Isolation and evolutionary analysis of Australasian topotype of bluetongue virus serotype 4 from India

Bluetongue (BT ) is a Culicoides ‐borne disease caused by several serotypes of bluetongue virus (BTV ). Similar to other insect‐borne viral diseases, distribution of BT is limited to distribution of Culicoides species competent to transmit BTV . In the tropics, vector activity is almost year long, and hence, the disease is endemic, with the circulation of several serotypes of BTV , whereas in temperate areas, seasonal incursions of a limited number of serotypes of BTV from neighbouring tropical areas are observed. Although BTV is endemic in all the three major tropical regions (parts of Africa, America and Asia) of the world, the distribution of serotypes is not alike. Apart from serological diversity, geography‐based diversity of BTV genome has been observed, and this is the basis for proposal of topotypes. However, evolution of these topotypes is not well understood. In this study, we report the isolation and characterization of several BTV ‐4 isolates from India. These isolates are distinct from BTV ‐4 isolates from other geographical regions. Analysis of available BTV seg‐2 sequences indicated that the Australasian BTV ‐4 diverged from African viruses around 3,500 years ago, whereas the American viruses diverged relatively recently (1,684 CE ). Unlike Australasia and America, BTV ‐4 strains of the Mediterranean area evolved through several independent incursions. We speculate that independent evolution of BTV in different geographical areas over long periods of time might have led to the diversity observed in the current virus population.     

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.     

A novel Bluetongue virus serotype 3 strain in Tunisia,November 2016

Since 1998, southern Europe has experienced multiple incursions of different serotypes and topotypes of Bluetongue virus, a vector‐borne transmitted virus, the causative agent of Bluetongue (BT), a major disease of ruminants. Some of these incursions originated from northern Africa, likely because of wind‐blown dissemination of infected midges. In this report, we describe the detection and whole genome characterization of a novel BTV‐3 strain identified in a symptomatic sheep in Tunisia. Sequences were immediately deposited with the GenBank Database under Accession Nos KY432369‐KY432378. Alert and preparedness are requested to face the next vector seasons in northern Africa and the potential incursion of this novel strain in southern Europe.     

Bluetongue Virus in Lebanon

Since 2000, several incursions of bluetongue virus (BTV) occurred in the Mediterranean Basin involving European and surrounding Countries. The Middle East represents one of the most important gateways for the access of BTV in Europe. Limited data on the BTV situation in this area are available. In this perspective, an epidemiological survey on the presence of BTV in Lebanon was conducted. Of the 181 serum samples tested, 97 (mean = 53.6%; 95% CI: 46.3–60.7) resulted positive when tested for the presence of BTV antibodies by c‐ELISA, of these 42 (mean = 42%; 95% CI: 32.8–51.8) serum samples were from sheep and 55 (mean = 67.9%; 95% CI: 57.1–77.1) serum samples were from goats. Fourteen blood samples (14/110; mean = 12.7%; 95% CI: 7.8–20.3), 6 (6/66; mean = 9.1%; 95% CI: 4.4–18.5) from sheep and 8 (8/44; mean = 18.2%; 95% CI: 9.6–32.0) from goats, were positive by qRT‐PCR. The results with serum‐neutralization assay and typing performed by RT‐PCR confirmed that six BTV serotypes are currently circulating in Lebanon, and these serotypes are as follows: 1, 4, 6, 8, 16 and 24. This study is the first report that confirms the presence and circulation of BTV in Lebanon.     

The possible route of introduction of bluetongue virus serotype 3 into Sicily by windborne transportation of infected Culicoides spp.

In October 2017, the first outbreak of bluetongue virus serotype 3 (BTV‐3) began in Italy, specifically in western Sicily. The route of entrance remains unclear, although since 2016 the same strain had been circulating only 150 km away, on the Tunisian peninsula of Cape Bon. The present analysis assessed the feasibility that wind could have carried BTV‐3‐infected Culicoides spp. from Tunisia to Sicily. An advection‐deposition‐survival (ADS) model was used to estimate when and where Culicoides spp. were likely to be introduced prior to the first BTV‐3 report in Italy. Additionally, the Hybrid Single‐Particle Lagrangian Integrated Trajectory (HYSPLIT) model was used to support ADS outputs. The modelling suggests that during September 2017, strong wind currents and suitable climatic conditions could have allowed the transportation of Culicoides spp. from BTV‐3‐infected areas in Tunisia into Sicily. ADS simulations suggest that particles could have reached the province of Trapani in western Sicily on 2 and 12 September. These simulations suggest the feasibility of aerial transportation of infected Culicoides spp. from Tunisia into Sicily. They demonstrate the suitability of the ADS model for retrospective studies of long‐range transportation of insects across large water bodies, which may enhance the early detection of vectorial disease introduction in a region.     

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.     

Phylogenetic Characterization Genome Segment 2 of Bluetongue Virus Strains Belonging to Serotypes 5, 7 and 24 Isolated for the First Time in China During 2012 to 2014

Bluetongue is endemic in China, and Bluetongue virus (BTV) strains belonging to eight different serotypes (BTV‐1, BTV‐2, BTV‐3, BTV‐4, BTV‐9, BTV‐12, BTV‐15 and BTV‐16) had been isolated between 1996 and 1997. However, there has been a long pause in investigating the epidemiology of BTV infection since then. During 2012–2014, eight BTV strains belonging to serotypes 5, 7 and 24 were isolated for the first time in Yunnan and Guangdong provinces from the blood of sentinel animals. Phylogenetic analyses of genome segment 2 of these Chinese BTV strains grouped them into nucleotypes E, F and A, respectively, along with the reference strains of the same serotype. For each serotype, Chinese strains cluster together closely to form a China's sublineage. In addition, these strains were most closely related to strains from Africa, indicating that they may share a recent common ancestry with African strains. To our knowledge, this is the first time that BTV‐5, BTV‐7 and BTV‐24 strains have been isolated in South‐East Asia. These data will be beneficial for understanding the BTV epidemiology and improving diagnostic assays and control measures against bluetongue in China and its neighbouring countries in the Asia–Pacific region.     

Bluetongue Serotype 2 and 9 Modified Live Vaccine Viruses as Causative Agents of Abortion in Livestock: A Retrospective Analysis in Italy

The recent outbreak caused by Schmallenberg virus, which affected sheep, goats and cattle in Europe, highlighted the importance of having a robust surveillance plan capable of monitoring abortions and malformations in the livestock offspring. In this context, bluetongue viruses (BTVs) represented and represent one of the major threats to the European livestock industry. Aiming to improve the understanding on BTV cross placental transmission and serotype involvement, in this retrospective study foetal spleens and/or brains of 663 ovines, 429 bovines, 155 goats and 17 buffaloes were tested for the presence of BTV by virus isolation. BTV vaccine strains were isolated from 31 foetuses (2.4%; 95% CI: 1.7–3.4%): 24 (3.6%; 95% CI: 2.4–5.3%) from ovine foetal tissues; 6 (1.4%; 95% CI: 0.6–3.0%) from bovine foetal tissues and 1 (0.6%; 95% CI: 0.2–3.5%) from the spleen of a caprine foetus. All foetuses were from animals vaccinated with either BTV‐2 or BTV‐2, and BTV‐9 modified live vaccines (MLVs) produced by Onderstepoort Biological Products (OBP), South Africa. Among the 31 isolated vaccine strains, serotype 9 (n = 28) was more frequently isolated (P < 0.05) than serotype 2 (n = 3). In two cases infectious vaccine strains were found in the foetal tissues 2 months after the vaccine administration. Other pathogens known to be causative agents of abortion in ruminants were not detected nor isolated. This study demonstrates, for the first time, that BTV‐2 and BTV‐9 vaccine strains are able to cross the placental barrier of sheep, cattle and goats. BTV‐2 and BTV‐9 vaccine strains are able to infect foetuses and cause abortions or malformations depending on the period of pregnancy at the time of vaccination.     

Characterization of transboundary foot‐and‐mouth disease viruses in Nigeria and Cameroon during 2016

Continuous surveillance for foot‐and‐mouth disease (FMD) in endemic settings such as West Africa is imperative to support improved local and regional control plans, with the long‐term goal of regional eradication. This paper describes the genetic characterization of FMD viruses (FMDV) obtained from outbreaks in Nigeria (n = 45) and Cameroon (n = 15) during 2016 and from archival samples (n = 3) retrieved from a 2014 outbreak in Nigeria. These viruses were analysed in the context of previously published FMDV sequences from the region. Four FMDV serotypes: O, A, SAT1 and SAT2, were detected. Phylogenetic analyses of the VP1 coding sequences indicate the continuity of FMDV serotype O East Africa‐3 (O/EA‐3), serotype A AFRICA genotype G‐IV (A/AFRICA/G‐IV) and serotype South African Territories (SAT) 2 lineage VII (SAT2/VII). The FMDV SAT1 topotype X (SAT1/X), which emerged in Nigeria in 2015, continued to be associated with outbreaks in the region during 2016, and SAT1 is reported for the first time from Cameroon. Additionally, a re‐emergence or re‐introduction of the serotype O West Africa (O/WA) topotype in Nigeria is described herein. Our findings indicate a consistent, pan‐serotypic relationship between FMDV strains detected in Cameroon and Nigeria. Additionally, FMDV strains from West Africa obtained in this study were genetically related to those occurring in East and North Africa. These phylogenetic relationships suggest that animal movements (pastoralism and/or trade) are important factors for virus spread across the African continent. These data provide critical baselines which are a necessary component of Stages 0 and 1 of the Progressive Control Pathway of FMD (PCP‐FMD). Specifically, characterizing the existing virus strains (risk) provides the basis for the comprehensive risk‐based control plan which is the requisite criteria for Nigeria's transition to Stage 2 of PCP‐FMD, and for coordinated regional control of FMD.     

Circulation of bluetongue virus 8 in French cattle,before and after the re‐emergence in 2015

Bluetongue virus serotype 8 (BTV ‐8) re‐emerged in Central France in August 2015. The viral strain identified is nearly identical to the one that circulated during the 2006/2009 massive outbreak throughout Europe. To address the question of an undetected BTV ‐8 circulation on the French territory, a serological study was conducted on young cattle along a transect of seven departments, three of them located in areas where the virus presence had been confirmed by RT ‐PCR by winter 2015/2016. Sera from 2,565 animals were collected during the winters preceding and following the re‐emergence, with 414 animals being sampled in each of the two consecutive years. All samples were tested by competitive ELISA (IDV et) and, when enough serum was available, ELISA ‐positive samples were confirmed by seroneutralization tests. In areas with infected holdings, seropositive animals were found before the re‐emergence (N  = 14 of 511), significantly more on the following year (N  = 17 of 257), and eight animals (N  = 158) seroconverted over 2015. Seropositive animals were also detected as early as winter 2014/2015 in one department without known infected holdings (N  = 12 of 150), and in winter 2015/2016 in three of them (N  = 21 of 555), where seven animals (N  = 154) seroconverted over 2015. These results suggest that BTV ‐8 may have spread at low levels before the re‐emergence, even in areas considered virus‐free. Unfortunately, whole blood from the seropositive animals was not available to definitely confirm the virus presence by RT ‐PCR .     

Seroprevalence of Bluetongue Virus in small ruminants in Balochistan province,Pakistan

Bluetongue (BT ), caused by bluetongue virus (BTV ), is a vector‐borne disease of small ruminants that has the potential to spread across international borders. Despite large populations of susceptible animals and borders with BTV endemic countries, little is known of the disease burden and prevalent serotypes in the province of Balochistan in Pakistan. We conducted a cross‐sectional study to determine seroconversion and prevalent serotypes in selected districts of the province using a competitive enzyme‐linked immunosorbent assay (cELISA ) and real‐time polymerase chain reaction (RT –PCR ). Sera (n  = 876) were collected from clinically healthy sheep and goats originating from the districts of Quetta (n  = 300), Mastung (n  = 201), Killa Saifullah (n  = 75) and Kech (n  = 300). None of the study herds (n  = 97) were seronegative for BTV , and at the individual level, the overall prevalence of BTV seroconversion was 47.26% (n  = 414/876, 95% CI  = 43.92%–50.63%). A higher percentage of goats (50.87%, 95% CI  = 45.99%–55.73%) were seropositive for anti‐VP 7 immunoglobulins (IgG) than sheep (44.21%, 95% CI  = 39.81%–48.70%). Odds ratios of seroconversion for goats were associated with breed type (χ² = 16.84, p  =  .01), parity (χ² = 23.66, p  =  .00) and presence of vector (χ² = 2.63, p  =  .10), whereas for sheep, it was associated with breed type (χ² = 13.80, p  =  .01) and parity (χ² = 53.40, p  =  .00). Serotype 8 was the most prevalent (26.82%, 95% CI  = 14.75%–43.21%) followed by an equal prevalence of serotypes 2 and 9 (7.31%, 95% CI  = 1.91%–21.01%). To the best of our knowledge, this is the first study conducted in Balochistan province and the results indicate that there is a necessity to initiate intervention strategies to control BT disease burden not only in this region of Pakistan but also in adjacent areas of the neighbouring countries, Iran and Afghanistan.     

Emergence of bluetongue virus serotype 4 in mainland France in November 2017

In November 2017, a 15‐day‐old calf located in France (Haute‐Savoie department) was found positive for bluetongue virus (BTV ) RNA by RT ‐PCR . Laboratory investigations allowed the isolation and identification of the serotype: BTV ‐4. The analysis of the full viral genome showed that all the 10 genome segments were closely related to BTV ‐4 strains involved in a large BT outbreak in the Balkan Peninsula, in Italy since 2014 and in Corsica since the end of October 2016. These results together with epidemiological data suggest that BTV ‐4 has been introduced to mainland France from Corsica or Italy where BTV ‐4 outbreaks have been reported in summer and autumn 2016. This is the first report of the introduction of BTV ‐4 in mainland France.     

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.     

Characterization of bluetongue virus serotype 28

Bluetongue virus (Reoviridae; Orbivirus, BTV), which is usually transmitted by biting midges, affects wild and domestic ruminants worldwide, thereby causing an economically important disease. Recently, a putative new BTV strain was isolated from contaminated vaccine batches. In this study, we investigated the genomic and clinical characteristics of this isolate, provisionally designated BTV‐28. Phylogenetic analysis of BTV‐28 segment 2 (Seg‐2) showed that it is related to Seg‐2 from BTV serotypes 4, 10, 11, 17, 20 and 24, sharing 64%–66% identity in nucleotide sequences (nt) and 59%–62% in amino acid (aa) sequences of BTV VP2. BTV‐28 Seg‐6 is related to the newly reported XJ1407 BTV isolate, sharing 76.70% nt and 90.87% aa sequence identity. Seg‐5 was most closely related to a South African BTV‐4 strain, and all other segments showed close similarity to BTV‐26. Experimental infection by injection of 6‐month‐old ewes caused clinical signs in all injected animals, lasting from 2 to 3 days to several weeks post‐infection, including high body temperature, conjunctivitis, nasal discharge and rhinitis, facial oedema, oral hyperaemia, coronitis, cough, depression and tongue cyanosis. Naïve control animals, placed together with the infected sheep, displayed clinical signs and were positive for viral RNA, but their acute disease phase was shorter than that of BTV‐injected ewes. Control animals that were kept in a separated pen did not display any clinical signs and were negative for viral RNA presence throughout the experiment. Seroconversion was observed in the injected and in one of the two contact‐infected animals. These findings demonstrate that BTV‐28 infection of sheep can result in clinical manifestation, and the clinical signs detected in the contact animals suggest that it might be directly transmitted between the mammalian hosts.     

Bluetongue virus serotype 27: Experimental infection of goats,sheep and cattle with three BTV‐27 variants reveal atypical characteristics and likely direct contact transmission BTV‐27 between goats

Bluetongue virus (BTV) hitherto consisted of 26 recognized serotypes, of which all except BTV‐26 are primarily transmitted by certain species of Culicoides biting midges. Three variants of an additional 27th bluetongue virus serotype (BTV‐27v01‐v03) were recently detected in asymptomatic goats in Corsica, France, 2014–2015. Molecular characterization revealed genetic differences between the three variants. Therefore, in vivo characteristics were investigated by experimental infection of a total of 15 goats, 11 sheep and 4 cattle with any one of the three variants in separated animal trials. In goat trials, BTV‐naïve animals of the same species were kept in a facility where direct contact was unhindered. Of the 15 inoculated goats, 13 and 14 animals were found positive for BTV‐RNA and antibodies (Ab), respectively, until the end of the experiments. Surprisingly, BTV‐Ab levels as measured with ELISA and neutralization test (SNT) were remarkably low in all seropositive goats. Virus isolation from whole‐blood was possible at the peak of viremia until 49 dpi. Moreover, detection of BTV‐27v02‐RNA and Ab in one contact goat indicated that—similar to BTV‐26—at least one of three BTV‐27 variants may be transmitted by contact between goats. In the field, BTV‐27 RNA can be detected up to 6 months in the whole‐blood of BTV‐27‐infected Corsican goats. In contrast, BTV RNA was not detected in the blood of cattle or sheep. In addition, BTV‐27 Abs were not detected in cattle and only a transient increase in Ab levels was observed in some sheep. None of the 30 animals showed obvious BT‐like clinical signs. In summary, the phenotypes observed for BTV‐27v01‐v03 phenotypes correspond to a mixture of characteristics known for BTV‐25 and 26.     

Evidence of reduced viremia,pathogenicity and vector competence in a re‐emerging European strain of bluetongue virus serotype 8 in sheep

The outbreak of bluetongue virus (BTV) serotype 8 (BTV‐8) during 2006–2009 in Europe was the most costly epidemic of the virus in recorded history. In 2015, a BTV‐8 strain re‐emerged in France which has continued to circulate since then. To examine anecdotal reports of reduced pathogenicity and transmission efficiency, we investigated the infection kinetics of a 2007 UK BTV‐8 strain alongside the re‐emerging BTV‐8 strain isolated from France in 2017. Two groups of eight BTV‐naïve British mule sheep were inoculated with 5.75 log₁₀TCID₅₀/ml of either BTV‐8 strain. BTV RNA was detected by 2 dpi in both groups with peak viraemia occurring between 5–9 dpi. A significantly greater amount of BTV RNA was detected in sheep infected with the 2007 strain (6.0–8.8 log₁₀ genome copies/ml) than the re‐emerging BTV‐8 strain (2.9–7.9 log₁₀ genome copies/ml). All infected sheep developed BTV‐specific antibodies by 9 dpi. BTV was isolated from 2 dpi to 12 dpi for 2007 BTV‐8‐inoculated sheep and from 5 to 10 dpi for sheep inoculated with the remerging BTV‐8. In Culicoides sonorensis feeding on the sheep over the period 7–12 dpi, vector competence was significantly higher for the 2007 strain than the re‐emerging strain. Both the proportion of animals showing moderate (as opposed to mild or no) clinical disease (6/8 vs. 1/8) and the overall clinical scores (median 5.25 vs. 3) were significantly higher in sheep infected with the 2007 strain, compared to those infected with the re‐emerging strain. However, one sheep infected with the re‐emerging strain was euthanized at 16 dpi having developed severe lameness. This highlights the potential of the re‐emerging BTV‐8 to still cause illness in naïve ruminants with concurrent costs to the livestock industry.     

Sequences of Genes Encoding Type‐Specific and Group‐Specific Antigens of an Indian Isolate of Bluetongue Virus Serotype 10 (BTV‐10) and Implications for their Origin

Bluetongue, a transboundary disease, is endemic in several tropical countries and is caused by bluetongue virus (BTV). The origin and movement of BTV can be predicted by comparing nucleotide sequences of its segmented RNA genome. Such analyses have been useful in evaluating the source of the virus responsible for recent incursion of BTV into previously unreported areas. Besides several serotypes, genetically related BTV strains circulate in each endemic area, but such clusters of strains have been reported to be distinct from one geographical region to another. We obtained partial or complete sequences of the open reading frames encoded by VP2, VP6, VP7, NS1 and NS2 genes of a BTV‐10 isolate of India and compared them with other BTV‐10 sequences available in public database. Sequences of all the five genes showed >99% identity to BTV‐10 prototype, vaccine strain and vaccine‐like virus isolates from the USA. Our results suggest that Indian BTV‐10 virus analysed in this study possibly originated from the United States.     

Complete genome sequence of bluetongue virus serotype 4 that emerged on the French island of Corsica in December 2016

In November 2016, sheep located in the south of Corsica island exhibited clinical signs suggestive of bluetongue virus (BTV) infection. Laboratory analyses allowed to isolate and identify a BTV strain of serotype 4. The analysis of the full viral genome showed that all the 10 genomic segments were closely related to those of the BTV‐4 present in Hungary in 2014 and involved in a large BT outbreak in the Balkan Peninsula. These results together with epidemiological data suggest that BTV‐4 has been introduced to Corsica from Italy (Sardinia) where BTV‐4 outbreaks have been reported in autumn 2016. This is the first report of the introduction in Corsica of a BTV strain previously spreading in eastern Europe.