varicella-Zoster Virus (VZV) using IgM-EIA and/or conventional PCR. Virus isolation was attempted on Vero, MRC-5, and WI-38 cell lines. PCR-positive products were sequenced for phylogenetic analysis to identify circulating VZV clades. Results Of the 195 suspected cases, 159 (81.53 %) were confirmed by serological or molecular methods. Majority of the cases (n = 152, 77.94 %) occurred in individuals under 18 years of age, with higher incidence among males (n = 119) than females (n = 76). Of 58 diverse clinical specimens, 43 showed VZV DNA. Sequencing of representative PCR products showed circulation of VZV clade-5 in 29 cases and clade-9 in a single case. Unfortunately, attempts at VZV isolation were not successful. Conclusion The study confirms a high laboratory-confirmed rate of varicella among suspected cases in India, with children being the most affected. Molecular data identified VZV clade-5 as primary circulating genotype. These findings highlight a significant burden of chickenpox and provide molecular evidence to support inclusion of the varicella vaccine in India’s Universal Immunization Program.

Highly pathogenic avian influenza (HPAI) clade 2.3.4.4b H5Nx viruses are a major zoonotic concern. Clade 2.3.4.4b was first reported in India during the HPAI H5N8 outbreaks in zoological parks, in 2016. Recently, in June 2024, clade 2.3.4.4b HPAI H5N1 virus outbreaks were reported from Kerala. The genetic and molecular characterization of this H5N1 virus revealed that it is a novel reassortant comprising of internal genes derived from LPAI viruses reported from Kerala; and some of European origin. The exact spatio-temporal origins of these viruses were unknown. The present study was undertaken to trace the possible source of the clade 2.3.4.4b HPAI H5N1 and H5N8 viruses reported from India. Molecular clock and phylogeography analyses were carried out using BEAST v1.10.4 for the global and regional datasets of clade 2.3.4.4b H5Nx virus hemagglutinin gene sequences downloaded from the GISAID database. Preliminary phylogeography analysis using the global dataset revealed direct linkages of South Asia with the Middle East, North Africa, Japan, Korea, and China in the transmission network. Further analysis using regional datasets revealed that the H5N1 isolates reported from Kerala in 2024 had been introduced to India from East Asia, around 2022. The two reported H5N8 viruses from 2016 showed separate introductions from East Asian countries, including China and Japan respectively, around the year 2015. In the present study we report the spatio-temporal origins of clade 2.3.4.4b HPAI H5Nx viruses from South Asia. The analyses would be useful in furthering our understanding of this pathogen of human and animal health importance.

India has reported highly pathogenic avian influenza (HPAI) H5N1 virus outbreaks since 2006, with the first human case reported in 2021. These included viruses belonging to the clades 2.2, 2.2.2, 2.2.2.1, 2.3.2.1a, and 2.3.2.1c. There are currently no data on the gene pool of HPAI H5N1 viruses in India. Molecular clock and phylogeography analysis of the HA and NA genes; and phylogenetic analysis of the internal genes of H5N1 viruses from India were carried out. Sequences reported from 2006 to 2015; and sequences from 2021 that were available in online databases were used in the analysis. Five separate introductions of H5N1 viruses into India were observed, via Indonesia or Korea (2002), Bangladesh (2009), Bhutan (2010), and China (2013, 2018) (clades 2.2, 2.2.2, 2.2.2.1, 2.3.2.1a, 2.3.2.1c, and 2.3.4.4b). Phylogenetic analysis revealed eight reassortant genotypes. The H5N1 virus isolated from the human case showed a unique reassortant genotype. Amino acid markers associated with adaptation to mammals were also present. This is the first report of the spatio-temporal origins and gene pool analysis of H5N1 viruses from India, highlighting the need for increased molecular surveillance.

Background & objectives Genetic analysis of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) strains circulating in India during 2020-2022 was carried out to understand the evolution of potentially expanding and divergent clades. Methods SARS-CoV-2 sequences (n=612) randomly selected from among the sequences of samples collected through a nationwide network of Virus Research Diagnostic Laboratories during 2020 (n=1532) and Indian sequences available in Global Initiative on Sharing All Influenza Data during March 2020-March 2022 (n=53077), were analyzed using the phylo-geo haplotype network approach with reference to the Wuhan prototype sequence. Results On haplotype analysis, 420 haplotypes were revealed from 643 segregating sites among the sequences. Haplotype sharing was noted among the strains from different geographical regions. Nevertheless, the genetic distance among the viral haplotypes from different clades could differentiate the strains into distinct haplo groups regarding variant emergence. Interpretation & conclusions The haplotype analysis revealed that the G and GR clades were co-evolved and an epicentrefor the evolution of the GH, GK and GRA clades. GH was more frequently identified in northern parts of India than in other parts, whereas GK was detected less in north India than in other parts. Thus, the network analysis facilitated a detailed illustration of the pathways of evolution and circulation of SARS-CoV-2 variants.

BackgroundNeutralization of virus infectivity has been suggested as the key mechanism of immunological defence against rotavirus infection. In India, in addition to Rotarix™ and RotaTeq®, two new vaccines i.e. ROTAVAC®; a monovalent vaccine and ROTASIIL®; a bovine-human reassortant multivalent vaccine are included under National Immunization programme. The aim of the study was to determine the comparative analysis of the neutralizing antibody (NAbs) responses against the most common RV types (G1–G4, G9 and G12) among ROTAVAC® and ROTASIIL® vaccinated infants.MethodsConvalescent serum samples were collected from either ROTAVAC® or ROTASIIL® vaccinated healthy infants. Neutralizing antibody titre against reference human RV strains, i.e., G1P[8] (KU), G2P[4] (S2), G3P[8] (YO), G4P[6] (ST-3), G9P[8] (F-45) and G12P[11] (NIV-1740121) was determined.ResultsAmong RotaSIIL® vaccinated infants, Geometric Mean Titres (GMTs) of NAbs against homotypic strains were significantly higher (p< 0.01) compared to heterotypic strains. Moreover, significantly higher NAbs were generated against RV strains, G1P[8] and G9P[8] compared to G3P[8], G2P[4] and G4P[6] (p< 0.01 for each comparison). In contrast, only 30.5% (n=11) infants revealed serum cross NAbs to fully heterotypic RV strain; G12P[11]. The neutralization profile of ROTAVAC® vaccines indicated that 80.6% of the recipients seroconverted to the partial homotypic G9P[8] strain. Interestingly, 77.4% vaccine recipients were seropositive against heterotypic strain G1P[8], while considerably lower heterotypic response to RV G2P[4], G3P[8] and G4P[6] strains was observed. Surprisingly, only 35.48% of sera, cross-neutralized the partial heterotypic strain, G12P[11]. A comparative analysis of the log GMTs between two groups indicated significantly higher serum NAbs against the evaluated strains; G1P[8], G3P[8], G4P[6] and G9P[8] amongst subjects who received RotaSIIL® vaccine compared to those of ROTAVAC®.ConclusionThe study highlights that a multivalent (RotaSIIL) vaccine may confer broader coverage against common rotaviruses. Also, both ROTAVAC® and RotaSIIL® vaccines exhibited relatively low cross-neutralization against RV G12P[11] strain.

Interleukin 1 receptor antagonist (IL1RN) is a competitive inhibitor of interleukin 1 (IL-1). Natural killer cells (NK cells) contribute to the elimination of viruses by their antiviral effector function, which depends on a balance between inhibitory and activating receptor genes such as NKG2D and NKG2A. Using polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) assays, the association of intronic single-nucleotide polymorphisms (SNPs) in these genes with viral infection were assessed in 111 patients with hepatitis E virus (HEV) infection and 222 HEV-naive healthy controls. An SNP in the IL1RN (VNTR) gene revealed allele 2 to be associated with protection against HEV infection (IL1RN *1/*1 vs. IL1RN *2/*2, OR = 0.26, 95

Human adenoviruses (HAdVs) are the viral agents responsible for a wide spectrum of acute and chronic diseases. HAdVs are the most important etiological agents of acute gastroenteritis (AGE) and are identified as the major contributor to the deaths of diarrheal children globally. The significant rise in HAdV infections in rotavirus-vaccinated children documented in multiple studies demands continuous monitoring of HAdV strains. After the inclusion of rotavirus vaccines in the immunization schedule of India, public health research regarding prevalence, etiology, and risk factors is highly necessary for evidence-based policies and their implementation to sustain diarrhea prevention programs. In the present study, children admitted for AGE between 2013 and 2016 in seven different hospitals in Maharashtra and Gujrat states of Western India were subjected for investigation. HAdVs were found in 5.2% of the fecal specimens with the dominance of species-F (52.4%) strains, followed by the occurrence of non-enteric adenoviruses of species A (17.4%), C (11.4%), B (8.2%), and D (3.2%). The species-F strains were predominant in Ahmadabad (78.5%), Mumbai (61.5%), and Surat (57.1%) cities, followed by species-A strains. In Pune city, species B strains were detected in all HAdV patients, with none of the species A strains. Clinically, patients infected with enteric and non-enteric HAdV strains were indistinguishable. However, a high viral load was observed in species-F specimens as compared to non-species-F. The present study on fecal specimens collected in the pre-rotavirus vaccination era from hospitalized AGE patients will be important for future comparative analysis to know the exact impact of vaccination in children of Western India.

The neutralizing antibody (Nt-Ab) response to vaccine and wild-type measles viruses (MeV) was studied in suspected measles cases reported during the years 2012–2016. The neutralization activity against MeV A, D4 and D8 genotypes was studied on sera (Panel A; n = 68 (measles-immunized) and Panel B; n = 50 (unvaccinated)) that were either laboratory confirmed or not confirmed by the presence of IgM antibodies. Additionally, the Nt-Ab response in Panel A was measured against the MeV vaccine and four wild-type viruses. Neutralization results were compared using homology modeling and molecular dynamics simulation (MDS) of MeV-hemagglutinin (H) and fusion (F) proteins. Overall, the Nt-Ab titres for MeV-A were found to be significantly lower than MeV-D4 and MeV-D8 viruses for Panel A. No major difference was noted in Nt-Ab titres between MeV-D8 viruses (Jamnagar and New Delhi), whereas MeV-D4 (Sindhudurg and Bagalkot (BGK) viruses) showed significant differences between Nt-Ab titres for Panel B. Interestingly, the substitutions observed in epitopes of H-protein, L249P and G316A are observed to be unique to MeV-BGK. MDS of H-protein revealed significant fluctuations in neutralizing epitopes due to L249P substitution. The majority of the clinically suspected cases showed Nt-Abs to MeV wild-types. Higher IgG antibody avidity and Nt-Ab titres were noted in IgM-negatives than in IgM-positives cases, indicating reinfection or breakthrough. MDS revealed reduced neutralization due to decreased conformational flexibility in the H-epitope.

We have evaluated the association of polymorphisms in the intronic variable-number tandem repeat (VNTR) regions of the human NKG2D, NKG2A, and IL-1RN genes with resistance and/or susceptibility to SARS-CoV-2 infection in a total of 209 patients with SARS-CoV-2 infection (125 asymptomatic patients and 84 symptomatic patients with mild symptoms) and 355 healthy controls, using the PCR-RFLP method. The genotypic and allelic frequency distributions for an IL-1RN (VNTR) single-nucleotide polymorphism (SNP) were found to be comparable among the patient groups. Overall, in SARS-CoV-2 patients, NKG2A (rs2734440) showed a protective association in the codominant [(A/A vs. A/G): (OR = 0.53, 95% CI = 0.34-0.83, p = 0.006)], recessive [(A/A vs. A/G+G/G): (OR = 0.6, 95% CI = 0.39-0.92, p = 0.02)] and over-dominant [(A/A+G/G vs. A/G): (OR = 0.57, 95% CI = 0.38-0.84, p = 0.005)] models. Similarly, NKG2D (rs7980470) showed a protective association in the codominant [(A/A vs. A/G): (OR = 0.46, 95% CI = 0.3-0.7, p = 0.0003), codominant (A/A vs. G/G): (OR = 0.54, 95% CI = 0.31-0.71, p = 0.027)], recessive [(A/A vs. A/G+G/G): (OR = 0.47, 95% CI = 0.32-0.7, p = 0.0001) and over-dominant [(A/A+G/G vs. A/G): (OR = 0.56, 95% CI = 0.38-0.82, p = 0.003)] models. At the allelic level, there was a higher frequency of the "G" allele of NKG2D (rs7980470) in healthy controls than in patients with SARS-CoV-2 infection, suggesting that individuals with the "G" allele in the intronic region of NKG2D are likely to be protected against SARS-CoV-2 infection. Overall, our data suggest that polymorphisms in the host NKG2D and NKG2A genes have a protective role in SARS-CoV-2 infection, although the functional impact of these polymorphisms on control of SARS-CoV-2 infection remains unknown.

Species C rotaviruses (RVC) are the second most common rotavirus species known to cause gastroenteritis in humans and pigs and with occurrence documented in cattle, dogs, ferrets, and sloth bears. Despite the host-specific nature of RVC genotypes, cross-species transmission, reassortment, and recombination events are also documented. In the present study, we inferred the evolutionary history of globally circulating RVC strains, including time scale stasis, the most probable ancestral country, and the most probable source host using Bayesian methods implemented in BEAST v.1.8.4. The human-derived RVC strains were majorly monophyletic and further grouped into two lineages. The RVC strains derived from pigs were monophyletic for the VP1 and the remaining genes were classified into 2 to 4 groups based on the high posterior support. The root mean age for all the genes indicated the circulation of RVC for over 800 years. Overall, the time to Most Recent Common Ancestor of human RVC strains dated back to the beginning of the 20th century. The VP7 and NSP2 genes had the lowest rates of evolution compared to other genes. The majority of the genes of RVC showed their origin in Japan except for VP7 and VP4 genes in South Korea. The phylogeographic analysis with the country as a trait showed the role of Japan, China, and India in the dispersion of the virus. In the current study, significant transmission links between different hosts were analyzed for the first time using the host as a trait. Significant transmission links between pigs and other animal species as well as humans indicate possible transmission from the pig as a source host and suggest monitoring of proximity with animals.

The COVID-19 pandemic has severely affected public health system and surveillance of other communicable diseases across the globe.The lockdown, travel constraints and COVID phobia turned down the number of people with illness visiting to the clinics or hospitals.Besides this, the heavy workload of SARS-CoV-2 diagnosis has led to the reduction in differential diagnosis of other diseases.Consequently, it added to the underlying burden of many diseases which remained under-diagnosed.Amidst the pandemic, the rise of emerging and re-emerging infectious diseases was observed worldwide and reported to the World Health Organization i.e.

Enterically transmitted waterborne hepatitis E (HE) caused due to hepatitis E virus (HEV) prevails as a significant public health problem endemic to India. Due to short-term viremia/fecal excretion and poor in vitro transmissibility of HEV, HE diagnosis depends on detection of specific IgM antibodies in serum. Present study evaluated performances of two in-house and six commercial IgM detection enzyme-linked immunosorbent assays (ELISAs) using sera collected from volunteers/acute hepatitis patients (n = 716). The in-house ELISAs were based on complete and truncated open reading frame 2 (ORF2) proteins containing neutralizing epitope/s region of genotype 1 HEV (ORF2p, 1–660 amino acid (a.a.) and T1NEp, 458–607 a.a., respectively). The commercial ELISAs included Wantai (China), MP Diagnostics (MPD) (Singapore), DIA.PRO Diagnostics (Italy), MBS (Italy), abia (Germany), and ImmunoVision (USA). T1NE ELISA showed 97.0% positive percent agreement (PPA), 99.4% negative percent agreement (NPA), and 98.6% concordance (κ = 0.97, P = 0.0000) with ORF2 ELISA. ORF2, T1NE, Wantai, and MPD ELISAs agreed on results for 88% of sera tested. Two percent sera showed reactivity in each combination of three and two of aforementioned four ELISAs. Remaining 8% sera were single ELISA reactive. PPA and NPA value ranges were 76.3–99.0% and 84.8–99.5%, respectively. Pairwise concordances between all the eight ELISAs ranged from 88.0 to 100% (κ: 0.74–1.00). Both the in-house ELISAs agreed better with Wantai over MPD ELISA. In conclusion, both ORF2 and T1NE ELISAs were equally efficient in diagnosing HEV infections. T1NEp proved to be an excellent tool in HE sero-diagnosis and is worth exploring in development of simple rapid tests. • In-house ELISA based on bacterially expressed neutralizing epitope/s region protein • In-house ELISA based on complete ORF2 protein expressed in insect cells • Comparison of two in-house and six commercial anti-HEV IgM antibody detection ELISAs

Objective: The Government of India (GoI) is working towards measles and rubella (MR) elimination in a phased manner. Serological and molecular confirmation of cases in research laboratories is crucial for measles and rubella (MR) elimination goal-2023. Methods: A study was designed to understand the qualitative and quantitative correlation of IgM, IgG, IgG-avidity and Nt-antibodies to Measles virus (MeV) and Rubella virus (RuV) in fever with skin-rash cases aged between 1-15 years. For this purpose, sera collected and referred during 2014-16 were used. Results: A total of 42%, 86.0% and 97.3% cases show MeV-specific IgM, IgG and Nt antibodies, respectively and 22.7%, 59.3% and 72.3% show RuV-specific IgM, IgG and Nt antibodies, respectively. Amongst laboratoryconfirmed cases, 69.0% (low) and 26.2% (high) have IgG antibody-avidity for measles, whereas 62.5% (low) and 31.3% (high) have IgG antibody avidity for rubella. In the 'non-measles and non-rubella' cases, 11.9% show low and 84.3% cases have high avidity for measles, whereas low avidity for rubella was detected in 12.7% and high avidity in 84.6% cases, indicating acute or past/reinfection. Overall, MeV-specific Nt and RuV-specific IgG antibody-avidity positivity was highest compared to IgM. Conclusions: Large percentage of cases have Nt antibodies to MeV and IgG antibody-avidity to RuV compared to IgM antibodies. Acute infection confirmed by low IgG avidity was 37.2% and 23.9% for measles and rubella, respectively. Measles and Rubella past/reinfection was observed in 58.6% and 72.6% cases, respectively. Thus the present study indicates usefulness of various serological tests for case confirmation.

The low pathogenic avian influenza H9N2 virus is a significant zoonotic agent and contributes genes to highly pathogenic avian influenza (HPAI) viruses. H9N2 viruses are prevalent in India with a reported human case. We elucidate the spatio-temporal origins of the H9N2 viruses from India. A total of 30H9N2 viruses were isolated from poultry and environmental specimens (years 2015-2020). Genome sequences of H9N2 viruses (2003-2020) from India were analyzed, revealing several substitutions. We found five reassortant genotypes. The HA, NA and PB2 genes belonged to the Middle-Eastern B sublineage; NP and M to the classical G1 lineage; PB1, PA and NS showed resemblance to genes from either HPAI-H7N3/H5N1 viruses. Molecular clock and phylogeography revealed that the introduction of all the genes to India took place around the year 2000. This is the first report of the genesis and evolution of the H9N2 viruses from India, and highlights the need for surveillance.

Background & objectives: Several phylogenetic classification systems have been devised to trace the viral lineages of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, inconsistency in the nomenclature limits uniformity in its epidemiological understanding. This study provides an integration of existing classifications and describes evolutionary trends of the SARS-CoV-2 strains circulating in India. Methods: The whole genomes of 330 SARS-CoV-2 samples were sequenced using next-generation sequencing (NGS). Phylogenetic and sequence analysis of a total of 3014 Indian SARS-CoV-2 sequences from 20 different States/Union Territories (January to September 2020) from the Global Initiative on Sharing All Influenza Data (GISAID) database was performed to observe the clustering of Nextstrain and Phylogenetic Assignment of Named Global Outbreak LINeages (Pangolin) lineages with the GISAID clades. The identification of mutational sites under selection pressure was performed using Mixed Effects Model of Evolution and Single-Likelihood Ancestor Counting methods available in the Datamonkey server. Results: Temporal data of the Indian SARS-CoV-2 genomes revealed that except for Uttarakhand, West Bengal and Haryana that showed the circulation of GISAID clade O even after July 2020, the rest of the States showed a complete switch to GR/GH clades. Pangolin lineages B.1.1.8 and B.1.113 identified within GR and GH clades, respectively, were noted to be indigenous evolutions. Sites identified to be under positive selection pressure within these clades were found to occur majorly in the non-structural proteins coded by ORF1a and ORF1b. Interpretation & conclusions: This study interpreted the geographical and temporal dominance of SARS-CoV-2 strains in India over a period of nine months based on the GISAID classification. An integration of the GISAID, Nextstrain and Pangolin classifications is also provided. The emergence of new lineages B.1.1.8 and B.1.113 was indicative of host-specific evolution of the SARS-CoV-2 strains in India. The hotspot mutations such as those driven by positive selection need to be further characterized.

Four gastroenteritis viruses were responsible for 54% of the acute gastroenteritis (AGE) cases in children hospitalized between May 2017 and December 2019 in Pune city of Maharashtra state, Western India. The majority (79%) of the children were <2 years of age. The prevalence of Rotavirus A (RVA) was 30.5% followed by 14.3% for norovirus, 8.4% for adenovirus, and 5.5% for astrovirus. The severity of the disease was highest in patients with coinfections compared with the patients with a single infection or negative for all (p = 0.024). Genotyping analysis showed that the majority of the RVA-positive samples (66%) could be typed as G3P[8], 63.6% of the norovirus as GII.4 Sydney [P16], 44% of the adenovirus as type 41%, and 56.2% of the astrovirus as astrovirus type 1. The almost equivalent prevalence of rotavirus and nonrotaviruses and acute gastroenteritis (AGE) cases without known etiology in around 46% of the cases was noted in the present study. Our data highlight that after the recent inclusion of rotavirus vaccines as a part of the National Immunization schedule in India, conducting extensive AGE surveillance in children should include nonrotaviruses such as norovirus.

Sir, The single-stranded RNA genome of the 2019 novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) about 29.9 kb in length and encoding about 9860 amino acids, was annotated to possess 14 open reading frames (ORFs) and 27 proteins12. The orf1ab and orf1a genes at the 5´-terminus of the genome encode the pp1ab and pp1a proteins, respectively, together form 15 non-structural proteins (nsps), nsp1-nsp10 and nsp12-nsp16. The 3´-terminus of the genome encodes four structural proteins, the spike surface glycoprotein (S), the small envelope protein (E), membrane protein (M) and nucleocapsid protein (N). There are eight accessory proteins denoted as 3a, 3b, p6, 7a, 7b, 8b, 9b and ORF142. The epidemiology of the SARS-CoV-2 since its emergence in December 2019 has been ever expanding, with increase in the number of cases and its spread globally34. The number of SARS-CoV-2 cases in India as on March 31, 2020 was 1,071, with mortality crossing 294. In this context, it is vital to understand the genetic nature of circulating SARS-CoV-2. In India, as per the guideline of the Ministry of Health and Family Welfare, suspected samples of SARS-CoV-2 were collected and tested at the designated Viral Research and Diagnostic Laboratories (VRDL)5. As a part of this activity, a total of 15 SARS-CoV-2 positive specimens were obtained during the first week of March 2020, from Italian tourists and travellers from Italy and their contact cases in India. Further, in an effort to screen Indian nationals in Iran to enable their evacuation, during March 5 to 17, 2020, throat swabs were collected from 1,920 individuals; of whom 281 were positive. In addition, a team of Indian doctors visited Italy and collected a total of 380 swabs of Indian citizens; of whom four positive specimens were identified. In an earlier study, the authors identified the first three cases of SARS-CoV-2 in Kerala, India, as imported cases from Wuhan, China, and presented the first two full-genome sequences along with the potential B-cell and T-cell epitopes on the spike protein6. Further, in another study, the SARS-CoV-2 viruses were isolated in Vero CCL-81 cells7. The present study was undertaken to understand and compare the genetic makeup of representative samples of the imported cases of SARS-CoV-2 to India from Wuhan, China, those of Italian tourists in India and the Indians evacuated from Iran and Italy. Throat swab/nasal swab specimens collected from the 1,920 individuals in Iran were tested at the Indian Council of Medical Research-National Institute of Virology (ICMR-NIV) Pune, using real-time reverse transcription-polymerase chain reaction (RT-PCR) protocols to detect RdRp (1), RdRp (2), E and N genes as described elsewhere8. Next-generation sequencing (NGS) was performed on a total of 41 SARS-CoV-2 positive clinical samples from Italy and Iran. Table I presents the details of the full genomes obtained (n=19) as a part of this study as well as the two earlier genomes retrieved from the Kerala samples (n=2) from those who had the travel history from China67.Table I: Cycle threshold (Ct) values of real-time reverse transcription-polymerase chain reaction (RT-PCR) for the E gene of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) along with the per cent of the reads mapped and the genome size recovered for the clinical samplesMultiple sequence alignment of 21 full genomes obtained and 1563 full-genome sequences (Supplementary Table (available from http://www.ijmr.org.in/articles/2020/151/2/images/IndianJMedRes_2020_151_2_255_283159_sm5.pdf)) available at the Global Initiative on Sharing All Influenza Data (GISAID) database (as of March 26, 2020) was carried out in MAFFT v.7.4509. The phylogenetic tree was constructed using MEGA v.610, employing the neighbour-joining method with the composite likelihood method and 1000 bootstrap replications. An initial tree was constructed based on a total of 1586 sequences. This tree was used to reduce the dataset to 121 sequences, on the basis of country and the genetic variant identified based on the GISAID classification. Comparison of the sequences of this study with respect to the Wuhan Hu-1 reference strain was done to identify unique mutations, if any. Phylogenetic trees based on full-genome sequences deposited and available at GISAID revealed the diversification and the clustering of sequences into groups, based on the genetic variants. Specific amino acid substitutions in the nsp3 region, spike protein and ORF8, in general, lead to the formation of V, G and S genetic variants/clades, respectively. The S clade corresponds to the C28144T nucleotide polymorphism that results in a non-synonymous substitution Leu84Ser in ORF8. Clades V, G and a group of unclassified strains possess mainly C28144 and are referred to as the L type11. The phylogenetic analyses of the study strains and the other global sequences revealed that the SARS-CoV-2 sequences derived from Italy (n=8) in this study, clustered in clade G, while the SARS-CoV-2 sequences (n=11) of Indians evacuated from Iran belonged to the unclassified group which also included one of the SARS-CoV-2 sequences imported from Wuhan (hCoV-19/India/1-27/2020) (Figure). The other sequences imported from Wuhan (hCoV-19/India/1-31/2020) possessed Leu84Ser in ORF8b, classifying it in clade S.Figure: Phylogenetic tree of selected representative full-genome sequences of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-derived from coronavirus disease 2019 positive patients' clinical samples who had travel history of Wuhan, China, Italy and Iran by neighbour-joining method. Strains sequenced at ICMR-NIV are shown in magenta colour. The clades as per Global Initiative on Sharing All Influenza Data (GISAID) nomenclature are indicated in blue (clade G), red (clade V), green (clade S) and black (unclassified).The sequences of Italy origin were noted to segregate into at least two subgroups. The percentage nucleotide divergence (PND) within these sequences was found to be 0.01 per cent. The SARS-CoV-2 sequences from the Italian tourists (n=6) showed relatedness to other European SARS-CoV-2 sequences from Scotland, Finland, England, Spain, Ireland and the Czech Republic along with a Shanghai, China, strain as the outgroup (Figure). Two other sequences (hCoV-19/India/3118/2020 and hCoV-19/India/3239/2020) clustered more closely with sequences from Belgium and Switzerland. The two sequences (hCoV-19/India/31/2020 and hCoV-19/India/32/2020) from the Agra contacts of the Italy-returned Delhi based individuals were more distinct and showed clustering in a strongly supported subgroup consisting of strains from Brazil and the European countries including Switzerland, Germany, France, Hungary and The Netherlands. The variable amino acid sites based on the alignment of the 21 sequences of this study with respect to Wuhan Hu-1 strain are shown in Table II. All the Italy-origin sequences possessed the substitution D7711G/D614G in the S protein, characteristic of the G clade, along with another mutation P4715L (nsp12-323) that is also shared with many other countries. Mutation S1515F (nsp3-697) was specific to the Italian cohort strain; D8726G (M-3) was specific to hCoV-19/India/3118/2020 and hCoV-19/India/3239/2020 (Indian contacts of an Indian citizen having travel history to Italy), similar to sequences from Scotland, Belgium, Finland, Switzerland and England. The mutations, R9455K and G9456R (N-203 and 204), were found to be specific to the two strains, hCoV-19/India/31/2020 and hCoV-19/India/32/2020 but shared with a few more countries. A recent study has identified the earliest Italian importation of SARS-CoV-2 to a case from Shanghai, China, and has also identified at least two circulating variants in Italy12. Thus, it is likely that the former strain (Italian cohort) has its origin from China, whereas the latter strain (contacts in Agra, n=2) appears to have been from a European cluster involving an entry into Germany that preceded the first cases in Italy by almost a month1213.Table II: Variable amino acid positions in the Indian full-genome sequencesAnalysis of the strains from the SARS-CoV-2 positives in Iran (Figure) showed that these sequences (n=11) clustered with other strains having a global spread inclusive of Canada, USA, several European countries, New Zealand, Australia and Southeast Asian countries noted in this group (moderate support of 64%). The PND among these study sequences was found to be 0.24 per cent. Common mutations shared among SARS-CoV-2 sequences in the group included R207C (nsp2-27), V378I (nsp2-198), M2796I (nsp4-33) and L3606F (nsp6-37). A mutation V9082F (ORF7a-74) was unique to four of the study sequences (hCoV-19/India/1073/2020, hCoV-19/India/1093/2020, hCoV-19/India/1115/2020 and hCoV-19/India/1100/2020) that clustered with a strain from Kuwait, KU12. The KU12 strain was also noted to possess this mutation. To date, there are no other sequences from Iran in the GISAID database. However, a phylogenetic study14 of full-genome sequences has identified distinct SARS-CoV-2 link to travellers returning from Iran to Australia and New Zealand. Some of these representative sequences were included in this study as well. In terms of the overall divergence of SARS-CoV-2, the strains in this study were 99.97 per cent identical to the earliest strain Wuhan Hu-1. However, it is vital to track the evolutionary dynamics of the strains vis-à-vis the strains circulating globally and monitor any specific changes in the functional sites of the major viral proteins. Delineation of circulating strains into three major evolving clades has been reflected in GISAID, with clade G apparently being one of the dominant ones. From the start of the pandemic, severity or transmission patterns have not been associated with any clade in particular. A limitation of this study was the non-availability of full genomes from other parts of India. This would enable a pan-India comparison of the circulating strains in the country. Overall, the present study revealed genetic variants in India that were similar to strains circulating in the specific regions of their origin. Continued surveillance of SARS-CoV-2 strains in India is warranted to get the complete picture of all circulating strains and identify changes that could be associated with increased virulence. Acknowledgment Authors thank Prof. (Dr) Balram Bhargava, Director-General, Indian Council of Medical Research (ICMR) & Secretary, Department of Health Research (DHR), Ministry of Health & Family Welfare (MoHFW), New Delhi for the support. Authors acknowledge the support from Dr P. Ravindran, Director, Emergency Medical Response (EMR), MoHFW, Dr R. Lakshminarayan, ICMR and the team from the DHR, MoHFW, for the logistic support. The National Centre for Disease Control (NCDC) team is acknowledged for sample collection from Italy. Shri Santosh Jadhav, Bioinformatics Group, ICMR-National Institute of Virology, Pune, is thanked for his inputs.

Rotavirus infections in neonates are generally nosocomial, and differ from pediatric infections both clinically and epidemiologically. These infections are predominantly asymptomatic and often associated with unusual strains. Globally, so far limited data is available on rotavirus infections in neonates admitted at Neonatal Intensive Care Unit. The aim of the present study is to determine the prevalence of rotavirus among neonates and to study their genetic characteristics. Stool specimens (n = 701) collected from neonates (n = 621) admitted during April 2016 to March 2018 mainly for prematurity, low birth weight and associated respiratory distress syndrome from two hospitals from Pune were tested for rotavirus, genotyped and representative strains were sequenced for the genes encoding outer capsid proteins, VP7 and VP4. Rotavirus was detected in 24.31% neonates. Majority of rotavirus infected neonates (98.68%) were asymptomatic. Peak rotavirus antigen detection (91.38%) occurred during the first 2 weeks of admission. Low, very low and normal birth weight neonates with gestational age >= 28 weeks had significantly higher rotavirus infection than those with extreme low birth weight with gestational age < 28 weeks. Rotaviral infections occurred almost evenly throughout the year without an apparent peak in colder months. Predominance of unusual G12P[11] strains (97.1%) was observed. Phylogenetic analysis of the partial VP7 coding gene revealed all G12 strains clustered in lineage III and shared 96.94%-100% (nucleotide) and 96.26%-100% (amino acid) identities among themselves, and 95.69%-98.98% (nucleotide) and 94.77%-98.98% (amino acid) with other lineage III G12 strains respectively. Similarly VP4 partial gene sequences of P[11] study strains shared 97.5%-100% (nucleotide and amino acid) identities among themselves and highest 93.34%-94.53% (nucleotide) and 93.57%-94.64% (amino acid) identity with vaccine strain 116E, G9P[11]. The study highlights high frequency of unusual G12P[11] strains among neonates for the first time in western India and reaffirms limited strain diversity in this population. The knowledge of neonatal strains is important for estimating the efficacies of rotavirus vaccines.

The number of whole-genome sequences of human rotavirus C (RVC) strains available in public databases is recently increasing. Thus far from India only a single whole genome of human RVC of a sporadic case was available. In this study, nearly full-length genome sequencing and phylogenetic analyses of three RVC strains isolated from three different gastroenteritis outbreaks during 2010-2014 in Western India was carried out. Further, an intra-genotypic lineage classification system for human RVCs based on the nucleotide divergence cut-off values was proposed by using the algorithm of the Rotavirus Classification Working Group. Two lineages could be defined for all the genes except the VP7 gene and the M3 VP3 genotype. Provisional classification of the lineages indicated the absence of reassortment events in the genomic constellation of Indian strains, contrary to earlier reports. The comparatively higher variability of the NSP1, NSP3, NSP5 and M2 VP3 genotype, emphasizes their utility in lineage classification.