Optimization of an allelic discrimination real-time RT-PCR assay for detection of H275Y oseltamivir resistance gene mutation among influenza A(H1N1)pdm09 patients from 2020 to 2022.

Influenza virus is known to cause mild to severe respiratory infections and is also prone to genetic mutations. Of all the mutations, neuraminidase (NA) gene mutations are a matter of concern, as most approved antivirals target this protein. During the 2020 influenza season, an emergence of mutation in the NA gene, affecting the binding of the World Health Organization (WHO)-recommended probes to the specific site of the NA gene, was reported by our group. As a result of this mutation, the WHO-recommended allelic discrimination real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay was unable to detect wild-type (H275) or mutant oseltamivir-resistant (Y275) strains of influenza A(H1N1)pmd09 viruses. In the current study, the WHO-recommended probes were redesigned according to the mutation in the probe binding site. Fifty undetermined samples (2020-2021) from the previous study were retested with the newly designed probes and found to be positive for H275 and/or Y275. The results obtained were similar to the Sanger sequencing results from the previous study, suggesting that the redesigned probes were efficient in discriminating between wild-type and mutant-type viruses. Furthermore, 133 samples from 2022, making a total of 183 samples (2020-2022), were tested using improved allelic discrimination real-time RT-PCR, and the overall prevalence rate of oseltamivir resistance in 2020-2022 was found to be 0.54%.

Cytokine profile, neutralisation potential and viral replication dynamics in sera of chikungunya patients in India: a cross-sectional study.

Background: Chikungunya disease (CHIKD) is a threat to global health, as it impairs the quality of life of an infected individual ranging from months to years. A systematic evaluation of the serological, virological, and immunological aspects of the circulating viruses and their impact on the host response is imperative for better understanding of the evolving disease dynamics. Methods: Serum samples were collected from 196 acute CHIKD patients from ten tertiary care hospitals across India during 2016-2021. Out of 196 patients, paired convalescent samples were collected from 51 patients (one-month post-onset of symptoms). The serum samples were profiled for cytokines and neutralisation capacity. Further, chikungunya virus (CHIKV) was isolated from the acute sera and the replication kinetics of the clinical isolates was evaluated. Findings: Serological analysis indicated that neutralisation could be correlated to seroconversion in the convalescent phase but not found significant in acute phase. In the acute phase samples, there was a correlation between elevated serum levels of IFN-γ, IP-10, MCP-1 and MIG and disease severity. During convalescent phase, pro-inflammatory markers such as IL-6, IL-1ß, IL-9 and IP-10 were found to be elevated with a corresponding decline in the secretion of anti-inflammatory cytokines such as IL-4 and IL-10, which correlated with persistent arthralgia. Analysis of replication of the clinical isolates revealed that 68.4% of viruses were fast-growing in the Vero cells (cytopathic effect [CPE] observed within 24 h post-infection), and their corresponding acute serum samples showed an elevated secretion of IFN-α, IL-1RA, IL-17F, IL-9, MCP-1 and MIP-1α. Interpretation: This study provides an important overview of neutralisation capabilities and cytokine responses along with virus pathogenesis associated with CHIKV infections in India. Funding: Biotechnology Industry Research Assistance Council (BIRAC).

Repurposing of approved antivirals against dengue virus serotypes: an in silico and in vitro mechanistic study.

Dengue is an emerging, mosquito-borne viral disease of international public health concern. Dengue is endemic in more than 100 countries across the world. However, there are no clinically approved antivirals for its cure. Drug repurposing proves to be an efficient alternative to conventional drug discovery approaches in this regard, as approved drugs with an established safety profile are tested for new indications, which circumvents several time-consuming experiments. In the present study, eight approved RNA-dependent RNA polymerase inhibitors of Hepatitis C virus were virtually screened against the Dengue virus polymerase protein, and their antiviral activity was assessed in vitro. Schrödinger software was used for in silico screening, where the compounds were passed through several hierarchical filters. Among the eight compounds, dasabuvir was finally selected for in vitro cytotoxicity and antiviral screening. Cytotoxicity profiling of dasabuvir in Vero cells revealed changes in cellular morphology, cell aggregation, and detachment at 50 µM. Based on these results, four noncytotoxic concentrations of dasabuvir (0.1, 0.25, 0.5, and 1 µM) were selected for antiviral screening against DENV-2 under three experimental conditions: pre-infection, co-infection, and post-infection treatment, by plaque reduction assay. Viral plaques were reduced significantly (p < 0.05) in the co-infection and post-infection treatment regimens; however, no reduction was observed in the pretreatment group. This indicated a possible interference of dasabuvir with NS5 RdRp, as seen from in silico interaction studies, translating into a reduction in virus plaques. Such studies reiterate the usefulness of drug repurposing as a viable strategy in antiviral drug discovery. In this drug repurposing study, dasabuvir, a known anti-hepatitis C drug, was selected through virtual screening and assessed for its anti-dengue activity.

Detection of H275Y oseltamivir resistance gene mutation among Influenza A(H1N1)pdm09 patients by allelic discrimination real-time RT-PCR.

Influenza viruses can mutate genetically and cause a range of respiratory ailments. The H275Y mutation in the neuraminidase (NA) gene reduces the effectiveness of oseltamivir, a widely used drug for the treatment of Influenza A and B virus infection. The World Health Organization (WHO) recommends single-nucleotide polymorphism assays to detect this mutation. The present study aims to estimate the prevalence of H275Y mutation conferring oseltamivir resistance in Influenza A(H1N1)pdm09 virus among hospitalized patients from June 2014 to December 2021. Following the WHO protocol, allelic discrimination real-time RT-PCR was performed for 752 samples. Out of the 752 samples, 1 tested positive for Y275 gene mutation by allelic discrimination real-time RT-PCR. In samples of years 2020 and 2021, neither the H275 nor Y275 genotype was detected. Sequencing of the NA gene of all negative samples showed a mismatch between the NA sequence and the probes used in the allelic discrimination assay. Also, Y275 mutation was detected in only 1 sample from 2020. The prevalence of oseltamivir resistance was estimated as 0.27% among the Influenza A(H1N1)pdm09 patients during 2014-2021. The study highlights that the WHO-recommended probes for detecting H275Y mutation may not be useful to detect 2020 and 2021 circulating strains of Influenza A(H1N1)pdm09, emphasizing the need for continuous monitoring of mutations in the influenza virus.

The emergence of human metapneumovirus G gene duplication in hospitalized patients with respiratory tract infection, India, 2016-2018.

BACKGROUND: Human metapneumovirus (HMPV) belongs to the family Pneumoviridae. It is one of the emerging respiratory viruses causing both upper and lower respiratory tract illnesses. HMPV has two genotypes: A and B. These genotypes are classified into lineage A1, A2, B1 and B2. Lineage-A2 is further classified as A2a, A2b and A2c. Similarly, B2 is classified as B2a and B2b. Studies have shown the circulation of A2b, B1 and B2 lineages in India. However, a limited amount of data is available on the current circulating genotypes of HMPV in India. METHODS: Throat swab samples positive for HMPV by real-time RT- PCR, archived at Manipal Institute of Virology as a part of a hospital-based acute febrile illness surveillance study, was used from April 2016 to August 2018 by purposive sampling method. We performed the conventional reverse transcriptase-polymerase chain reaction for twenty samples targeting the G gene and then subjected them to sequencing. Phylogenetic analysis was done using MEGA X software by the Maximum Likelihood method. RESULTS: All the twenty sequences belonged to the A2c subgroup. Phylogenetic analysis showed that strains from the study have genetic relation with circulating strains in Japan, China and Croatia. Seven out of the twenty sequences showed 180-nucleotide duplication and eleven sequences showed 111-nucleotide duplication. Two sequences did not show any duplications. CONCLUSION: In the current study, we report that A2c is the sub-lineage in India from April 2016 to August 2018. This study is the first retrospective study reporting the circulation of the A2c sub-lineage among adults in India with 180- and 111-nucleotide duplications in the G gene of human metapneumovirus.

Trends in Influenza Infections in Three States of India from 2015-2021: Has There Been a Change during COVID-19 Pandemic?

The COVID-19 pandemic and public health response to the pandemic has caused huge setbacks in the management of other infectious diseases. In the present study, we aimed to (i) assess the trends in numbers of samples from patients with influenza-like illness and severe acute respiratory syndrome tested for influenza and the number and proportion of cases detected from 2015−2021 and (ii) examine if there were changes during the COVID-19 period (2020−2021) compared to the pre-COVID-19 period (2015−2019) in three states of India. The median (IQR) number of samples tested per month during the pre-COVID-19 period was 653 (395−1245), compared to 27 (11−98) during the COVID-19 period (p value < 0.001). The median (IQR) number of influenza cases detected per month during the pre-COVID-19 period was 190 (113−372), compared to 29 (27−30) during the COVID-19 period (p value < 0.001). Interrupted time series analysis (adjusting for seasonality and testing charges) confirmed a significant reduction in the total number of samples tested and influenza cases detected during the COVID-19 period. However, there was no change in the influenza positivity rate between pre-COVID-19 (29%) and COVID-19 (30%) period. These findings suggest that COVID-19-related disruptions, poor health-seeking behavior, and overburdened health systems might have led to a reduction in reported influenza cases rather than a true reduction in disease transmission.

Genetic characterization of hemagglutinin (HA) gene of influenza A viruses circulating in Southwest India during 2017 season.

Molecular surveillance of influenza viruses is essential for early detection of novel variants. The aim of the present study was to analyze the hemagglutinin gene of influenza A(H1N1)pdm09 and A(H3N2) viruses circulating during the 2017 season. To investigate the genetic diversity of hemagglutinin gene of influenza A(H1N1)pdm09 and A(H3N2) viruses from 2017 season, ten samples from each subtype were sequenced and analyzed. The season was predominated by influenza A(H1N1)pdm09 viruses. Ten samples were sequenced from each subtype and all sequenced influenza A(H1N1)pdm09 and A(H3N2) viruses belonged to clades 6B.1 and 3C.2a, respectively. Sequence analysis of H1 gene in comparison to 2010-2016 vaccine strain showed mutations K166Q and S188T (K180Q and S202T here) that most likely resulted in antigenic drift and emergence of variant viruses. H3 gene substitutions N137K, N187K, I422V, and G500E that define clade 3C.2a1 were detected during analysis of sequences in comparison to 2017-2018 vaccine strain of northern hemisphere. These substitutions contributed to the change of WHO's recommendation of the 2018-2019 vaccine strain for northern hemisphere. The results of this study provide insights about the continuous genetic variability of the HA gene.

Hemagglutination Inhibition Antibody Response Following Influenza A(H1N1)pdm09 Virus Natural Infection: A Cross-Sectional Study from Thirthahalli, Karnataka, India.

Influenza viruses are major respiratory pathogens that cause seasonal epidemics and occasional pandemics. Immune response to influenza viruses is majorly targeted against the hemagglutinin antigen. A laboratory-based retrospective cross-sectional study was conducted on 50 acute and 50 follow-up samples to assess the immune response to influenza A(H1N1)pdm09 virus after natural infection and detect the presence of pre-existing antibodies against influenza A(H3N2) and influenza B viruses. Two-fourfold rise in hemagglutination-inhibition (HAI) titer was observed in 100% of the follow-up samples for influenza A(H1N1)pdm09 virus. No change in HAI titers for influenza A(H3N2) and influenza B viruses was observed.

HSV susceptibility to acyclovir - genotypic and phenotypic characterization.

BACKGROUND: Infections due to drug-resistant herpes simplex viruses (HSV) represent an important clinical concern, especially in immunocompromised patients. The present study was aimed at detecting acyclovir (ACV) susceptibility in HSV clinical samples. METHODS: A total of 13 HSV-positive clinical samples (5 HSV-1 and 8 HSV-2) recovered from patients (1 immunocompromised and 12 of unknown immune status) were included in the study. The genotypic analysis involved an initial UL23 (thymidine kinase) gene sequencing, followed by a confirmatory phenotypic assay using plaque reduction technique. RESULTS: Two novel amino acid changes, A37V and H283N, were detected in HSV-1 positive clinical samples, which were found to be susceptible to acyclovir (half maximal effective concentration = 1.5 µM) by plaque reduction assay. CONCLUSIONS: These two novel amino acid changes could be therefore considered as natural polymorphisms, a phenomenon widely associated with the HSV-UL23 gene.

Molecular characterization of neuraminidase genes of influenza A(H3N2) viruses circulating in Southwest India from 2009 to 2013.

Molecular characterization of neuraminidase (NA) gene of 25 influenza A(H3N2) virus isolates (2009-2013) archived at the Manipal Centre for Virus Research was carried out. The annual rate of amino acid substitutions in the N2 gene of influenza A(H3N2) virus isolates was 0.2-0.6%. Out of the 25 NA sequences analyzed, catalytic site mutations were observed in three isolates. Two of the mutations (D151G and E276G) were detected in functional catalytic residues, and an E227V mutation was detected in the framework residues. To the best of our knowledge, NA inhibitor resistance associated with the mutations E276G and E227V has not been reported. However, the mutation D151G, which is commonly associated with culturing of influenza A(H3N2) virus in Madin-Darby canine kidney (MDCK) cells, has been reported to result in a reduction in virus susceptibility to NA inhibitor drugs. Our study also detected mutations in antigenic residues. Some of the mutations (except D197G, K249E, A250T, S334C, and H347R/N) remained conserved in isolates of succeeding seasons. Antigenic residue mutations (D197G and S334C) have not been reported globally to date. The effect of these catalytic and antigenic mutant residues on drug and antibody binding was analyzed using three-dimensional structural analysis and biochemical assays. Antigenic variability of influenza A(H3N2) viruses is a major concern, and vaccine failures are mainly due to genetic variations in the HA gene. Our study documents that genetic changes in N2 occur at a slower rate, and this information is useful for the consideration and standardization of NA in influenza vaccines.

Detection of D151G/N mutations in the neuraminidase gene of influenza A (H3N2) viruses by real-time RT-PCR allelic discrimination assay.

Single nucleotide polymorphisms (SNPs) at D151 position of neuraminidase (NA) gene of influenza A (H3N2) virus has been associated with drug resistance and increased binding affinity. NA-D151G/N-substitutions of influenza A (H3N2) viruses are frequently induced and selected by culturing in Madin-Darby canine kidney (MDCK) cell lines. It is important to consider and exclude D151G/N mutants after isolation of influenza virus in MDCK cell line; since, the substitutions can highly influence the results of experimental research. The study aims to develop an allelic discrimination real-time reverse transcriptase polymerase chain reaction (RT-PCR) for the screening of D151G/N mutants. Thirty-six influenza A (H3N2) virus isolates were included and screened for D151G/N mutants using allelic discrimination assay. Out of the 36 isolates, 11 isolates (30.5%) were detected as heterozygous for D and G/N substitutions. Twenty-one (58.3%) isolates were identified as homozygous wild type and four isolates (11.1%) were undetermined. Isolates with substitutions at D151 position were sequenced by Sanger sequencing method. The present study demonstrates a rapid and convenient method for primary screening of the mutation after culturing of the influenza virus in MDCK cell lines in order to avoid potential misinterpretations of results and improve the quality of experimental research.

Genetic analysis of neuraminidase gene of influenza A(H1N1)pdm09 virus circulating in Southwest India from 2009 to 2012.

Genetic analysis of neuraminidase gene sequences in 23 archived isolates of influenza A(H1N1)pdm09 virus, isolated during the 2009-2012 influenza seasons, was carried out to determine the genetic variability. Amino acid substitutions were observed at the rates of 0.3-0.7% per year. The catalytic site consisting of 8 functional and 11 framework residues were found conserved in 20 isolates and mutated in three (E228G, E278G, and N295T) isolates. To the best of our knowledge the three catalytic site mutants observed in our study have not been reported elsewhere to date. Similarly, mutations in the antigenic sites (K217E, K254E, V267A, and D451E except I263V) are discussed for the first time through this article. The effect of these mutations on drug and antibody binding were analyzed using biochemical and structural studies. Detailed studies on the neuraminidase gene are sparse and our study may serve as an appropriate platform to gain insights about the evolution of influenza virus, thereby facilitating drugs/vaccines design and development. J. Med. Virol. 89:202-212, 2017. © 2016 Wiley Periodicals, Inc.

Influenza virus neuraminidase (NA): a target for antivirals and vaccines.

Influenza, the most common infectious disease, poses a great threat to human health because of its highly contagious nature and fast transmissibility, often leading to high morbidity and mortality. Effective vaccination strategies may aid in the prevention and control of recurring epidemics and pandemics associated with this infectious disease. However, antigenic shifts and drifts are major concerns with influenza virus, requiring effective global monitoring and updating of vaccines. Current vaccines are standardized primarily based on the amount of hemagglutinin, a major surface antigen, which chiefly constitutes these preparations along with the varying amounts of neuraminidase (NA). Anti-influenza drugs targeting the active site of NA have been in use for more than a decade now. However, NA has not been approved as an effective antigenic component of the influenza vaccine because of standardization issues. Although some studies have suggested that NA antibodies are able to reduce the severity of the disease and induce a long-term and cross-protective immunity, a few major scientific issues need to be addressed prior to launching NA-based vaccines. Interestingly, an increasing number of studies have shown NA to be a promising target for future influenza vaccines. This review is an attempt to consolidate studies that reflect the strength of NA as a suitable vaccine target. The studies discussed in this article highlight NA as a potential influenza vaccine candidate and support taking the process of developing NA vaccines to the next stage.