BackgroundRespiratory viral illnesses among children are a prominent cause of morbidity and mortality in the developing world. The aim of this study is to understand the seasonal pattern and surge of respiratory viruses among the Nicobarese tribe.MethodsRespiratory specimens were collected from both ARI and SARI cases attended the BJR district hospital in Car Nicobar Island, India, between 2021 and 2022. Respiratory viruses were identified from the specimens by using the qRT-PCR assay. Meteorological parameters were collected and evaluated using Microsoft Excel and SPSS 21. The significant association between the surge of respiratory viruses and each climatic parameter was evaluated.ResultsIn this hospital-based cross-sectional study, 471 ILI cases were enrolled, and 209 of these were positive for respiratory viral infections. Of these respiratory virus infections, 201 (96.2%) were infected with a single respiratory virus infection, and 8 (3.8%) had mixed viral infections. Fever, cough, and chills were the most common symptoms of respiratory illness among this indigenous population. There was a significant link between respiratory viruses and influenza-like illness in children (below 5 years and 6 to 15 years).ConclusionThis prevalence study revealed that viral respiratory infections were more common in children than adults. Among these respiratory viruses, respiratory syncytial virus A (RSV) and influenza B virus were predominantly reported among tribal children up to age five years. In the year 2021, these viruses were recorded frequently during the winter season. Climate factors such as high humidity, high precipitation, moderate temperature, and moderate rainfall are found to be correlated with respiratory viral infections. This study implicates important information for preventing a further outbreak of respiratory viral infections in Car Nicobar Island.

Introduction:The Indian Council of Medical Research has set up a nationwide network of 28 laboratories for simultaneous surveillance of influenza virus and SARS-CoV-2 in ILI/SARI patients, using an in-house developed and validated multiplex real-time RTPCR assay. The aim of this study was to ensure the quality of testing by these laboratories by implementing an external quality assessment program (EQAP).Methods:For this EQAP, a proficiency test (PT) panel comprising tissue-culture or egg-grown influenza virus and SARS-CoV-2 was developed. The PT panel was distributed to all the participant laboratories, which tested the panel and submitted the qualitative results online to the EQAP provider. The performance of the laboratories was evaluated on qualitative criteria but cycle threshold (Ct) values were also gathered for each sample.Results:On a qualitative basis, all the laboratories achieved the criteria of 90% concordance with the results of the PT panel provider. Ct values of different samples across the laboratories were within ≤ ±3 cycles of the corresponding mean values of the respective sample. The results of this EQAP affirmed the quality and reliability of testing being done for simultaneous surveillance of influenza virus and SARS-CoV-2 in India.

Background:SARS-CoV-2 was first reported in China in December 2019 and quickly spread across the world. Non-pharmaceutical interventions (NPIs) are the key to control the transmission of respiratory viruses. To stop the spread, NPI is widely recommended and is still followed by most countries.Methods:At the National Influenza Center of the Indian Council of Medical Research-National Institute of Virology (ICMR-NIV), the surveillance of severe acute respiratory illness and acute respiratory illness cases for influenza and other respiratory viruses is in place. In this study, we analyzed surveillance data on respiratory viruses and/or SARS-CoV-2 testing from January 2017 to December 2021. Multiplex real-time PCR was used to detect the respiratory viruses.Results:Our findings indicate that during the pandemic, the positivity for influenza A and B, metapneumovirus, parainfluenza virus, respiratory syncytial virus, and human coronavirus declined significantly.Conclusion:The annual distinct seasonal outbreaks of influenza, RSV, and other respiratory viruses as observed during the pre-COVID-19 period were not observed during the COVID-19 pandemic in years 2020 and 21. Social distancing, lock-downs, and non-pharmaceutical interventions may play an important role in the reduction of respiratory viruses. Understanding the seasonal respiratory virus decline could help public health experts prepare for future respiratory virus pandemics.

Pertussis, commonly known as whooping cough, is one of the most poorly controlled vaccine-preventable diseases in the world. South-East Asia is estimated to contribute the most to childhood disease burden while this remains largely unexplored in India. The clinical diagnosis of pertussis in young children is a challenge as the classical four-stage presentation with paroxysmal cough or whoop may be absent. It is also difficult to differentiate from other respiratory infections which can cause pertussis-like illness. Children below two years with prolonged cough illness attending an urban pediatric center in western India, were evaluated for pertussis and viral infections by molecular methods. Bordetella pertussis and B. holmesii were confirmed in three each of 45 suspected cases, and RSV-A and hMPV were the most common viruses that were detected. These organisms can mimic mild cases of pertussis and need to be considered in differential diagnosis of prolonged cough illness in young children. The accurate etiology of prolonged cough illness needs to be detected and documented to ensure appropriate management and accurate estimates of disease burden.

As the global severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic expands, genomic epidemiology and whole genome sequencing are being used to investigate its transmission and evolution. Against the backdrop of the global emergence of "variants of concern" (VOCs) during December 2020 and an upsurge in a state in the western part of India since January 2021, whole genome sequencing and analysis of spike protein mutations using sequence and structural approaches were undertaken to identify possible new variants and gauge the fitness of the current circulating strains. Phylogenetic analysis revealed that newly identified lineages B.1.617.1 and B.1.617.2 were predominantly circulating. The signature mutations possessed by these strains were L452R, T478K, E484Q, D614G and P681R in the spike protein, including within the receptor-binding domain (RBD). Of these, the mutations at residue positions 452, 484 and 681 have been reported in other globally circulating lineages. The structural analysis of RBD mutations L452R, T478K and E484Q revealed that these may possibly result in increased ACE2 binding while P681R in the furin cleavage site could increase the rate of S1-S2 cleavage, resulting in better transmissibility. The two RBD mutations, L452R and E484Q, indicated decreased binding to select monoclonal antibodies (mAbs) and may affect their neutralization potential. Further in vitro/in vivo studies would help confirm the phenotypic changes of the mutant strains. Overall, the study revealed that the newly emerged variants were responsible for the second wave of COVID-19 in Maharashtra. Lineage B.1.617.2 has been designated as a VOC delta and B.1.617.1 as a variant of interest kappa, and they are being widely reported in the rest of the country as well as globally. Continuous monitoring of these and emerging variants in India is essential.

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.

The WHO has recommended reverse transcriptionpolymerase chain reaction (RT-PCR) for the confirmation of coronavirus disease 2019 (COVID-19) diagnosis. Real-time RT-PCR assays with automated extraction systems are required to process large numbers of specimens. Corman et al1 have reported three real-time RT-PCR assays [based on the RNAdependent RNA polymerase (RdRp) gene, envelope (E) gene and nucleocapsid (N) gene] for detecting beta coronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)1, and additionally, Chu et al2 have reported two real-time RT-PCR assays based on ORF 1b and N gene that are highly conserved among Sarbeco viruses.