Search

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. [ABSTRACT FROM AUTHOR]

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. • Avian influenza H9N2 viruses are of zoonotic concern with pandemic potential. • There is no data on the genesis and detailed gene pool analysis of H9N2 in India. • We report their phylogeography, molecular and evolutionary characterization. • The H9N2 viruses show gradual shift towards mammalian adaptation. • Reassortments of H9N2 have occurred with HPAI H5N1 and H7N3 viruses. [ABSTRACT FROM AUTHOR]

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 K M 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. [Display omitted] • Influenza virus neuraminidase protein (NA) plays a key role in replication. • NA enzyme kinetics are important to determine the activity of the enzyme. • We report combination of novel mutations near the NA active site of an H9N2 strain. • The mutations altered surface properties leading to increased substrate binding. • Enhanced virus elution was observed due to these changes in the virus NA. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

• NA gene sequencing of 67 HPAI H5N1 viruses revealed NA mutations in nine viruses. • A novel NA I117T mutation of HPAI H5N1 virus conferred resistance to NA inhibitors. • NAI susceptibility studied using fluorometric MUNANA assays and in ovo assays. • The I117T and I117V variants showed different levels of NAI susceptibility. • Natural occurrence of NAI-resistant I117T-mutant H5N1 virus is a cause of concern. Occurrence of avian influenza (AI) with Neuraminidase (NA) mutations which confer reduced neuraminidase inhibitor (NAI) susceptibility has remained a cause of concern. The susceptibility to NAIs of 67 highly pathogenic avian influenza H5N1 viruses isolated during 2006–2012 in India was tested in phenotypic fluorescence-based NA inhibition assay, sequence analysis and in ovo. One isolate showed a novel NA I117T amino acid substitution (N2 numbering) and eight isolates showed previously known NAI-resistance marker mutations (I117V, E119D, N294S, total 9/67). The overall incidence of resistant variants was 13.4%. The novel I117T substitution reduced oseltamivir susceptibility by 18.6-fold and zanamivir susceptibility by 11.8-fold, compared to the wild type AI H5N1virus, thus showed cross-resistance to both oseltamivir and zanamivir in NA inhibition assays. However, the other two isolates with I117V substitution were sensitive to both the NAIs. In addition, the comparison of growth of the I117T and I117V variants in presence of NAI's in the in ovo assays exhibited difference in growth levels. The present study reports the natural occurrence of a novel I117T mutation in AI H5N1 virus conferring cross-resistance to oseltamivir and zanamivir highlighting the urgent need of antiviral surveillance of AI viruses. [ABSTRACT FROM AUTHOR]

An outbreak of Hand, Foot and Mouth Disease (HFMD) was reported in Navi Mumbai in July-October 2018. Of 15 HFMD cases, two had recurrences within a month while three had lesions extending to trunk. Coxsackie virus A6 and A16 were detected from 13 cases (CV-A6 from 10 cases and CV-A16 from 3 cases) indicating co-circulation of these viruses.. The present study highlights an urgent need of HFMD surveillance. [ABSTRACT FROM AUTHOR]

A letter is presented regarding avian surveys conducted in India from 1954 to 1981 to determine the role of wild and migratory birds in transmission of arboviruses.

Background: In January 2011, human cases with hemorrhagic manifestations in the hospital staff were reported from a tertiary care hospital in Ahmadabad, India. This paper reports a detailed epidemiological investigation of nosocomial outbreak from the affected area of Ahmadabad, Gujarat, India. Principal Findings: Samples from 3 suspected cases, 83 contacts, Hyalomma ticks and livestock were screened for Crimean-Congo hemorrhagic fever (CCHF) virus by qRT-PCR of which samples of two medical professionals (case C and E) and the husband of the index case (case D) were positive for CCHFV. The sensitivity and specificity of indigenous developed IgM ELISA to screen CCHFV specific antibodies in human serum was 75.0% and 97.5% respectively as compared to commercial kit. About 17.0% domestic animals from Kolat, Ahmadabad were positive for IgG antibodies while only two cattle and a goat showed positivity by qRT-PCR. Surprisingly, 43.0% domestic animals (Buffalo, cattle, sheep and goat) showed IgG antibodies in the adjoining village Jivanpara but only one of the buffalo was positive for CCHFV. The Hyalomma anatolicum anatolicum ticks were positive in PCR and virus isolation. CCHFV was isolated from the blood sample of case C, E in Vero E-6 cells and Swiss albino mice. In partial nucleocapsid gene phylogeny from CCHFV positive human samples of the years 2010 and 2011, livestock and ticks showed this virus was similar to Tajikistan (strain TAJ/H08966), which belongs in the Asian/middle east genetic lineage IV. Conclusions: The likely source of CCHFV was identified as virus infected Hyalomma ticks and livestock at the rural village residence of the primary case (case A). In addition, retrospective sample analysis revealed the existence of CCHFV in Gujarat and Rajasthan states before this outbreak. An indigenous developed IgM ELISA kit will be of great use for screening this virus in India. Author Summary: A nosocomial outbreak of CCHFV occurred in January 2011, in a tertiary care hospital in Ahmadabad, Gujarat State in western India. Out of a total five cases reported, contact transmission occurred to three treating medical professionals, all of whom succumbed to the disease. The only survivor was the husband of the index case. These results highlight the importance of considering CCHFV as a potential aetiology for Hemorrhagic fever (HF) cases in India. This also underlines the need for strict barrier nursing and patient isolation while managing these patients. During the investigation presence of CCHFV RNA in Hyalomma anatolicum ticks and livestock were detected in the village from where the primary case (case A) was reported. Further retrospective investigation confirmed two CCHF human cases in Rajkot village 20 kilometres to the west of Ahmadabad in 2010, and CCHFV presence in the livestock 200 kilometres to the north in the neighbouring State Rajasthan. This report shows the presence of CCHFV in human, ticks and animals in Gujarat, India. The fact of concern is the spread of this disease from one state to another due to trading of livestock. [ABSTRACT FROM AUTHOR]

Avian influenza (AI) H9N2 has been reported from poultry in India. A seroepidemiological study was undertaken among poultry workers to understand the prevalence of antibodies against AI H9N2 in Pune, Maharashtra, India. A total of 338 poultry workers were sampled. Serum samples were tested for presence of antibodies against AI H9N2 virus by hemagglutination inhibition (HI) and microneutralization (MN) assays. A total of 249 baseline sera from general population from Pune were tested for antibodies against AI H9N2 and were negative by HI assay using ≥40 cut-off antibody titre. Overall 21 subjects (21/338 = 6.2%) were positive for antibodies against AI H9N2 by either HI or MN assays using ≥40 cut-off antibody titre. A total of 4.7% and 3.8% poultry workers were positive for antibodies against AI H9N2 by HI and MN assay respectively using 40 as cut-off antibody titre. This is the first report of seroprevalence of antibodies against AI H9N2 among poultry workers in India. [ABSTRACT FROM AUTHOR]

Introduction: Hemagglutination (HA) and hemagglutination inhibition (HI) assays are conventionally used for detection and identification of influenza viruses. HI assay is also used for detection of antibodies against influenza viruses. Primarily turkey or chicken erythrocytes [red blood cells (RBCs)] are used in these assays, as they are large, nucleated, and sediment fast, which makes it easy to determine the titer. Human influenza viruses agglutinate RBCs from chicken, human, and guinea pig, but not from horse. Human influenza viruses bind preferentially to sialic acid (SA) linked to galactose (Gal) by α 2, 6 linkage (SA α 2, 6-Gal), whereas avian influenza (AI) viruses bind preferentially to SA α 2, 3-Gal linkages. With this background, the present study was undertaken to study erythrocyte binding preferences and receptor specificities of AI viruses isolated from India. Materials and methods: A total of nine AI virus isolates (four subtypes) from India and three reference AI strains (three subtypes) were tested in HA and HI assays against mammalian and avian erythrocytes. The erythrocytes from turkey, chicken, goose, guinea pig and horse were used in the study. The receptor specificity determination assays were performed using goose and turkey RBCs. The amino acids present at 190 helix, 130 and 220 loops of the receptor-binding domain of the hemagglutinin protein were analyzed to correlate amino acid changes with the receptor specificity. Results: All tested highly pathogenic avian influenza (HPAI) H5N1 viruses reacted with all five types of RBCs in the HA assay; AI H9N2 and H5N2 viruses did not react with horse RBCs. For H5N1 viruses guinea pig and goose RBCs were best for both HA and HI assays. For H9N2 viruses, guinea pig, fowl and turkey RBCs were suitable. For other tested AI subtypes, avian and guinea pig RBCs were better. Eight isolates of H5N1, one H4N6 and one H7N1 virus showed preference to avian sialic acid receptors. Importantly, two isolates of HPAI H5N1, H9N2 and H11N1 viruses showed receptor specificity preference to both avian and mammalian sialic acid (α-2, 3 and α-2, 6) receptors. Conclusions: Use of different types of RBCs resulted in titer variations in HA and HI assays. This showed that RBCs giving optimum HA and HI titers would increase sensitivity of detection and would be more appropriate for identification and antigenic analysis of AI viruses. Analysis of 16 amino acids in the receptor-binding domain of the hemagglutinin of HPAI H5N1 viruses revealed that the only variation observed was in S221P amino acid position. Two H5N1 viruses showed S221P amino acid change, out of which only one H5N1 virus showed preference to α 2, 6 sialic acid receptor. One H5N1 virus isolate with amino acid S at 221 position, showed preference to α 2,3 as well as α 2,6 sialic acid receptors. This indicated that factor(s) other than S221P mutation in the hemagglutinin are probably involved in determining receptor specificity of H5N1 viruses. This is the first report of receptor specificity and erythrocyte binding preferences of AI viruses from India. [ABSTRACT FROM AUTHOR]

Introduction: More than 70 outbreaks of the highly pathogenic avian influenza (HPAI) H5N1 have been reported in poultry in the western and north-eastern parts of India. Therefore, in view of the recent HPAI H5N1 outbreaks in poultry, active AI surveillance encompassing wild, resident, migratory birds and poultry was undertaken during 2009-2011 in the State of West Bengal. Methods: A total of 5722 samples were collected from West Bengal; 3522 samples (2906 fecal droppings + 616 other environmental samples) were from migratory birds and 2200 samples [1604 tracheal, cloacal swabs, environmental samples, tissue samples + 596 blood (serum)] were from domestic ducks and poultry. All tracheal, cloacal and environmental samples were processed for virus isolation. Virus isolates were detected using hemagglutination assay and identified using hemagglutination inhibition (HI) and reverse transcriptase polymerase chain reaction (RT-PCR) assays. Sequencing and phylogenetic analysis of partial region of the hemagglutinin and neuraminidase genes was done. Intravenous pathogenicity index assays were performed in chickens to assess pathogenicity of AI virus isolates. Serum samples were tested for detection of antibodies against AI viruses using HI assay. Results: A total of 57 AI H9N2, 15 AI H4N6 and 15 Newcastle Disease (NDV) viruses were isolated from chickens, from both backyard and wet poultry markets; AI H4N6 viruses were isolated from backyard chickens and domestic ducks. Characterization of AI H9N2 and H4N6 viruses revealed that they were of low pathogenicity. Domestic ducks were positive for antibodies against H5 and H7 viruses while chickens were positive for presence of antibodies against AI H9N2 and NDV. Conclusions: In the current scenario of HPAI H5N1 outbreaks in West Bengal, this report shows presence of low pathogenic AI H9N2 and H4N6 viruses in chickens and domestic ducks during the period 2009-2011. This is the first report of isolation of H4N6 from India. Antibodies against AI H5 and H7 in ducks highlight the probable role of domestic ducks in the transmission of AI viruses. Human infections of H9N2 have been reported from China and Hong Kong. This necessitates implementation of prevention and control measures to limit the spread of AI viruses. [ABSTRACT FROM AUTHOR]

Background: The world has recently overcome the first influenza pandemic of the 21st century caused by a novel H1N1 virus (pH1N1) which is a triple reassortant comprising genes derived from avian, human, and swine influenza viruses and antigenically quite different from seasonal H1N1 strains. Although the case fatality rates have decreased in many developed countries, the situation is still alarming in many developing countries including India where considerable numbers of new cases are appearing everyday. There is still a high morbidity and mortality of susceptible adult as well as young population without having underlying health issues due to the influenza infection. Results: To achieve a better understanding of the risk posed by the pH1N1 and to understand its pathogenicity, we studied the host gene expression response to Indian isolate of pH1N1 infection and compared it with seasonal H1N1 infection. The response was studied at four different time points (4, 8, 16 and 24 h) post infection (hpi) in A549 cells using microarray platform. We found that pH1N1 induces immune response earlier than seasonal H1N1 viruses, but at the later stages of infection there is a suppression of host immune responses. The infection with pH1N1 resulted in considerable decrease in the expression of cytokine and other immune genes namely IL8, STAT1, B2 M and IL4 compared to seasonal H1N1. Conclusion: We propose that the inability to induce strong innate immune response could be a reason for the high transmissibility, pathogenicity and mortality caused by pH1N1 virus. [ABSTRACT FROM AUTHOR]

Background & objectives: An outbreak of influenza was investigated between June 24 and July 30, 2009 in a residential school at Panchgani, Maharashtra, India. The objectives were to determine the aetiology, study the clinical features in the affected individuals and, important epidemiological and environmental factors. The nature of public health response and effectiveness of the control measures were also evaluated. Methods: Real time reverse transcriptase polymerase chain reaction was performed on throat swabs collected from 82 suspected cases to determine the influenza types (A or B) and sub-types [pandemic (H1N1) 2009, as well as seasonal influenza H1N1, H3N2]. Haemagglutination inhibition assay was performed on serum samples collected from entire school population (N = 415) to detect antibodies for pandemic (H1N1) 2009, seasonal H1N1, H3N2 and influenza B/Yamagata and B/Victoria lineages. Antibody titres ≥ 10 for pandemic (H1N1) 2009 and ≥ 20 for seasonal influenza A and B were considered as positive for these viruses. Results: Clinical attack rate for influenza-like illness was 71.1 per cent (295/415). The attack rate for pandemic (H1N1) 2009 cases was 42.4 per cent (176/415). Throat swabs were collected from 82 cases, of which pandemic (H1N1) 2009 virus was detected in 15 (18.3%), influenza type A in (6) 7.4 per cent and influenza type B only in one case. A serosurvey carried out showed haemagglutination inhibition antibodies to pandemic (H1N1) 2009 in 52 per cent (216) subjects in the school and 9 per cent (22) in the community. Interpretation & conclusion: Our findings confirmed an outbreak of pandemic (H1N1) 2009 due to local transmission among students in a residential school at Panchgani, Maharashtra, India. [ABSTRACT FROM AUTHOR]

Background: In India, Pune was one of the badly affected cities during the influenza A (H1N1) 2009 pandemic. We undertook serosurveys among the risk groups and general population to determine the extent of pandemic influenza A (H1N1) 2009 virus infections. Methods: Pre-pandemic sera from the archives, collected during January 2005 to March 2009, were assayed for the determination of baseline seropositivity. Serosurveys were undertaken among the risk groups such as hospital staff, general practitioners, school children and staff and general population between 15th August and 11th December 2009. In addition, the PCR-confirmed pandemic influenza A (H1N1) 2009 cases and their household contacts were also investigated. Haemagglutination-inhibition (HI) assays were performed using turkey red blood cells employing standard protocols. A titre of ≥1:40 was considered seropositive. Results: Only 2 (0.9%) of the 222 pre-pandemic sera were positive. The test-retest reliability of HI assay in 101 sera was 98% for pandemic H1N1, 93.1% for seasonal H1N1 and 94% for seasonal H3N2. The sera from 48 (73.8%) of 65 PCR-confirmed pandemic H1N1 cases in 2009 were positive. Seropositivity among general practitioners increased from 4.9% in August to 9.4% in November and 15.1% in December. Among hospital staff, seropositivity increased from 2.8% in August to 12% in November. Seropositivity among the schools increased from 2% in August to 10.7% in September. The seropositivity among students (25%) was higher than the school staff in September. In a general population survey in October 2009, seropositivity was higher in children (9.1%) than adults (4.3%). The 15-19 years age group showed the highest seropositivity of 20.3%. Seropositivity of seasonal H3N2 (55.3%) and H1N1 (26.4%) was higher than pandemic H1N1 (5.7%) (n = 2328). In households of 74 PCR-confirmed pandemic H1N1 cases, 25.6% contacts were seropositive. Almost 90% pandemic H1N1 infections were asymptomatic or mild. Considering a titre cut off of 1:10, seropositivity was 1.5-3 times as compared to 1:40. Conclusions: Pandemic influenza A (H1N1) 2009 virus infection was widespread in all sections of community. However, infection was significantly higher in school children and general practitioners. Hospital staff had the lowest infections suggesting the efficacy of infection-control measures. [ABSTRACT FROM AUTHOR]

Background: A focal H5N1 outbreak in poultry was reported from Manipur, a north-eastern state, of India, in 2007. The aim of this study was to genetically characterize the Manipur isolate to understand the relationship with other H5N1 isolates and to trace the possible source of introduction of the virus into the country. Results: Characterization of the complete genome revealed that the virus belonged to clade 2.2. It was distinctly different from viruses of the three EMA sublineages of clade 2.2 but related to isolates from wild migratory waterfowl from Russia, China and Mongolia. The HA gene, had the cleavage site GERRRRKR, earlier reported in whooper swan isolates from Mongolia in 2005. A stop codon at position 29 in the PB1-F2 protein could have implications on the replication efficiency. The acquisition of polymorphisms as seen in recent isolates of 2005-07 from distinct geographical regions suggests the possibility of transportation of H5N1 viruses through migratory birds. Conclusion: Considering that all eight genes of the earlier Indian isolates belonged to the EMA3 sublineage and similar strains have not been reported from neighbouring countries of the subcontinent, it appears that the virus may have been introduced independently. [ABSTRACT FROM AUTHOR]

A letter to the editor is presented which discusses the seroprevalence of H1N1 pandemic influenza in 2011 and seasonal influenza viruses among pigs in Maharashtra and Gujarat State in India.

Antiviral susceptibility screening of avian influenza (AI) H9N2 viruses is crucial considering their role at the animal-human interface and potential to cause human infections. The Matrix 2 (M2) inhibitors (amantadine and rimantadine) have been used for prophylaxis and treatment of influenza A virus infections, however, resistance to these drugs has been widely reported. Information about amantadine susceptibility of H9N2 viruses from India is scanty. Matrix genes of 48H9N2 viruses isolated from India during 2009–2017 were sequenced and M2 trans-membrane region sequences were screened for mutations which are known to confer resistance to amantadine namely, L26F, V27A, A30 T/V, S31N and G34E. All the viruses isolated during the year 2009 were sensitive to amantadine. However, resistance started to appear since the year 2010 and all the viruses isolated from the year 2015 onwards showed presence of molecular markers conferring resistance to amantadine. Majority of the resistant viruses exhibited S31 N mutation. Four isolates showed presence of V27A + S31 N dual mutations. Comparison of the M2 sequences from other Asian countries showed different patterns of amantadine resistance wherein phylogenetic analysis of the M genes of the strains from Pakistan formed a separate cluster. In conclusion, the present study reports prevalence and gradual increase of amantadine resistance among AI H9N2 viruses in India, emphasizing the importance of the antiviral surveillance. • Amantadine resistance mutations in avian influenza H9N2 reported worldwide. • Reports on H9N2 amantadine susceptibility from India are scanty. • H9N2 isolates from India from 2009 to 2017 were screened for resistance markers. • All viruses from the year 2015 onwards showed amantadine resistance markers. • Highlights importance of antiviral surveillance of avian influenza. [ABSTRACT FROM AUTHOR]

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., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

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.

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.

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., Competing Interests: None

Background & Objectives: The susceptibility of influenza viruses to neuraminidase inhibitors (NAIs) is studied using enzyme-based assays, sequence analysis and in vitro and in vivo studies. Oseltamivir carboxylate (OC) is the active prodrug of the NAI oseltamivir. There is lack of information on the use of embryonated chicken eggs for studying susceptibility of highly pathogenic avian influenza (HPAI) H5N1 viruses to antiviral drugs. The aim of the present study was to assess the use of 10 day old embryonated chicken eggs for studying antiviral susceptibility of HPAI H5N1 viruses., Methods: Two HPAI H5N1 viruses isolated from India were used in the study. Fluorescence-based NAI assay was performed to determine antiviral susceptibility of these viruses. In ovo antiviral assays were carried out using 10 day old embryonated chicken eggs. The virus dilutions were incubated with 14 μg/ml of OC and inoculated in the allantoic cavity. In the eggs, 50 per cent egg infectious dose (EID 50 ) titres as well as mortality were quantitated., Results: The two viruses used were susceptible to OC in the NAI assay. It was found that there was a significant drop in EID 50 titres; however, no significant protection from mortality after OC treatment was observed., Interpretation & Conclusions: By measuring viral titres, the egg model was suitable to study the susceptibility of HPAI viruses to antiviral drugs along with NAI assay. The present study highlights the use of eggs as a model to study susceptibility of HPAI viruses to OC., Competing Interests: None

The healthcare workers having seroprotection at 3 weeks (n = 127) following Pandemic H1N1 2009 influenza vaccination were followed up for antibody persistence. Seroprotection at 12 mo (60.2%) was significantly lower as compared with 3 weeks (74.7%), 3 mo (77.8%) and 6 mo (75.4%). The vaccine provided seroprotection up to one year.

An avian influenza (AI) surveillance was undertaken in Maharashtra state, India during the period 2010-2011. There are no reports of AI surveillance in emus from India. A total of 202 blood samples and 467 tracheal and cloacal swabs were collected from eight emu farms. A hemagglutination inhibition (HI) assay was performed for detection of antibodies against AI H5N1, H7N1, H9N2, and avian paramyxovirus type 1 (APMV-1) viruses. A microneutralization (MN) assay was performed to confirm the presence of neutralizing antibodies against AI H9N2 and to compare with HI assays. A total of 28.2% and 28.7% of samples were positive for antibodies against AI H9N2 by HI and MN assays, respectively, using > or = 1:40 as a cut-off titer; 15.3% samples were positive for APMV-1 by HI assay using a > or = 1:10 cut-off titer. Seropositivity of AI H9N2 was nil in the grower (<1 yr) age group and highest (78%) in the breeder (2-3 yr) age group, whereas seropositivity against APMV-1 was observed in all age groups. Performance of both HI and MN assays was similar, suggesting the utility of using the MN assay along with HI assay for surveillance studies. This is the first report of the seroprevalence of AI H9N2 and APMV-1 in emus in India.

Widespread infection of highly pathogenic avian influenza A H5N1 was reported from backyard and commercial poultry in West Bengal (WB), an eastern state of India in early 2008. Infection gradually spread to Tripura, Assam and Sikkim, the northeastern states, with 70 outbreaks reported between January 2008 and May 2009. Whole genome sequence analysis of three isolates from WB, one isolate from Tripura along with the analysis of hemagglutinin (HA) and neuraminidase (NA) genes of 17 other isolates was performed during this study. In the HA gene phylogenetic tree, all the 2008-09 Indian isolates belonged to EMA3 sublineage of clade 2.2. The closest phylogenetic relationship was found to be with the 2007-09 isolates from Bangladesh and not with the earlier 2006 and 2007 Indian isolates implying a third introduction into the country. The receptor-binding pocket of HA1 of two isolates from WB showed S221P mutation, one of the markers predicted to be associated with human receptor specificity. Two substitutions E119A (2 isolates of WB) and N294S (2 other isolates of WB) known to confer resistance to NA inhibitors were observed in the active site of neuraminidase. Several additional mutations were observed within the 2008-09 Indian isolates indicating genetic diversification. Overall, the study is indicative of a possible endemicity in the eastern and northeastern parts of the country, demanding active surveillance specifically in view of the critical mutations that have been observed in the influenza A H5N1 viruses.

An outbreak of highly pathogenic avian influenza A (H5N1) virus in poultry was reported from Nandurbar and Jalgaon districts of Maharashtra and adjoining areas of Uchhal in Gujarat and Burhanpur in Madhya Pradesh in India from January to April, 2006. In the present study, the full genome of two previously uncharacterized strains of H5N1 viruses isolated at the National Institute of Virology (NIV), Pune, from post-mortem tissues of chicken collected from Navapur, Nandurbar district during the outbreak, has been presented. All the genes belong to clade 2.2 of the Z genotype and are close to the 2006 isolates from Iran, Afghanistan, Mongolia, Italy, and Krasnodar. In a study reported earlier, based on the partial gene sequences of HA, the authors (Pattnaik et al.) hypothesized that the viruses in Jalgaon and Navapur, causing outbreaks 12 days apart, were introduced at different times from different sources. However, our Navapur isolates are closer to the isolate reported from Jalgaon than that from Navapur. Molecular markers suggest that the isolates are sensitive to both drugs Oseltamivir and Amantadine. Amino acid residues responsible for pathogenesis, glycosylation, and receptor binding have also been discussed. The relationship between the Indian viruses and those in the East Africa/West-Asia flyway of migratory birds and the position of Nandurbar in this route suggests that the viruses in India may have been introduced through migratory birds although the role of trade as a possible route of introduction of the virus cannot be ruled out.