Search

Hop stunt viroid (HSVd), a small, single stranded, circular, non-coding infectious RNA known to cause infection in various economically important crop plants. In the present investigation, a study was conducted in the southern part of Karnataka districts of India to detect the possible association of HSVd infection in mulberry plants. A total of 41 mulberry plants showing typical viroid-like symptoms along with asymptomatic samples were collected and screened using conventional Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) using a specific set of HSVd-Fw/ HSVd-Re primers. Out of 41 samples, the study confirmed the presence of HSVd in six samples of mulberry collected from Ramanagara (1 sample), Chikkaballapur (3 samples) and Doddaballapura (2 samples) regions with an expected HSVd amplicon size of ∼ 290-300 nucleotides. The mechanical transmission of HSVd was also confirmed on cucumber (cv. Suyo) seedlings through bioassay, which was reconfirmed by RT-PCR. The amplicons were cloned, sequenced, and the representative nucleotide sequences were deposited in the NCBI GenBank. Subsequently, molecular phylogenetic analysis showed that HSVd mulberry isolates from this study were most closely related to grapevine isolates, indicating a common origin. On the other hand, it was shown to belong to a different group from mulberry isolates so far reported from Iran, Italy, Lebanon, and China. The secondary structure analysis of HSVd mulberry Indian isolates exhibited substitutions in the terminal left, pathogenicity, and variable regions compared to those of the Indian grapevine isolates. As far as this study is concerned, HSVd was detected exclusively in some mulberry plants with viral-like symptoms, but the pathogenesis and symptom expression needs to be further investigated to establish the relationship between HSVd and the disease symptoms in the mulberry plants., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Purpose: Acute Hemorrhagic conjunctivitis (AHC) is associated with CVA24v. Recently there was a severe outbreak of conjunctivitis in months of July and August 2023 in India. This study emphasizes the identification of the distinct mutations in the CVA24v strains, which were isolated during the AHC outbreak and could have potentially played a role in the high transmission of AHC in India during the 2023 outbreak., Methods: A total of 71 conjunctivitis patients aged 1-75 years comprising 47 males and 24 females who attended Ophthalmology department of a tertiary care hospital of easternIndia were studied.RNA was extracted from all conjunctival swab samples and converted into cDNA. Subsequently, the viral 5' UTR was amplified and the PCR positive samples were subjected to sequencing. The newly isolated viral 5' UTR sequences were aligned with other worldwide sequences using the Clustal W tool to conduct mutational analysis. A phylogenetic tree was built using the MEGA software for viral genotype identification., Results: All of the current outbreak strains belonged to genotype IV of CVA24v. The present outbreak strains formed a distinct clade in the phylogenetic tree and were different from previously reported Indian strains. Two persistent mutations, specifically in domain IV (T213C) and domain V (C475T), were exclusively detected within the internal ribosome entry site (IRES) of the 5' UTR of the current strains causing the outbreak. These two alterations have previously been shown to impact the virulence of another enterovirus (CV B3), but they have not been described in CVA24v until now., Conclusion: Finding of the present study highlights the possibility and the significance of the aforementioned two mutations in enhancing the transmissibility of the newer CVA24v strains. Hence, these two distinct mutations should be investigated further for developing antiviral therapies to combat future AHC outbreaks associated with CVA24v., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Baijayantimala Mishra reports financial support was provided by DHR-ICMR. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Indian Association of Medical Microbiologists. Published by Elsevier B.V. All rights reserved.)

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 × 10 4 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., (© 2024 Wiley Periodicals LLC.)

Background Objectives: West Bengal is a dengue-endemic State in India, with all four dengue serotypes in co-circulation. The present study was conceived to determine the changing trends of circulating dengue virus (DENV) serotypes in five consecutive years (2015-2019) using a geographic information system (GIS) during the dengue season in West Bengal, India., Methods: Molecular serotyping of dengue NS1 sero-reactive serum samples from individuals with ≤5 days of fever was performed using conventional nested reverse transcriptase-PCR. GIS techniques such as Getis-Ord Gi* hotspot analysis and heatmap were used to elucidate dengue transmission based on the received NS1-positive cases and vector data analysis was used to point out risk-prone areas., Results: A total of 3915 dengue NS1 sero-positive samples were processed from most parts of West Bengal and among these, 3249 showed RNA positivity. The major circulating serotypes were DENV 3 (63.54%) in 2015, DENV 1 (52.79%) in 2016 and DENV 2 (73.47, 76.04 and 47.15%) in 2017, 2018 and 2019, respectively. Based on the NS1 positivity, dengue infections were higher in males than females and young adults of 21-30 yr were mostly infected. Getis-Ord Gi* hotspot cluster analysis and heatmap indicate that Kolkata has become a hotspot for dengue outbreaks and serotype plotting on maps confirms a changing trend of predominant serotypes during 2015-2019 in West Bengal., Interpretation Conclusions: Co-circulation of all the four dengue serotypes was observed in this study, but only one serotype became prevalent during an outbreak. Representation of NS1-positive cases and serotype distribution in GIS mapping clearly showed serotypic shift in co-circulation. The findings of this study suggest the need for stringent surveillance in dengue-endemic areas to limit the impact of dengue and implement better vector-control strategies., (Copyright © 2024 Copyright: © 2024 Indian Journal of Medical Research.)

Among recently prevalent tick-borne infections in India, Kyasanur Forest Virus Disease (KFD) is an important public health concern. During last decade the emergence of cases apart from endemic zone raised concern about case positivity. Early diagnosis is therefore very important in disease management and primary containment. This study, aimed to develop a simplified viral RNA extraction in combination to dry down format of novel isothermal assay for (Reverse Transcription- Polymerase Spiral reaction) specific and rapid identification of Kyasanur Forest Disease Virus targeting viral envelope gene. The one step method was optimized by magnetic bead based viral RNA extraction followed by isothermal RT-PSR assay in heat bath at 63⁰C for 60 minutes. Further, visual results interpretation was done by color change of Hydroxy Naphthol Blue dye. The detection limit of the assay was found 10 RNA copies/rxn with comparable to silica column based viral RNA combined real time qPCR. No cross reactivity was observed with other closely related flaviviruses. The assay was evaluated with clinical samples has shown >99% concordance between two methods. This is the first report of sample extraction coupled isothermal detection of KFD in a simplified manner without a need of any hi-end equipment. The assay developed here has potential to use as an alternate for field-based detection in resource limited settings for KFD., Competing Interests: Declaration of Competing Interest The authors declare that they do not have any conflicts of interests associated with the research presented here, (Copyright © 2023. Published by Elsevier B.V.)

Mumps is a vaccine-preventable disease, and research on the vaccine's efficacy has recently indicated declining efficacy that has failed to protect against primary infections or reinfections, leading to a global resurgence in nations that use mumps vaccine in their national immunization programmes (NIPs). Lack of reports on its infection, documentation and published studies prevents it from being recognized as a public health issue in India. The waning of immunity is ascribed to the changes between the circulating and vaccine strains. The goal of the current study was to describe the circulating MuV strains in the Dibrugarh district of Assam, India, from 2016 to 2019. Blood samples were examined for IgM antibodies, and throat swab samples were put through Taqman assay for molecular detection. The small hydrophobic (SH) gene was targeted for genotyping through sequencing, and its genetic variations and phylogenetic analysis were carried out. Mumps RNA was found in 42 cases, and Mumps IgM in 14, of which 60% (25/42) of the cases were male and 40% (17/42) were female mostly affecting children between the ages of 6 and 12. Sequence and phylogeny analyses of SH gene revealed Genotypes C (83%) and G (17%) were simultaneously circulating during the study period. The study offers crucial genetic baseline information for the creation of Mumps prevention and control measures. Therefore, based on the research, it is clear that developing an effective vaccination strategy should take into account all currently prevalent genotypes in order to provide better protection against the disease's comeback., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Regulated oxidative stress (OS) is important during pregnancy. Sporadic studies suggest the significance of deregulated OS in hepatitis E virus (HEV) infected pregnancy, but with limited reactive oxygen species (ROS) or antioxidant markers. The present novel study, therefore, aimed to evaluate the significance of ROS-antioxidant imbalance and resulting altered OS in HEV infected pregnancy complications like preterm delivery (PTD) and outcome. Difference in serum levels of ROS and antioxidant panel of markers were evaluated by ELISA for HEV immunoglobulin M RNA positive genotype 1 cases (including acute [acute viral hepatitis, AVH] and fulminant [fulminant hepatic failure, FHF] cases) and healthy term delivery subjects, and analyzed statistically. Direct ROS marker H 2 O 2 levels and indirect OS marker for DNA damage 8-hydroxy-2'-deoxyguanosine was significantly increased in HEV-cases compared to controls, and was associated and prognostic factor for PTD and fetal death in HEV cases. A comparatively lower total serum antioxidant capacity was observed in the FHF cases compared to the control subjects and the AVH cases. Glutathione (GSH) levels and superoxide dismutase (SOD) activity were significantly associated with PTD in the FHF sub-cohorts (p = 0.017) and AVH sub-cohorts (p < 0.001), respectively, and was associated with poor prognosis in HEV cases. The serum H 2 O 2 levels were found to be negatively correlated with SOD activity (p = 0.016) and GSH levels (p = 0.001) in the HEV-AVH cases; and positively correlated with the viral load in HEV cases (p = 0.023). The ROS-antioxidant imbalance resulting OS plays a detrimental associative role in HEV infected pregnancy complications like PTD and adverse pregnancy outcomes; and holds therapeutic significance., (© 2023 Wiley Periodicals LLC.)

Purpose: The purpose of this study was to evaluate the prevalence of SARS-CoV-2 in human postmortem ocular tissues of asymptomatic donors and its implications on our eye banking protocols., Methods: The expression of SARS-CoV-2 RNA was assessed by reverse transcription-polymerase chain reaction in corneal rims and conjunctival tissues from 100 donors who were found suitable for transplantation as per the donor screening guidelines of the Global Alliance of Eye Bank Associations. The donor's clinical history and cause of death were assessed for secondary analysis., Results: Of 200 ocular tissues (100 corneal and 100 conjunctival) from the same 1 eye of 100 surgical-intended donors, between September 2020 and April 2021, the overall positivity rate for SARS-CoV-2 was ∼1% (2/200). Both the ocular samples that tested positive were conjunctival biopsies (2/100, 2%), whereas corneal samples were negative (0/100, 0%) in both donors. The causes of donor death were trauma in 51 donors, suicide in 33, cardiac arrest in 7, electric shock in 5, metabolic cause in 2, malignancy in 1, and snake bite in 1. None of the donors had a medical history suggestive of COVID infection or possible contact. None of the recipients from the donors were reported to have any systemic adverse event after keratoplasty until the follow-up of 6 weeks., Conclusions: The overall prevalence of SARS-CoV-2 was 1% (2% for conjunctival and 0% for corneal samples, P value = 0.5) in the donors who were found suitable for cornea recovery and transplantation. The findings of exceptionally low positive rates in our samples validate the criticality of history-based donor screening and do not support the necessity of postmortem PCR testing as a criterion for procurement and subsequent use for corneal transplantation., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Purpose: Hepatitis E virus (HEV) is responsible for >50% of acute viral hepatitis (AVH) in developing countries. It has 4 major genotypes and various subtypes which vary in geographical distribution, clinical manifestations and epidemiological patterns. This study was conducted to characterise HEV isolates from north India to study the effect of host and viral factors on HEV infection., Methods: Serum samples collected from 536 AVH patients admitted to Department of Medicine, King George's Medical University, Lucknow from July 2016 to June 2017 were screened for anti HEV IgM, anti HAV IgM, HBsAg and anti HCV antibodies using commercial ELISA kits. Samples either positive for anti HEV IgM antibodies (n ​= ​204) or negative for all 4 hepatotropic viruses (n ​= ​37) were enrolled and tested by real time PCR for HEV RNA. HEV RNA positive samples with high viral load were further subjected to nested PCR for amplification of capsid gene. Sequencing and phylogenetic analysis were performed. HEV strains isolated from this study were deposited to GenBank under accession numbers MG571274 to MG571283., Results: Anti HEV IgM positivity was observed among 38% clinically suspected AVH cases. HEV RNA was detected in 31.8% seropositive HEV cases and additional 3 seronegative cases. Males outnumbered females and the most affected age group was of young adults. Maximum number of cases were seen during the months of June to September. Phylogenetic analysis showed that HEV strains in our study belonged to genotype 1a. Mortality in HEV infected pregnant females was 23.5% as compared to 2.4% in non-pregnant females. Adverse fetal outcome was recorded in 51% of HEV infected pregnancies., Conclusions: HEV genotype 1a is prevalent in our setting. HEV during pregnancy is associated with adverse maternal and fetal outcome., Competing Interests: Declaration of competing interest None., (Copyright © 2021 Indian Association of Medical Microbiologists. Published by Elsevier B.V. All rights reserved.)

Severe acute respiratory syndrome coronavirus (SARS-CoV-2) has affected all inhabited continents, and India is currently experiencing a devastating second wave of coronavirus disease-2019 (COVID-19). Here, we examined the duration of clearance of SARS-CoV-2 in respiratory samples from 207 infected cases by real-time reverse-transcription polymerase chain reaction (RT-PCR). A substantial proportion of COVID-19 positive cases with cycle threshold (Ct) values more than or equal to 31 (45.7%) were subsequently tested negative for SARS-CoV-2 RNA within 7 days of initial detection of the viral load. A total of 60% of all the patients with COVID-19, irrespective of their Ct values, cleared SARS-CoV-2 RNA within 14 days of the initial detection. Longitudinal assessment of RT-PCR test results in individuals requiring 15-30 days to clear SARS-CoV-2 RNA showed a significant reduction of the viral load in samples with high or intermediate viral loads (Ct values ≤ 25 and between 26 and 30, respectively) but the follow-up group with low viral RNA (Ct values ≥ 31) exhibited a stable viral load. Together, these results suggest that COVID-19 positive cases with Ct values more than or equal to 31 require reduced duration to clear SARS-CoV-2, and thus, a shorter isolation period for this group might be considered to facilitate adequate space in the COVID Care Centres and reduce the burden on healthcare infrastructure., (© 2021 Wiley Periodicals LLC.)

To meet the unprecedented requirement of diagnostic testing for SARS-CoV-2, a large number of diagnostic kits were authorized by concerned authorities for diagnostic use within a short period of time during the initial phases of the ongoing pandemic. We undertook this study to evaluate the inter-test agreement and other key operational features of 5 such commercial kits that have been extensively used in India for routine diagnostic testing for COVID-19. The five commercial kits were evaluated, using a panel of positive and negative respiratory samples, considering the kit provided by National Institute of Virology, Indian Council of Medical Research (2019-nCoV Kit) as the reference. The positive panel comprised of individuals who fulfilled the 3 criteria of being clinically symptomatic, having history of contact with diagnosed cases and testing positive in the reference kit. The negative panel included both healthy and disease controls, the latter being drawn from individuals diagnosed with other respiratory viral infections. The same protocol of sample collection, same RNA extraction kit and same RT-PCR instrument were used for all the kits. Clinical samples were collected from a panel of 92 cases and 60 control patients, who fulfilled our inclusion criteria. The control group included equal number of healthy individuals and patients infected with other respiratory viruses (n = 30, in each group). We observed varying sensitivity and specificity among the evaluated kits, with LabGun COVID-19 RT-PCR kit showing the highest sensitivity and specificity (94% and 100% respectively), followed by TaqPath COVID-19 Combo and Allplex 2019-nCoV assays. The extent of inter-test agreement was not associated with viral loads of the samples. Poor correlation was observed between Ct values of the same genes amplified using different kits. Our findings reveal the presence of wide heterogeneity and sub-optimal inter-test agreement in the diagnostic performance of the evaluated kits and hint at the need of adopting stringent standards for fulfilling the quality assurance requirements of the COVID-19 diagnostic process., (© 2021. The Author(s).)

Purpose: Hepatitis C virus is a major cause of chronic hepatitis with seven known genotypes. Uttarakhand is a north Indian state in the Sub-Himalayan region where the genotypic distribution of HCV is largely unknown. This study was undertaken in order to assess the pattern of genotype and subtype and understand the risk factors leading to transmission of Hepatitis C virus in this understudied region., Methods: Anti-HCV reactive cases were selected for determination of the circulating genotypes. Viral RNA was confirmed by real-time PCR. Strains were amplified and sequenced using Sanger's methods. Phylogenetic tree was constructed to determine the genotype., Results: Genotype 3 was found to be the predominant genotype majority being subtype 3a and 3b followed by genotype 1. Subtypes 3g and genotype 4a were also observed. Major risk factor found was parenteral injection therapy from unregistered medical practitioners for minor ailments., Conclusions: Findings of our study will help in tailoring management and prevention protocols for HCV for the people of this region., Competing Interests: Declaration of competing interest The author(s) declare that they have no conflicting interests., (Copyright © 2021 Indian Association of Medical Microbiologists. Published by Elsevier B.V. All rights reserved.)

After the 2005-2009 chikungunya epidemic, intermittent outbreaks were reported in many parts of India. The outbreaks were caused by either locally circulating strains or imported viruses. Virus transmission routes can be traced by complete genome sequencing studies. We investigated two outbreaks in 2014 and 2019 in Kerala, India. Chikungunya virus (CHIKV) was isolated from the samples, and whole genomes were sequenced for a 2014 isolate and a 2019 isolate. Phylogenetic analysis revealed that the isolates formed a separate group with a 2019 isolate from Pune, Maharashtra, and belonged to the East/Central/South African (ECSA) genotype, Indian subcontinent sublineage of the Indian Ocean Lineage (IOL). A novel mutation at amino acid position 76 of the E2 gene was observed in the group. The phylogenetic results suggest that the outbreaks might have been caused by a virus that had been circulating in India since 2014. A detailed study is needed to investigate the evolution of CHIKV in India., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Porcine astrovirus (PAstV) is distributed worldwide and has been reported to cause diarrhea in pigs. PAstV belongs to the family Astroviridae and genus Mamastrovirus. PAstVs are divided into five diverse genotypes (PAstV1-PAstV5) on the basis of phylogenetic analysis of a part of the RNA-dependent RNA polymerase (RdRp) gene and the capsid gene. However, knowledge regarding the clinical significance and molecular characteristics of PAstV in Haryana, India, is limited. In this study, we investigated the presence of PAstV by RT-PCR of the partial RdRp gene in 110 rectal swabs collected from diarrheic pigs in different parts of Haryana, India. Of these, 35 samples (31.8%) tested positive for PAstV, with the highest positivity observed among weaning piglets 3 to 9 weeks of age (47.7%, 21/44), followed by fattening pigs 9 to 24 weeks of age (28.5%, 8/28). Phylogenetic analysis of the partial RdRp gene revealed circulation of four different genotypes (PAstV1, PAstV2, PAstV4, and PAstV5) in Haryana, with PAstV1 being the predominant genotype. To the best of our knowledge, this is the first report of the presence of PAstV1 and PAstV5 in the pig population of India. The PAstV sequences revealed high genetic variability and genetic heterogeneity in a relatively confined area., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

The present study was conducted from July 1, 2020 to September 25, 2020 in a dedicated coronavirus disease 2019 (COVID-19) hospital in Delhi, India to provide evidence for the presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus in atmospheric air and surfaces of the hospital wards. Swabs from hospital surfaces (patient's bed, ward floor, and nursing stations area) and suspended particulate matter in ambient air were collected by a portable air sampler from the medicine ward, intensive care unit, and emergency ward admitting COVID-19 patients. By performing reverse-transcriptase polymerase chain reaction (RT-PCR) for E-gene and RdRp gene, SARS-CoV-2 virus was detected from hospital surfaces and particulate matters from the ambient air of various wards collected at 1 and 3-m distance from active COVID-19 patients. The presence of the virus in the air beyond a 1-m distance from the patients and surfaces of the hospital indicates that the SARS-CoV-2 virus has the potential to be transmitted by airborne and surface routes from COVID-19 patients to health-care workers working in COVID-19 dedicated hospital. This warrants that precautions against airborne and surface transmission of COVID-19 in the community should be taken when markets, industries, educational institutions, and so on, reopen for normal activities., (© 2021 Wiley Periodicals LLC.)

Background: Thirty-four CCHF cases (17 fatal; 17 survived) were confirmed from Gujarat state, India during the year 2019. We aimed to find out the viral load, antibody kinetics, cytokine profile and phylogenetic analysis between fatal and non- fatal cases., Methods: Thirty four cases were included in this study. Blood and urine samples were collected from all the cases on the day of admission to hospital. Non-fatal cases were followed weekly for understanding the profile of viral kinetics, anti-CCHFV IgM and IgG antibodies. We also quantified the cytokines in both fatal and non-fatal cases. For epidemiological correlation, livestock were screened for anti-CCHF IgG antibodies and the tick pool specimens were tested by real time RT-PCR. Virus isolation was attempted on tick pools and human specimens and phylogenetic analysis performed on human and ticks complete genome sequences., Results: CCHF cases were detected throughout year in 2019 with the peak in August. Out of 34 cases, eight secondary CCHF cases were reported. Cases were predominantly detected in males and in 19-45 years age group (55.88%). The persistence of viremia was observed till 76th POD (post onset date) in one case whereas anti-CCHFV IgM and IgG was detected amongst these cases from the 2nd and 20th POD respectively. Positivity observed amongst livestock and tick pools were was 21.57% and 7.4% respectively. The cytokine analysis revealed a significant increase in the level of serum IL-6, IL-10 and IFN-γ during the acute phase of the infection, but interestingly IL-10 lowered to normal upon clearance of the virus in the clinically recovered case. Fatal cases had high viral RNA copy numbers. Bleeding from one or two mucosal sites was significantly associated with fatality (OR-16.47;p-0.0034 at 95% CI). We could do CCHF virus isolation from two cases. Phylogenetic analysis revealed circulation of re-assortment of Asian-West African genotypes in humans and ticks., Conclusions: The persistence of CCHF viral RNA was detected till 76th POD in one of the survivors. The circulation of a re-assortment Asian-West African genotype in a CCHF case is also reported first time from India., Competing Interests: The authors have declared that no competing interests exist.

Covid-19 disease caused by SARS-CoV-2 is still being transmitted in developed and developing countries irrespective of healthcare setups. India with 1.3 billion people in the world is severely affected by Covid-19 with 11.3 million cases and 157 000 deaths so far. We have assessed the mismatches in WHO recommended rRT-PCR assays primer and probe binding regions against SARS-CoV-2 Indian genome sequences through in-silico bioinformatics analysis approach. Primers and probe sequences belonging to CN-CDC-ORF1ab from China and HKU-ORF1b from Hong Kong targeting ORF1ab gene while NIH-TH-N from Thailand, HKU-N from Hong Kong and US-CDCN-2 from USA targeting N genes displayed accurate matches (>98.3%) with the 2019 novel corona virus sequences from India. On the other hand, none of the genomic sequences displayed exact match with the primer/probe sequences belonging to Charité-ORF1b from Germany targeting ORF1ab gene. We think it will be worthwhile to release this information to the clinical and medical communities working in Indian Covid-19 frontline taskforce to tackle the recently emerging Covid-19 outbreaks as of March-2021., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The adverse relationship between viral hepatitis and pregnancy in developing countries had been interpreted as a reflection of retrospectively biased hospital-based data collection by the West. However, the discovery of hepatitis E virus (HEV) as the etiological agent of an epidemic of non-A, non-B hepatitis in Kashmir, and the documenting of the increased incidence and severity of hepatitis E in pregnancy via a house-to-house survey, unmasked this unholy alliance. In the Hepeviridae family, HEV-genotype (gt)1 from genus Orthohepevirus A has a unique open reading frame (ORF)4-encoded protein which enhances viral polymerase activity and viral replication. The epidemics caused by HEV-gt1, but not any other Orthohepevirus A genotype, show an adverse relationship with pregnancy in humans. The pathogenesis of the association is complex and at present not well understood. Possibly multiple factors play a role in causing severe liver disease in the pregnant women including infection and damage to the maternal-fetal interface by HEV-gt1; vertical transmission of HEV to fetus causing severe fetal/neonatal hepatitis; and combined viral and hormone related immune dysfunction of diverse nature in the pregnant women, promoting viral replication. Management is multidisciplinary and needs a close watch for the development and management of acute liver failure. (ALF). Preliminary data suggest beneficial maternal outcomes by early termination of pregnancy in patients with lower grades of encephalopathy.

Background: Dengue virus causes the dengue fever as well as hemorrhagic fever in tropical and sub-tropical countries. It is now endemic in most parts of the South East Asia. Full-genome information of dengue virus 3 is not available from Yemen., Methods: In this study, the dengue virus 3 was detected by diagnostic tools like serology and RT-PCR in the samples isolated from a patient in Yemen. The full-genome was sequenced, and the identity, phylogenetic relationship and recombination analysis was performed by using BioEdit, MEGA X and RDP4 softwares., Results: The full-genome of the Yemen isolate was found to be 10,643 nt long with 3390 amino acids. The Yemen dengue virus 3 isolate showed the sequence similarity (98.5-92.4%) with dengue virus 3 isolates from China, Pakistan, India and Bangladesh respectively. The significant non-synonymous substitutions of amino acid in Yemen isolate were observed with selected isolates. The phylogenetic tree of Yemen isolate formed a unique clade within genotype III and sub-clade into lineage III. The Dengue virus isolate from Jeddah formed separated cluster with lineage IV., Conclusions: This reveals the unique genetic variability among DENV-3 serotypes from Jeddah and earlier reported isolates from other regions., (Copyright © 2021 King Fahd Medical Research Center. Published by Elsevier Ltd.. All rights reserved.)

Hand, Foot, and Mouth disease (HFMD) is a mild exanthematous and febrile disease occurs in children aged ≤10 years old. The present study highlights clinical, epidemiological characteristics, distribution of enterovirus (EV) types, and sub genotypes in HFMD cases reported during 2017 to 2018 in Western India. A total of 93 clinical samples collected from 68 HFMD cases were included. The presence of EV-RNA was determined by 5'UTR based nested reverse transcription polymerase chain reaction followed by molecular typing, sub genotyping by VP1/2A junction or VP1, full VP1 gene amplification, and phylogenetic analysis. The study reports 80.64% (75/93) EV positivity and 94.66% (71/75) typing rate, with a predominant circulation of CVA16 and CVA6 strains. Sequence analysis revealed the presence of coxsackievirus (CV)A16 (57.7%), CVA6 (40.8%), and Echo1 (1.4%) strains. EV infections were predominantly observed in children aged 1 to 3 years old (43.9%). Although cases were reported throughout the year, peaked in July (15.8%) and August (24.6%) months and persisted till September (19.3%). All the CVA16 and CVA6 positive strains were genotyped using full VP1 gene amplification. All CVA16 Indian strains (n = 41) were clustered with rarely reported B1c sub genotype and CVA6 strains (n = 29) with E2 sub-lineage. The study highlights the genetic characteristics of circulating CVA16, CVA6, and Echo1 strains in HFMD cases from Western India. The emergence of CVA16 B1c genotype and sub-lineage E2 of CVA6 strains and their constant circulation further demands systemic surveillance studies on HFMD from different parts of India to facilitate the rapid diagnosis of CVA16 and CVA6 strains using the molecular and serological based approach and for intervention strategies., (© 2020 Wiley Periodicals LLC.)

Dengue (DEN) is the most common cause of mosquito-borne endemic viral diseases in the tropical and subtropical countries. DEN outbreaks associated with multiple dengue virus (DV) serotypes have been regularly reported in different parts of India. This study was done during DEN outbreaks in 2015 to 2016 in UP and Bihar where DEN-2 was found as the only prevalent serotype. DV-2 was the only serotype amplified in serotype-specific reverse-transcription polymerase chain reaction from sera of 210 (65.21%) out of 322 DV NS1 antigen-positive patients. Further genetic analysis based on full-length envelope (E) protein sequence derived from patient's sera as well as DV isolate showed the circulation of lineages I and III of DV-2 cosmopolitan genotype during 2015 and lineage II during 2016. Finally, the phylogenetic analysis using the E gene sequence revealed that these DV-2 strains have a close genetic relationship with the recently reported DV-2 genotypes from DEN outbreaks reported from different parts of north India. These results showed the circulation of cosmopolitan genotype of DV-2 in eastern Uttar Pradesh and western Bihar, India. The genetic database generated on circulating DV strains in this study will be useful as reference for disease surveillance and strengthening laboratory diagnosis protocols., (© 2020 Wiley Periodicals LLC.)

Background: The prevalence of HCV infection is high and it is a major cause of liver-related morbidity and mortality in hemodialysis and renal transplant patients. Diagnosis of hepatitis C virus (HCV) infection requires both HCV antibody screening and confirmatory nucleic acid testing (NAT). Hepatitis C virus core antigen (HCVcAg) is a reliable direct viral marker to identify active HCV infection., Aim: To assess the clinical utility of HCV core antigen to identify active HCV infection in hemodialysis and renal transplant patients., Methods: A representative total of 231 plasma samples with a predominance of low viral load were included for HCVcAg testing and its performance characteristics were compared with the gold standard HCV RNA., Results: Comparison of HCVcAg with HCV RNA showed an excellent specificity of 99% (95% CI: 94.7 to 100%) and sensitivity of 80.62% (95% CI: 73.59 to 87.7%). Likewise, the PPV and NPV of HCVcAg were 99.1% (95% CI: 93.7% to 99.9%) and 80.2% (95% CI: 74% to 85.2%) respectively. The correlation between HCVcAg and HCV RNA was found to be good (R2 = 0.86, p<0.0001). Among common Indian HCV genotypes (1, 3 & 4), good correlation was observed between HCV RNA and HCVcAg (R2 = 0.81, p <0.0001)., Conclusions: It is the first Indian study to show that HCVcAg is a reliable, cost-effective direct marker to identify active HCV infection in hemodialysis and renal transplant patients. Implementation of HCVcAg testing could improve the accessibility to efficacious and affordable disease management in hemodialysis and renal transplant patients. In HCVcAg negative cases, sequential testing with anti-HCV antibody followed by HCV RNA could be a reliable and cost-effective approach., Competing Interests: The authors declare that they have no competing interests.

There are more than 350 real-time polymerase chain reaction (RT-PCR) coronavirus disease-2019 (COVID-19) testing kits commercially available but these kits have not been evaluated for pooled sample testing. Thus, this study was planned to compare and evaluate seven commercially available kits for pooled samples testing. Diagnostic accuracy of (1) TRUPCR SARS-CoV-2 Kit (Black Bio), (2) TaqPath RT-PCR COVID-19 Kit (Thermo Fisher Scientific), (3) Allplex 2019-nCOV Assay (Seegene), (4) Patho detect COVID-19 PCR kit (My Lab), (5) LabGun COVID-19 RT-PCR Kit (Lab Genomics, Korea), (6) Fosun COVID-19 RT-PCR detection kit (Fosun Ltd.), (7) Real-time Fluorescent RT-PCR kit for SARS CoV-2 (BGI) was evaluated on precharacterised 40 positive and 10 negative COVID-19 sample pools. All seven kits detected all sample pools with low C t values (<30); while testing weak positive pooled samples with high C t value (>30); the TRUPCR Kit, TaqPath Kit, Allplex Assay, and BGI RT-PCR kit showed 100% sensitivity, specificity, and accuracy. However, the Fosun kit, LabGun Kit, and Patho detect kit could detect only 90%, 85%, and 75% of weakly positive samples, respectively. We conclude that all seven commercially available RT-PCR kits included in this study can be used for routine molecular diagnosis of COVID-19. However, regarding performing pooled sample testing, it might be advisable to use those kits that performed best regarding positive identification in samples' pool, that is TRUPCR SARS-CoV-2 Kit, TaqPath RT-PCR COVID-19 Kit, Allplex 2019-nCOV Assay, and BGI Real-time RT-PCR kit for detecting SARS CoV-2., (© 2020 Wiley Periodicals LLC.)

Newcastle disease virus (NDV) is endemic in Bangladesh and is a major threat to commercial poultry operations. While complete fusion (F) genes are recommended for molecular characterization and classification of NDV isolates, heretofore, only partial F gene data have been available for Bangladeshi NDVs. To this end, we obtained the full-length F gene coding sequences of 11 representative NDVs isolated in Bangladesh between 2010 and 2017. In addition, one of the viruses (MK934289/chicken/Bangladesh/C161/2010) was used in an experimental infection of chickens to establish the viral pathotype and study gross and microscopic lesions. Phylogenetic analysis provided evidence that all studied Bangladeshi isolates belong to genotype XIII.2 of class II NDVs. Six of the viruses were isolated between 2010 and 2017 and grouped together with isolates from neighbouring India during 2013-2016. Another four Bangladeshi isolates (2010-2016) formed a separate monophyletic branch within XIII.2 and showed high nucleotide distance from the isolates from India and the other six Bangladeshi viruses within the sub-genotype; however, none of these groups fulfils all classification criteria to be named as a separate sub-genotype. The eleventh Bangladeshi virus studied here (C162) was genetically more distant from the remaining isolates. It out-grouped the viruses from sub-genotypes XIII.2.1 and XIII.2.2 and showed more than 9.5 % nucleotide distance from all genotype XIII sub-genotypes. This isolate may represent an NDV variant that is evolving independently from the other viruses in the region. The experimental infection in chickens revealed that the tested isolate (C161) is a velogenic viscerotropic virus. Massive haemorrhages, congestion and necrosis in different visceral organs, and lymphoid depletion in lymphoid tissues, typical for infection with velogenic NDV, were observed. Our findings demonstrate the endemic circulation of sub-genotype XIII.2 in Southcentral Asia and further genetic diversification of these viruses in Bangladesh and neighbouring India. This constant evolution of the viruses may lead to the establishment of new genetic groups in the region. Additional historical and prospective virus and surveillance data from the region and neighbouring countries will allow a more detailed epidemiological inference.

The rapid emergence of coronavirus disease 2019 (COVID-19) as a global pandemic affecting millions of individuals globally has necessitated sensitive and high-throughput approaches for the diagnosis, surveillance, and determining the genetic epidemiology of SARS-CoV-2. In the present study, we used the COVIDSeq protocol, which involves multiplex-PCR, barcoding, and sequencing of samples for high-throughput detection and deciphering the genetic epidemiology of SARS-CoV-2. We used the approach on 752 clinical samples in duplicates, amounting to a total of 1536 samples which could be sequenced on a single S4 sequencing flow cell on NovaSeq 6000. Our analysis suggests a high concordance between technical duplicates and a high concordance of detection of SARS-CoV-2 between the COVIDSeq as well as RT-PCR approaches. An in-depth analysis revealed a total of six samples in which COVIDSeq detected SARS-CoV-2 in high confidence which were negative in RT-PCR. Additionally, the assay could detect SARS-CoV-2 in 21 samples and 16 samples which were classified inconclusive and pan-sarbeco positive respectively suggesting that COVIDSeq could be used as a confirmatory test. The sequencing approach also enabled insights into the evolution and genetic epidemiology of the SARS-CoV-2 samples. The samples were classified into a total of 3 clades. This study reports two lineages B.1.112 and B.1.99 for the first time in India. This study also revealed 1,143 unique single nucleotide variants and added a total of 73 novel variants identified for the first time. To the best of our knowledge, this is the first report of the COVIDSeq approach for detection and genetic epidemiology of SARS-CoV-2. Our analysis suggests that COVIDSeq could be a potential high sensitivity assay for the detection of SARS-CoV-2, with an additional advantage of enabling the genetic epidemiology of SARS-CoV-2., Competing Interests: The authors have declared that no competing interests exist.

We report clinical profile of hundred and nine patients with SARS CoV-2 infection, and whole genome sequences (WGS) of seven virus isolates from the first reported cases in India, with various international travel histories. Comorbidities such as diabetes, hypertension, and cardiovascular disease were frequently associated with severity of the disease. WBC and neutrophil counts showed an increase, while lymphocyte counts decreased in patients with severe infection suggesting a possible neutrophil mediated organ damage, while immune activity may be diminished with decrease in lymphocytes leading to disease severity. Increase in SGOT, SGPT and blood urea suggests the functional deficiencies of liver, heart, and kidney in patients who succumbed to the disease when compared to the group of recovered patients. The WGS analysis showed that these isolates were classified into two clades: I/A3i, and A2a (four according to GISAID: O, L, GR, and GH). Further, WGS phylogeny and travel history together indicate possible transmission from Middle East and Europe. Three S protein variants: Wuhan reference, D614G, and Y28H were identified predicted to possess different binding affinities to host ACE2., Competing Interests: The authors have declared that no competing interests

Background: despite being in the 5th month of pandemic, knowledge with respect to viral dynamics, infectivity and RT-PCR positivity continues to evolve., Aim: to analyse the SARS CoV-2 nucleic acid RT-PCR profiles in COVID-19 patients., Design: it was a retrospective, observational study conducted at COVID facilities under AIIMS, New Delhi., Methods: patients admitted with laboratory confirmed COVID-19 were eligible for enrolment. Patients with incomplete details, or only single PCR tests were excluded. Data regarding demographic details, comorbidities, treatment received and results of SARS-CoV-2 RT-PCR performed on nasopharyngeal and oropharyngeal swabs, collected at different time points, was retrieved from the hospital records., Results: a total of 298 patients were included, majority were males (75·8%) with mean age of 39·07 years (0·6-88 years). The mean duration from symptom onset to first positive RT-PCR was 4·7 days (SD 3·67), while that of symptom onset to last positive test was 17·83 days (SD 6·22). Proportions of positive RT-PCR tests were 100%, 49%, 24%, 8·7% and 20·6% in the 1st, 2nd, 3rd, 4th and >4 weeks of illness. A total of 12 symptomatic patients had prolonged positive test results even after 3 weeks of symptom onset. Age > = 60 years was associated with prolonged RT-PCR positivity (statistically significant)., Conclusion: this study showed that the average period of PCR positivity is more than 2 weeks in COVID-19 patients; elderly patients have prolonged duration of RT-PCR positivity and requires further follow up., (© The Author(s) 2020. Published by Oxford University Press on behalf of FEMS.)

The diagnosis of coronavirus disease-19 (COVID-19) relies on the detection of severe acute respiratory syndrome coronavirus 2 (SARS CoV-2) RNA by real-time reverse-transcription polymerase chain reaction in respiratory samples. Rapid increase in the COVID-19 cases across the world requires fast and efficient testing as testing capacity is a bottleneck in diagnosis. In this context, pooling strategy can be opted for rapid testing in a cost-effective manner. In this study, the authors have optimized and compared the effect of pooling (5 and 10 samples) before and after nucleic acid extraction. It was concluded that there was no significant difference in the SARS CoV-2 RNA detection in the pools prepared at sample or RNA level. Even after pooling, 10-fold dilution was detectable with 3-cycle threshold value change in both type of pools when compared with individual samples. Hence, sample pool size of 10 can be used in low-prevalent areas, and testing capacity can be substantially increased., Competing Interests: Declaration of competing interest None., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background & Objectives: Since its first recognition in Wuhan, China, in December 2019, the SARS-CoV-2 has spread rapidly across the world. Though SARS-CoV-2 spreads mainly via the droplets of respiratory secretions, it was also detected in stool samples of patients, indicating active infection of the gastrointestinal tract. Presence of SARS-CoV-2 RNA in sewage samples was reported in February 2020, raising the possibility of using environmental water surveillance to monitor SARS-CoV-2 activity in infected areas. The aim of this study was to standardize the methodology for detection of SARS-CoV-2 from sewage and explore the feasibility of establishing supplementary surveillance for COVID-19., Methods: Sewage specimens were collected from six sites in Mumbai, India, using the grab sample method and processed using polyethylene glycol (PEG)-dextran phase separation method for virus concentration. Real-time reverse transcription-polymerase chain reaction (RT-PCR) assay was used to detect the presence of SARS-CoV-2 RNA., Results: A total of 20 sewage samples collected from six different wards in Mumbai city, before the spread of SARS-CoV-2 infections and during May 11-22, 2020, were processed using the phase separation method. The WHO two-phase PEG-dextran method was modified during standardization. SARS-CoV-2 was found to concentrate in the middle phase only. All samples collected before March 16, 2020 were SARS-CoV-2 negative. Viral RNA was detected in sewage samples collected during the ongoing COVID-19 pandemic in all the six wards., Interpretation & Conclusions: PEG-dextran phase separation method was effectively used to concentrate SARS-CoV-2 from domestic waste waters to detection levels. It would be feasible to initiate sewage surveillance for SARS-CoV-2 to generate data about the viral transmission in various epidemiologic settings., Competing Interests: None

Introduction: Hepatitis A virus causes an acute infection mainly in young children. The present study was carried out to characterize the nature of hepatitis A virus (HAV) involved in an outbreak of jaundice in children., Methodology: Serum and stool samples from five children were sampled from among 26 clinically diagnosed jaundice cases. HAV IgM ELISA and PCR were used for confirmatory diagnosis and molecular characterization by direct amplicon sequencing and analysis., Results: All the serum samples collected from the symptomatic cases were found to be positive for Anti-HAV IgM ELISA as were all the serum samples and stool samples using semi-nested PCR. Phylogenetic analysis revealed that the HAV involved in the outbreak belonged to genotype IIIA., Conclusions: The infection was caused by HAV genotype IIIA. Improved access to clean drinking water, sanitation around drinking water sources and routine chlorination of drinking water in poor and developing countries are needed, as well as childhood HAV vaccination under regular immunization programs in endemic countries., Competing Interests: No Conflict of Interest is declared, (Copyright (c) 2020 Rajamani Barathidasan, Kandasamy Vanathy, Kaliaperumal Venkatesh, Ferdina Marie Sharmila, Rahul Dhodapkar.)

Background: Prevalence of IgG antibodies against SARS-CoV-2 infection provides essential information for deciding disease prevention and mitigation measures. We estimate the seroprevalence of SARS-CoV-2 specific IgG antibodies in District Srinagar., Methods: 2906 persons >18 years of age selected from hospital visitors across District Srinagar participated in the study. We tested samples for the presence of SARS-CoV-2 specific IgG antibodies using a chemiluminescent microparticle immunoassay-based serologic test., Results: Age- and gender-standardized seroprevalence was 3.6% (95% CI 2.9% to 4.3%). Age 30-69 years, a recent history of symptoms of an influenza-like-illness, and a history of being placed under quarantine were significantly related to higher odds of the presence of SARS-CoV-2 specific IgG antibodies. The estimated number of SARS-CoV-2 infections during the two weeks preceding the study, adjusted for test performance, was 32602 with an estimated (median) infection-to-known-case ratio of 46 (95% CI 36 to 57)., Conclusions: The seroprevalence of SARS-CoV-2 specific IgG antibodies is low in the District. A large proportion of the population is still susceptible to the infection. A sizeable number of infections remain undetected, and a substantial proportion of people with symptoms compatible with COVID-19 are not tested., Competing Interests: The authors have declared that no competing interests exist.

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) was first identified in Wuhan, China late in 2019. Nine months later (Sept. 23, 2020), the virus has infected > 31.6 million people around the world and caused > 971.000 (3.07 %) fatalities in 220 countries and territories. Research on the genetics of the SARS-CoV-2 genome, its mutants and their penetrance can aid future defense strategies. By analyzing sequence data deposited between December 2019 and end of May 2020, we have compared nucleotide sequences of 570 SARS-CoV-2 genomes from China, Europe, the US, and India to the sequence of the Wuhan isolate. During worldwide spreading among human populations, at least 10 distinct hotspot mutations had been selected and found in up to > 80 % of viral genomes. Many of these mutations led to amino acid exchanges in replication-relevant viral proteins. Mutations in the SARS-CoV-2 genome would also impinge upon the secondary structure of the viral RNA molecule and its repertoire of interactions with essential cellular and viral proteins. The increasing frequency of SARS-CoV-2 mutation hotspots might select for dangerous viral pathogens. Alternatively, in a 29.900 nucleotide-genome, there might be a limit to the number of mutable and selectable sites which, when exhausted, could prove disadvantageous to viral survival. The speed, at which novel SARS-CoV-2 mutants are selected and dispersed around the world, could pose problems for the development of vaccines and therapeutics., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

•Most of the COVID-19 cases in Nepal are in the Southern districts of Nepal bordering India with travel histories to India.•Very few positive cases of COVID-19 are detected in Nepal which could either be due to early national lockdown.•Low PCR positivity rates could also be due to inefficiency of the PCR methods.•Whole genomes of 93 clinical samples from COVID-19 patients were analyzed to find the primer and probe binding sites.•Mutations in probe binding sites were found which could impact PCR efficiency resulting in false negative results., Competing Interests: None., (© 2020 Elsevier Ltd. All rights reserved.)

Crimean Congo hemorrhagic fever (CCHF) is a zoonotic viral disease presenting with fever and hemorrhagic manifestations in humans. After several outbreaks of CCHF being reported from Gujarat since 2011 till 2019 and from Rajasthan in 2014 and 2015, the present study reports the CCHF outbreak which was recorded from five human cases in three districts Jodhpur, Jaisalmer, and Sirohi of Rajasthan state since August 2019 till November 2019. A high percent of positivity was recorded in livestock animal samples for the CCHFV IgG antibody. CCHF virus (CCHFV) positive human blood samples and Hyalomma tick pool samples were sequenced using next-generation sequencing method. Two different M segment genotypes, encoding glycoprotein precursor, were identified from tick pools in the study: first from Asian and second from African lineage. The L gene (polymerase) and the S gene (nucleocapsid) clustered in the Asian lineage. The present study illustrates the existence of two different CCHFV lineages being circulating within the Hyalomma tick pools in the Rajasthan state, India. We also observed 3.56% amino acid changes between the death and the survived case of CCHFV in the M gene. This report also sets an alarm to enhance human, tick and livestock surveillance in other districts of Rajasthan and nearby states of India. Biosafety measures, barrier nursing along with the availability of personal protective equipment and ribavirin drug will always be a mainstay in preventing nosocomial infection for proper case management., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: Corona virus disease 2019 (COVID-19) which initially started as a cluster of pneumonia cases in the Wuhan city of China has now become a full-blown pandemic. Timely diagnosis of COVID-19 is the key in containing the pandemic and breaking the chain of transmission. In low- and middle-income countries availability of testing kits has become the major bottleneck in testing. Novel methods like pooling of samples are the need of the hour., Objective: We undertook this study to evaluate a novel protocol of pooling of RNA samples/elutes in performance of PCR for SARS CoV-2 virus., Study Design: Extracted RNA samples were randomly placed in pools of 8 on a 96 well plate. Both individual RNA (ID) and pooled RNA RT-qPCR for the screening E gene were done in the same plate and the positivity for the E gene was seen., Results: The present study demonstrated that pool testing with RNA samples can easily detect even up to a single positive sample with Ct value as high as 38. The present study also showed that the results of pool testing is not affected by number of positive samples in a pool., Conclusion: Pooling of RNA samples can reduce the time and expense, and can help expand diagnostic capabilities, especially during constrained supply of reagents and PCR kits for the diagnosis of SARS-CoV-2 infection., Competing Interests: The authors have declared that no competing interests exist.

In the current COVID-19 crisis, many national healthcare systems are confronted with a huge demand for mass testing and an acute shortage of diagnostic resources. Considering group testing as a viable solution, this pilot study was carried out to find the maximum number of samples that can be pooled together to accurately detect one positive sample carrying the severe acute respiratory syndrome-coronavirus 2 viral RNA from different pools. We made different pool sizes ranging from 5 to 30 samples. Three positive samples, covering the common range of polymerase chain reaction (PCR) threshold cycle values (an indirect indicator of viral load) observed in our patients, were selected, and different pools were made with known negative samples. The pools underwent real-time qualitative PCR for the determination of effective maximum pool size. It was observed that up to 20-sample pools of all positive samples could accurately be detected in terms of both E gene and RdRp gene, leading to considerable conservation of resources, time and workforce. However, while deciding the optimal pool size, the infection level in that particular geographical area and sensitivity of the test assay used (limit of detection) have to be taken into account., Competing Interests: None

Background & Objectives: Public health and diagnostic laboratories are facing huge sample loads for COVID-19 diagnosis by real-time reverse transcription-polymerase chain reaction (RT-PCR). High sensitivity of optimized real-time RT-PCR assays makes pooled testing a potentially efficient strategy for resource utilization when positivity rates for particular regions or groups of individuals are low. We report here a comparative analysis of pooled testing for 5- and 10-sample pools by real-time RT-PCR across 10 COVID-19 testing laboratories in India., Methods: Ten virus research and diagnostic laboratories (VRDLs) testing for COVID-19 by real-time RT-PCR participated in this evaluation. At each laboratory, 100 nasopharyngeal swab samples including 10 positive samples were used to create 5- and 10-sample pools with one positive sample in each pool. RNA extraction and real-time RT-PCR for SARS-CoV-2-specific E gene target were performed for individual positive samples as well as pooled samples. Concordance between individual sample testing and testing in the 5- or 10-sample pools was calculated, and the variation across sites and by sample cycle threshold (C t ) values was analyzed., Results: A total of 110 each of 5- and 10-sample pools were evaluated. Concordance between the 5-sample pool and individual sample testing was 100 per cent in the C t value ≤30 cycles and 95.5 per cent for C t values ≤33 cycles. Overall concordance between the 5-sample pooled and individual sample testing was 88 per cent while that between 10-sample pool and individual sample testing was 66 per cent. Although the concordance rates for both the 5- and 10-sample pooled testing varied across laboratories, yet for samples with C t values ≤33 cycles, the concordance was ≥90 per cent across all laboratories for the 5-sample pools., Interpretation & Conclusions: Results from this multi-site assessment suggest that pooling five samples for SARS-CoV-2 detection by real-time RT-PCR may be an acceptable strategy without much loss of sensitivity even for low viral loads, while with 10-sample pools, there may be considerably higher numbers of false negatives. However, testing laboratories should perform validations with the specific RNA extraction and RT-PCR kits in use at their centres before initiating pooled testing., Competing Interests: None

Background & Objectives: SARS-CoV-2 (Severe acute respiratory syndrome coronavirus-2) is evolving with the progression of the pandemic. This study was aimed to investigate the diversity and evolution of the coronavirus SARS-CoV-2 with progression of the pandemic over time and to identify similarities and differences of viral diversity and evolution across geographical regions (countries)., Methods: Publicly available data on type definitions based on whole-genome sequences of the SARS-CoV-2 sampled during December and March 2020 from 3636 infected patients spread over 55 countries were collected. Phylodynamic analyses were performed and the temporal and spatial evolution of the virus was examined., Results: It was found that (i) temporal variation in frequencies of types of the coronavirus was significant; ancestral viruses of type O were replaced by evolved viruses belonging to type A2a; (ii) spatial variation was not significant; with the spread of SARS-CoV-2, the dominant virus was the A2a type virus in every geographical region; (iii) within a geographical region, there was significant micro-level variation in the frequencies of the different viral types, and (iv) the evolved coronavirus of type A2a swept rapidly across all continents., Interpretation & Conclusions: SARS-CoV-2 belonging to the A2a type possesses a non-synomymous variant (D614G) that possibly eases the entry of the virus into the lung cells of the host. This may be the reason why the A2a type has an advantage to infect and survive and as a result has rapidly swept all geographical regions. Therefore, large-scale sequencing of coronavirus genomes and, as required, of host genomes should be undertaken in India to identify regional and ethnic variation in viral composition and its interaction with host genomes. Further, careful collection of clinical and immunological data of the host can provide deep learning in relation to infection and transmission of the types of coronavirus genomes., Competing Interests: None

Group A rotavirus (RVA) is recognized as a major cause of severe gastroenteritis in newborn calves and goat kids. We estimated the proportion of ruminants infected with rotavirus and identified the circulating genotypes in cattle and goats in Bangladesh. Between May 2009 and August 2010, fecal samples were collected from 520 cattle and goats presenting with diarrhea at three government veterinary hospitals in three districts of Bangladesh. All samples were screened for RVA RNA using real-time, one-step, reverse transcription polymerase chain reaction (qRT-PCR). Of the 520 animals tested, 11.7% (61) were positive for RVA RNA, with 6.2% (15/241) and 16.5% (46/279) positivity in cattle and goats, respectively. RVA positive samples were further characterized by nucleotide sequence analysis of two structural protein gene fragments, VP7 (G genotype), and VP4 (P genotype). Among 17 successfully sequenced strains, G8 (17.9%) was the most prevalent G-genotype followed by G10 (8%) and G6 (1.6%). P[1] (11.3%) was the most frequently detected P-genotype followed by P[11] (3.2%) and P[15] (1.6%). The most common VP7/VP4 combinations for cattle were G10P[11], G10P[15], and G6P[11], and for goat, G8P[1], and G10P[1]. Phylogenetic analysis of the RVA strains showed clustering with bovine and caprine strains from neighboring India. The study adds to our understanding of the genetic diversity of bovine and caprine rotavirus strains in Bangladesh. Our findings highlight the importance of rotavirus surveillance in cattle and goat populations, which may serve as a potential source for genetic reassortment and zoonotic transmission., Competing Interests: Declaration of Competing Interest Google and Rockefeller Foundation played no role in the study design nor in the collection, analysis, and interpretation of data, nor in the decision to submit the manuscript for publication. None of the authors has any other financial or personal relationships that could inappropriately influence or bias the content of the paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background & Objectives: Hepatitis A is prevalent worldwide and is among the leading cause of acute viral hepatitis in India. Major geographical differences in endemicity of hepatitis A are closely related to hygienic and sanitary conditions and other indicators of the level of socio-economic development. The present study was aimed to know the seropositivity prevalence and predominant circulating strain of HAV in a north India., Methods: Patients with acute viral hepatitis were enrolled. Blood samples were collected over a period of one year from June 2016 to May 2017. Serum samples were tested for anti-immunoglobulin M (IgM) HAV antibodies. The seropositive samples were analyzed for HAV-RNA by real-time reverse transcription-polymerase chain reaction (RT-PCR). Samples detected on molecular assay were subjected to conventional semi-nested RT-PCR for VP1 gene. Further sequencing of amplified RT-PCR products was done, and data were analyzed., Results: A total of 1615 patients were enrolled, and serum samples were collected and tested. The male:female ratio was 1.3:1 with a mean age of 24.31±17.02 yr (range 0-83 yr). Among these, 128 (7.93%) were positive for anti-HAV IgM antibodies; 41.63 per cent of seropositive patients were in their childhood or early adolescent age group. Of all seropositive samples, 59 (46.09%) were positive for HAV RNA. Genotyping sequencing of 10 representative strains was carried out, and the circulating genotype was found to be IIIA. The nucleotide sequences showed homology among the strains., Interpretation & Conclusions: Our results showed that hepatitis A was a common disease in children with IIIA as a circulating genotype in this region. In approximately 50 per cent of cases, HAV RNA could be detected. Higher number of HAV IgM-seropositive cases was observed during monsoon period., Competing Interests: None

The Kyasanur Forest Disease (KFD) has become a major public health problem in the State of Karnataka, India where the disease was first identified and in Tamil Nadu, Maharashtra, Kerala, and Goa covering the Western Ghats region of India. The incidence of positive cases and distribution of the Kyasanur Forest Disease virus (KFDV) in different geographical regions raises the need to understand the evolution and spatiotemporal transmission dynamics. Phylogeography analysis based on 48 whole genomes (46 from this study) and additionally 28 E-gene sequences of KFDV isolated from different regions spanning the period 1957-2017 was thus undertaken. The mean evolutionary rates based the E-gene was marginally higher than that based on the whole genomes. A subgroup of KFDV strains (2006-2017) differing from the early Karnataka strains (1957-1972) by ~2.76% in their whole genomes and representing spread to different geographical areas diverged around 1980. Dispersal from Karnataka to Goa and Maharashtra was indicated. Maharashtra represented a new source for transmission of KFDV since ~2013. Significant evidence of adaptive evolution at site 123 A/T located in the vicinity of the envelope protein dimer interface may have functional implications. The findings indicate the need to curtail the spread of KFDV by surveillance measures and improved vaccination strategies.

Background & Objectives: Since December 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has globally affected 195 countries. In India, suspected cases were screened for SARS-CoV-2 as per the advisory of the Ministry of Health and Family Welfare. The objective of this study was to characterize SARS-CoV-2 sequences from three identified positive cases as on February 29, 2020., Methods: Throat swab/nasal swab specimens for a total of 881 suspected cases were screened by E gene and confirmed by RdRp (1), RdRp (2) and N gene real-time reverse transcription-polymerase chain reactions and next-generation sequencing. Phylogenetic analysis, molecular characterization and prediction of B- and T-cell epitopes for Indian SARS-CoV-2 sequences were undertaken., Results: Three cases with a travel history from Wuhan, China, were confirmed positive for SARS-CoV-2. Almost complete (29,851 nucleotides) genomes of case 1, case 3 and a fragmented genome for case 2 were obtained. The sequences of Indian SARS-CoV-2 though not identical showed high (~99.98%) identity with Wuhan seafood market pneumonia virus (accession number: NC 045512). Phylogenetic analysis showed that the Indian sequences belonged to different clusters. Predicted linear B-cell epitopes were found to be concentrated in the S1 domain of spike protein, and a conformational epitope was identified in the receptor-binding domain. The predicted T-cell epitopes showed broad human leucocyte antigen allele coverage of A and B supertypes predominant in the Indian population., Interpretation & Conclusions: The two SARS-CoV-2 sequences obtained from India represent two different introductions into the country. The genetic heterogeneity is as noted globally. The identified B- and T-cell epitopes may be considered suitable for future experiments towards the design of vaccines and diagnostics. Continuous monitoring and analysis of the sequences of new cases from India and the other affected countries would be vital to understand the genetic evolution and rates of substitution of the SARS-CoV-2., Competing Interests: None

The present paper describes first full genome sequence of the Garlic virus D (GarV-D) from northern India with a genome size of 8425 bp long ssRNA. The infected leaves and bulbs of garlic variety Yamuna Safed (G-282) plants suspected for GarV-D infection were collected with the aim to identify contagion virus during March, 2018. The total RNA was extracted from the pooled garlic plants using TRIzol reagent and sequenced using an Illumina HiSeq 2000 platform. BLASTn search in the NCBI database identified contagion as GarV-D (MK518067). It shared 83.63-85.83% nucleotide sequence identities with other (GarV-D) isolates from Argentina (KF550407, KF555653, KR819505) and 83.15% with isolates from China (MF795136, MF363012). Keywords: Allium sativum; Allexivirus; Garlic virus D; India.

Direct massively parallel sequencing of SARS-CoV-2 genome was undertaken from nasopharyngeal and oropharyngeal swab samples of infected individuals in Eastern India. Seven of the isolates belonged to the A2a clade, while one belonged to the B4 clade. Specific mutations, characteristic of the A2a clade, were also detected, which included the P323L in RNA-dependent RNA polymerase and D614G in the Spike glycoprotein. Further, our data revealed emergence of novel subclones harbouring nonsynonymous mutations, viz. G1124V in Spike (S) protein, R203K, and G204R in the nucleocapsid (N) protein. The N protein mutations reside in the SR-rich region involved in viral capsid formation and the S protein mutation is in the S 2 domain, which is involved in triggering viral fusion with the host cell membrane. Interesting correlation was observed between these mutations and travel or contact history of COVID-19 positive cases. Consequent alterations of miRNA binding and structure were also predicted for these mutations. More importantly, the possible implications of mutation D614G (in S D domain) and G1124V (in S 2 subunit) on the structural stability of S protein have also been discussed. Results report for the first time a bird's eye view on the accumulation of mutations in SARS-CoV-2 genome in Eastern India.

Foot-and-mouth disease (FMD) affects the livestock industry in a transboundary manner. It is essential to understand the FMD phylodynamics to assist in the disease-eradication. FMD critically affects the Sri Lankan cattle industry causing substantial economic losses. Even though many studies have covered the serotyping and genotyping of FMD virus (FMDV) in Sri Lanka, there is a significant knowledge gap exists in understanding the FMDV phylodynamics in the country. In the present study, the VP1 genomic region of FMD viral isolates belonging to serotype C from Sri Lanka and other South Asian countries were sequenced. All the published VPI sequences of serotype C and most of the published VP1 sequences for lineage ME-SA/Ind-2001d of serotype O from Sri Lanka, India, and other South Asian countries were retrieved. The datasets of serotype C and serotype O were separately analyzed using Bayesian, maximum likelihood, and phylogenetic networking methods to infer the transboundary movements and evolutionary aspects of the FMDV incursions in Sri Lanka. A model-based approach was used to detect any possible recombination events of FMDV incursions. Our results revealed that the invasions of the topotype ASIA of serotype C and the lineage ME-SA/Ind-2001d have a similar pattern of transboundary movement and evolution. The haplotype networks and phylogenies developed in the present study confirmed that FMDV incursions in Sri Lanka mainly originate from the Indian subcontinent, remain quiet after migration, and then cause outbreaks in a subsequent year. Since there are no recombination events detected among the different viral strains across serotypes and topotypes, we can assume that the incursions tend to show the independent evolution compared to the ancestral viral populations. Thus, we highlight the need for thorough surveillance of cattle/ruminants and associated product-movement into Sri Lanka from other regions to prevent the transboundary movement of FMDV., Competing Interests: The authors have declared that no competing interests exist.

Purpose: Hepatitis E virus (HEV) is an emerging pathogen causing acute viral hepatitis worldwide. Clinical manifestations often occur in young adults with an increased mortality rate among pregnant women. HEV genotypes 1 and 4 are mainly reported among humans and swines, respectively., Aims: The aim was to study the currently circulating genotypes of HEV in India., Materials and Methods: A retrospective cross-sectional study was carried out at Manipal Institute of Virology to know the circulating genotypes of hepatitis E, spanning over 5 years from August 2014 to September 2018. The serum samples screened serologically positive and confirmed positive for active infection by real-time reverse transcriptase-polymerase chain reaction (PCR) (Real Star ® HEV RT-PCR Kit 2.0, Altona Diagnostics, GmbH, Hamburg, Germany) were further subjected to nested conventional PCR targeting the RdRp gene of non-structural ORF1 region. The purified PCR product was sequenced in BigDye Terminator v3.1 Cycle Sequencing Kit (Life Technologies, Thermo Fisher Scientific, USA). The chromatograms obtained by sequencing were analysed using Sequencher 5.4.6, and HEV FASTA sequences were compared with reference sequences for HEV in GenBank Nucleotide Blast., Results: During the study period, there were 317 cases of laboratory-confirmed cases of acute viral hepatitis comprising 202, 70, 43 and 2 cases of hepatitis A, E, B and C, respectively. Serum samples of 70 acute hepatitis cases were positive for anti-hepatitis E IgM. According to the clinical case classification, there were 66 cases of acute viral hepatitis and four cases of fulminant hepatic liver failure. The mean age of the patients was 30.3 years (standard deviation = 12.5). The samples from various parts of India were genotyped as 1a., Conclusion: The HEV genotypes 1a was observed to be the currently circulating strain in the regions studied., Competing Interests: None

Introduction: Human rhinovirus (HRV) and Enterovirus (ENV) are the major causes of childhood acute respiratory tract infections (ARTIs). This study sought to understand the distribution pattern of HRV subgroups, their seasonality and association with respiratory complications in patients at a tertiary care hospital., Results: Of the total 332 ARTI samples, 82 (24.7%) were positive for ENV/HRV. Twenty positive samples were processed further for phylogenetic analysis. Ten of the 20 samples were identified to be HRVs (70% HRV A and 30% HRV C) and nine were enteroviruses. HRV A clustered near three distinct HRV types (A12, A78 and A82). Four of the HRV strains (represented as SEQ 137 rhino, SEQ 282 rhino, SEQ 120 rhino and SEQ 82 rhino) had high sequence similarity. HRV C showed seasonality and was associated with disease severity., Conclusion: The genotyping and phylogenetic analysis of the HRVs in the current study shows its circulatory pattern, association with risk factors and evolutionary dynamics., Competing Interests: None

Objective: To characterize the in vitro replication fitness, viral diversity, and phylogeny of dengue viruses (DENV) isolated from Indian patients., Methods: DENV was isolated from whole blood collected from patients by passaging in cell culture. Passage 3 viruses were used for growth kinetics in C6/36 mosquito cells. Parallel efforts also focused on the isolation of DENV RNA from plasma samples of the same patients, which were processed for next-generation sequencing., Results: It was possible to isolate 64 clinical isolates of DENV, mostly DENV-2. Twenty-five of these were further used for growth curve analysis in vitro, which showed a wide range of replication kinetics. The highest viral titers were associated with isolates from patients with dengue with warning signs and severe dengue cases. Full genome sequences of 21 DENV isolates were obtained. Genome analysis mapped the circulating DENV-2 strains to the Cosmopolitan genotype., Conclusions: The replication kinetics of isolates from patients with mild or severe infection did not differ significantly, but the viral titers varied by two orders of magnitude between the isolates, suggesting differences in replication fitness among the circulating DENV-2., (Copyright © 2018 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background & Objectives: Influenza virological surveillance is an essential tool for the early detection of novel genetic variants of epidemiologic and clinical significance. This study was aimed to genetically characterize A(H1N1)pdm09 virus circulating in 2017 and to compare it with the global data., Methods: The regional/State Viral Research and Diagnostic Laboratories (VRDLs) provided influenza diagnosis for referred clinical samples and shared influenza A(H1N1)pdm09 positives with the Indian Council of Medical Research-National Institute of Virology (ICMR-NIV), Pune, India, for hemagglutinin (HA) gene phylogenetic analysis. Sites at Manipal, Jaipur and Dibrugarh performed the sequencing and shared the sequence data for analysis. The antiviral susceptibility of influenza viruses was assessed for known molecular marker H275Y at the ICMR-NIV, Pune., Results: All the eight VRDLs had well-established influenza diagnostic facilities and showed increased activity of influenza A(H1N1)pdm09 during 2017. Phylogenetic analysis showed that the viruses from the different regions of the country were similar to A/Michigan/45/2015 strain which was the 2017-2018 recommended vaccine strain and were clustered with the globally circulating clade 6B.1 with signature mutations S84N, S162N and I216T. The clade 6B.1 showed further subgrouping with additional mutations S74R, S164T and I295V; however, there was no significant association between the presence of these mutations and severity of disease due to influenza. All the study viruses were sensitive to oseltamivir., Interpretation & Conclusions: During the study period, all the study sites reported globally circulating A/Michigan/45/2015 vaccine strain of influenza A(H1N1)pdm09 viruses and remained sensitive to oseltamivir. Further genetic and antigenic characterization of influenza viruses is recommended to address public health concerns., Competing Interests: None