5' UTR m(6)A Promotes Cap-Independent Translation.

Protein translation typically begins with the recruitment of the 43S ribosomal complex to the 5' cap of mRNAs by a cap-binding complex. However, some transcripts are translated in a cap-independent manner through poorly understood mechanisms. Here, we show that mRNAs containing N(6)-methyladenosine (m(6)A) in their 5' UTR can be translated in a cap-independent manner. A single 5' UTR m(6)A directly binds eukaryotic initiation factor 3 (eIF3), which is sufficient to recruit the 43S complex to initiate translation in the absence of the cap-binding factor eIF4E. Inhibition of adenosine methylation selectively reduces translation of mRNAs containing 5'UTR m(6)A. Additionally, increased m(6)A levels in the Hsp70 mRNA regulate its cap-independent translation following heat shock. Notably, we find that diverse cellular stresses induce a transcriptome-wide redistribution of m(6)A, resulting in increased numbers of mRNAs with 5' UTR m(6)A. These data show that 5' UTR m(6)A bypasses 5' cap-binding proteins to promote translation under stresses.

m6A enhances the phase separation potential of mRNA.

N6-methyladenosine (m6A) is the most prevalent modified nucleotide in mRNA1,2, with around 25% of mRNAs containing at least one m6A. Methylation of mRNA to form m6A is required for diverse cellular and physiological processes3. Although the presence of m6A in an mRNA can affect its fate in different ways, it is unclear how m6A directs this process and why the effects of m6A can vary in different cellular contexts. Here we show that the cytosolic m6A-binding proteins-YTHDF1, YTHDF2 and YTHDF3-undergo liquid-liquid phase separation in vitro and in cells. This phase separation is markedly enhanced by mRNAs that contain multiple, but not single, m6A residues. Polymethylated mRNAs act as a multivalent scaffold for the binding of YTHDF proteins, juxtaposing their low-complexity domains and thereby leading to phase separation. The resulting mRNA-YTHDF complexes then partition into different endogenous phase-separated compartments, such as P-bodies, stress granules or neuronal RNA granules. m6A-mRNA is subject to compartment-specific regulation, including a reduction in the stability and translation of mRNA. These studies reveal that the number and distribution of m6A sites in cellular mRNAs can regulate and influence the composition of the phase-separated transcriptome, and suggest that the cellular properties of m6A-modified mRNAs are governed by liquid-liquid phase separation principles.

Unravelling the genomic origins of lumpy skin disease virus in recent outbreaks.

Lumpy skin disease virus (LSDV) belongs to the genus Capripoxvirus and family Poxviridae. LSDV was endemic in most of Africa, the Middle East and Turkey, but since 2015, several outbreaks have been reported in other countries. In this study, we used whole genome sequencing approach to investigate the origin of the outbreak and understand the genomic landscape of the virus. Our study showed that the LSDV strain of 2022 outbreak exhibited many genetic variations compared to the Reference Neethling strain sequence and the previous field strains. A total of 1819 variations were found in 22 genome sequences, which includes 399 extragenic mutations, 153 insertion frameshift mutations, 234 deletion frameshift mutations, 271 Single nucleotide polymorphisms (SNPs) and 762 silent SNPs. Thirty-eight genes have more than 2 variations per gene, and these genes belong to viral-core proteins, viral binding proteins, replication, and RNA polymerase proteins. We highlight the importance of several SNPs in various genes, which may play an essential role in the pathogenesis of LSDV. Phylogenetic analysis performed on all whole genome sequences of LSDV showed two types of variants in India. One group of the variant with fewer mutations was found to lie closer to the LSDV 2019 strain from Ranchi while the other group clustered with previous Russian outbreaks from 2015. Our study highlights the importance of genomic characterization of viral outbreaks to not only monitor the frequency of mutations but also address its role in pathogenesis of LSDV as the outbreak continues.

Comparative immune profiling in survivors of the 2023 Nipah outbreak in Kerala state, India.

Immune profiling of Nipah virus (NiV) infection survivors is essential for advancing our understanding of NiV pathogenesis, improving diagnostic and therapeutic strategies, and guiding public health efforts to prevent future outbreaks. There is currently limited data available on the immune response to NiV infection. We aimed to elucidate the specific immune mechanisms involved in protection against NiV infection by analyzing the immune profiles of survivors of the Nipah outbreak in Kerala, India 2023. Immune cell populations were quantified and compared between survivors (up to 4 months post onset day of illness) and healthy controls. Statistical analysis was performed to explore associations between immune profiles and clinical outcomes. Immune signatures common to all three cases were: a heretofore undescribed persistent lymphopenia including the CD4+ Treg compartment with the relative expansion of memory Tregs; trends indicative of global leukopenic modulation were observed in monocytes and granulocytes including an expansion of putatively immunosuppressive low-density granulocytes described recently in the context of severe COVID-19; altered mucosal homing with respect to integrin beta-7 (ITGB7) expressing subsets; increased mobilization of activated T-cells (CD4+ and CD8+) and plasmablasts in the early phase of infection. Comparative analysis based on clinical presentation and outcome yielded lower initial viremia, increased activated T-cell responses, expanded plasmablasts, and restoration of ITGB7 expressing CD8+ T-cells as possible protective signatures. This longitudinal study delineates putative protective signatures associated with milder NiV disease. It emphasizes the need for the development of immunotherapeutic interventions such as monoclonal antibodies to blunt early viremia and ameliorate pathogenesis.

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.

Reversible methylation of m6Am in the 5' cap controls mRNA stability.

Internal bases in mRNA can be subjected to modifications that influence the fate of mRNA in cells. One of the most prevalent modified bases is found at the 5' end of mRNA, at the first encoded nucleotide adjacent to the 7-methylguanosine cap. Here we show that this nucleotide, N6,2'-O-dimethyladenosine (m6Am), is a reversible modification that influences cellular mRNA fate. Using a transcriptome-wide map of m6Am we find that m6Am-initiated transcripts are markedly more stable than mRNAs that begin with other nucleotides. We show that the enhanced stability of m6Am-initiated transcripts is due to resistance to the mRNA-decapping enzyme DCP2. Moreover, we find that m6Am is selectively demethylated by fat mass and obesity-associated protein (FTO). FTO preferentially demethylates m6Am rather than N6-methyladenosine (m6A), and reduces the stability of m6Am mRNAs. Together, these findings show that the methylation status of m6Am in the 5' cap is a dynamic and reversible epitranscriptomic modification that determines mRNA stability.

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.

m(6)A RNA methylation promotes XIST-mediated transcriptional repression.

The long non-coding RNA X-inactive specific transcript (XIST) mediates the transcriptional silencing of genes on the X chromosome. Here we show that, in human cells, XIST is highly methylated with at least 78 N6-methyladenosine (m6A) residues-a reversible base modification of unknown function in long non-coding RNAs. We show that m6A formation in XIST, as well as in cellular mRNAs, is mediated by RNA-binding motif protein 15 (RBM15) and its paralogue RBM15B, which bind the m6A-methylation complex and recruit it to specific sites in RNA. This results in the methylation of adenosine nucleotides in adjacent m6A consensus motifs. Furthermore, we show that knockdown of RBM15 and RBM15B, or knockdown of methyltransferase like 3 (METTL3), an m6A methyltransferase, impairs XIST-mediated gene silencing. A systematic comparison of m6A-binding proteins shows that YTH domain containing 1 (YTHDC1) preferentially recognizes m6A residues on XIST and is required for XIST function. Additionally, artificial tethering of YTHDC1 to XIST rescues XIST-mediated silencing upon loss of m6A. These data reveal a pathway of m6A formation and recognition required for XIST-mediated transcriptional repression.

Metagenomic analysis of bacterial communities during estrous cycle in Bos indicus.

The present study, using 16 s rRNA sequencing of the V3-V4 hypervariable region, was aimed to check diversity of vaginal microbiota throughout different stages of the estrous cycle in Bos indicus, with attention to changes in progesterone hormone and microorganism diversity. Metagenomic research was conducted on vaginal swabs obtained from nine healthy Indian Gir cows at estrus (day 0), metestrus (day 04), diestrus (day 12), and proestrus (day 16) phases of the estrous cycle. The findings revealed that the diestrus phase has a different bacterial diversity than the other three estrous cycle phases, implying that progesterone affects bacterial diversity. Proteobacteria and Firmicutes were the most abundant phyla at the phylum level, accounting for 94% of bacterial diversity. Actinobacteriota, Patescibacteria, Cyanobacteria, and Bacteroidota were among the less prevalent phyla observed in all samples. After statistical analysis, Bacillaceae, Alcaligenes, Enterobacteriaceae, and Morganellaceae families were more significant. The Enterobacteriaceae family was found to be lower in the diestrus phase than in the other three phases; in contrast, all statistically significant genera were high at the diestrus phase. The luteal stage had higher levels of Micrococcus, Stenotrophomonas, UGC-010, Massilia, and Methylobacillus than the follicular stage, and statistical analysis revealed substantial difference between the luteal and follicular stages. Lactobacillus genus was present in both the estrus and diestrus phases. This study represents an important step toward the understanding of microbial diversity within different stages of the estrous cycle of Indian cows.

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.

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.

Recent Progress in Devices Based on Magnetoelectric Composite Thin Films.

The strain-driven interfacial coupling between the ferromagnetic and ferroelectric constituents of magnetoelectric (ME) composites makes them potential candidates for novel multifunctional devices. ME composites in the form of thin-film heterostructures show promising applications in miniaturized ME devices. This article reports the recent advancement in ME thin-film devices, such as highly sensitive magnetic field sensors, ME antennas, integrated tunable ME inductors, and ME band-pass filters, is discussed. (Pb1-xZrx)TiO3 (PZT), Pb(Mg1/3Nb2/3)O3-PbTiO3 (PMN-PT), Aluminium nitride (AlN), and Al1-xScxN are the most commonly used piezoelectric constituents, whereas FeGa, FeGaB, FeCo, FeCoB, and Metglas (FeCoSiB alloy) are the most commonly used magnetostrictive constituents in the thin film ME devices. The ME field sensors offer a limit of detection in the fT/Hz1/2 range at the mechanical resonance frequency. However, below resonance, different frequency conversion techniques with AC magnetic or electric fields or the delta-E effect are used. Noise floors of 1-100 pT/Hz1/2 at 1 Hz were obtained. Acoustically actuated nanomechanical ME antennas operating at a very-high frequency as well as ultra-high frequency (0.1-3 GHz) range, were introduced. The ME antennas were successfully miniaturized by a few orders smaller in size compared to the state-of-the-art conventional antennas. The designed antennas exhibit potential application in biomedical devices and wearable antennas. Integrated tunable inductors and band-pass filters tuned by electric and magnetic field with a wide operating frequency range are also discussed along with miniaturized ME energy harvesters.

Potential Induced Radioactivity in Materials Processed with X-ray Energy Above 5 MeV.

Section 5.1.2 of ANSI/AAMI/ISO 11137-1 states that "the potential for induced radioactivity in product shall be assessed." This article describes how compliance with this requirement may be achieved using qualified test methods. Materials of consideration are conceptually discussed, and results of testing conducted on products processed with a 7.5-MeV X-ray irradiation process are provided. As X-ray becomes more widely used in healthcare sterilization, having standard assessment protocols for activation coupled with a shared database of material test results will benefit manufacturers seeking to utilize this innovative technology.

Laboratory surveillance of chikungunya in Madhya Pradesh, India (2016-2017).

Background & objectives: Chikungunya (CHIK) is a neglected, re-emerging arboviral disease. Limited information on CHIK-confirmed cases during interepidemic period is available from India. This surveillance study was conducted in Madhya Pradesh (MP), India, during the years 2016-2017, to provide information about CHIK cases. Methods: Blood samples collected from patients suspected having CHIK were tested by immunoglobulin (Ig) IgM ELISA or real time reverse transcription-polymerase chain reaction (rRT-PCR) for the detection of CHIK virus (CHIKV)-specific IgM antibodies or viral RNA, respectively. Partial envelope-1 gene sequencing was done. Clinical and demographic data were collected and analyzed. Results: Of the 4019 samples tested, 494 (12.2%) were found positive for CHIKV infection. The positivity was detected in both rural and urban areas. The mean age of CHIK-positive cases was 33.12±18.25 yr. Headache and joint pain were the most prominent symptoms, 34.6 per cent (171/494) of the CHIK cases required hospitalization and six patients with CHIKV infection died. The East/Central/South African genotype of CHIKV was found to be circulating in the study area. Interpretation & conclusions: Our study recorded a higher CHIK positivity during 2016-2017 in comparison to earlier reports from MP, India. A high proportion of CHIK cases required hospitalization and deaths were also reported, which indicated the severity of the disease in the study area. In-depth molecular analysis of the virus and other risk factors is essential to understand the trends in disease severity.

Crimean Congo hemorrhagic fever serosurvey in humans for identifying high-risk populations and high-risk areas in the endemic state of Gujarat, India.

BACKGROUND: Crimean Congo Hemorrhagic Fever (CCHF) is a highly infectious zoonotic disease of humans transmitted by Hyalomma ticks. Earlier studies have shown CCHF seroprevalence in livestock throughout India, yet sporadic outbreaks have been recorded mostly from the Gujarat state of India since 2011. Occupational vulnerability to CCHF for animal handlers, veterinarians, abattoir workers, and healthcare workers has been documented. The current study was planned to determine the seroprevalence of CCHF with an intention to identify the high -risk population and high -risk areas from Gujarat state, India. METHODS: Based on the socio-clinical data, the human population of Gujarat was divided into eight categories viz. A: CCHF affected person/house/close contact, B: Neighborhood contacts, C: Animal handlers, D: General population, E: Farmers, F: Abattoir workers, G: Veterinarian, H: Healthcare workers. A total of 4978 human serum samples were collected from 33 districts of Gujarat during year 2015, 2016 and 2017. All the samples were screened for the presence of anti-CCHFV IgG using indigenously developed anti-CCHFV IgG ELISA. Univariate regression analysis was performed to recognize significant risk factors for CCHF seropositivity. RESULTS: Twenty-five serum samples were found to be positive with an overall CCHF human seropositivity of 0.5% (95% CI 0.30-0.74%). Gender predisposition to CCHF prevalence was observed in males (OR: 2.80; p-value: 0.020). The risk for seropositivity increased sevenfold when a person was in contact or neighbor with a CCHF case (OR 7.02; p-value: < 0.0001). No significant difference in seropositivity was observed within different age groups. Veterinarians, healthcare workers, and control group were found to be seronegative for CCHF. CONCLUSIONS: In-spite of CCHF sporadic outbreaks reported in Gujarat, the seropositivity for CCHF in the state was low as compared to other endemic countries. Males, close contacts and neighbors were identified as a high-risk population for CCHF infection. To recognize the high-risk area, tick screening and animal serosurvey would be a wiser choice. The study also suggests circulation and under diagnoses of CCHFV in the naïve regions of Gujarat.

Identification and characterization of novel mosquito-borne (Kammavanpettai virus) and tick-borne (Wad Medani) reoviruses isolated in India.

In 1954, a virus named Wad Medani virus (WMV) was isolated from Hyalomma marginatum ticks from Maharashtra State, India. In 1963, another virus was isolated from Sturnia pagodarum birds in Tamil Nadu, India, and named Kammavanpettai virus (KVPTV) based on the site of its isolation. Originally these virus isolates could not be identified with conventional methods. Here we describe next-generation sequencing studies leading to the determination of their complete genome sequences, and identification of both virus isolates as orbiviruses (family Reoviridae). Sequencing data showed that KVPTV has an AT-rich genome, whereas the genome of WMV is GC-rich. The size of the KVPTV genome is 18 234 nucleotides encoding proteins ranging 238-1290 amino acids (aa) in length. Similarly, the size of the WMV genome is 16 941 nucleotides encoding proteins ranging 214-1305 amino acids in length. Phylogenetic analysis of the VP1 gene, along with the capsid genes VP5 and VP7, revealed that KVPTV is likely a novel mosquito-borne virus and WMV is a tick-borne orbivirus. This study focuses on the phylogenetic comparison of these newly identified orbiviruses with mosquito-, tick- and Culicoides-borne orbiviruses isolated in India and other countries.