Structural and functional characterization of avian influenza H9N2 virus neuraminidase with a combination of five novel mutations.

The influenza virus neuraminidase (NA) protein is responsible for actively cleaving the sialic acid (SA) bound to the viral hemagglutinin. In the present study, we identified a combination of five novel amino acid substitutions in the NA, conferring increased substrate binding and altered surface characteristics to a low pathogenic avian influenza (LPAI) H9N2 virus strain. The H9N2 strain reported from India, A/Environmental/India/1726265/2017 (H9N2-1726265) showed the combination of amino acid substitutions T149I, R249W, G346A, W403R and G435R, which were in the vicinity of the enzyme active site cavity. The strain A/chicken/India/99321/2009 (H9N2-99321) did not show these substitutions and was used for comparison. Virus elution was studied using turkey red blood cells (tRBCs). NA enzyme kinetics assays were carried out using the MUNANA substrate, which is an SA analogue. Homology modelling and molecular docking were performed to determine alterations in the surface characteristics and substrate binding. H9N2-1726265 showed enhanced elution from tRBCs. Enzyme kinetics revealed a lower KM of H9N2-1726265 (111.5 µM) as compared to H9N2-99321 (135.2 µM), indicating higher substrate binding affinity of H9N2-1726265, due to which the NA enzyme cleaved the SA more efficiently, leading to faster elution. Molecular docking revealed a greater number of binding interactions of H9N2-1726265 to SA as compared to H9N2-99321 corroborating the greater substrate binding affinity. Changes in the surface charge, hydrophobicity, and contour, were observed in H9N2-1726265 NA due to the five substitutions. Thus, the novel combination of five amino acids near the sialic acid binding site of NA, resulted in altered surface characteristics, higher substrate binding affinity, and virus elution.

The Immunogenicity of Monovalent Oral Poliovirus Vaccine Type 1 (mOPV1) and Inactivated Poliovirus Vaccine (IPV) in the EPI Schedule of India.

BACKGROUND: In 2016, the Global Polio Eradication Initiative (GPEI) recommended the cessation of using type 2 oral poliovirus vaccine (OPV) and OPV, with countries having to switch from the trivalent to bivalent OPV (bOPV) with the addition of inactivated poliovirus vaccine (IPV) in their routine immunization schedule. The current GPEI strategy 2022-2026 includes a bOPV cessation plan and a switch to IPV alone or a combination of vaccine schedules in the future. The focus of our study was to evaluate the immunogenicity of monovalent OPV type 1 (mOPV1) with IPV and IPV-only schedules. METHODS: This was a three-arm, multi-center randomized-controlled trial conducted in 2016-2017 in India. Participants, at birth, were randomly assigned to the bOPV-IPV (Arm A) or mOPV1-IPV (Arm B) or IPV (Arm C) schedules. Serum specimens collected at birth and at 14, 18, and 22 weeks old were analyzed with a standard microneutralization assay for all the three poliovirus serotypes. RESULTS: The results of 598 participants were analyzed. The type 1 cumulative seroconversion rates four weeks after the completion of the schedule at 18 weeks were 99.5% (97.0-99.9), 100.0% (97.9-100.0), and 96.0% (92.0-98.1) in Arms A (4bOPV + IPV), B (4mOPV1 + IPV), and C (3IPV), respectively. Type 2 and type 3 seroconversions at 18 weeks were 80.0% (73.7-85.1), 76.9% (70.3-82.4); 93.2% (88.5-96.1), 100.0% (98.0-100.0); and 81.9% (75.6-86.8), 99.4% (96.9-99.9), respectively, in the three arms. CONCLUSIONS: This study shows the high efficacy of different polio vaccines for serotype 1 in all three schedules. The type 1 seroconversion rate of mOPV1 is non-inferior to bOPV. All the vaccines provide high type-specific immunogenicity. The program can adopt the use of different vaccines or schedules depending on the epidemiology from time to time.

Phylogeography and gene pool analysis of highly pathogenic avian influenza H5N1 viruses reported in India from 2006 to 2021.

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.

Utility of glutaraldehyde-fixed turkey red blood cells for influenza virus detection after 18 months of storage.

Turkey red blood cells (tRBCs) are an essential reagent used in the laboratory diagnosis of influenza viruses. Fresh tRBCs when stored at 4 °C have a shelf life of less than a week. Previous studies have shown the utility of glutaraldehyde-fixed tRBCs, with an increased shelf life, for use in hemagglutination (HA) assays. In the present study, we report their functionality after storage for 18 months, at -80 °C. Three influenza A subtypes, namely, H3N2, H1N1 and H5N1, were used in the study. Hemagglutination assay was performed using freshly prepared 0.5 % tRBCs suspension and stored 1 % glutaraldehyde-fixed tRBCs. There was no significant difference in the HA titers obtained using fresh and stored tRBCs. The validation of the HA assay was carried out, to determine the specificity, linearity, precision, accuracy, and robustness of the assay. All of the titers were within the acceptable range, indicating the validity of the HA assay using stored tRBCs. Hemagglutination inhibition assay was also performed to compare the antibody titers obtained using stored and fresh tRBCs. The stored RBCs also gave equivalent antibody titers, as compared to the fresh tRBCs. Thus, the present study demonstrates the utility of glutaraldehyde-fixed tRBCs after one and a half years of storage.

Spatio-temporal distribution & seasonality of highly pathogenic avian influenza H5N1 & H5N8 outbreaks in India, 2006-2021.

Background & objectives: The highly pathogenic avian influenza (HPAI) H5N1 and H5N8 viruses have been one of the leading causes of avian diseases worldwide, resulting in severe economic losses and posing potential zoonotic risk. There are no reports on the correlation of the seasonality of H5N1 and H5N8 viruses with the migratory bird season in India, along with the species affected. The present report describes the distribution and seasonality of HPAI outbreaks in India from 2006 to 2021. Methods: The data on the occurrence and locations of outbreaks in India and affected bird species were collated from the Food and Agriculture Organization of the United Nations database and grouped by month and year. The distribution and seasonality of HPAI H5N1 and H5N8 viruses were analyzed. Results: A total of 284 H5N1 outbreaks were reported since 2006, with a surge in 2021. The initial outbreaks of H5N1 were predominantly in poultry. Since 2016, 57 outbreaks of H5N8 were also reported, predominantly in wild birds. Most of the outbreaks of HPAI were reported from post monsoon onwards till pre-summer season (i.e. between October and March) with their peak in winter, in January. Apart from poultry, the bird species such as owl, Indian peafowl, lesser adjutant, crows and wild migratory birds such as demoiselle crane, northern pintail and bar-headed goose were positive for HPAI. Interpretation & conclusions: Such studies on the seasonality of HPAI outbreaks would help in the development of prevention and control strategies. The recent human infections of H5N1 and H9N2 viruses highlight the need to strengthen surveillance in wild, resident, migratory birds and in poultry along with One Health studies in India.

The evolution, characterization and phylogeography of avian influenza H9N2 viruses from India.

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.

Replication of SARS-CoV-2 in cell lines used in public health surveillance programmes with special emphasis on biosafety.

Background & objectives: Polio, measles, rubella, influenza and rotavirus surveillance programmes are of great public health importance globally. Virus isolation using cell culture is an integral part of such programmes. Possibility of unintended isolation of SARS-CoV-2 from clinical specimens processed in biosafety level-2 (BSL-2) laboratories during the above-mentioned surveillance programmes, cannot be ruled out. The present study was conducted to assess the susceptibility of different cell lines to SARS-CoV-2 used in these programmes. Methods: Replication of SARS-CoV-2 was studied in RD and L20B, Vero/hSLAM, MA-104 and Madin-Darby Canine Kidney (MDCK) cell lines, used for the isolation of polio, measles, rubella, rotavirus and influenza viruses, respectively. SARS-CoV-2 at 0.01 multiplicity of infection was inoculated and the viral growth was assessed by observation of cytopathic effects followed by real-time reverse transcription-polymerase chain reaction (qRT-PCR). Vero CCL-81 cell line was used as a positive control. Results: SARS-CoV-2 replicated in Vero/hSLAM, and MA-104 cells, whereas it did not replicate in L20B, RD and MDCK cells. Vero/hSLAM, and Vero CCL-81 showed rounding, degeneration and detachment of cells; MA-104 cells also showed syncytia formation. In qRT-PCR, Vero/hSLAM and MA-104 showed 106 and Vero CCL-81 showed 107 viral RNA copies per µl. The 50 per cent tissue culture infectious dose titres of Vero/hSLAM, MA-104 and Vero CCL-81 were 105.54, 105.29 and 106.45/ml, respectively. Interpretation & conclusions: Replication of SARS-CoV-2 in Vero/hSLAM and MA-104 underscores the possibility of its unintended isolation during surveillance procedures aiming to isolate measles, rubella and rotavirus. This could result in accidental exposure to high titres of SARS-CoV-2, which can result in laboratory acquired infections and community risk, highlighting the need for revisiting biosafety measures in public health laboratories.

Seroprevalence of SARS-CoV-2 infection among frontline police personnel in Mumbai, India.

Introduction: A serosurvey was designed to ascertain the extent of infection among police personnel in Mumbai, India, during the last week of January 2021, at the end of the first wave of the COVID-19 pandemic, and just before the introduction of vaccination. Methods: The survey was carried out to screen for SARS-CoV-2 among 3232 police personnel. Of the 3176 participants willing for blood sample collection, 3077 personnel were found to be eligible for testing antibodies against the SARS-CoV-2 virus using the Roche Diagnostics' Elecsys Anti-SARS-CoV-2 assay. Results: The overall seroprevalence was 74.1% (95% CI 72.5-75.6). Males (75.1%, 73.4-76.8) had significantly higher seroprevalence than females (69.8%, 66.0-73.3), 18-39 years age group (76.4%, 74.4-78.3) than 40-59 years age group (70.6%, 67.9-73.1), non-officers (75.2%, 73.5-76.7) than officers (63.8%, 58.2-69.0), and personnel without comorbidities (75.0%, 73.3-76.6) than with comorbidities (69.7%, 65.6-73.5). Additionally, personnel with resident members positive for COVID-19 (89.6%, 84.7-93.1), personnel having reported COVID-19 earlier (95.5%, 93.8-96.7), and personnel having PCR positivity earlier (96.4%, 94.7-97.6) had significantly higher seroprevalences than others. All other variables, including diabetes and blood glucose status, lipid levels and thyroid enzymes, were not significantly associated with the seroprevalence levels. Conclusions: Almost three-fourths of frontline police personnel had evidence of past COVID-19 infection at the end of the first wave in January 2021, just before the introduction of COVID-19 vaccination. These frontline non-healthcare essential workers are an important risk group, and amenable to rapid serosurveys. These findings may help in estimating transmission status in the general community, along with disease burden, aiding prioritization of healthcare services.

Expansion of the measles and rubella laboratory network, India.

Objective: To expand the measles and rubella laboratory network of India by integrating new laboratories. Methods: In collaboration with the World Health Organization (WHO), the Indian government developed a 10-step scheme to systematically expand the number of laboratories performing serological and molecular testing for measles and rubella. The Indian Council of Medical Research and WHO identified suitable laboratories based on their geographical location, willingness, preparedness, past performance and adherence to national quality control and quality assurance mechanisms. The 10-step scheme was initiated with training on measles and rubella diagnostic assays followed by testing of both measles and rubella serology and molecular unknown panels, cross-verification with reference laboratories and ended with WHO on-site accreditation. Findings: After extensive training, technical support, funding and monitoring, all six selected laboratories attained passing scores of 90.0% or more in serological and molecular proficiency testing of measles and rubella. Since 2018, the laboratories are a part of the measles and rubella network of India. Within 12 months of initiation of independent reporting, the six laboratories have tested 2287 serum samples and 701 throat or nasopharyngeal swabs or urine samples. Conclusion: The process led to strengthening and expansion of the network. This proficient laboratory network has helped India in scaling up serological and molecular testing of measles and rubella while ensuring high quality testing. The collaborative model developed by the Indian government with WHO can be implemented by other countries for expanding laboratory networks for surveillance of measles and rubella as well as other infectious diseases.

Immunogenicity of Fractional Dose Inactivated Poliovirus Vaccine in India.

INTRODUCTION: Following the withdrawal of Sabin type 2 from trivalent oral poliovirus vaccine (tOPV) in 2016, the introduction of ≥1 dose of inactivated poliovirus vaccine (IPV) in routine immunization was recommended, either as 1 full dose (0.5mL, intramuscular) or 2 fractional doses of IPV (fIPV-0.1mL, intradermal). India opted for fIPV. We conducted a comparative assessment of IPV and fIPV. METHODS: This was a 4-arm, open-label, multicenter, randomized controlled trial. Infants were enrolled and vaccines administered according to the study design, and the blood was drawn at age 6, 14, and 18 weeks for neutralization testing against all 3 poliovirus types. RESULTS: Study enrolled 799 infants. The seroconversion against type 2 poliovirus with 2 fIPV doses was 85.8% (95% confidence interval [CI]: 80.1%-90.0%) when administered at age 6 and 14 weeks, 77.0% (95% CI: 70.5-82.5) when given at age 10 and 14 weeks, compared to 67.9% (95% CI: 60.4-74.6) following 1 full-dose IPV at age 14 weeks. CONCLUSION: The study demonstrated the superiority of 2 fIPV doses over 1 full-dose IPV in India. Doses of fIPV given at 6 and 14 weeks were more immunogenic than those given at 10 and 14 weeks. Clinical Trial Registry of India (CTRI). Clinical trial registration number was CTRI/2017/02/007793.

Selection and application of biological safety cabinets in diagnostic and research laboratories with special emphasis on COVID-19.

The ongoing coronavirus disease (COVID-19) pandemic is a global public health emergency. Adherence to biosafety practices is mandatory to protect the user as well as the environment, while handling infectious agents. A biological safety cabinet (BSC) is the most important equipment used in diagnostic and research laboratories in order to safeguard the product, the person, and the environment. The World Health Organization has emphasized the use of validated BSCs in order to ensure quality of the results. There are different classes of BSCs that are used in various work environments based on the need. It is imperative to use appropriate levels of biosafety and types of BSCs in laboratories based on the risk assessment of the pathogen used. During the development of COVID-19 laboratories and training of laboratory staff, we came across several queries about the functions and selection of BSCs and realized that the knowledge about the detailed information on selections and applications of BSCs is scanty. There are several guidelines regarding the biosafety aspects for diagnostic and research laboratories handling infectious pathogens from national and international agencies. However, there is no detailed information on the use of appropriate types of BSCs and their functions in the context of Severe Acute Respiratory Syndrome-Coronavirus 2 (SARS-CoV-2). In view of this, the present paper describes in detail the selection and applications of BSCs, which could be useful for laboratories handling or planning to handle SARS-CoV-2 and suspected samples.

Validation of a redesigned pan-poliovirus assay and real-time PCR platforms for the global poliovirus laboratory network.

Surveillance and detection of polioviruses (PV) remain crucial to monitoring eradication progress. Intratypic differentiation (ITD) using the real-time RT-PCR kit is key to the surveillance workflow, where viruses are screened after cell culture isolation before a subset are verified by sequencing. The ITD kit is a series of real-time RT-PCR assays that screens cytopathic effect (CPE)-positive cell cultures using the standard WHO method for virus isolation. Because ITD screening is a critical procedure in the poliovirus identification workflow, validation of performance of real-time PCR platforms is a core requirement for the detection of poliovirus using the ITD kit. In addition, the continual update and improvement of the ITD assays to simplify interpretation in all platforms is necessary to ensure that all real-time machines are capable of detecting positive real-time signals. Four platforms (ABI7500 real-time systems, Bio-Rad CFX96, Stratagene MX3000P, and the Qiagen Rotor-Gene Q) were validated with the ITD kit and a redesigned poliovirus probe. The poliovirus probe in the real-time RT-PCR pan-poliovirus (PanPV) assay was re-designed with a double-quencher (Zen™) to reduce background fluorescence and potential false negatives. The updated PanPV probe was evaluated with a panel consisting of 184 polioviruses and non-polio enteroviruses. To further validate the updated PanPV probe, the new assay was pilot tested in five Global Polio Laboratory Network (GPLN) laboratories (Madagascar, India, Philippines, Pakistan, and Democratic Republic of Congo). The updated PanPV probe performance was shown to reduce background fluorescence and decrease the number of false positives compared to the standard PanPV probe.

Phylogenetic classification of the whole-genome sequences of SARS-CoV-2 from India & evolutionary trends.

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.

A novel reassortant avian influenza H4N6 virus isolated from an environmental sample during a surveillance in Maharashtra, India.

Background & objectives: Low pathogenic avian influenza (LPAI) viruses cause mild clinical illness in domestic birds. Migratory birds are a known reservoir for all subtypes of avian influenza (AI) viruses. The objective of the study was to characterize AI H4N6 virus isolated from an environmental sample during surveillance in Maharashtra, India. Methods: AI surveillance in wild migratory birds was conducted during the winter migratory bird season (2016-2017) in Pune, India. AI H4N6 virus was isolated from the faecal droppings of a wild migratory waterbird. Virological and molecular characterization of the isolated virus was carried out. Virus titration, haemagglutination inhibition assay, receptor specificity assay, intravenous pathogenicity index and neuraminidase inhibition assays were performed. Full genome sequencing, molecular and phylogenetic analyses were also conducted. Results: The virus was found to be of low pathogenicity, with avian type receptor specificity, and was susceptible to neuraminidase inhibitors. Phylogenetic and molecular analysis revealed that the present virus is a result of extensive reassortment with AI H8N4, H6N2, H4N3 and H3N6, predominantly as donor viruses among others. Interpretation & conclusions: This is the first report of the isolation and characterization of an LPAI H4N6 virus from an environmental sample from India. The present study showed that the H4N6 virus is a novel reassortant and divergent as compared with the reported H4N6 viruses from poultry in India, indicating independent introduction. This highlights the role of wild and migratory birds in the transmission of AI viruses and necessity of such studies at the human-animal interface.

Application of frozen and stored glutaraldehyde-fixed turkey red blood cells for hemagglutination and hemagglutination inhibition assays for the detection and identification of influenza viruses.

INTRODUCTION: Hemagglutination (HA) and hemagglutination inhibition (HI) assays are conventionally used for the detection and identification of influenza viruses, using red blood cells (RBCs) from mammalian and avian sources. However, there could be limitations for availability of fresh RBCs due to situations such as pandemics, public health emergencies, outbreaks in avian species, lack of animal facilities, animal ethics concerns; or resource-constrained laboratories, and laboratories which do not carry out HA and HI assays routinely. Turkey RBCs (tRBCs) are widely used for HA and HI assays of influenza viruses. The present study explored the possibility of the use of glutaraldehyde-fixed tRBCs, which could be stored at -80 ºC and readily used for HA and HI assays. MATERIALS AND METHODS: A total of nine subtypes of human and avian influenza viruses, A H1N1, H3N2, H4N6, H5N1, H6N1, H7N9, H9N2, H11N1 and type B, were used in the study. Turkey RBCs were fixed with glutaraldehyde. The HA and HI assays were performed three times by two different operators using fresh and glutaraldehyde fixed tRBCs. The significance of difference in HA and HI titers between fixed and fresh RBCs was compared using 't-test'. The performance of fixed RBCs was evaluated before and after storing at -80 ºC for three weeks. RESULTS: There was no significant difference (p > 0.05) between mean HA and HI titers using fresh and glutaraldehyde-fixed turkey RBCs. In addition, the HA and HI titers using fixed tRBCs before and after storing at -80 ºC were equivalent, indicating suitability of the fixed and stored RBCs. CONCLUSIONS: This is the first report of the use of fixed and stored tRBCs for HA and HI assays of influenza viruses, highlighting their applicability as a ready-to-use reagent for laboratory diagnosis of influenza.

Steps, implementation and importance of quality management in diagnostic laboratories with special emphasis on coronavirus disease-2019.

A well-established and functional quality management system is an integral part of any diagnostic laboratory. It assures the reliability and standards of the laboratory function. A pandemic situation such as that caused by the influenza H1N1 2009 virus or the recent severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) increases the demands on the public health system, and the need to build, upgrade and expand the number of diagnostic laboratories. The Coronavirus disease-19 (COVID-19) pandemic caused by the SARS-CoV-2 unleashed a public health emergency of an unprecedented scale. The need has been highlighted for the accreditation of tests relating to COVID-19 by the National Accreditation Board for Testing and Calibration Laboratories (NABL) or any agencies approved by the World Health Organization (WHO) or Indian Council of Medical Research. The implementation of quality system in diagnostic laboratories would ensure accurate, reliable and efficient test results at par with the international standards. The functional aspects of a laboratory such as a well-defined organogram, standard operating procedures, good laboratory practices, quality controls, human resources, equipment management, reagents, inventory of records, proper communication need to be addressed to assure quality. Biosafety considerations should include the guidelines laid out by the WHO, the Institutional Biosafety Committee and the Department of Biotechnology, Government of India for carrying out diagnostic work in the laboratory. Currently, there are 1922 laboratories, operational for COVID-19 diagnosis in India. Considering the urgency of testing, the NABL has expedited the process of accreditation and issued accreditation to 818 laboratories. The adherence to the practicable aspects of quality described in this article would help in establishing quality in COVID-19 testing laboratories.

A real-time polymerase chain reaction-based approach for qualitative estimation of viral RNA in organ tissues of coxsackievirus A-16-infected neonatal mice.

Hand, foot and mouth disease (HFMD) is a paediatric disease associated with enteroviruses (EVs). Among EVs, coxsackievirus A-16 (CVA-16) strain is currently in circulation and causing outbreaks in India. Neonatal mice (Institute of Cancer Research) strains were infected with CVA-16 strain isolated from HFMD patients to conduct pathological and molecular studies. Infected organs were harvested as per time points. A real-time polymerase chain reaction was used for qualitative estimation of viral RNA in organ tissues of infected mice. Skeletal muscle, brain tissue and cardiac tissues were the major target sites of CVA-16 tropism. The first-ever study was conducted on CVA-16 strains using the current approach in India.

Analysis of influenza virus-induced perturbation in autophagic flux and its modulation during Vitamin D3 mediated anti-apoptotic signaling.

Vitamin D3/Calcitriol supplementation in humans is associated with reduced incidence and severity during influenza A virus (IAV) infection. Apoptosis in response to IAV infection is a major contributor to host cell death and tissue damage; however, its modulation by Vitamin D3 remains unclear. In this study, we demonstrate the efficacy of Vitamin D3 in preventing apoptosis induction by pandemic influenza A (H1N1)pdm09 virus in human alveolar cells (A549). Human alveolar epithelial cell line A549 was used to assess the cytotoxic effects of IAV infection. Immunoblotting and fluorescence microscopy were used to study apoptosis and autophagy. The results of the present study demonstrate that IAV induces apoptosis by subversion of host autophagy via down-regulating components of autophagic machinery involved in autophagosome-lysosome fusion and lysosomal activity. Vitamin D3 restores the autophagic flux inhibited by IAV by upregulating the expression of Syntaxin-17 (STX17) and V-type proton ATPase subunit (ATP6V0A2) thereby causing a concomitant decrease in cellular apoptosis via a Vitamin D3 receptor (VDR) dependent mechanism. The present study suggests that Vitamin D3 is a potentially useful agent for limiting IAV-induced cellular injury via its pro-autophagic action.

Isolation and molecular characterization of coxsackievirus A6 and coxsackievirus A16 from a case of recurrent Hand, Foot and Mouth Disease in Mumbai, Maharashtra, India, 2018.

Hand, Foot and Mouth Disease (HFMD) is caused by multiple Enterovirus (EV) serotypes mainly coxsackievirus A6 (CV-A6), coxsackievirus A16 (CV-A16) and Enterovirus 71 (EV-A71). Recurrent HFMD infections are rarely reported. An unusual rise in HFMD cases was reported in Mumbai during May-June 2018. Stool and throat swab specimens were referred from seven children from two hospitals for laboratory diagnosis. The age group of cases ranged from 9 months to 5 years with median age 13 months. Out of seven cases, three were males and four females. One 13-month-old female case was reported twice within 21 days. Stool, throat swab specimens were tested by pan enterovirus RT-PCR and also by virus isolation using human rhabdomyosarcoma cell line for detection of Enteroviruses. Out of seven HFMD cases, CV-A6 and CV-A16 viruses were isolated from five and two cases respectively. The phylogenetic analysis of CV-A6 viruses showed their similarity with CV-A6 viruses from Finland and China, whereas the two CV-A16 isolates showed similarity with those from Japan, France, China, Sarawak and Thailand. For the recurrent HFMD case, CV-A6 and CV-A16 were isolated from the stool specimens collected during the first and second episodes, respectively. There are no reports of isolation and molecular characterization of CV-A6 and CV-A16 viruses from recurrent HFMD cases. The present study reports molecular characterization of two Enterovirus serotypes CV-A6 and CV-A16 from a recurrent HFMD case, highlighting need of virological and molecular surveillance of HFMD.