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.

Clinical presentation, viral kinetics, and management of human monkeypox cases from New Delhi, India 2022.

We describe the clinical and demographic characteristics, virological follow-up, and management of five confirmed monkeypox cases from New Delhi, India without any international travel history. The viral load kinetics and viral clearance were estimated in oropharyngeal swabs (OPS), nasopharyngeal swabs (NPS), EDTA blood, serum, urine, and various lesion specimens on every fourth day of follow-up ranging from 5 to 24 post onset day (POD) of illness. All five cases presented with mild to moderate-grade intermittent fever, myalgia, and lesions on the genitals, groins, lower limb, trunk, and upper limb. Four cases had non-tender firm lymphadenopathy. No secondary complications or sexually transmitted infections were recorded in these cases except for the presence of viral hepatitis B infection marker hepatitis B virus surface antigen (HBsAg) in one case. All the cases were mild and had a good recovery. A higher viral load was detected in lesion fluid (POD 9), followed by lesion roof (POD 9), urine (POD 5), lesion base (POD 5), and OPS/NPS (POD 5). The monkeypox virus (MPXV) DNA was detected in clinical samples from 5th to 24th POD. These monkeypox cases without international travel history suggest the underdiagnosed monkeypox infection in the community. This emphasizes the need for active surveillance of MPXV in the high-risk population such as men having sex with men and female sex workers.

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.

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.

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.

A case control investigation of COVID-19 associated mucormycosis in India.

BACKGROUND: Increased occurrence of mucormycosis during the second wave of COVID-19 pandemic in early 2021 in India prompted us to undertake a multi-site case-control investigation. The objectives were to examine the monthly trend of COVID-19 Associated Mucormycosis (CAM) cases among in-patients and to identify factors associated with development of CAM. METHODS: Eleven study sites were involved across India; archived records since 1st January 2021 till 30th September 2021 were used for trend analysis. The cases and controls were enrolled during 15th June 2021 to 30th September 2021. Data were collected using a semi-structured questionnaire. Among 1211 enrolled participants, 336 were CAM cases and 875 were COVID-19 positive non-mucormycosis controls. RESULTS: CAM-case admissions reached their peak in May 2021 like a satellite epidemic after a month of in-patient admission peak recorded due to COVID-19. The odds of developing CAM increased with the history of working in a dusty environment (adjusted odds ratio; aOR 3.24, 95% CI 1.34, 7.82), diabetes mellitus (aOR: 31.83, 95% CI 13.96, 72.63), longer duration of hospital stay (aOR: 1.06, 95% CI 1.02, 1.11) and use of methylprednisolone (aOR: 2.71, 95% CI 1.37, 5.37) following adjustment for age, gender, occupation, education, type of houses used for living, requirement of ventilatory support and route of steroid administration. Higher proportion of CAM cases required supplemental oxygen compared to the controls; use of non-rebreather mask (NRBM) was associated as a protective factor against mucormycosis compared to face masks (aOR: 0.18, 95% CI 0.08, 0.41). Genomic sequencing of archived respiratory samples revealed similar occurrences of Delta and Delta derivates of SARS-CoV-2 infection in both cases and controls. CONCLUSIONS: Appropriate management of hyperglycemia, judicious use of steroids and use of NRBM during oxygen supplementation among COVID-19 patients have the potential to reduce the risk of occurrence of mucormycosis. Avoiding exposure to dusty environment would add to such prevention efforts.

Neutralization assays for SARS-CoV-2: Implications for assessment of protective efficacy of COVID-19 vaccines.

The WHO emergency use-listed (EUL) COVID-19 vaccines were developed against early strains of SARS-CoV-2. With the emergence of SARS-CoV-2 variants of concern (VOCs) - Alpha, Beta, Gamma, Delta and Omicron, it is necessary to assess the neutralizing activity of these vaccines against the VOCs. PubMed and preprint platforms were searched for literature on neutralizing activity of serum from WHO EUL vaccine recipients, against the VOCs, using appropriate search terms till November 30, 2021. Our search yielded 91 studies meeting the inclusion criteria. The analysis revealed a drop of 0-8.9-fold against Alpha variant, 0.3-42.4-fold against Beta variant, 0-13.8-fold against Gamma variant and 1.35-20-fold against Delta variant in neutralization titres of serum from the WHO EUL COVID-19 vaccine recipients, as compared to early SARS-CoV-2 isolates. The wide range of variability was due to differences in the choice of virus strains selected for neutralization assays (pseudovirus or live virus), timing of serum sample collection after the final dose of vaccine (day 0 to 8 months) and sample size (ranging from 5 to 470 vaccinees). The reasons for this variation have been discussed and the possible way forward to have uniformity across neutralization assays in different laboratories have been described, which will generate reliable data. Though in vitro neutralization studies are a valuable tool to estimate the performance of vaccines against the backdrop of emerging variants, the results must be interpreted with caution and corroborated with field-effectiveness studies.

Development of Nipah virus-specific IgM & IgG ELISA for screening human serum samples.

Background & objectives: Nipah virus (NiV) is a zoonotic paramyxovirus that causes fatal encephalitis in humans. Enzyme Linked Immunosorbent Assay (ELISA) is a safe, sensitive, specific, and affordable diagnostic tool that can be used during screening of large-scale epidemiological investigations. Development and evaluation of IgM and IgG ELISA for screening serum samples of NiV suspected cases would also help in planning public health interventions. Methods: An IgM capture (MAC) ELISA and an indirect IgG ELISA were developed using NiV antigen to detect IgM and IgG antibodies against NiV in human sera. The sensitivity, specificity, and cross-reactivity of the assays were evaluated using NiV IgM, IgG positive, negative human sera and measles, mumps, rubella, Crimean-Congo haemorrhagic fever, Kyasanur forest disease IgM, IgG positive sera, respectively. Results: The developed anti-NiV IgM and IgG ELISAs have shown specificity of 99.28 per cent and sensitivity of 100 per cent compared to reference test from Centers for Disease Control and Prevention, USA. Assays demonstrated negative predictive value of 100 per cent and positive predictive value as 90 and 93.94 per cent for anti-Nipah IgM ELISA and IgG ELISA respectively with test accuracy of 99.33 per cent. Interpretation & conclusions: Timely diagnosis of NiV is crucial for the management of cases, which could prevent further spread of infection in the community. IgM ELISA can be used as primary diagnostic tool followed by polymerase chain reaction. These assays have advantages of its applicability during outbreak investigations and surveillance activities at hospital or onsite laboratories with basic biosafety practices.

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

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

Detection of Nipah virus in Pteropus medius in 2019 outbreak from Ernakulam district, Kerala, India.

BACKGROUND: In June 2019, Nipah virus (NiV) infection was detected in a 21-year-old male (index case) of Ernakulum, Kerala, India. This study was undertaken to determine if NiV was in circulation in Pteropus species (spp) in those areas where the index case had visit history in 1 month. METHODS: Specialized techniques were used to trap the Pteropus medius bats (random sampling) in the vicinity of the index case area. Throat and rectal swabs samples of 141 bats along with visceral organs of 92 bats were collected to detect the presence of NiV by real-time reverse transcriptase-polymerase chain reaction (qRTPCR). Serum samples of 52 bats were tested for anti-NiV Immunoglobulin (Ig) G antibodies by Enzyme-Linked Immunosorbent Assay (ELISA). The complete genome of NiV was sequenced by next-generation sequencing (NGS) from the tissues and swab samples of bats. RESULTS: One rectal swab sample and three bats visceral organs were found positive for the NiV. Interestingly, 20.68% (12/58) of Pteropus were positive for anti-NiV IgG antibodies. NiV sequences of 18,172; 17,200 and 15,100 nucleotide bps could be retrieved from three Pteropus bats. CONCLUSION: A distinct cluster of NiV sequences, with significant net-evolutionary nucleotide divergence, was obtained, suggesting the circulation of new genotype (I-India) in South India. NiV Positivity in Pteropus spp. of bats revealed that NiV is circulating in many districts of Kerala state, and active surveillance of NiV should be immediately set up to know the hotspot area for NiV infection.

The species distribution of ticks & the prevalence of Kyasanur forest disease virus in questing nymphal ticks from Western Ghats of Kerala, South India.

Background & objectives: Kyasanur forest disease (KFD) is a zoonotic tick-borne disease across the Western Ghats of India. With the discovery of a cluster of human KFD cases in the Wayanad district of Kerala, the present study was focused on detecting KFD virus (KFDV) in tick populations. To manage this disease, it is necessary to understand the diversity of the tick species and factors influencing the distribution, abundance and prevalence of infected ticks in Wayanad district. Methods: Surveys were conducted from November 2016 to May 2018 in four forest ranges of Wayanad district. Ticks were collected by the dragging method and were identified to species level and assayed for virus detection using real-time polymerase chain reaction. Results: A total of 25,169 ticks were collected from 64 sites. Of the identified species, Haemaphysalis spinigera was the most abundant (56.64%), followed by H. turturis 9047 (35.94%), H. bispinosa 999 (3.96%), Amblyomma integrum 691 (2.74%), H. kyasanurensis (0.55%), Rhipicephalus sanguineus (0.08%), Hyalomma marginatum (0.02%), H. cuspidata (0.01%), R.microplus (0.01%) and Dermacentor auratus (0.003%). The nymphal stage was predominant from December to February having peak activity in January. A total of 572 pools were screened for the presence of KFDV, of which 21 pools were positive. The infection rates in H. spinigera and H. turturis tick were 2.62 and 1.04 per cent, respectively. Interpretation & conclusions: The circulation of KFDV was detected and its correlation with the prevalence in ticks near the fragmented forest and teak plantation areas of Wayanad district. Residents and visitors of these regions may become vulnerable to tick bites and to an increased risk of KFD as the distribution of established, infected tick populations continues to expand.

Experiences of Indian Council of Medical Research with tick-borne zoonotic infections: Kyasanur Forest disease & Crimean-Congo haemorrhagic fever in India with One Health focus.

Emergence and re-emergence of several pathogens have been witnessed by this century in the form of outbreaks, epidemics and pandemics. In India, the influencing factor that promotes dissemination of emerging and re-emerging viral infections is the biogeographical zones: a megadiverse country, characterized by varied geographical, climatic conditions and ever-changing socio-economical and geopolitical issues. These influence the movement of humans and animals and add layers of complexity for the identification and timely management of infectious diseases. This review focuses on two tick-borne infections: Crimean-Congo haemorrhagic fever (CCHF) and Kyasanur forest disease (KFD). In the last two decades, these viruses have emerged and caused outbreaks in different parts of India. KFD virus was initially identified in 1957 and was known to be endemic in Karnataka State while CCHF virus was first identified during 2010 in Gujarat State, India. These viruses have managed to emerge in new areas within the last decade. With changing epidemiology of these arboviruses, there is a probability of the emergence of these viruses from new areas in future. The investigations on these two diseases under the One Health focus involved early detection, quickly developing diagnostic tools, identifying stakeholders, capacity building by developing collaboration with major stakeholders to understand the epidemiology and geographical spread in domestic animal reservoirs and tick vectors in the affected areas, developing laboratory network, providing diagnostic reagents and biosafety and laboratory diagnosis training to the network laboratories to control these diseases.

Performance evaluation of Truenat™ Beta CoV & Truenat™ SARS-CoV-2 point-of-care assays for coronavirus disease 2019.

Background & objectives: The rapid diagnosis of coronavirus disease 2019 (COVID-19) is a significant step towards the containment of the virus. The surge of COVID-19 cases in India and across the globe necessitates a rapid and sensitive molecular assay. Rapid point-of-care (PoC) assays (Truenat Beta CoV and Truenat SARS-CoV-2 assays) for the diagnosis of COVID-19 have been developed which are expected to shorten the turnaround time of reporting of results and also can be used for field investigations of COVID-19. The objectives of the study were to validate the performance of Truenat Beta CoV and Truenat SARS-CoV-2 PoC assays for the detection of SARS-CoV-2 infected cases with reference to analytical sensitivity, precision/inter-machine variation, clinical sensitivity and clinical specificity. Methods: The rapid PoC screening and confirmatory assays were prospectively validated at the State Level Virus Research and Diagnostic Laboratory at Bangalore Medical College and Research Institute, Bengaluru, under technical supervision by the Indian Council of Medical Research-National Institute of Virology (ICMR-NIV), Pune. Real-time reverse transcription-polymerase chain reaction (rRT-PCR)was considered as the reference standard against which the rapid assays were validated for all samples tested based on analytical sensitivity, precision/inter-machine variation, clinical sensitivity and clinical specificity. Results: Truenat Beta CoV and Truenat SARS-CoV-2 assays showed concordant results when compared with the reference standard rRT-PCR. These PoC assays exhibited 100 per cent sensitivity, specificity, positive predictive value and negative predictive value. Interpretation & conclusions: Truenat Beta CoV and Truenat SARS-CoV-2 assays showed concordance with the reference standard assay and may be recommended for screening and confirmation of SARS-CoV-2 in the field settings.

Comparison of the immunogenicity & protective efficacy of various SARS-CoV-2 vaccine candidates in non-human primates.

BACKGROUND & OBJECTIVES: The COVID-19 pandemic has emerged as a global public health crisis and research groups worldwide are engaged in developing vaccine candidates to curb its transmission, with a few vaccines having progressed to advanced stages of clinical trials. The aim of this systematic review was to compare immunogenicity and protective efficacy of various SARS-CoV-2 vaccine candidates tested in non-human primate (NHP) models. METHODS: Literature on effect of SARS-CoV-2 vaccines in NHP models reported on PubMed and preprint platforms (medRxiv and bioRxiv) till October 22, 2020, was searched with the following terms: coronavirus vaccine, COVID-19 vaccine, SARS-CoV-2 vaccine, nonhuman primate, and rhesus macaque. RESULTS: Our search yielded 19 studies, which reported immune response elicited by 18 vaccine candidates in NHP. All the vaccines induced detectable neutralizing antibody (NAb) titres in the serum of vaccinated animals, with some showing effective viral clearance from various organs. The vaccinated animals also showed nil to mild histopathological changes in their lungs compared to placebo groups in the trials that performed necropsy. INTERPRETATION & CONCLUSIONS: Our findings highlighted onset of quick immunogenicity and protective efficacy of mRNA-1273, followed by Ad26.CoV2.S, NVX-CoV2373, BNT162b2, RBD and BBV152 vaccine candidates in preclinical trials as compared to the others. NHP data also showed correlation with clinical trial data available for a few vaccines. Preclinical trials of COVID-19 vaccine candidates in NHPs yielded promising results, with some candidates faring better than others.

Molecular characterization of varicella zoster virus isolated from clinical samples in India.

Background & objectives: Varicella zoster virus (VZV) strains are classified into six different clades based on the sequencing of its genome. Clades 4 and 5 are reported from India based on the single-nucleotide polymorphism (SNP). Till now, multiple clade circulations using partial sequences have been reported from India due to the lack of availability of the full VZV genome sequence. This study conducted a genome sequencing of VZV in India to identify circulating clade. Methods: Four clinical samples obtained from symptomatic patients tested positive for VZV by real-time PCR were used. These four samples were preferred to retrieve the genomic VZV sequence using the next-generation sequencing method. A reference-based assembly method was used to retrieve the genome of VZV, which was further analyzed. Results: At the least, 98 per cent of the whole-genome sequences were recovered from the four samples. The VZV sequences obtained in this study formed a separate monophyletic branch with clade 5, indicating it to be evolved from a distinct ancestor. The nucleotide-based analysis revealed 13 different SNP mutations and one multiple nucleotide variation in the VZV sequences when compared to one of the clade 5 genomes having accession number: DQ457052.1. Interpretation & conclusions: The present study described approximately 98 per cent of the genome sequence of VZV from India. The availability of these genomic sequences will lead to enrichment in the clinical genomic data set from India. The available data would help in the development of diagnostic methods along with evolutionary analysis. We hypothesize the existence of a new sub-clade that belongs to clade 5 and propose further experiments to confirm these results.

Characterization of Novel Reoviruses Wad Medani Virus (Orbivirus) and Kundal Virus (Coltivirus) Collected from Hyalomma anatolicum Ticks in India during Surveillance for Crimean Congo Hemorrhagic Fever.

In 2011, ticks were collected from livestock following an outbreak of Crimean Congo hemorrhagic fever (CCHF) in Gujarat state, India. CCHF-negative Hyalomma anatolicum tick pools were passaged for virus isolation, and two virus isolates were obtained, designated Karyana virus (KARYV) and Kundal virus (KUNDV), respectively. Traditional reverse transcription-PCR (RT-PCR) identification of known viruses was unsuccessful, but a next-generation sequencing (NGS) approach identified KARYV and KUNDV as viruses in the Reoviridae family, Orbivirus and Coltivirus genera, respectively. Viral genomes were de novo assembled, yielding 10 complete segments of KARYV and 12 nearly complete segments of KUNDV. The VP1 gene of KARYV shared a most recent common ancestor with Wad Medani virus (WMV), strain Ar495, and based on nucleotide identity we demonstrate that it is a novel WMV strain. The VP1 segment of KUNDV shares a common ancestor with Colorado tick fever virus, Eyach virus, Tai Forest reovirus, and Tarumizu tick virus from the Coltivirus genus. Based on VP1, VP6, VP7, and VP12 nucleotide and amino acid identities, KUNDV is proposed to be a new species of Coltivirus Electron microscopy supported the classification of KARYV and KUNDV as reoviruses and identified replication morphology consistent with other orbi- and coltiviruses. The identification of novel tick-borne viruses carried by the CCHF vector is an important step in the characterization of their potential role in human and animal pathogenesis.IMPORTANCE Ticks and mosquitoes, as well Culicoides, can transmit viruses in the Reoviridae family. With the help of next-generation sequencing (NGS), previously unreported reoviruses such as equine encephalosis virus, Wad Medani virus (WMV), Kammavanpettai virus (KVPTV), and, with this report, KARYV and KUNDV have been discovered and characterized in India. The isolation of KUNDV and KARYV from Hyalomma anatolicum, which is a known vector for zoonotic pathogens, such as Crimean Congo hemorrhagic fever virus, Babesia, Theileria, and Anaplasma species, identifies arboviruses with the potential to transmit to humans. Characterization of KUNDV and KARYV isolated from Hyalomma ticks is critical for the development of specific serological and molecular assays that can be used to determine the association of these viruses with disease in humans and livestock.

Experiential learnings from the Nipah virus outbreaks in Kerala towards containment of infectious public health emergencies in India.

Nipah virus (NiV) outbreak occurred in Kozhikode district, Kerala, India in 2018 with a case fatality rate of 91% (21/23). In 2019, a single case with full recovery occurred in Ernakulam district. We described the response and control measures by the Indian Council of Medical Research and Kerala State Government for the 2019 NiV outbreak. The establishment of Point of Care assays and monoclonal antibodies administration facility for early diagnosis, response and treatment, intensified contact tracing activities, bio-risk management and hospital infection control training of healthcare workers contributed to effective control and containment of NiV outbreak in Ernakulam.

Proactive preparedness for Cat Que virus: An Orthobunyavirus existing in India.

Background & objectives: The presence of Cat Que virus (CQV) in Culex mosquitoes and pigs has been reported in China and Vietnam. Due to the spread of similar species of the Culex mosquitoes in India, there is a need to understand the replication kinetics of this virus in mosquito models. As a part of preparedness and to identify the presence of this CQV in humans and swine, this study was carried out to develop diagnostic tests. Methods: Serological and molecular diagnostic assays were developed for testing the mosquito population, human and swine serum samples. In this line, RNA-dependent RNA polymerase (L), glycoprotein (M) and nucleocapsid (S) genes-based reverse transcription-polymerase chain reaction (RT-PCR) assays were developed for CQV. Real-time RT-PCR was used for screening of retrospectively collected human serum samples (n=1020) with acute febrile illness during 2014-2017. Simultaneously, an in-house anti-CQV swine and human IgG ELISAs were also developed to detect anti-CQV IgG antibody. Human serum samples (n=883) with post-onset of disease (POD) >4 days and swine serum samples (n=459) were tested for the presence of anti-CQV IgG antibodies. CQV NIV 612,045 isolate was used for susceptibility and replication kinetics experiment using three different species of mosquitoes to understand its behaviour in Indian mosquitoes. Results: All human serum samples (n=1020) screened for the presence of CQV using real-time RT-PCR were found to be negative. Anti-CQV IgG antibody positivity was recorded in two of 883 human serum samples tested. Virus susceptibility experiments indicated that three species of mosquito, namely Aedes aegypti, Culex quinquefasciatus and Cx. tritaeniorhynchus supported multiplication of CQV by intrathoracic as well as artificial membrane/oral feeding routes. Interpretation & conclusions: Anti-CQV IgG antibody positivity in human serum samples tested and the replication capability of CQV in mosquitoes indicated a possible disease causing potential of CQV in Indian scenario. Screening of more human and swine serum samples using these assays is required as a proactive measure for understanding the prevalence of this neglected tropical virus.

Biorisk assessment for infrastructure & biosafety requirements for the laboratories providing coronavirus SARS-CoV-2/(COVID-19) diagnosis.

Novel coronavirus infection [coronavirus disease 2019 (COVID-19)] has spread to more than 203 countries of various regions including Africa, America, Europe, South East Asia and Western Pacific. The WHO had declared COVID-19 as the global public health emergency and subsequently as pandemic because of its worldwide spread. It is now one of the top-priority pathogens to be dealt with, because of high transmissibility, severe illness and associated mortality, wide geographical spread, lack of control measures with knowledge gaps in veterinary and human epidemiology, immunity and pathogenesis. The quick detection of cases and isolating them has become critical to contain it. To meet the increasing demand of the diagnostic services, it is necessary to enhance and expand laboratory capabilities since existing laboratories cannot meet the emerging demand. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a BSL-2 (Biosafety Level 2) agent and needs to be handled in biosafety cabinet using standard precautions. This review highlights minimum requirements for the diagnostic laboratories opting testing of material for the diagnosis of COVID-19 and associated biorisk to the individuals and to the community.