Development of a multiplexed Luminex assay for simultaneous detection of enteric viruses in cattle.

Viral and bacterial gastroenteritis and diarrhea have long been a problem in livestock with devastating effects on animal health and production causing a heavy financial burden on producers. Therefore, the bead-based multiplex detection assay was created for simultaneous detection of three livestock viral diarrheic agents viz. bovine rotavirus (BRV), bovine coronavirus (BCoV) and bluetongue virus (BTV). The primers and probes for triplex MAGPIX assay for simultaneous detection of three enteric viruses were designed and the assay was optimized for hybridization temperature, primer-probe and bead concentrations. The newly developed MAGPIX assay was used to determine the prevalence of these diarrhea-associated viruses by testing 200 fecal samples collected from Haryana state of India during 2018-2019. The limit of detection of the developed triplex assay was 1 × 105, 1 × 104, and 1 × 105 RNA copies for BRV, BCoV, and BTV, respectively, being lower than the reverse transcription-quantitative polymerase chain reaction (RT-qPCR). However, it was higher than the conventional RT-PCR, showing it to be more sensitive. The newly developed MAGPIX assay was a rapid, cost-effective and high throughput diagnostic tool for identification of three major entero-pathogenic diarrhea associated viruses, either alone or in tandem, with the aim to prevent and control viral diarrhea in animals.

Prevalence of porcine viral respiratory diseases in India.

The pig industry is growing rapidly in India and contributes a major share of growth in the livestock sector. Over the last few years, there is a gradual increase in the adoption of pigs for production by economically weaker sections of the country. However, this production is affected by many respiratory diseases which are responsible for significant economic loss. The occurrence and impact of these diseases are still under-documented. The four important pathogens including porcine circovirus type 2 (PCV2), porcine reproductive and respiratory syndrome virus (PRRSV), swine influenza A viruses (SIV) and classical swine fever virus (CSFV) are documented here. These diseases are highly devastating in nature and frequent outbreaks have been reported from different parts of the country. The rapid and specific diagnosis, effective prevention and control measures are required for the eradication of these diseases which is urgently required for the growth of the pig industry. This review highlights the prevalence, epidemiology, diagnostics and information gaps on important respiratory viral pathogens of pigs reported from different parts of India. This review also emphasizes the importance of these viral diseases and the urgent need to develop vaccines and effective measures for the eradication of these diseases.

Co-infection of bluetongue virus serotypes 12 and 16 in sheep from Haryana, India.

World Organization for Animal Health has listed bluetongue (BT) under notifiable diseases. The BT is an arboviral infectious disease of domestic and wild ruminants caused by the bluetongue virus (BTV). Southern states of India had remained the point of attention for BT since first presence in 1964 in Maharashtra. Recently, northern states of India have also been reported positive for BTV in small ruminants. The present study reported the dual infection of BTV serotypes, BTV-12 and -16 in sheep population from Sirsa district of Haryana in the year 2016. After detection and serotyping with Seg-2 specific real time polymerase chain reaction (PCR), the Seg-2 and Seg-6 of BTV were PCR amplified and sequenced. On phylogenetic analysis it was detected to be clustered in nucleotype G and nucleotype B specific for BTV-12 and BTV-16, respectively. This was the first report of BTV-16 from Haryana. The results signified the co-infection of two different serotypes in an animal from a single outbreak.

Development of real-time RT-PCR systems for detection and quantitation of bovine enteric viral pathogens.

The enteric viruses in animals are responsible for severe and devastating losses to the livestock owners with a profound negative impact on animal, health, welfare, and productivity. These viruses are usually transmitted via the feco-oral route and primarily infect the digestive tract of the humans, bovines and different mammals as well as birds. Some of the important enteric viruses in ruminants are: Rotavirus A (RVA), Peste des petits virus (PPRV), Norovirus (NV), Bovine corona virus (BoCV) and Bluetongue virus (BTV). In the present study, sensitive, specific and reliable TaqMan probe-based RT-qPCRs were developed and standardized for the rapid detection and quantification of enteric viruses from fecal samples. The assays result in efficient amplification of the RVA, BTV and BoCV RNA with a limit of detection (LoD) of 5, 5 and 4 copies, respectively, which is 1000 times more sensitive than the traditional gel-based RT-PCR. The reproducibility of each assay was satisfactory, thus allowing for a sensitive and accurate measurement of the viral RNA load in clinical samples. In conclusion, real time PCR developed for these viruses are highly specific and sensitive technique for the detection of diarrheic viral pathogens of cattle and buffalo.

VP2 Gene-Based Molecular Evolutionary Patterns of Major Circulating Bluetongue Virus Serotypes Isolated during 2014-2018 from Telangana and Andhra Pradesh States of India.

INTRODUCTION: Bluetongue disease is an economically important viral disease of livestock caused by bluetongue virus (BTV) having multiple serotypes. It belongs to the genus Orbivirus of family Reoviridae and subfamily Sedoreovirinae. The genome of BTV is 10 segmented dsRNA that codes for 7 structural and 4 nonstructural proteins, of which VP2 was reported to be serotype-specific and a major antigenic determinant. OBJECTIVE: It is important to know the circulating serotypes in a particular geographical location for effective control of the disease. The present study unravels the molecular evolution of the circulating BTV serotypes during 2014-2018 in Telangana and Andhra Pradesh states of India. METHODS: Multiple sequence alignment with available BTV serotypes in GenBank and phylogenetic analysis were performed for the partial VP2 sequences of major circulating BTV serotypes during the study period. RESULTS: The multiple sequence alignment of circulating serotypes with respective reference isolates revealed variations in antigenic VP2. The phylogenetic analysis revealed that the major circulating serotypes were grouped into eastern topotypes (BTV-1, BTV-2, BTV-4, and BTV-16) and Western topotypes (BTV-5, BTV-12, and BTV-24). CONCLUSION: Our study strengthens the need for development of an effective vaccine, which can induce the immune response for a range of serotypes within and in between topotypes.

Contrasting selective patterns across the segmented genome of bluetongue virus in a global reassortment hotspot.

For segmented viruses, rapid genomic and phenotypic changes can occur through the process of reassortment, whereby co-infecting strains exchange entire segments creating novel progeny virus genotypes. However, for many viruses with segmented genomes, this process and its effect on transmission dynamics remain poorly understood. Here, we assessed the consequences of reassortment for selection on viral diversity through time using bluetongue virus (BTV), a segmented arbovirus that is the causative agent of a major disease of ruminants. We analysed ninety-two BTV genomes isolated across four decades from India, where BTV diversity, and thus opportunities for reassortment, are among the highest in the world. Our results point to frequent reassortment and segment turnover, some of which appear to be driven by selective sweeps and serial hitchhiking. Particularly, we found evidence for a recent selective sweep affecting segment 5 and its encoded NS1 protein that has allowed a single variant to essentially invade the full range of BTV genomic backgrounds and serotypes currently circulating in India. In contrast, diversifying selection was found to play an important role in maintaining genetic diversity in genes encoding outer surface proteins involved in virus interactions (VP2 and VP5, encoded by segments 2 and 6, respectively). Our results support the role of reassortment in driving rapid phenotypic change in segmented viruses and generate testable hypotheses for in vitro experiments aiming at understanding the specific mechanisms underlying differences in fitness and selection across viral genomes.

Quantitative RT-PCR assays for identification and typing of the Equine encephalosis virus.

Equine encephalosis (EE) is an acute, arthropod-borne, noncontagious, febrile disease of equids. The clinical signs of EE are similar to milder forms of African horse sickness (AHS) and the two diseases can be easily confused. The Equine encephalosis virus (EEV) is a distinct virus species within the genus Orbivirus, family Reoviridae, with ten linear segments of dsRNA genome. Seven distinct serotypes of EEV have been recognised on the basis of sequence analyses of Seg-2. The need for differential diagnosis of similar forms of EE and AHS warranted the development of molecular diagnostic methods for specific detection and identification of EEV. We report the development of quantitative real-time RT-PCR assay for detection of any member of the EEV species targeting the highly conserved EEV Seg-9. Similar serotype-specific qRT-PCR assays were designed for each of the seven EEV serotypes targeting genome Seg-2, encoding the serotype determining VP2 protein. These assays were evaluated using different EEV serotypes and other closely related orbiviruses. They were shown to be EEV virus species-specific, or EEV type-specific capable of detecting 1 to 13 copies of viral RNA in clinical samples. The assays failed to detect RNA from closely related orbiviruses, including AHSV and Peruvian horse sickness virus (PHSV) isolates.

Molecular analysis of genome segment-3 of bluetongue virus serotype 12 isolates from Haryana.

AIM: The present study was designed to characterize the genome segment 3 (Seg-3) of bluetongue virus (BTV) serotype 12 isolates from different outbreaks of Bluetongue disease in Haryana, India. MATERIALS AND METHODS: Blood and swab samples were collected from goat and sheep suspected to be suffering of BT from different outbreaks from Gurugram, Sirsa, Hisar, and Karnal districts of Haryana. The samples were grown in insect and mammalian cell lines. After preliminary identification, serotyping was done using BTV type-specific quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assays. Sequencing was performed using terminal and walking internal primers specific for Seg-3 on ABI Capillary Sequencer 3130 using a "BigDye cycle sequencing kit." The obtained sequence data were analyzed with various bioinformatic tools. RESULTS: Real-time PCR results confirmed the samples to be positive for BTV-12. The Seg-3 of Indian isolates was most closely related to that of a south Indian isolate of BTV-12 from Andhra Pradesh (KC662614) with 97% nucleotide identity. CONCLUSIONS: The study confirmed the circulation of BTV-12 in Haryana, India. The variations shown in genome Seg-3 of BTV-12 isolates may have some significance and need to be further explored.

A comprehensive study on seroprevalence of bluetongue virus in Haryana state of India.

AIM: The aim of present study was to determine seroprevalence of bluetongue virus (BTV) in Haryana state of India. MATERIALS AND METHODS: A total of 803 serum samples, 408 of cattle and 395 of buffalo origin, respectively, were collected from different villages of Haryana. Sampling was done randomly to obtain unbiased results. The samples were evaluated by a competitive enzyme-linked immunosorbent assay for the presence of BTV antibodies. RESULTS: Overall seroprevalence of BTV antibody in cattle and buffaloes for all 21 districts of Haryana state was found to be 75.49% and 92.91%, respectively. The prevalence of BTV in different agroclimatic zones ranged between 72-77% and 90-94% for cattle and buffalo, respectively. In buffaloes, the BTV seroprevalence was comparatively higher than in cattle. CONCLUSION: The study showed that BTV is circulating in cattle and buffalo populations in the Northern part of India.

Development and Evaluation of Real Time RT-PCR Assays for Detection and Typing of Bluetongue Virus.

Bluetongue virus is the type species of the genus Orbivirus, family Reoviridae. Bluetongue viruses (BTV) are transmitted between their vertebrate hosts primarily by biting midges (Culicoides spp.) in which they also replicate. Consequently BTV distribution is dependent on the activity, geographic distribution, and seasonal abundance of Culicoides spp. The virus can also be transmitted vertically in vertebrate hosts, and some strains/serotypes can be transmitted horizontally in the absence of insect vectors. The BTV genome is composed of ten linear segments of double-stranded (ds) RNA, numbered in order of decreasing size (Seg-1 to Seg-10). Genome segment 2 (Seg-2) encodes outer-capsid protein VP2, the most variable BTV protein and the primary target for neutralising antibodies. Consequently VP2 (and Seg-2) determine the identity of the twenty seven serotypes and two additional putative BTV serotypes that have been recognised so far. Current BTV vaccines are serotype specific and typing of outbreak strains is required in order to deploy appropriate vaccines. We report development and evaluation of multiple 'TaqMan' fluorescence-probe based quantitative real-time type-specific RT-PCR assays targeting Seg-2 of the 27+1 BTV types. The assays were evaluated using orbivirus isolates from the 'Orbivirus Reference Collection' (ORC) held at The Pirbright Institute. The assays are BTV-type specific and can be used for rapid, sensitive and reliable detection / identification (typing) of BTV RNA from samples of infected blood, tissues, homogenised Culicoides, or tissue culture supernatants. None of the assays amplified cDNAs from closely related but heterologous orbiviruses, or from uninfected host animals or cell cultures.

Full-Genome Sequencing as a Basis for Molecular Epidemiology Studies of Bluetongue Virus in India.

Since 1998 there have been significant changes in the global distribution of bluetongue virus (BTV). Ten previously exotic BTV serotypes have been detected in Europe, causing severe disease outbreaks in naïve ruminant populations. Previously exotic BTV serotypes were also identified in the USA, Israel, Australia and India. BTV is transmitted by biting midges (Culicoides spp.) and changes in the distribution of vector species, climate change, increased international travel and trade are thought to have contributed to these events. Thirteen BTV serotypes have been isolated in India since first reports of the disease in the country during 1964. Efficient methods for preparation of viral dsRNA and cDNA synthesis, have facilitated full-genome sequencing of BTV strains from the region. These studies introduce a new approach for BTV characterization, based on full-genome sequencing and phylogenetic analyses, facilitating the identification of BTV serotype, topotype and reassortant strains. Phylogenetic analyses show that most of the equivalent genome-segments of Indian BTV strains are closely related, clustering within a major eastern BTV 'topotype'. However, genome-segment 5 (Seg-5) encoding NS1, from multiple post 1982 Indian isolates, originated from a western BTV topotype. All ten genome-segments of BTV-2 isolates (IND2003/01, IND2003/02 and IND2003/03) are closely related (>99% identity) to a South African BTV-2 vaccine-strain (western topotype). Similarly BTV-10 isolates (IND2003/06; IND2005/04) show >99% identity in all genome segments, to the prototype BTV-10 (CA-8) strain from the USA. These data suggest repeated introductions of western BTV field and/or vaccine-strains into India, potentially linked to animal or vector-insect movements, or unauthorised use of 'live' South African or American BTV-vaccines in the country. The data presented will help improve nucleic acid based diagnostics for Indian serotypes/topotypes, as part of control strategies.

Genetic characterization of the tick-borne orbiviruses.

The International Committee for Taxonomy of Viruses (ICTV) recognizes four species of tick-borne orbiviruses (TBOs): Chenuda virus, Chobar Gorge virus, Wad Medani virus and Great Island virus (genus Orbivirus, family Reoviridae). Nucleotide (nt) and amino acid (aa) sequence comparisons provide a basis for orbivirus detection and classification, however full genome sequence data were only available for the Great Island virus species. We report representative genome-sequences for the three other TBO species (virus isolates: Chenuda virus (CNUV); Chobar Gorge virus (CGV) and Wad Medani virus (WMV)). Phylogenetic comparisons show that TBOs cluster separately from insect-borne orbiviruses (IBOs). CNUV, CGV, WMV and GIV share low level aa/nt identities with other orbiviruses, in 'conserved' Pol, T2 and T13 proteins/genes, identifying them as four distinct virus-species. The TBO genome segment encoding cell attachment, outer capsid protein 1 (OC1), is approximately half the size of the equivalent segment from insect-borne orbiviruses, helping to explain why tick-borne orbiviruses have a ~1 kb smaller genome.

Genome Sequence of Bluetongue Virus Type 2 from India: Evidence for Reassortment between Outer Capsid Protein Genes.

Southern Indian isolate IND1994/01 of bluetongue virus serotype 2 (BTV-2), from the Orbivirus Reference Collection at the Pirbright Institute (http://www.reoviridae.org/dsRNA_virus_proteins/ReoID/btv-2.htm#IND1994/01), was sequenced. Its genome segment 6 (Seg-6) [encoding VP5(OCP2)] is identical to that of the Indian BTV-1 isolate (IND2003/05), while Seg-5 and Seg-9 are closely related to isolates from South Africa and the United States, respectively.

Development and evaluation of loop-mediated isothermal amplification assay for rapid detection of Capripoxvirus.

AIM: The present study was undertaken to develop a nucleic acid-based diagnostic assay loop-mediated isothermal amplification assay (LAMP) targeting highly conserved genomic regions of Capripoxvirus (CaPVs) and its comparative evaluation with real-time polymerase chain reaction (PCR). MATERIAL AND METHODS: Lyophilized vaccine strain of sheeppox virus (SPPV) was used for optimization of LAMP assay. The LAMP assay was designed using envelope immunogenic protein (P32) coding gene targeting highly conserved genomic regions of CaPV responsible for causing sheep pox, goat pox, and lumpy skin disease in sheep, goat and cattle respectively. Serial tenfold dilution of SPPV recombinant plasmid DNA was used for a calculating limit of detection. Analytical sensitivity and specificity were performed. RESULTS: The test described is quick (30 min), sensitive and specific for detection of CaPVs. The described assay did not show any cross-reactivity to other related viruses that cause apparently similar clinical signs. It was found to be ten times more sensitive than conventional PCR however, 100 times less sensitive than quantitative PCR (qPCR). LAMP assay results were monitored by color change method using picogreen dye and agarose gel electrophoresis. CONCLUSION: LAMP assay can be a very good alternative for CaPV detection to other molecular techniques requiring sophisticated equipments.

A quantitative real-time reverse transcription PCR (qRT-PCR) assay to detect genome segment 9 of all 26 bluetongue virus serotypes.

Bluetongue (BT) is an arboviral disease, which can often be fatal in naïve sheep and white tailed deer, but is usually less severe, or unapparent in other ruminants. Twenty-six bluetongue virus (BTV) serotypes have been recognised so far, two of which (BTV-25 and BTV-26) were recently identified by phylogenetic comparisons of genome-segment/outer-capsid protein VP2 (subsequently confirmed by serological 'virus-neutralisation' assays). Rapid, sensitive, reliable and quantitative diagnostic-assays for detection and identification of BTV represent important components of effective surveillance and control strategies. The BTV genome comprises 10 linear segments of dsRNA. We describe a 'TaqMan' fluorescence-probe based quantitative real-time RT-PCR assay, targeting the highly conserved genome-segment-9 (encoding the viral-helicase 'VP6' and NS4). The assay detected Seg-9 from isolates of all 26 BTV types, as well as from clinical samples derived from BTV-6w and BTV-8w outbreaks (in Europe), BTV-25 from Switzerland, BTV-26 from Kuwait, BTV-1w, BTV-4w and BTV-8w from Spain, BTV-4w, BTV-8, BTV-10 and BTV-16 from Brazil. Assay efficiency was evaluated with RNA derived from the reference strain of BTV-1w [RSArrrr/01] and was 99.6%, detecting down to 4 copies per reaction. Samples from uninfected insect or mammalian cell-cultures, hosts-species (uninfected sheep blood) or vector-insects, all gave negative results. The assay failed to detect RNA from heterologous but related Orbivirus species (including the nine African horse sickness virus [AHSV] and seven epizootic haemorrhagic disease virus [EHDV] serotypes).

Full genome characterization of the culicoides-borne marsupial orbiviruses: Wallal virus, Mudjinbarry virus and Warrego viruses.

Viruses belonging to the species Wallal virus and Warrego virus of the genus Orbivirus were identified as causative agents of blindness in marsupials in Australia during 1994/5. Recent comparisons of nucleotide (nt) and amino acid (aa) sequences have provided a basis for the grouping and classification of orbivirus isolates. However, full-genome sequence data are not available for representatives of all Orbivirus species. We report full-genome sequence data for three additional orbiviruses: Wallal virus (WALV); Mudjinabarry virus (MUDV) and Warrego virus (WARV). Comparisons of conserved polymerase (Pol), sub-core-shell 'T2' and core-surface 'T13' proteins show that these viruses group with other Culicoides borne orbiviruses, clustering with Eubenangee virus (EUBV), another orbivirus infecting marsupials. WARV shares <70% aa identity in all three conserved proteins (Pol, T2 and T13) with other orbiviruses, consistent with its classification within a distinct Orbivirus species. Although WALV and MUDV share <72.86%/67.93% aa/nt identity with other orbiviruses in Pol, T2 and T13, they share >99%/90% aa/nt identities with each other (consistent with membership of the same virus species - Wallal virus). However, WALV and MUDV share <68% aa identity in their larger outer capsid protein VP2(OC1), consistent with membership of different serotypes within the species - WALV-1 and WALV-2 respectively.

Real time RT-PCR assays for detection and typing of African horse sickness virus.

Although African horse sickness (AHS) can cause up to 95% mortality in horses, naïve animals can be protected by vaccination against the homologous AHSV serotype. Genome segment 2 (Seg-2) encodes outer capsid protein VP2, the most variable of the AHSV proteins. VP2 is also a primary target for AHSV specific neutralising antibodies, and consequently determines the identity of the nine AHSV serotypes. In contrast VP1 (the viral polymerase) and VP3 (the sub-core shell protein), encoded by Seg-1 and Seg-3 respectively, are highly conserved, representing virus species/orbivirus-serogroup-specific antigens. We report development and evaluation of real-time RT-PCR assays targeting AHSV Seg-1 or Seg-3, that can detect any AHSV type (virus species/serogroup-specific assays), as well as type-specific assays targeting Seg-2 of the nine AHSV serotypes. These assays were evaluated using isolates of different AHSV serotypes and other closely related orbiviruses, from the 'Orbivirus Reference Collection' (ORC) at The Pirbright Institute. The assays were shown to be AHSV virus-species-specific, or type-specific (as designed) and can be used for rapid, sensitive and reliable detection and identification (typing) of AHSV RNA in infected blood, tissue samples, homogenised Culicoides, or tissue culture supernatant. None of the assays amplified cDNAs from closely related heterologous orbiviruses, or from uninfected host animals or cell cultures.

Isolation and molecular characterization of contagious pustular dermatitis virus from Rajasthan, India.

This article describes the isolation and identification of contagious pustular dermatitis virus/orf virus (ORFV) from an outbreak of contagious pustular dermatitis (orf) in flocks of goats, in the north western region of India (Rajasthan). The virus was isolated in Vero cell cultures from scab and swab suspensions and has been identified using GIF/IL-2 and B2L gene specific primers in PCR and sequencing. The virus showed high nucleotide identity with previously reported Chinese, far eastern, Brazilian and Indian isolates. This report described the use of molecular tools for fast, reliable and confirmatory diagnosis of ORFV infection.

Full genome sequence of a Western reference strain of bluetongue virus serotype 16 from Nigeria.

The genome of NIG1982/10, a Nigerian bluetongue virus serotype 16 (BTV-16) strain, was sequenced (19,193 bp). Comparisons to BTV strains from other areas of the world show that all 10 genome segments of NIG1982/10 are derived from a western lineage (w), indicating that it represents a suitable reference strain of BTV-16w.