Emergence of Recombinant Subclade D3/Y in Coxsackievirus A6 Strains in Hand-Foot-and-Mouth Disease (HFMD) Outbreak in India, 2022.

Coxsackievirus-A6 (CV-A6) is responsible for more severe dermatological manifestations compared to other enteroviruses such as CV-A10, CV-A16, and EV-A71, causing HFMD in children and adults. Between 2005 and 2007, the recombinant subclade D3/RF-A started to expand globally, and a CV-A6 pandemic started. The study aimed to conduct whole-genome sequencing (WGS) of an isolated CV-A6 strain from currently circulating HFMD cases from India in 2022. Gene-specific RT-PCR and sequencing were used to perform molecular characterization of the isolated virus. Confirmation of these isolates was also performed by transmission electron microscopy and WGS. Among eleven positive clinical enterovirus specimens, eight CV-A6 strains were successfully isolated in the RD cell line. Isolates confirmed the presence of the CV-A6 strain based on VP1 and VP2 gene-specific RT-PCR. Sequences of isolates were clustered and identified as the novel CV-A6 strain of the D3/Y sub-genotype in India. The studies revealed that the D3/Y sub-genotype is being introduced into Indian circulation. The predicted putative functional loops found in VP1 of CV-A6 showed that the nucleotide sequences of the amino acid were a remarkably conserved loop prediction compatible with neutralizing linear epitopes. Therefore, this strain represents a potential candidate for vaccine development and antiviral studies.

Clinico-epidemiological presentations and management of Nipah virus infection during the outbreak in Kozhikode district, Kerala state, India 2023.

India experienced its sixth Nipah virus (NiV) outbreak in September 2023 in the Kozhikode district of Kerala state. The NiV is primarily transmitted by spillover events from infected bats followed by human-to-human transmission. The clinical specimens were screened using real-time RT-PCR, and positive specimens were further characterized using next-generation sequencing. We describe here an in-depth clinical presentation and management of NiV-confirmed cases and outbreak containment activities. The current outbreak reported a total of six cases with two deaths, with a case fatality ratio of 33.33%. The cases had a mixed presentation of acute respiratory distress syndrome and encephalitis syndrome. Fever was a persistent presentation in all the cases. The Nipah viral RNA was detected in clinical specimens until the post-onset day of illness (POD) 14, with viral load in the range of 1.7-3.3 × 104 viral RNA copies/mL. The genomic analysis showed that the sequences from the current outbreak clustered into the Indian clade similar to the 2018 and 2019 outbreaks. This study highlights the vigilance of the health system to detect and effectively manage the clustering of cases with clinical presentations similar to NiV, which led to early detection and containment activities.

Neutralizing antibody responses to SARS-CoV-2 Omicron variants: Post six months following two-dose & three-dose vaccination of ChAdOx1 nCoV-19 or BBV152.

BACKGROUND OBJECTIVES: The Omicron sub-lineages are known to have higher infectivity, immune escape and lower virulence. During December 2022 - January 2023 and March - April 2023, India witnessed increased SARS-CoV-2 infections, mostly due to newer Omicron sub-lineages. With this unprecedented rise in cases, we assessed the neutralization potential of individuals vaccinated with ChAdOx1 nCoV (Covishield) and BBV152 (Covaxin) against emerging Omicron sub-lineages. METHODS: Neutralizing antibody responses were measured in the sera collected from individuals six months post-two doses (n=88) of Covishield (n=44) or Covaxin (n=44) and post-three doses (n=102) of Covishield (n=46) or Covaxin (n=56) booster dose against prototype B.1 strain, lineages of Omicron; XBB.1, BQ.1, BA.5.2 and BF.7. RESULTS: The sera of individuals collected six months after the two-dose and the three-dose demonstrated neutralizing activity against all variants. The neutralizing antibody (NAbs) level was highest against the prototype B.1 strain, followed by BA5.2 (5-6 fold lower), BF.7 (11-12 fold lower), BQ.1 (12 fold lower) and XBB.1 (18-22 fold lower). INTERPRETATION CONCLUSIONS: Persistence of NAb responses was comparable in individuals with two- and three-dose groups post six months of vaccination. Among the Omicron sub-variants, XBB.1 showed marked neutralization escape, thus pointing towards an eventual immune escape, which may cause more infections. Further, the correlation of study data with complete clinical profile of the participants along with observations for cell-mediated immunity may provide a clear picture for the sustained protection due to three-dose vaccination as well as hybrid immunity against the newer variants.

Surveillance of Nipah virus in Pteropus medius of Kerala state, India, 2023.

Introduction: Since 2018, the Indian state of Kerala has reported four Nipah virus (NiV) disease outbreaks, raising concerns about NiV spillover from bats to the human population. Considering this, a cross-sectional study was undertaken in the Pteropus medius bat population around the Nipah virus-affected regions of Kozhikode, Kerala, India, during February, July, and September 2023. Methods: Throat swabs, rectal swabs, and organ samples were collected from bats to test for NiV using the real-time reverse transcriptase polymerase chain reaction (RT-PCR), while serum samples were screened for anti-Nipah IgG antibodies through ELISA. Results: An overall seroprevalence of 20.9% was observed in 272 P. medius bats tested. The throat and rectal swab samples of 321 bats were negative for NiV RNA. However, 4 of 44 P. medius bats tested positive for NiV in their liver/spleen samples. The partial N gene retrieved showed more than 99% similarity with the earlier reported NiV genome from Kerala state, India. Discussion: The findings of the study caution that there is a spillover risk in the region and necessary precautions should be taken.

Cellular Immune Responses Against γ-Inactivated Antigen in the Recovered Cases of Kyasanur Forest Disease.

Kyasanur Forest Disease Virus (KFDV) is a tick-borne flavivirus that causes life-threatening hemorrhagic fever in humans with case fatality rates of 3-5%. Relatively little is known about the mechanism of its pathogenesis or host immune responses to KFDV infection. Here, we investigated KFDV-specific cellular immune responses in the recovered cases of Kyasanur Forest Disease (KFD). Peripheral blood mononuclear cells of the recovered KFD cases and healthy controls were exposed to γ-inactivated KFDV antigen ex vivo. The proliferation index was determined using an enzyme-linked immunosorbent assay-based lymphoproliferative assay. The frequencies of CD4+ and CD8+ T cells expressing intracellular interferon (IFN)-γ in response to stimulation with γ-inactivated KFDV antigen were determined using flow cytometry. A significant increase in lymphoproliferation and a high frequency of CD4+ and CD8+ T cells secreting IFN-γ against γ-inactivated KFDV antigen were found in the recovered KFD group compared to the healthy control group. In conclusion, the study indicated the generation of cellular immune responses in individuals who recovered from KFD and can be used as indicators of cellular immunity in KFD vaccine studies.

Clinical, epidemiological, and molecular investigation of Kyasanur forest disease from Karnataka state, India during 2018-2019.

BACKGROUND: In this study, we carried out an investigation of Kyasanur Forest Disease (KFD) suspected human cases reported in Karnataka state, India from December 2018 to June 2019. METHODS: The clinical samples of KFD suspected cases (n = 1955) from 14 districts of Karnataka were tested for KFD using real-time RT-PCR and IgM ELISA. Further, the KFD-negative samples were tested for IgM antibodies against dengue and chikungunya viruses. Monkey samples (n = 276) and tick pools (n = 11582) were also screened using real-time RT-PCR. KFD-positive samples were further analysed using next-generation sequencing along with clinico-epidemiological analysis. RESULTS: Of all, 173 (8.8%) cases tested positive for KFD either by real-time RT-PCR (n = 124), IgM ELISA (n = 53) or both tests (n = 4) from seven districts. Among KFD-negative cases, IgM antibody positivity was observed for dengue (2.6%), chikungunya (5.8%), dengue and chikungunya coinfection (3.7%). KFD cases peaked in January 2019 with fever, conjunctivitis, and myalgia as the predominant symptoms and a mortality of 4.6%. Among confirmed cases, 41% received a single dose and 20% received two doses of the KFD vaccine. Of the seven districts with KFDV positivity, Shivamogga and Hassan districts reported KFD viral RNA positivity in humans, monkeys, and ticks. Sequencing analysis of 2019 cases demonstrated a difference of less than 1.5% amino acid compared to prototype KFDV. CONCLUSION: Although the KFD has been endemic in many districts of Karnataka state, our study confirms the presence of KFDV for the first time in two new districts, i.e. Hassan and Mysore. A comparative analysis of KFDV infection among the KFD-vaccinated and non-vaccinated populations demonstrated an insignificant difference.

Cost estimation of COVID Kavach test developed in 2020: A National Reference Laboratory initiative during early phase of Indian pandemic mitigation.

BACKGROUND OBJECTIVES: A new indigenously developed technology, coronavirus disease (COVID) Kavach, an IgG immunoglobulin-based enzyme-linked immunosorbent assay (ELISA) kit, was developed in 2020 by the Indian Council of Medical Research-National Institute of Virology (ICMR-NIV), Pune, India. The primary objective of this study was to determine the total cost of development of COVID Kavach IgG ELISA and estimate the unit cost (UC) as well. METHODS: The total development cost (TDC) of COVID Kavach and its UC during the early phase of pandemic mitigation were estimated through a micro-costing approach from provider's perspective. An activity-based bottom-up costing approach was used to facilitate data collection from all resources, and analysis was performed using Microsoft Excel version 2016. The micro-costing data were utilized to interpret the breakdown of cost across all inputs and different levels of activity. RESULTS: The TDC of COVID Kavach was estimated to be JOURNAL/ijmer/04.03/02223309-202310000-00007/363FF04/v/2023-11-25T134903Z/r/image-tiff 2,884,032 (US$ 38,265). The UC of providing test results for exposure to severe acute respiratory syndrome corona virus-2 (SARS-CoV-2) was estimated to be JOURNAL/ijmer/04.03/02223309-202310000-00007/363FF04/v/2023-11-25T134903Z/r/image-tiff 300 (US$ 4) during July 2020. The capital and recurrent cost were incurred around 5-10 per cent and 90-95 per cent, respectively, in both the development and UC of COVID Kavach. The major portion of funds (70-80%) was utilized for procurement of laboratory consumables, followed by human resources (8-12%) in the development as well as for UC of COVID Kavach. INTERPRETATION CONCLUSIONS: The estimates from this study can be useful for conducting economic evaluations, which will help in deciding upon the subsidy in government health facilities. The data may be useful to set up laboratory facilities analogous to the National Reference Laboratory located at the ICMR-NIV, Pune and for allotting sufficient budget to develop such assays in government-funded laboratories.

Whole-genome sequencing and phylogenetic analysis of coxsackievirus-A16 strains causing hand, foot and mouth disease (HFMD) in India.

Hand, foot and mouth disease (HFMD) is a common childhood infectious disease, caused by enteroviruses (EVs), which can present with typical or atypical lesions. The illness is self-limiting, but it can also have serious complications. Since 1997, HFMD infections have become endemic and have increased to epidemic proportions across the Asia Pacific region, including India. Coxsackievirus-A16 (CV-A16) outbreaks occurred in India from 2005 onwards, although the clinical symptoms were noticeably different during this period. Understanding the population dynamics of enteroviruses that cause HFMD is crucial in the post-polio era because one of the circulating strain may replace another as the dominant strain. The aim of this study is to describe the genetic features of the CV-A16 strains isolated from hand, foot and mouth disease (HFMD) patients in India. Reverse transcription PCR (RT-PCR) and cell-culture-based isolation of CV-A16 was done from the 55 clinical samples. The entire genome of the CV-A16 isolate was performed from the seven isolates. After the sequences were analysed, a phylogenetic tree was created using bioinformatics tools. The total genomic length obtained was 7411 base pairs (bp). Nucleotide similarity across various regions, including 5'UTR, P1, P2 and 3'UTR, ranged from 87.0-97.9 %, 77.0-95.4 %, 80.3-96.9 %, and 77.9-96.2 %, respectively. Correspondingly, similarities in the VP1 region's nucleotide and amino acid sequences were 91.4-96.4 % and 99.3-99.7 %, respectively. Phylogenetic analysis highlighted that CV-A16 strains identified in Pune, Maharashtra, were grouped within the same cluster. The analysed CV-A16 isolates in this study aligned with subgenotype B1c. These findings have far-reaching implications for the surveillance, prevention and management of HFMD and CV-A16. Monitoring the dynamics of CV-A16 strains, informed by the genetic characteristics identified here, will significantly impact strategies aimed at tackling HFMD and its associated public health challenges.

Broadly Reactive SARS-CoV-2-Specific T-Cell Response and Participation of Memory B and T Cells in Patients with Omicron COVID-19 Infection.

January 2022 onward, India witnessed a sudden increase in Omicron COVID-19 infections, having a mild course that prompted us to identify the key host factors/immune molecules modulating disease course/outcomes. The current study evaluated the percentages of lymphocyte subsets by flowcytometry, SARS-CoV-2 specific T-cell immune response by ELISPOT, estimation of plasma cytokine/chemokine levels on a Bio-plex Multiplex Immunoassay System and anti-SARS-CoV-2 IgG levels by enzyme-linked immunosorbent assay in 19 mild Omicron infected patients, 45 mild SARS-CoV-2 (2020) patients and 36 uninfected controls from India. Natural killer cells, B and memory B cells were high in vaccinated and total Omicron-infected patients groups compared to the mild SARS-CoV-2 (2020) patient group, while CD8+ T cells were high in total Omicron-infected patients group compared to the uninfected control group (p < 0.05 each). Omicron-infected patients had T-cell response against SARS-CoV-2 whole virus, S1 proteins (wild type and delta variant) in 10 out of 17 (59%), 10 out of 17 (59%), and 8 out of 17 (47%), respectively. The current study of Omicron-infected patients elucidates broadly reactive antibody, T-cell response, and participation of memory B and T cells induced by vaccination/natural infection. The limited effect of Omicron's mutations on T-cell response is suggestive of protection from severity. Pro-inflammatory IL-6, IFN-γ, chemokines CCL-2, CCL-3, CCL-4, CCL-5, and IL-8 as potential biomarkers of Omicron infection may have future diagnostic importance. The cellular immune response data in Omicron-infected patients with parental Omicron lineage could serve as a starting point to define the readouts of protective immunity against circulating Omicron subvariants.

First detection of Varicella Zoster Virus clade 9 cases in India during mpox surveillance.

BACKGROUND: The multi-country mpox outbreak across the globe has led to the systematic surveillance of mpox cases in India. During the surveillance of mpox, we encountered cases of Varicella Zoster Virus (VZV) in suspected mpox cases amongst children & adults. This study focused on the genomic characterization of VZV in India. METHODS: A total of 331 mpox suspected cases were tested for VZV through real-time PCR, and the positive samples were subjected to next-generation sequencing to retrieve the whole genome of VZV using CLC genomics software. Phylogenetic analysis has been done in MEGA 11.0 software to identify circulating clades. RESULT: Of the 331 suspected cases, 28 cases with vesicular rashes were found to be positive for VZV. The maximum genome could be retrieved from the clinical specimens of 16 cases with coverage greater than 98% when mapped with reference strain Dumas (NC 001348). The phylogenetic analyses of these sequences determined the circulation of clades 1, 5, and 9 in India. Further, the sequence analysis demonstrated non-synonymous single nucleotide polymorphism (SNPs) among specific ORF of VZV including ORF 14, ORF 22, ORF 36, ORF 37 and ORF 51. Although clade 1 and 5 has been reported earlier, the circulation of clade 9 of VZV has been determined for the first time in India. CONCLUSION: Although the circulation of different clades of VZV was reported from India, the presence of clade 9 was detected for the first time during the mpox surveillance.

Comparative pathogenicity of BA.2.12, BA.5.2 and XBB.1 with the Delta variant in Syrian hamsters.

Omicron variant is evolving into numerous sub variants with time and the information on the characteristics of these newly evolving variants are scant. Here we performed a pathogenicity evaluation of Omicron sub variants BA.2.12, BA.5.2 and XBB.1 against the Delta variant in 6-8-week-old Syrian hamster model. Body weight change, viral load in respiratory organs by real time RT-PCR/titration, cytokine mRNA quantification and histopathological evaluation of the lungs were performed. The intranasal infection of the BA.2.12, BA.5.2 and XBB.1 variants in hamster model resulted in body weight loss/reduced weight gain, inflammatory cytokine response and interstitial pneumonia with lesser severity compared to the Delta variant infection. Among the variants studied, BA.2.12 and XBB.1 showed lesser viral shedding through the upper respiratory tract, whereas the BA.5.2 showed comparable viral RNA shedding as that of the Delta variant. The study shows that the Omicron BA.2 sub variants may show difference in disease severity and transmissibility amongst each other whereas the overall disease severity of the Omicron sub variants studied were less compared to the Delta variant. The evolving Omicron sub variants and recombinants should be monitored for their properties.

Genomic characterization, transcriptome analysis, and pathogenicity of the Nipah virus (Indian isolate).

Nipah virus (NiV) is a high-risk pathogen which can cause fatal infections in humans. The Indian isolate from the 2018 outbreak in the Kerala state of India showed ~ 4% nucleotide and amino acid difference in comparison to the Bangladesh strains of NiV and the substitutions observed were mostly not present in the region of any functional significance except for the phosphoprotein gene. The differential expression of viral genes was observed following infection in Vero (ATCC® CCL-81™) and BHK-21 cells. Intraperitoneal infection in the 10-12-week-old, Syrian hamster model induced dose dependant multisystemic disease characterized by prominent vascular lesions in lungs, brain, kidney and extra vascular lesions in brain and lungs. Congestion, haemorrhages, inflammatory cell infiltration, thrombosis and rarely endothelial syncitial cell formation were seen in the blood vessels. Intranasal infection resulted in respiratory tract infection characterised by pneumonia. The model showed disease characteristics resembling the human NiV infection except that of myocarditis similar to that reported by NiV-Malaysia and NiV-Bangladesh isolates in hamster model. The variation observed in the genome of the Indian isolate at the amino acid levels should be explored further for any functional significance.

Host immune responses in aged rhesus macaques against BBV152, an inactivated SARS-CoV-2 vaccine, and cross-neutralization with beta and delta variants.

The magnitude and duration of immune response to COVID-19 vaccination in older adults are known to be adversely affected due to immunosenescence and inflammaging. The threat of emerging variants warrants studies on immune response in older adults to primary vaccination and booster doses so as to understand the effectiveness of vaccines in countering the threat of emerging variants. Non-human primates (NHPs) are ideal translational models, as the immunological responses in NHPs are similar to those in humans, so it enables us to understand host immune responses to the vaccine. We initially studied humoral immune responses in aged rhesus macaques employing a three-dose regimen of BBV152, an inactivated SARS-CoV-2 vaccine. Initially, the study investigated whether the third dose enhances the neutralizing antibody (Nab) titer against the homologous virus strain (B.1) and variants of concern (Beta and Delta variants) in aged rhesus macaques immunized with BBV152, adjuvanted with Algel/Algel-IMDG (imidazoquinoline). Later, we also attempted to understand cellular immunity in terms of lymphoproliferation against γ-inactivated SARS-CoV-2 B.1 and delta in naïve and vaccinated rhesus macaques after a year of the third dose. Following the three-dose regimen with 6 µg of BBV152 with Algel-IMDG, animals had increased Nab responses across all SARS-CoV-2 variants studied, which suggested the importance of booster dose for the enhanced immune response against SARS-CoV-2-circulating variants. The study also revealed the pronounced cellular immunity against B.1 and delta variants of SARS-CoV-2 in the aged rhesus macaques even after a year of vaccination.

Molecular characterization & recombination analysis of complete enterovirus-88 isolated from acute flaccid paralysis cases in India.

Background & objectives: Focus on non-polio enteroviruses (NPEVs) causing acute flaccid paralysis (AFP) due to myelitis has increased with the containment of the poliovirus. Enterovirus-B88 (EV-B88) has been associated with the AFP cases in Bangladesh, Ghana, South Africa, Thailand and India. In India, EV-B88 infection was linked to AFP a decade ago; however, to date, no complete genome has been made available. In this study, the complete genome sequence of EV-B88 was identified and reported from two different States (Bihar and Uttar Pradesh) in India using the next-generation sequencing technique. Methods: Virus isolation was performed on the three AFP suspected cases as per the WHO-recommended protocol. Samples showing cytopathic effects in the human Rhabdocarcinoma were labelled as NPEVs. Next-generation sequencing was performed on these NPEVs to identify the aetiological agent. The contiguous sequences (contigs) generated were identified, and reference-based mapping was performed. Results: EV-B88 sequences retrieved in our study were found to be 83 per cent similar to the EV-B88 isolate from Bangladesh in 2001 (strain: BAN01-10398; Accession number: AY843306.1). Recombination analyses of these samples demonstrate recombination events with sequences from echovirus-18 and echovirus-30. Interpretation & conclusions: Recombination events in the EV-B serotypes are known, and this work reconfirms the same for EV-B88 isolates also. This study is a step in increasing the awareness about EV-B88 in India and emphasizes future studies to be conducted in the identification of other types of EV present in India.

Needle-free injection system delivery of ZyCoV-D DNA vaccine demonstrated improved immunogenicity and protective efficacy in rhesus macaques against SARS-CoV-2.

The apprehension of needles related to injection site pain, risk of transmitting bloodborne pathogens, and effective mass immunization have led to the development of a needle-free injection system (NFIS). Here, we evaluated the efficacy of the NFIS and needle injection system (NIS) for the delivery and immunogenicity of DNA vaccine candidate ZyCoV-D in rhesus macaques against SARS-CoV-2 infection. Briefly, 20 rhesus macaques were divided into 5 groups (4 animals each), that is, I (1 mg dose by NIS), II (2 mg dose by NIS), III (1 mg dose by NFIS), IV (2 mg dose by NFIS) and V (phosphate-buffer saline [PBS]). The macaques were immunized with the vaccine candidates/PBS intradermally on Days 0, 28, and 56. Subsequently, the animals were challenged with live SARS-CoV-2 after 15 weeks of the first immunization. Blood, nasal swab, throat swab, and bronchoalveolar lavage fluid specimens were collected on 0, 1, 3, 5, and 7 days post infection from each animal to determine immune response and viral clearance. Among all the five groups, 2 mg dose by NFIS elicited significant titers of IgG and neutralizing antibody after immunization with enhancement in their titers postvirus challenge. Besides this, it also induced increased lymphocyte proliferation and cytokine response. The minimal viral load post-SARS-CoV-2 challenge and significant immune response in the immunized animals demonstrated the efficiency of NFIS in delivering 2 mg ZyCoV-D vaccine candidate.