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.

Delta variant SARS-CoV-2 infections in pediatric cases during the second wave in India.

BACKGROUND: During October 2020, Delta variant was detected for the first time in India and rampantly spread across the globe. It also led to second wave of pandemic in India which affected millions of people. However, there is limited information pertaining to the SARS-CoV-2 strain infecting the children in India. METHODS: Here, we assessed the SARS-CoV-2 lineages circulating in the pediatric population of India during the second wave of the pandemic. Clinical and demographic details linked with the nasopharyngeal/oropharyngeal swabs (NPS/OPS) collected from SARS-CoV-2 cases (n = 583) aged 0-18 year and tested positive by real-time RT-PCR were retrieved from March to June 2021. RESULTS: Symptoms were reported among 37.2% of patients and 14.8% reported to be hospitalized. The E gene CT value had significant statistical difference at the point of sample collection when compared to that observed in the sequencing laboratory. Out of these 512 sequences 372 were VOCs, 51 were VOIs. Most common lineages observed were Delta, followed by Kappa, Alpha and B.1.36, seen in 65.82%, 9.96%, 6.83% and 4.68%, respectively in the study population. CONCLUSION: Overall, it was observed that Delta strain was the leading cause of SARS-CoV-2 infection in Indian children during the second wave of the pandemic. We emphasize on the need of continuous genomic surveillance in SARS-CoV-2 infection even amongst children.

Zika a Vector Borne Disease Detected in Newer States of India Amidst the COVID-19 Pandemic.

Background: During the second wave of the COVID-19 pandemic, outbreaks of Zika were reported from Kerala, Uttar Pradesh, and Maharashtra, India in 2021. The Dengue and Chikungunya negative samples were retrospectively screened to determine the presence of the Zika virus from different geographical regions of India. Methods: During May to October 2021, the clinical samples of 1475 patients, across 13 states and a union territory of India were screened and re-tested for Dengue, Chikungunya and Zika by CDC Trioplex Real time RT-PCR. The Zika rRTPCR positive samples were further screened with anti-Zika IgM and Plaque Reduction Neutralization Test. Next generation sequencing was used for further molecular characterization. Results: The positivity was observed for Zika (67), Dengue (121), and Chikungunya (10) amongst screened cases. The co-infections of Dengue/Chikungunya, Dengue/Zika, and Dengue/Chikungunya/Zika were also observed. All Zika cases were symptomatic with fever (84%) and rash (78%) as major presenting symptoms. Of them, four patients had respiratory distress, one presented with seizures, and one with suspected microcephaly at birth. The Asian Lineage of Zika and all four serotypes of Dengue were found in circulation. Conclusion: Our study indicates the spread of the Zika virus to several states of India and an urgent need to strengthen its surveillance.

SARS-CoV-2 Kappa Variant Shows Pathogenicity in a Syrian Hamster Model.

Objectives: The emergence of SARS-CoV-2 lineage B.1.617 variants in India has been associated with a surge in the number of daily infections. We investigated the pathogenic potential of Kappa (B.1.617.1) variant in Syrian golden hamsters. Methods: Two groups of Syrian golden hamsters (18 each) were inoculated intranasally with SARS-CoV-2 isolates, B.1 (D614G) and Kappa variant, respectively. The animals were monitored daily for the clinical signs and body weight. Throat swab, nasal wash, and organ samples (lungs, nasal turbinate, trachea) were collected and screened using SARS-CoV-2-specific RT-qPCR. Histopathologic evaluation of the lung samples was performed. Results: The hamsters infected with the Kappa variant demonstrated increased body weight loss compared to the B.1 lineage isolate. The highest viral RNA load was observed in the nasal turbinate and lung specimens of animals infected with both variants. A significantly higher sgRNA load was observed in the nasal swabs (7 DPI), trachea (3 DPI), and lungs (3 DPI) of hamsters infected with the Kappa variant. Neutralizing antibody response generated in the B.1 lineage-infected hamster sera were comparable against both B.1 and Kappa variant in contrast to Kappa variant-infected hamsters, which showed lower titers against B.1 lineage isolate. Gross and microscopic evaluation of the lung specimens showed severe lung lesions in hamsters infected with Kappa variant compared to B.1. Conclusions: The study demonstrates pathogenicity of Kappa variant in hamsters evident with reduced body weight, high viral RNA load in lungs, and pronounced lung lesions. Both Kappa variant- and B.1-infected hamsters produced neutralizing antibodies against both variants studied.

Pathogenicity of SARS-CoV-2 Omicron (R346K) variant in Syrian hamsters and its cross-neutralization with different variants of concern.

BACKGROUND: SARS-CoV-2 Omicron variant is rampantly spreading across the globe. We assessed the pathogenicity and immune response generated by BA.1.1 sub-lineage of SARS-CoV-2 [Omicron (R346K) variant] in 5 to 6-week old Syrian hamsters and compared the observations with that of Delta variant infection. METHODS: Virus shedding, organ viral load, lung disease and immune response generated in hamsters were sequentially assessed. FINDINGS: The disease characteristics of the Omicron (R346K) variant were found to be similar to that of the Delta variant infection in hamsters like viral replication in the respiratory tract and interstitial pneumonia. The Omicron (R346K) infected hamsters demonstrated lesser body weight reduction and viral RNA load in the throat swab and nasal wash samples in comparison to the Delta variant infection. The viral load in the lungs and nasal turbinate samples and the lung disease severity of the Omicron (R346K) infected hamsters were found comparable with that of the Delta variant infected hamsters. Neutralizing antibody response against Omicron (R346K) variant was detected from day 5 and the cross-neutralization titre of the sera against other variants showed severe reduction ie., 7 fold reduction against Alpha and no titers against B.1, Beta and Delta. INTERPRETATION: This preliminary data shows that Omicron (R346K) variant infection can produce moderate to severe lung disease similar to that of the Delta variant and the neutralizing antibodies produced in response to Omicron (R346K) variant infection shows poor neutralizing ability against other co-circulating SARS-CoV-2 variants like Delta which necessitates caution as it may lead to increased cases of reinfection. FUNDING: This study was supported by Indian Council of Medical Research as an intramural grant (COVID-19) to ICMR-National Institute of Virology, Pune.

First case of Zika virus infection during an outbreak of chikungunya in a rural region of Maharashtra state, India.

BACKGROUND: In July 2021, an outbreak of chikungunya virus (CHIKV) was reported in a rural region of Maharashtra state, India. METHODS: Serum samples of symptomatic cases (n=33) were screened for dengue virus (DENV), CHIKV and Zika virus (ZIKV) by molecular and serological assays. RESULTS: The first case of ZIKV infection from Maharashtra was detected and confirmed by molecular and serological assays. Complete genome sequencing revealed that the ZIKV sequence belongs to the Asian genotype and had a closer homology with pre-epidemic strains present before 2007. CONCLUSIONS: ZIKV surveillance needs to be strengthened in the regions experiencing dengue and chikungunya outbreaks.

Isolation and Genomic Characterization of SARS-CoV-2 Omicron Variant Obtained from Human Clinical Specimens.

Due to the failure of virus isolation of the Omicron variant in Vero CCL-81 from the clinical specimens of COVID-19 cases, an initial in vivo and subsequent in vitro approach was utilized for the isolation of the virus. A total of 74 oropharyngeal/nasopharyngeal specimens were collected from SARS-CoV-2 positive international travellers and a contact case at Delhi and Mumbai, India. All the specimens were sequenced using next-generation sequencing and simultaneously inoculated onto Vero CCL-81 cells for virus isolation. Subsequently, two omicron positive specimens were inoculated into Syrian hamsters for two passages. The initial passage of the positive hamster specimens was inoculated onto Vero CCL-81 cells. The clinical specimens, hamster specimens, and Vero CCL-81 passages were sequenced to assess the mutational changes in different host species. The replication of the Omicron variant in hamsters was confirmed with the presence of a high viral load in nasal turbinate and lung specimens of both passages. The successful isolation of the virus from hamster specimens with Vero CCL-81 was observed with cytopathic effect in infected cells and high viral load in the cell suspension. The genome analysis revealed the presence of L212C mutation, Tyrosine 69 deletion, and C25000T nucleotide change in spike gene of hamster passage sequences and an absence of V17I mutation in E gene in hamster passage sequences, unlike human clinical specimen and Vero CCL-81 passages. No change was observed in the furin cleavage site in any of the specimen sequences, suggesting intact pathogenicity of the virus isolate. Our data demonstrated successful isolation of the Omicron variant with the in vivo method first followed by in vitro method. The virus isolate could be used in the future to explore different aspects of the Omicron variant.

Nipah Virus Outbreak in Kerala State, India Amidst of COVID-19 Pandemic.

We report here a Nipah virus (NiV) outbreak in Kozhikode district of Kerala state, India, which had caused fatal encephalitis in a 12-year-old boy and the outbreak response, which led to the successful containment of the disease and the related investigations. Quantitative real-time reverse transcription (RT)-PCR, ELISA-based antibody detection, and whole genome sequencing (WGS) were performed to confirm the NiV infection. Contacts of the index case were traced and isolated based on risk categorization. Bats from the areas near the epicenter of the outbreak were sampled for throat swabs, rectal swabs, and blood samples for NiV screening by real-time RT-PCR and anti-NiV bat immunoglobulin G (IgG) ELISA. A plaque reduction neutralization test was performed for the detection of neutralizing antibodies. Nipah viral RNA could be detected from blood, bronchial wash, endotracheal (ET) secretion, and cerebrospinal fluid (CSF) and anti-NiV immunoglobulin M (IgM) antibodies from the serum sample of the index case. Rapid establishment of an onsite NiV diagnostic facility and contact tracing helped in quick containment of the outbreak. NiV sequences retrieved from the clinical specimen of the index case formed a sub-cluster with the earlier reported Nipah I genotype sequences from India with more than 95% similarity. Anti-NiV IgG positivity could be detected in 21% of Pteropus medius (P. medius) and 37.73% of Rousettus leschenaultia (R. leschenaultia). Neutralizing antibodies against NiV could be detected in P. medius. Stringent surveillance and awareness campaigns need to be implemented in the area to reduce human-bat interactions and minimize spillover events, which can lead to sporadic outbreaks of NiV.

Protective Immunity of the Primary SARS-CoV-2 Infection Reduces Disease Severity Post Re-Infection with Delta Variants in Syrian Hamsters.

The Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) Delta variant has evolved to become the dominant SARS-CoV-2 lineage with multiple sub-lineages and there are also reports of re-infections caused by this variant. We studied the disease characteristics induced by the Delta AY.1 variant and compared it with the Delta and B.1 variants in Syrian hamsters. We also assessed the potential of re-infection by these variants in Coronavirus disease 2019 recovered hamsters 3 months after initial infection. The variants produced disease characterized by high viral load in the respiratory tract and interstitial pneumonia. The Delta AY.1 variant produced mild disease in the hamster model and did not show any evidence of neutralization resistance due to the presence of the K417N mutation, as speculated. Re-infection with a high virus dose of the Delta and B.1 variants 3 months after B.1 variant infection resulted in reduced virus shedding, disease severity and increased neutralizing antibody levels in the re-infected hamsters. The reduction in viral load and lung disease after re-infection with the Delta AY.1 variant was not marked. Upper respiratory tract viral RNA loads remained similar after re-infection in all the groups. The present findings show that prior infection could not produce sterilizing immunity but that it can broaden the neutralizing response and reduce disease severity in case of reinfection.

Detection and isolation of SARS-CoV-2 Eta variant from the international travelers and local residents of India.

International travel has been the major source for the rapid spread of new SARS-CoV-2 variants across the globe. During SARS-CoV-2 genomic surveillance, a total of 212 SARS-CoV-2 positive clinical specimens were sequenced using next-generation sequencing. A complete SARS-CoV-2 genome could be retrieved from 90 clinical specimens. Of them, 14 sequences belonged to the Eta variant from clinical specimens of international travelers (n = 12) and local residents (n = 2) of India, and 76 belonged to other SARS-CoV-2 variants. Of all the Eta-positive specimens, the virus isolates were obtained from the clinical specimens of six international travelers. Many variants of interest have been found to cause substantial community transmission or cluster infections. The detection of this variant with lethal E484K mutation across the globe and India necessitates persistent genomic surveillance of the SARS-CoV-2 variants, which would aid in taking preventive action.

Isolation and characterization of SARS-CoV-2 Beta variant from UAE travelers.

BACKGROUND: The emergence of SARS-CoV-2 variants in places where the virus is uncontained poses a global threat from the perspective of public health and vaccine efficacy. Travel has been important factor for the easy spread of SARS-CoV-2 variants worldwide. India has also observed the importation of SARS-CoV-2 variants through international travelers. METHODS: In this study, we have collected the oropharyngeal and nasopharyngeal swab specimens from 58 individuals with travel history from United Arab Emirates (UAE), East, West and South Africa, Qatar, Ukraine and Saudi Arabia arrived in India during February-March 2021. The clinical specimens were initially screened for SARS-CoV-2 using Real time RT-PCR. All the specimens were inoculated on to Vero CCL-81 cells for virus isolation. The viral isolates were further sequenced using Next-Generation Sequencing. RESULTS: All 58 cases were tested positive for SARS-CoV-2 using Real time RT-PCR. Four specimens showed progressive infectivity with fusion of the infected cells with neighboring cells leading to large mass of cells. Replication competent virus was confirmed from culture supernatant of the passage 2 using Real time RT-PCR. Two plaque purified SARS-CoV-2 isolates demonstrated high viral RNA load of 3.8-7.5 × 1011 and 1.1-1.6 × 1011 at passage 4 and 5 respectively. Nucleotide variations along with amino acid changes were also observed among these two isolates at passage 2-5. All four cases were male with no symptoms and co-morbidity. The sequence analysis has shown two different clusters, first cluster with nucleotide deletions in the ORF1ab and the spike, while second cluster with deletions in spike region. The viral isolates demonstrated 99.88-99.96% nucleotide identity with the representative sequences of Beta variant (B.1.351). CONCLUSION: These findings suggest easier transmission of SARS-CoV-2 variants with human mobility through international travel. The isolated Beta variant would be useful to determine the protective efficacy of the currently available and upcoming COVID-19 vaccines in India.