Molecular characterization of equine infectious anaemia virus from a major outbreak in southeastern France

In 2009, a major outbreak of equine infectious anaemia (EIA ) was reported in the south‐east of France. This outbreak affected three premises located in the Var region where the index case, a 10‐year‐old mare that exhibited clinical signs consistent with EIA , occurred at a riding school. Overall, more than 250 horses were tested for EIAV (equine infectious anaemia virus) antibodies, using agar gel immunodiffusion test, and 16 horses were positive in three different holdings. Epidemiological survey confirmed that the three premises were related through the purchase/sale of horses and the use of shared or nearby pastures. Molecular characterization of viruses was performed by sequencing the full gag gene sequence (1,400 bp) of the proviral DNA s retrieved from the spleen of infected animals collected post‐mortem . Phylogenetic analysis confirmed epidemiological data from the field, as viruses isolated from the three premises were clustering together suggesting a common origin whereas some premises were 50 km apart. Moreover, viruses characterized during this outbreak are different from European strains described so far, underlying the high genetic diversity of EIAV in Europe.     

评价全血DNA二代测序共有序列在人类免疫缺陷病毒优势准种研究中的准确性

目的 评价二代测序共有序列对人类免疫缺陷病毒(HIV)优势准种分析的代表性和准确性.方法 共收集29个HIV感染病例的32份全血样本,其中3个病例在随访期间发生耐药,分别采集治疗前和耐药后的两份样本.提取样本DNA,分别用二代测序和一代测序方法测定HIV pol区扩增产物序列.利用软件Sequencher(4.10.1...     

First evidence of bovine immunodeficiency virus infection in Mexican cattle

This study set out to identify the presence of bovine immunodeficiency virus (BIV) in animals geographically located in Mexico. BIV was first discovered in the United States in a dairy cow with persistent lymphocytosis, lymphoid hyperplasia and lymphocytic encephalitis. Many studies indicate that BIV infection is globally distributed, but its presence in Mexico remains unknown. We collected 1,168 heparinized blood samples from cattle in ten states across the Mexican Republic, then separated plasma using centrifugation and tested for antibodies against BIV. We used an indirect ELISA based on the use of a synthetic peptide derived from transmembrane glycoprotein (gp45/TM). In order to identify the viral genome, we designed a synthetic gene as a PCR control, as well as a pair of oligonucleotides for amplifying a 519 bp product of the env gene which encodes the surface protein. Positive amplicons were purified and subjected to nucleotide sequencing. A total of 189 (28.94%) tested plasma samples suggest the presence of specific anti‐BIV antibodies in all states studied except for Chiapas. Additionally, PCR results identified six positive cows in the states of Puebla and Coahuila. BIV in these cows was confirmed via nucleotide sequencing and in silico analysis of these samples. This is the first report of the presence of BIV in Mexican cattle.     

Development of sensitive ddPCR assays to reliably quantify the proviral DNA reservoir in all common circulating HIV subtypes and recombinant forms

Introduction

The latent reservoir is the main barrier on the road to HIV cure, and clinical approaches towards eradication are often evaluated by their effect on proviral DNA. To ensure inclusiveness and representativeness in HIV cure studies, proviral DNA quantification assays that are able to detect all common circulating HIV clades are urgently needed. Here, three HIV DNA assays targeting three different genomic regions were evaluated for their sensitivity and subtype‐tolerance using digital PCR.

Methods

A subtype‐B‐specific assay targeting gag (GAG) and two assays targeting conserved sequences in ltr and pol (LTR and JO) were assessed for their sensitivity and subtype‐tolerance in digital PCR (Bio‐Rad QX200), using a panel of serially diluted subtype reference plasmids as well as a panel of clinical isolates. Both panels represent subtypes A, B, C, D, F, G and circulating recombinant forms (CRFs) AE and AG, which together are responsible for 94% of HIV infections worldwide.

Results

HIV subtype was observed to greatly affect HIV DNA quantification results. Robust regression analysis of the serially diluted plasmid panel showed that the GAG assay was only able to linearly quantify subtype B, D and G isolates (4/13 reference plasmids, average R² = 0.99), whereas LTR and JO were able to quantify all tested isolates (13/13 reference plasmids, respective average R² = 0.99 and 0.98). In the clinical isolates panel, isolates were considered detectable if all replicates produced a positive result. The GAG assay could detect HIV DNA in four out of five subtype B and one out of two subtype D isolates, whereas the LTR and JO assays detected HIV DNA in all twenty‐nine tested isolates. LTR and JO results were found to be equally precise but more precise than GAG.

Conclusions

The results demonstrate the need for a careful validation of proviral reservoir quantification assays prior to investigations into non‐B subtype reservoirs. The LTR and JO assays can sensitively and reliably quantify HIV DNA in a panel that represents the worldwide most prevalent subtypes and CRFs (A, B, C, D, AE, F, G and AG), justifying their application in future trials aimed at global HIV cure.

     

Isolation,Characterization and Virulence Potential of Tenacibaculum dicentrarchi in Salmonid Cultures in Chile

In this study, we isolated, identified and characterized isolates of Tenacibaculum dicentrarchi in Atlantic salmon (Salmo salar) farmed in Chile for the first time. In 2010 and 2014, mortalities were observed in Atlantic salmon (average weight 25–30 and 480–520 g, respectively) at an aquaculture centre in Puerto Montt, Chile. Severe tail rots, frayed fins and, in some cases, damaged gills were detected. Wet smear analyses of these lesions revealed a high occurrence of Gram‐negative, filamentous bacteria. Microbiological analysis of infected gill and tail tissues yielded six bacterial isolates. All were identified as T. dicentrarchi through polyphasic taxonomy, which included phenotypic characterization, 16S rRNA sequencing and multilocus sequence typing. The latter method revealed a close relationship of the Chilean genotype with the T. dicentrarchi type strain and two Norwegian Atlantic cod (Gadus morhua) isolates. The pathogenic potential of the TdChD05 isolate was assessed by challenging Atlantic salmon and rainbow trout (Oncorhynchus mykiss) for one hour, which resulted in mean cumulative mortality rates of 65% and 93%, respectively, as well as clinical signs 14 days post‐challenge. However, challenged Coho salmon (Oncorhynchus kisutch) presented no mortalities or clinical signs of infection. These findings indicate that the geographical and host distribution of T. dicentrarchi is wider than previously established and that this bacterium may have negative impacts on salmonid cultures.     

Sloths host Anhanga virus‐related phleboviruses across large distances in time and space

Sloths are genetically and physiologically divergent mammals. Phleboviruses are major arthropod‐borne viruses (arboviruses) causing disease in humans and other animals globally. Sloths host arboviruses, but virus detections are scarce. A phlebovirus termed Anhanga virus (ANHV) was isolated from a Brazilian Linnaeus's two‐toed sloth (Choloepus didactylus) in 1962. Here, we investigated the presence of phleboviruses in sera sampled in 2014 from 74 Hoffmann's two‐toed (Choloepus hoffmanni, n = 65) and three‐toed (Bradypus variegatus, n = 9) sloths in Costa Rica by broadly reactive RT‐PCR. A clinically healthy adult Hoffmann's two‐toed sloth was infected with a phlebovirus. Viral load in this animal was high at 8.5 × 10⁷ RNA copies/ml. The full coding sequence of the virus was determined by deep sequencing. Phylogenetic analyses and sequence distance comparisons revealed that the new sloth virus, likely representing a new phlebovirus species, provisionally named Penshurt virus (PEHV), was most closely related to ANHV, with amino acid identities of 93.1%, 84.6%, 94.7% and 89.0% in the translated L, M, N and NSs genes, respectively. Significantly more non‐synonymous mutations relative to ANHV occurred in the M gene encoding the viral glycoproteins and in the NSs gene encoding a putative interferon antagonist compared to L and N genes. This was compatible with viral adaptation to different sloth species and with micro‐evolutionary processes associated with immune evasion during the genealogy of sloth‐associated phleboviruses. However, gene‐wide mean dN/dS ratios were low at 0.02–0.15 and no sites showed significant evidence for positive selection, pointing to comparable selection pressures within sloth‐associated viruses and genetically related phleboviruses infecting hosts other than sloths. The detection of a new phlebovirus closely‐related to ANHV, in sloths from Costa Rica fifty years after and more than 3,000 km away from the isolation of ANHV confirmed the host associations of ANHV‐related phleboviruses with the two extant species of two‐toed sloths.     

First report on characterization and pathogenicity study of emerging Lactococcus garvieae infection in farmed rainbow trout,Oncorhynchus mykiss (Walbaum), from India

“ Warm water lactococcosis” in farm‐reared rainbow trout, Oncorhynchus mykiss (Walbaum) in the northern Himalayan region of India, caused by bacterium Lactococcus garvieae is described in this study. Nine bacterial isolates were recovered from the organs of haemorrhagic septicaemia rainbow trout and were subjected to biochemical and molecular identification. Cell surface characteristics and virulence of the bacterial isolates are also described. All the nine bacterial isolates had homogenous biochemical characteristics and were Gram‐positive, short chains forming (two to eight cells long), α‐haemolytic, non‐motile ovoid cocci. Partial 16S rDNA nucleotide sequence (~1,400 bp) of current isolates shared 99% identities with the 16S rDNA nucleotide sequence of L. garvieae R421, L. garvieae FMA 395 and L. garvieae CAU :1730. The identity of the bacterial isolates was further confirmed by PCR amplification of L . garvieae‐ specific ~1,100 bp fragment. Transmission electron microscopy (TEM ) of one representative isolate, L. garvieae RTCLI 04, indicates that the isolated strain lacks thick outer capsule and is of KG + (non‐capsulates) phenotype. An intraperitoneal and intramuscular injection (2.6 × 10⁵ CFU ml⁻¹) and also immersion in bacterial suspension @ of 2.6 × 10⁵ CFU ml⁻¹ to healthy rainbow trout juveniles (body weight: 27.5 ± 3.7 g) with L . garvieae RTCLI 04 caused 80%, 60% and 10% cumulative mortality in challenged fish, respectively, within 15 days post‐infection. The haemorrhagic septicaemic disease was reproduced experimentally. Histopathological examination of organs of experimentally infected fish revealed extensive degenerative and inflammatory changes in eye, kidney, gill and liver. PCR amplification of several putative virulence genes such as haemolysins, adhesins, LP xTG ‐containing surface proteins and adhesins cluster confirms the virulence of our Indian L. garvieae isolates. To the best of our knowledge, we are reporting for the first time that L. garvieae is associated with fatal haemorrhagic septicaemia in farmed rainbow trout in India.     

Past and future impact of next‐generation sequencing in head and neck cancer

Progress in sequencing technology is intrinsically linked to progress in understanding cancer genomics. The purpose of this review was to discuss the development from Sanger sequencing to next‐generation sequencing (NGS) technology. We highlight the technical considerations for understanding reports using NGS. We discuss the findings of studies in head and neck cancer using NGS as well as The Cancer Genome Atlas. Finally we discuss future routes for research utilizing this methodology and the potential impact of this. © 2015 Wiley Periodicals, Inc. Head Neck 38 : E2395–E2402, 2016     

Equine Herpesvirus‐1 Myeloencephalopathy,an Emerging Threat of Working Equids in Ethiopia

Although equine herpesvirus myeloencephalopathy (EHM) is a sporadic and relatively uncommon manifestation of equine herpesvirus‐1 (EHV‐1), it has the potential for causing devastating outbreaks in horses. Up till now, there were no reported EHM outbreaks in donkeys and mules. This study describes the isolation and molecular characterization of EHV‐1 from clinically EHM‐affected horses (n = 6), mules (n = 3) and donkeys (n = 82) in Ethiopia during outbreaks from May 2011 to December 2013. The incidence of EHM cases was higher from April to mid‐June. EHM in donkeys was more severe and death without clinical signs of paralysis, and recumbency was frequently observed. The main age of affected equines ranged from 7 to 10 years (n = 51; 56.0%), and females (n = 58; 63.7%) were more affected than males. The incidence of neuropathogenic (D₇₅₂) and non‐neuropathogenic (N₇₅₂) variants of EHV‐1 from EHM‐affected equines in Ethiopia was assessed by sequencing the DNA polymerase gene (ORF30) of the EHV‐1 isolates. The results indicated that from the total of 91 clinically affected equines, 90 (98.9%) of them had an ORF30 D₇₅₂ genotype. An ORF30 N₇₅₂ variant was only found in one donkey. Analysis of ORF68 as grouping marker for geographical differences showed that the Ethiopian EHV‐1 isolates belong to geographical group 4. Due to the fatal nature of EHV‐1 in donkeys, it would be interesting to examine the pathogenesis of EHM in this species. At present, there is no vaccine available in Ethiopia, and therefore, outbreaks of EHV‐1 should be controlled by proper management adaptations. In addition, it is important to test the efficacy of the commercial vaccines not only in horses, but also in donkeys and mules.     

Molecular Detection of Tick‐Borne Pathogens of the Family Anaplasmataceae in Brazilian Brown Brocket Deer (Mazama gouazoubira,Fischer, 1814) and Marsh Deer (Blastocerus dichotomus,Illiger, 1815)

Deer are important natural reservoir hosts of Anaplasmataceae. The present study used nested PCR and nucleotide sequencing to evaluate the occurrence of Anaplasmataceae species in 23 free‐living and six captive specimens of the cervids Mazama gouazoubira and Blastocerus dichotomus in Minas Gerais State, Brazil. Blood samples were tested for the presence of Ehrlichia and Anaplasma spp. using nPCR assays and sequencing of the msp4, msp1 and 16S rRNA genes. The identity of each sequence was confirmed by comparison with sequences available from GenBank using BLAST software. Of the animals investigated, 93.1% (27/29) were infected with haemoparasites including Anaplasma marginale (79.3%), Ehrlichia chaffeensis (3.4%), Anaplasma bovis (3.4%) and Anaplasma spp. (assigned to A. platys and A. phagocytophilum) (17.2%). Co‐infection occurred in 20% (6/29) of the deer examined. Four (13.8%) were infected with A. marginale and Anaplasma sp., one (3.4%) was infected with A. marginale and E. chaffeensis, and one (3.4%) was infected with A. marginale and A. bovis. The results of the present study suggest that cross‐protection does not occur in these deer. Immunological cross‐reaction occurs when sera are tested diagnostically because these bacteria are closely related taxonomically, reinforcing the importance of molecular diagnosis followed by nucleotide sequencing.     

Tuberculosis Caused by Mycobacterium orygis in Dairy Cattle and Captured Monkeys in Bangladesh: a New Scenario of Tuberculosis in South Asia

Mycobacterium orygis, commonly known as the oryx bacillus and a newly proposed Mycobacterium tuberculosis complex subspecies, was isolated from 18 cattle in a dairy farm and two captured rhesus monkeys in a zoo in Bangladesh. All the infected animals had tuberculosis lesions in their lungs, suggesting transmission and infection with M. orygis by an airborne route. The 20 isolates were analysed using a range of conventional and molecular typing methods, and RD‐deletion typing and sequencing of selected genes confirmed the isolates as M. orygis. Multiple‐locus variable‐number tandem repeat analysis (MLVA) allowed the isolates to be divided into three clusters based on the relatedness of their MLVA profiles. The two monkey isolates shared the same MLVA pattern with 15 of the cattle isolates, whereas the remaining three cattle isolates had different patterns, even though the latter animals had been kept in the same dairy farm. The diversity observed among isolates may suggest the bacteria have been established in this area for a long period. This study along with other recent findings that report the detection of M. orygis from animals as well as humans originating from South Asia potentially indicate endemic distribution of M. orygis in South Asia.     

TACR1 gene polymorphism and sex differences in postoperative nausea and vomiting

We hypothesised that the genetic effect of single nucleotide polymorphisms in the TACR1 gene, which encodes NK1 receptors, could influence the sex difference in postoperative nausea and vomiting. Thirty‐two selected single nucleotide polymorphisms were genotyped by the Sanger sequencing method in 200 patients who underwent lower abdominal surgery. The incidence and severity of postoperative nausea and vomiting were evaluated after surgery. The rs3755468‐SNP showed significant association with the incidence and severity of postoperative nausea and vomiting (p = 0.016). The TT haplotype defined by two single nucleotide polymorphisms, including the rs3755468‐SNP, was associated with reduced incidence and severity of postoperative nausea and vomiting in female patients (p = 0.03). The rs3755468‐SNP is located within the predicted oestrogen response element and a DNase I hypersensitive site. The single nucleotide polymorphisms in the TACR1 gene are associated with sex differences in postoperative nausea and vomiting and may help to elucidate the mechanisms underlying these differences.     

Impact of kidney transplantation on sleep apnea severity: A prospective polysomnographic study

Fluid overload has been associated with a high prevalence of sleep apnea (SA) in patients with end‐stage kidney disease (ESKD). In this prospective study, we hypothesized that improvement in kidney function and hydration status after kidney transplantation (Tx) may result in an improvement in SA severity. A total of 196 patients on the kidney Tx waiting list were screened for SA using home nocturnal polysomnography (PSG) to measure the apnea‐hypopnea index (AHI) and underwent bioimpedance to assess body composition. Of 88 participants (44.9%) with SA (AHI ≥ 15/h), 42 were reassessed 6 months post‐Tx and were compared with 27 control patients. There was a significant, but small, post‐Tx improvement in AHI (from 44.2 ± 24.3 to 34.7 ± 20.9/h, P = .02) that significantly correlated with a reduction in fluid overload (from 1.8 ± 2.0 to 1.2 ± 1.2 L, P = .02) and body water (from 54.9% to 51.6%, P = .003). A post‐Tx increase in body fat mass (from 26% to 30%, P = .003) possibly blunted the beneficial impact of kidney Tx on SA. All parameters remained unchanged in the control group. In conclusion, SA is a frequent condition in ESKD patients and partially improved by kidney Tx. We suggest that SA should be systematically assessed before and after kidney Tx. ClinicalTrials.gov Identifier: NCT02020642.     

Molecular epidemiological characterization of Brucella isolates from sheep and yaks in northwest China

Animal brucellosis is a re‐emerging disease in China with high prevalence in the northwest region. A total of 66 isolates of Brucella were recovered from sheep and yaks in the Inner Mongolia, Xinjiang, Qinghai and Gansu provinces of northwest China in 2015 and 2016. Using classical biotyping and the Brucella AMOS PCR assay, all isolates were identified as Brucella melitensis biovar 3 (n  = 58), B. melitensis biovar 1 (n  = 1), Brucella abortus (n  = 5), or Brucella suis biovar 3 (n  = 2), and B. melitensis biovar 3 was found to be mainly responsible for sheep brucellosis in northwest China. Multilocus variable‐number tandem‐repeat analysis (MLVA ) was used to identify the epidemiological relationships among the isolates and to assess their genetic diversity. Multilocus variable‐number tandem‐repeat analysis‐16 identified 46 genotypes in these populations, including 37 unique and nine shared genotypes. Multilocus variable‐number tandem‐repeat analysis‐11 showed that 71% of the isolates (47 of 66) were genotype 116 (1‐5‐3‐13‐2‐2‐3‐2‐4‐41‐8), a characteristic subgroup of the East Mediterranean group, showing that isolates from different geographical areas exhibit similar epidemiological characteristics in different regions and may be epidemiologically linked. Multilocus variable‐number tandem‐repeat analysis‐11 also revealed that an isolate from Inner Mongolia had a novel genotype, 369 (1‐5‐3‐13‐2‐2‐3‐2‐7‐41‐8). Multilocus variable‐number tandem‐repeat analysis‐16 genotyping of northwest China Brucella isolates allows a better understanding of the epidemiology of animal brucellosis in this region. This study is the first analysis of B. melitensis in Gansu province, and the results confirmed that in this province, isolates of this species are disorderly and unsystematic.     

Characterization of Foot‐and‐Mouth Disease Viruses Collected in Nigeria Between 2007 and 2014: Evidence for Epidemiological Links Between West and East Africa

This study describes the molecular characterization of 47 foot‐and‐mouth disease (FMD) viruses recovered from field outbreaks in Nigeria between 2007 and 2014. Antigen ELISA of viral isolates was used to identify FMD virus serotypes O, A and SAT 2. Phylogenetic analyses of VP1 nucleotide sequences provide evidence for the presence of multiple sublineages of serotype SAT 2, and O/EAST AFRICA 3 (EA‐3) and O/WEST AFRICA topotypes in the country. In contrast, for serotype A, a single monophyletic cluster of viruses has persisted within Nigeria (2009–2013). These results demonstrate the close genetic relatedness of viruses in Nigeria to those from other African countries, including the first formal characterization of serotype O/EA‐3 viruses in Nigeria. The introductions and persistence of certain viral lineages in Nigeria may reflect transmission patterns via nomadic pastoralism and animal trade. Continuous monitoring of field outbreaks is necessary to dissect the complexity of FMD epidemiology in sub‐Saharan Africa.     

Phylogenetic Analysis of Canine Parvovirus VP2 Gene in China

In this study, a total of 37 samples (58.0%) were found through PCR assay to be positive for canine parvovirus (CPV) of 66 suspected faecal samples of dogs collected from various cities throughout China. Eight CPV isolates could be obtained in the CRFK cell line. The sequencing of the VP2 gene of CPV identified the predominant CPV strain as CPV‐2a (Ser297Ala), with two CPV‐2b (Ser297Ala). Sequence comparison revealed homologies of 99.3–99.9%, 99.9% and 99.3–99.7% within the CPV 2a isolates, within the CPV 2b isolates and between the CPV 2a and 2b isolates, respectively. In addition, several non‐synonymous and synonymous mutations were also recorded. The phylogenetic tree revealed that most of the CPV strains from different areas in China were located in the formation of a large branch, which were grouped together along with the KU143‐09 strain from Thailand and followed the same evolution. In this study, we provide an updated molecular characterization of CPV 2 circulation in China.     

TCR Repertoire Analysis by Next Generation Sequencing Allows Complex Differential Diagnosis of T Cell–Related Pathology

Clonotype analysis is essential for complete characterization of antigen‐specific T cells. Moreover, knowledge on clonal identity allows tracking of antigen‐specific T cells in whole blood and tissue infiltrates and can provide information on antigenic specificity. Here, we developed a next generation sequencing (NGS)‐based platform for the highly quantitative clonotype characterization of T cells and determined requirements for the unbiased characterization of the input material (DNA, RNA, ex vivo derived or cell culture expanded T cells). Thereafter we performed T cell receptor (TCR) repertoire analysis of various specimens in clinical settings including cytomegalovirus (CMV), polyomavirus BK (BKV) reactivation and acute cellular allograft rejection. Our results revealed dynamic nature of virus‐specific T cell clonotypes; CMV reactivation was linked to appearance of new highly abundant antigen‐specific clonalities. Moreover, analysis of clonotype overlap between BKV‐, alloantigen‐specific T cell–, kidney allograft‐ and urine‐derived lymphocytes provided hints for the differential diagnosis of allograft dysfunction and enabled appropriate therapy adjustment. We believe that the established approach will provide insights into the regulation of virus‐specific/anti‐tumor immunity and has high diagnostic potential in the clinical routine.     

Non‐tuberculous Mycobacteria in Wild Boar (Sus scrofa) from Southern Spain: Epidemiological,Clinical and Diagnostic Concerns

Non‐tuberculous mycobacteria (NTM) are widely distributed in the environment, particularly in wet soil, marshland, rivers or streams, but also are causative agents of a wide variety of infections in animals and humans. Little information is available regarding the NTM prevalence in wildlife and their effects or significance in the bovine tuberculosis (bTB) epidemiology and diagnosis. This research shows the most frequently NTM isolated in lymph nodes of wild boar (Sus scrofa) from southern Spain, relating the NTM presence with the individual characteristics, the management of animals and the possible misdiagnosis of Mycobacterium bovis in concurrent infections. A total of 219 NTM isolates were obtained from 1249 wild boar mandibular lymph nodes sampled between 2007 and 2011. All but 75 isolates were identified by the PCR‐restriction analysis‐hsp65, and a partial sequencing of the 16S rDNA was carried out to identify the rest of the isolates. Results showed that Mycobacterium chelonae was the most frequently isolated NTM specie (133 isolates, 60.7%), followed by Mycobacterium avium (24 isolates, 11%). No relation was found regarding sex, body condition and management, but M. chelonae was more frequently detected in adults, whereas M. avium was more prevalent in subadults. The high NTM prevalence observed in the studied wild boar populations could make difficult the bTB diagnostic.     

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.