Virological, immunological and pathological findings of transplacentally transmitted bluetongue virus serotype 1 in IFNAR1-blocked mice during early and mid gestation.

Transplacental transmission (TPT) of wild-type Indian BTV-1 had never been experimentally proved. This study was first time investigated TPT of Indian BTV-1 (isolated from aborted and stillborn goat fetal spleens). The sequential pathology, virological and immune cell kinetics (CD4+, CD8+ T-lymphocytes and NK cells in spleen and PBMCs), and apoptosis in IFNAR1-blocked pregnant mice during early (infected on 1 GD) and mid (infected on 8 GD) gestation have been studied. There was higher rate of TPT during mid stage (71.43%) than early (57.14%) stage. In early stage reduced implantation sites, early embryonic deaths, abortions, and necro-haemorrhagic lesions had observed. Mid stage, congenital defects and neurological lesions in foetuses like haemorrhages, diffuse cerebral edema, necrotizing encephalitis and decreased bone size (Alizarin red staining) were noticed. BTV-1 antigen was first time demonstrable in cells of mesometrium, decidua of embryos, placenta, uterus, ovary, and brain of foetuses by immunohistochemistry and quantified by real-time qRT-PCR. BTV-inoculated mice were seroconverted by 7 and 5 dpi, and reached peak levels by 15 and 9 dpi in early and mid gestation, respectively. CD4+ and CD8+ cells were significantly decreased (increased ratio) on 7 dpi but subsequently increased on 15 dpi in early gestation. In mid gestation, increased CD8+ cells (decreased ratio) were observed. Apoptotic cells in PBMCs and tissues increased during peak viral load. This first time TPT of wild-type Indian BTV-1 deserves to be reported for implementation of control strategies. This model will be very suitable for further research into mechanisms of TPT, overwintering, and vaccination strategies.

Concurrent infection of Bluetongue and Peste-des-petits-ruminants virus in small ruminants in Haryana State of India.

Bluetongue (BT) and peste-des-petits-ruminants (PPR) are major transboundary diseases of small ruminant, which are endemic in India. Testing of bluetongue virus (BTV) and peste-des-petits-ruminants virus (PPRV) from recent outbreaks (2015-2016) in different regions of Haryana State of India revealed that 27.5% of the samples showed the presence of dual infection of BTV and PPRV. Analysis of Seg-2 of BTV (the serotype-determining protein) showed the presence of BTV-12w in several isolates. However, analysis of N gene fragment amplicons showed that viruses belong to lineage IV were most closely related to a pathogenic strain of PPRV from Delhi. This is the first report of co-circulation of PPRV lineage IV and bluetongue virus serotype 12 in the state.

Blood-feeding patterns of native mosquitoes and insights into their potential role as pathogen vectors in the Thames estuary region of the United Kingdom.

BACKGROUND: The range of vertebrate hosts on which species of mosquito blood-feed is an important parameter for identifying potential vectors and in assessing the risk of incursion and establishment of vector-borne pathogens. In the United Kingdom, studies of mosquito host range have collected relatively few specimens and used techniques that could only broadly identify host species. This study conducted intensive collection and analysis of mosquitoes from a grazing marsh environment in southeast England. This site provides extensive wetland habitat for resident and migratory birds and has abundant human nuisance biting mosquitoes. The aim was to identify the blood-feeding patterns of mosquito species present at the site which could contribute to the transmission of pathogens. METHODS: Twice-weekly collections of mosquitoes were made from Elmley Nature Reserve, Kent, between June and October 2014. Mosquitoes were collected using resting boxes, by aspiration from man-made structures and using a Mosquito Magnet Pro baited with 1-octen-3-ol. Blood-fed specimens were classified according to the degree of blood meal digestion using the Sella scale and vertebrate origin determined using sequencing of a fragment of the mitochondrial cytochrome C oxidase subunit I gene. Mosquitoes that were morphologically cryptic were identified to species level using multiplex PCR and sequencing methods. RESULTS: A total of 20,666 mosquitoes of 11 species were collected, and 2,159 (10.4%) were blood-fed (Sella scale II-VI); of these 1,341 blood-fed specimens were selected for blood meal analysis. Vertebrate origin was successfully identified in 964 specimens (72%). Collections of blood-fed individuals were dominated by Anopheles maculipennis complex (73.5%), Culiseta annulata (21.2%) and Culex pipiens form pipiens (10.4%). Nineteen vertebrate hosts comprising five mammals and 14 birds were identified as hosts for mosquitoes, including two migratory bird species. Feeding on birds by Culex modestus and Anopheles atroparvus populations in England was demonstrated. CONCLUSIONS: This study expands the vertebrate host range of mosquitoes in the Thames estuary region of the UK. Feeding on both resident and migratory bird species by potential arbovirus vectors including Cx. pipiens f. pipiens and Cx. modestus indicates the potential for enzootic transmission of an introduced arbovirus between migratory and local bird species by native mosquito species.

Development of Real-Time RT-PCR Assays for Detection and Typing of Epizootic Haemorrhagic Disease Virus.

Epizootic haemorrhagic disease virus (EHDV) is an emerging arboviral pathogen of wild and domestic ruminants worldwide. It is closely related to bluetongue virus (BTV) and is transmitted by adult females of competent Culicoides vector species. The EHDV genome consists of ten linear double-stranded (ds)RNA segments, encoding five non-structural and seven structural proteins. Genome-segment reassortment contributes to a high level of genetic variation in individual virus strains, particularly in the areas where multiple and distinct virus lineages co-circulate. In spite of the relatively close relationship between BTV and EHDV herd-immunity to BTV does not appear to protect against the introduction and infection of animals by EHDV. Although EHDV can cause up to 80% morbidity in affected animals, vaccination with the homologous EHDV serotype is protective. Outer-capsid protein VP2, encoded by Seg-2, is the most variable of the EHDV proteins and determines both the specificity of reactions with neutralizing antibodies and consequently the identity of the eight EHDV serotypes. In contrast, VP6 (the viral helicase), encoded by Seg-9, is highly conserved, representing a virus species/serogroup-specific antigen. We report the development and evaluation of quantitative (q)RT-PCR assays targeting EHDV Seg-9 that can detect all EHDV strains (regardless of geographic origin/topotype/serotype), as well as type-specific assays targeting Seg-2 of the eight EHDV serotypes. The assays were evaluated using orbivirus isolates from the 'Orbivirus reference collection' (ORC) at The Pirbright Institute and were shown to be EHDV pan-reactive or type-specific. They can be used for rapid, sensitive and reliable detection and identification (typing) of EHDV RNA from infected blood, tissue samples, homogenized Culicoides, or tissue culture supernatant. None of the assays detected RNA from closely related but heterologous orbiviruses, or from uninfected host animals or cell cultures. The techniques presented could be used for both surveillance and vaccine matching (serotype identification) as part of control strategies for incursions in wild and domestic animal species.

Dual Infection with Bluetongue Virus Serotypes and First-Time Isolation of Serotype 5 in India.

Bluetongue is endemic in India and has been reported from most Indian states. Of late, the clinical disease is most frequently seen in the states of Andhra Pradesh, Telangana (erstwhile Andhra Pradesh state), Tamil Nadu and Karnataka. Our analysis of diagnostic samples from bluetongue outbreaks during 2010-2011 from the state of Karnataka identified bluetongue virus (BTV) serotype 5 (BTV-5) for the first time in India. One of the diagnostic samples (CH1) and subsequent virus isolate (IND2010/02) contained both BTV-2 and BTV-5. Segment 2 (seg-2) sequence data (400 bp: nucleotides 2538-2921) for IND2010/02-BTV5, showed 94.3% nucleotide identity to BTV-5 from South Africa (Accession no. AJ585126), confirming the virus serotype and also indicating that Seg-2 was derived from a Western topotype, which is in contrast to serotype 2, that belongs to an Eastern topotype. BTV-5 has been recently reported from Africa, China, French islands and the Americas. Although the exact source of the Indian BTV-5 isolate is still to be confirmed, recent identification of additional exotic serotypes in India is of real concern and might add to the severity of the disease seen in these outbreaks.

Isolation of Bluetongue Virus 24 from India - An Exotic Serotype to Australasia.

Bluetongue (BT) is a viral disease of ruminants and is caused by different serotypes of bluetongue virus (BTV), which is transmitted by several species of Culicoides midges. The disease is endemic in tropical areas, and incursions have been observed in some of the temperate areas. Twenty-seven recognized serotypes of BTV have been reported so far. Some serotype viruses have been shown to circulate in certain geographical areas. BTV-24 has been reported from Africa, the Mediterranean and the Americas, whereas it is exotic to Australasia. Here, we report isolation of BTV-24 from India and show that it has high sequence homology in genome segment 2 with other Western isolates of BTV-24. Entry of this serotype into Australasian region is a cause of concern.

Reverse transcription loop-mediated isothermal amplification assays for rapid identification of eastern and western strains of bluetongue virus in India.

Bluetongue virus (BTV) infects all ruminants, including cattle, goats and camelids, causing bluetongue disease (BT) that is often severe in naïve deer and sheep. Reverse-transcription-loop-mediated-isothermal-amplification (RT-LAMP) assays were developed to detect eastern or western topotype of BTV strains circulating in India. Each assay uses four primers recognizing six distinct sequences of BTV genome-segment 1 (Seg-1). The eastern (e)RT-LAMP and western (w)RT-LAMP assay detected BTV RNA in all positive isolates that were tested (n=52, including Indian BTV-1, -2, -3, -5, -9, -10, -16, -21 -23, and -24 strains) with high specificity and efficiency. The analytical sensitivity of the RT-LAMP assays is comparable to real-time RT-PCR, but higher than conventional RT-PCR. The accelerated eRT-LAMP and wRT-LAMP assays generated detectable levels of amplified DNA, down to 0.216 fg of BTV RNA template or 108 fg of BTV RNA template within 60-90min respectively. The assays gave negative results with RNA from foot-and-mouth-disease virus (FMDV), peste des petits ruminants virus (PPRV), or DNA from Capripox viruses and Orf virus (n=10), all of which can cause clinical signs similar to BT. Both RT-LAMP assays did not show any cross-reaction among themselves. The assays are rapid, easy to perform, could be adapted as a 'penside' test making them suitable for 'front-line' diagnosis, helping to identify and contain field outbreaks of BTV.

African horse sickness outbreaks caused by multiple virus types in Ethiopia.

African horse sickness (AHS) is associated with high morbidity and mortality in equids, especially horses. A retrospective analysis was carried out concerning 737 AHS outbreaks that occurred during 2007-2010 in Ethiopia. A total of ten outbreaks were investigated in the study period. All four forms of the disease (pulmonary, cardiac, horse sickness fever and the combined form) were observed, with the cardiac form being the most prevalent. Multiple African horse sickness virus serotypes (AHSV-2, AHSV-4, AHSV-6, AHSV-8 and AHSV-9) were detected by molecular methods (type-specific real-time RT-PCR assays), and fourteen isolates were derived from blood and tissue samples collected during 2009-2010. This is the first report of AHSV-4, AHSV-6 or AHSV-8 in Ethiopia.

RNA segment 9 exists as a duplex concatemer in an Australian strain of epizootic haemorrhagic disease virus (EHDV): Genetic analysis and evidence for the presence of concatemers as a normal feature of orbivirus replication.

This paper reports a concatemeric RNA in a strain of epizootic haemorrhagic disease virus (EHDV) serotype 5. Sequencing showed that the concatemeric RNA contains two identical full-length copies of genome segment 9, arranged in series, which has apparently replaced the monomeric form of the segment. In vitro translation demonstrated that the concatemeric RNA can act as a viable template for VP6 translation, but that no double-sized protein is produced. Studies were also performed to assess whether mutations might be easily introduced into the second copy (which might indicate some potential evolutionary significance of a concatemeric RNA segment), however multiple (n=40) passages generated no changes in the sequence of either the upstream or downstream segments. Further, we present results that demonstrate the presence of concatemers or partial gene duplications in multiple segments of different orbiviruses (in tissue culture and purified virus), suggesting their generation is likely to be a normal feature of orbivirus replication.

A competitive ELISA for the detection of group-specific antibody to equine encephalosis virus.

A polyclonal antibody-based, group-specific, competitive ELISA (C-ELISA) for the detection of antibodies to equine encephalosis virus (EEV) was developed. The assay measures the competition between a specific guinea pig antiserum and a test serum, for a pre-titrated EEV antigen. The C-ELISA detected antibodies to the seven known EEV serotypes. Reference antisera raised against other arboviruses did not cross react with EEV antigen. Negative sera from horses in the United Kingdom were used to establish the baseline for a negative population. Negative and positive populations of South African horses, selected on the basis of virus neutralisation were assayed subsequently. Optimal test parameters, where sensitivity≅specificity≅100%, were calculated by two-graph receiver operator characteristic (TG-ROC) analysis to be at a cut-off value of 29.5% inhibition. Results show the EEV C-ELISA described to be sensitive, specific and reliable. Used in conjunction with ELISAs available for African horse sickness virus (AHSV), differential serological diagnosis between EEV and AHSV can be achieved.

Detection of a novel reassortant epizootic hemorrhagic disease virus (EHDV) in the USA containing RNA segments derived from both exotic (EHDV-6) and endemic (EHDV-2) serotypes.

Epizootic hemorrhagic disease virus (EHDV) is a Culicoides-transmitted orbivirus that infects domestic and wild ruminants and is provisionally thought to be distributed throughout Africa, North America, Australia, East Asia and the Middle East. Historically, of the seven proposed serotypes of EHDV, only EHDV-1 and EHDV-2 have been reported from North America. In 2006, EHDV isolates were recovered from moribund or dead white-tailed deer (Odocoileus virginianus) in Indiana and Illinois that could not be identified as either EHDV-1 or EHDV-2 by virus neutralization tests or by serotype-specific RT-PCR. Additional serological and genetic testing identified the isolates as EHDV-6, a serotype that, although originally described from Australia, has recently been recognized as an emerging pathogen of cattle in Morocco, Algeria and Turkey. In 2007 and 2008, EHDV-6 was isolated again from white-tailed deer, this time in Missouri, Kansas and Texas, suggesting that the virus is capable of overwintering and that it may become, or already is, endemic in a geographically widespread region of the USA. Genetic characterization of the virus indicates that it is a reassortant, such that the outer capsid proteins determining serotype specificity (VP2 and VP5) are derived from exotic EHDV-6, whilst the remaining structural and non-structural proteins are apparently obtained from indigenous EHDV-2 (Alberta).

The evolution of two homologues of the core protein VP6 of epizootic haemorrhagic disease virus (EHDV), which correspond to the geographical origin of the virus.

Epizootic haemorrhagic disease virus is a 10-segmented, double-stranded RNA virus. When these ten segments of dsRNA are run on 1% agarose, eastern (Australia, Japan) and western (North America, Africa, Middle-East) strains of the virus can be separated phenotypically based on the migration of genome segments 7-9. In western strains, segments 7-9 are roughly the same size and co-migrate as a single RNA band. In eastern strains, segment 9 is smaller, so while segments 7 and 8 co-migrate, the segment 9 RNA runs faster than its western homologue. Translation experiments demonstrated that these two segment 9 homologues are both functional and produce proteins (VP6) of different sizes-something that has not been reported in any other orbivirus species to date. Sequence analysis suggests that eastern and western versions of segment 9 (VP6) have likely evolved as a response to adaptive selection in different geographical regions via gene duplication and subsequent mutation. These significant findings are considered unusual given the conserved nature of VP6 and its presumed role as the viral helicase. It is not currently known what the biological relevance of each homologue is to the virus.

Genetic and phylogenetic analysis of the non-structural proteins NS1, NS2 and NS3 of epizootic haemorrhagic disease virus (EHDV).

Three unique non-structural (NS) proteins are produced by Epizootic haemorrhagic disease virus (EHDV) during infection of a host cell; NS1, NS2 and NS3. This study presents a complete genetic and phylogenetic analysis of these proteins (and the genes that code for them) to allow comparison of the selective pressures acting on each. Accession numbers, gene and protein sizes, ORF positions, G+C contents, terminal hexanucleotides, start and stop codons and phylogenetic relationships are all presented. Unlike the core, or outer-coat proteins, there are no characteristic genetic or phylogenetic traits common to all of the EHDV NS proteins; indicating that each is evolving under different selection pressures. These differences are discussed. Evidence of genetic recombination in genome segment 8 (coding for NS2) is also presented, together with evidence of gene duplication and mutation, suggesting the EHDV genome may have evolved using mechanisms such as these.

Genetic and phylogenetic analysis of the core proteins VP1, VP3, VP4, VP6 and VP7 of epizootic haemorrhagic disease virus (EHDV).

The core proteins of epizootic haemorrhagic disease virus (EHDV) have important roles to perform in maintaining the structure and function of the virus. A complete genetic and phylogenetic analysis was therefore performed on these proteins (and the genes that code for them) to allow comparison of the selective pressures acting on each. Accession numbers, gene and protein sizes, ORF positions, G+C contents, terminal hexanucleotides, start and stop codons and phylogenetic relationships are all presented. The inner core proteins (VP1, VP3, VP4 and VP6) were characterised by high levels of sequence conservation, and the ability to topotype isolates very strongly into eastern or western groups. This is particularly evident in genome segment 9 (VP6) which exists as two different sized homologues. VP7 did not topotype, but rather exhibited a more random, radial phylogeny suggestive of genetic drift. With the exception of VP6, all of the core proteins also showed high numbers of synonymous mutations in the third base position, suggesting they have been evolving for a long period of time. Interestingly, VP6 did not show this, and possible reasons for this are discussed.

Genetic and phylogenetic analysis of the outer-coat proteins VP2 and VP5 of epizootic haemorrhagic disease virus (EHDV): comparison of genetic and serological data to characterise the EHDV serogroup.

The outer-coat proteins, VP2 and VP5, of epizootic haemorrhagic disease virus (EHDV) are important for host cell binding during the initiation of infection. They are also known to determine virus serotype. This study presents a complete genetic and phylogenetic analysis of these proteins (and the genes that code for them) to allow comparison of the selective pressures acting on each and the correlation of genetic sequence data with serotype. Accession numbers, gene and protein sizes, ORF positions, G+C contents, terminal hexanucleotides, start and stop codons and phylogenetic relationships are all presented. The results show that VP2 is highly variable, is under great pressure to adapt and can be correlated with serotype. While also variable, VP5 appears to be under less adaptive pressure than VP2 but still shows some correlation with serotype. Seven serotypes of EHDV have been defined in this study, although the results do show that some serotypes are extremely closely related--and highlight the benefit of using both molecular and serologic analyses. Analysis of the terminal hexanucleotides showed that the 5' terminus is under greater purifying selection than the 3'. Evidence is also presented that both segments 2 and 6 (coding for VP2 and VP5 respectively) have grown via gene duplication and subsequent mutation.

Invasion of bluetongue and other orbivirus infections into Europe: the role of biological and climatic processes.

The invasion of multiple strains of the midge-borne bluetongue virus into southern Europe since the late 1990s provides a rare example of a clear impact of climate change on a vector-borne disease. However, the subsequent dramatic continent-wide spread and burden of this disease has depended largely on altered biotic interactions with vector and host communities in newly invaded areas. Transmission by Palearctic vectors has facilitated the establishment of the disease in cooler and wetter areas of both northern and southern Europe. This paper discusses the important biological and climatic processes involved in these invasions, and the lessons that must be drawn for effective risk management of bluetongue and other midge-borne viruses in Europe.

Design of primers and use of RT-PCR assays for typing European bluetongue virus isolates: differentiation of field and vaccine strains.

Bluetongue virus (BTV) is the causative agent of bluetongue, a disease of ruminant livestock that occurs almost worldwide between latitudes 3 degrees S and 5 degrees N. There are 24 serotypes of BTV (currently identified by serum neutralization assays). Since 1998, eight strains of six BTV serotypes (1, 2, 4, 8, 9 and 16) have invaded Europe. The most variable BTV protein is major outer-capsid component VP2, encoded by segment 2 (Seg-2) of the double-stranded RNA virus genome. VP2 represents the major target for neutralizing (and protective) antibodies that are generated in response to BTV infection, and is therefore the primary determinant of virus serotype. RT-PCR primers and assays targeting Seg-2 have been developed for rapid identification (within 24 h) of the six European BTV types. These assays are sensitive, specific and show perfect agreement with the results of conventional virus-neutralization methods. Previous studies have identified sequence variations in individual BTV genome segments that allow different isolates to be grouped on the basis of their geographical origins (topotypes). The assays described in this paper can detect any of the BTV isolates of the homologous serotype that were tested from different geographical origins (different Seg-2 topotypes). Primers were also identified that could be used to distinguish members of these different Seg-2 topotypes, as well as field and vaccine strains of most of the European BTV serotypes. The serotype-specific assays (and primers) showed no cross-amplification when they were evaluated with multiple isolates of the most closely related BTV types or with reference strains of the remaining 24 serotypes. Primers developed in this study will be updated periodically to maintain their relevance to current BTV distribution and epidemiology (http://www.iah.bbsrc.ac.uk/dsRNA_virus_proteins/ReoID/rt-pcr-primers.htm).