Epizootic Hemorrhagic Disease Virus Research Articles

Management of North American Culicoides Biting Midges: Current Knowledge and Research Needs.

Culicoides biting midges (Diptera: Ceratopogonidae) are biological vectors of two important viruses impacting North American ruminants--bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV). These viruses have been identified for over 60 years in North America, but we still lack an adequate understanding of the basic biology and ecology of the confirmed vector, Culicoides sonorensis, and know even less about other putative Culicoides vector species. The major gaps in our knowledge of the biology of Culicoides midges are broad and include an understanding of the ecology of juveniles, the identity of potential alternate vector species, interactions of midges with both pathogens and vertebrates, and the effectiveness of potential control measures. Due to these broad and numerous fundamental knowledge gaps, vector biologists and livestock producers are left with few options to respond to or understand outbreaks of EHD or BT in North America, or respond to emerging or exotic Culicoides-transmitted pathogens. Here we outline current knowledge of vector ecology and control tactics for North American Culicoides species, and delineate research recommendations aimed to fill knowledge gaps.

A Review of Knowledge Gaps and Tools for Orbivirus Research.

Although recognized as causing emerging and re-emerging disease outbreaks worldwide since the late 1800 s, there has been growing interest in the United States and Europe in recent years in orbiviruses, their insect vectors, and the diseases they cause in domestic livestock and wildlife. This is due, in part, to the emergence of bluetongue (BT) in northern Europe in 2006-2007 resulting in a devastating outbreak, as well as severe BT outbreaks in sheep and epizootic hemorrhagic disease (EHD) outbreaks in deer and cattle in the United States. Of notable concern is the isolation of as many as 10 new BT virus (BTV) serotypes in the United States since 1999 and their associated unknowns, such as route of introduction, virulence to mammals, and indigenous competent vectors. This review, based on a gap analysis workshop composed of international experts on orbiviruses conducted in 2013, gives a global perspective of current basic virological understanding of orbiviruses, with particular attention to BTV and the closely related epizootic hemorrhagic disease virus (EHDV), and identifies a multitude of basic virology research gaps, critical for predicting and preventing outbreaks.

Simultaneous detection of five notifiable viral diseases of cattle by single-tube multiplex real-time RT-PCR

Multiplexed real-time PCR (qPCR) assays enable the detection of several target genes in a single reaction, which is applicable for simultaneous testing for the most important viral diseases in samples obtained from ruminants with unspecific clinical symptoms. Here, reverse transcription qPCR (RT-qPCR) systems for the detection of bovine viral diarrhoea virus (BVDV) and bluetongue virus (BTV) were combined with an internal control system based on the beta-actin gene. Additionally, a background screening for three further major pathogens of cloven-hoofed animals reportable to the World Organisation for Animal Health, namely foot-and-mouth disease virus, epizootic haemorrhagic disease virus, and Rift Valley fever virus, was integrated using the identical fluorophore for the respective RT-qPCR assays.Every pathogen-specific assay had an analytical sensitivity of at least 100genome copies per reaction within the multiplex approach, and a series of reference samples and clinical specimens obtained from cattle, but also from small ruminants, were detected reliably. The qPCR systems integrated in the background screening were even not influenced by the simultaneous amplification of very high BVDV and BTV genome copy numbers.The newly developed multiplex qPCR allows the specific and sensitive detection of five of the most important diseases of ruminants and could be used in the context of monitoring programs or for differential diagnostics.

Genetic characterization of epizootic hemorrhagic disease virus strains isolated from cattle in Israel.

Epizootic hemorrhagic disease virus (EHDV), a member of the genus Orbivirus not reported previously in Israel, was isolated from Israeli cattle during a 'bluetongue-like' disease outbreak in 2006. To ascertain the origin of this new virus, three isolates from the outbreak were fully sequenced and compared with available sequences. Whilst the L2 gene segment clustered with the Australian EHDV serotype 7 (EHDV-7) reference strain, most of the other segments were clustered with EHDV isolates of African/Middle East origin, specifically Bahrain, Nigeria and South Africa. The M6 gene had genetic relatedness to the Australian/Asian strains, but with the limited data available the significance of this relationship is unclear. Only one EHDV-7 L2 sequence was available, and as this gene encodes the serotype-specific epitope, the relationship of these EHDV-7 L2 genes to an Australian EHDV-7 reflects the serotype association, not necessarily the origin. The genetic data indicated that the strains affecting Israel in 2006 may have been related to similar outbreaks that occurred in North Africa in the same year. This finding also supports the hypothesis that EHDV entered Israel during 2006 and was not present there before this outbreak.

Cytokine release and endothelial dysfunction: a perfect storm in orbivirus pathogenesis.

Although bluetongue viruses (BTV) and epizootic haemorrhagic disease viruses (EHDV) are closely related, there are differences in susceptibility to these viruses both between and within a species. White‑tailed deer are susceptible to disease by both BTV and EHDV, sheep are susceptible to BTV, but resistant to EHDV, and cattle can be infected with both viruses but disease is usually subclinical. Host genetics probably play a role in the disease outcome, but cytokine and endothelial responses are likely to determine if subclinical or clinical disease develops. Dendritic macrophages deliver virus to lymph nodes following the bite of an infected Culicoides. The virus then disseminates to many organs replicating in mononuclear phagocytes and endothelium. Initially, an interferon‑1 response probably determines if the disease develops. Replication in mononuclear cells and endothelium results in the release of cytokines and vasoactive mediators, and may result in endothelial cell death leading to the clinical features of fever, hyperaemia, exudation of fluid, and haemorrhage. Disease outcome may also be linked to virus binding Toll‑like receptor‑3 and upregulation of endothelial surface receptors potentiating cytokine release and allowing transmigration of inflammatory cells, respectively. Despite a wealth of information, host genetics involved in resistance to BTV and EHDV and how variations in cytokines and endothelial responses determine clinical outcome still need further elucidation.

Molecular evolution of American field strains of bluetongue and epizootic haemorrhagic disease viruses.

Recent Orbivirus occurrences in the Americas have been investigated using whole genome amplification and sequencing followed by phylogenetic analysis. The bluetongue virus (BTV) and epizootic haemorrhagic disease virus (EHDV) whole genomes were amplified without prior sequence knowledge and deep sequenced. This technology was applied to evaluate BTV‑3 isolates spanning 4 decades from Florida, Arkansas, Mississippi, South Dakota, Central America, and the Caribbean basin. The results of the dataset analysis are consistent with the hypothesis that these viruses were introduced into the United States from Central America and the Caribbean basin. A similar analysis has been performed on a recent BTV‑2 isolate from California. It indicates that the BTV‑2 strain was likely introduced into Florida and then moved South to the Caribbean and West to California. A historical (1955‑2012) molecular characterisation of EHDV strains was also completed, and subsequently used as reference sequence for comparison of genomes from recent 2012 cattle isolates associated with clinical disease. Finally, this analysis was performed on BTV‑11 isolated from 2 canine cases and demonstrated that the genome sequences of the virus isolates from these cases were almost identical. These studies indicate the value of this technology in understanding virus epidemiology and ecology.

Bovine Arboviruses in Culicoides Biting Midges and Sentinel Cattle in Southern Japan from 2003 to 2013.

Epizootic congenital abnormalities, encephalomyelitis and febrile illnesses in cattle caused by arthropod-borne viruses (arboviruses) are prevalent in Japan. Causative viruses including orthobunyaviruses, orbiviruses and rhabdovirus are thought to be transmitted by Culicoides biting midges. Recently, the incursions of several arboviruses, potentially Culicoides-borne, were newly confirmed in Japan. However, their spread pattern and exact vector species are currently uncertain. Attempts to isolate arboviruses from Culicoides biting midges and sentinel cattle were conducted in Kagoshima, located at the southernmost end of the main islands of Japan, a potentially high-risk area for incursion of arboviral diseases and outbreak of endemic ones. Seventy-eight isolates comprising Akabane, Peaton and Sathuperi viruses of the genus Orthobunyavirus of the family Bunyaviridae, bluetongue virus serotype 16, D'Aguilar virus, Bunyip Creek virus and epizootic haemorrhagic disease virus serotype 1 of the genus Orbivirus of the family Reoviridae, a potentially novel rhabdovirus of the genus Ephemerovirus and unidentified orbivirus-like viruses were obtained from Culicoides biting midges and sentinel cattle between 2003 and 2013. Akabane, Sathuperi, D'Aguilar and Bunyip Creek viruses were selectively isolated from Culicoides oxystoma, suggesting this vector's responsibility for these arbovirus outbreaks. The results of virus isolation also implied that C.tainanus, C.jacobsoni and C.punctatus are competent for the transmission of bluetongue virus serotype 16, Peaton virus and epizootic haemorrhagic disease virus serotype 1, respectively. Our monitoring in Culicoides biting midges and sentinel cattle detected the circulation of Akabane virus just prior to the accumulations of bovine congenital abnormalities and encephalomyelitis by it around study sites in 2003, 2006, 2008 and 2013. Silent circulations of the other arboviruses, including potentially new viruses, were also detected during the study period.

Apparent increase of reported hemorrhagic disease in the midwestern and northeastern USA.

We investigated temporal and spatial trends in reporting of hemorrhagic disease (HD) in the midwestern and northeastern US using a 33-yr (1980-2012) questionnaire-based data set. This data set was supported by an additional 19 yr (1994-2012) of bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV) isolation results from clinically affected white-tailed deer (Odocoileus virginianus) in these regions. Both the number of counties that were reported positive for HD and the northern latitudinal range of reported HD increased with time. A similar increase was observed with both the number of states annually reporting HD and the number of counties where HD was reported. Large-scale outbreaks occurred in 1988, 1996, 2007, and 2012, and the scale of these individual outbreaks also increased with time. The predominant virus isolated from these regions was EHDV-2, but the prevalence of EHDV-6, which was first detected in 2006, appears to be increasing. Temporally, the extent of regional HD reporting was correlated with regional drought conditions. The significance of increases in reported HD and the incursions and establishment of new BTV and EHDV in the US currently are unknown.

Development of reverse genetics for Ibaraki virus to produce viable VP6-tagged IBAV

Ibaraki virus (IBAV) is a member of the epizootic hemorrhagic disease virus (EHDV) serogroup, which belongs to the Orbivirus genus of the Reoviridae family. Although EHDV, including IBAV, represents an ongoing threat to livestock in the world, molecular mechanisms of EHDV replication and pathogenesis have been unclear. The reverse genetics (RG) system is one of the strong tools to understand molecular mechanisms of virus replication. Here, we developed a RG system for IBAV to identify the nonessential region of a minor structural protein, VP6, by generating VP6-truncated IBAV. Moreover, several tags were inserted into the truncated region to produce VP6-tagged IBAV. We demonstrated that all VP6-tagged IBAV could replicate in BHK cells in the absence of any helper VP6 protein. Further, tagged-VP6 proteins were first assembled into puncta in cells infected with VP6-tagged IBAV. Our data suggests that, in order to initiate primary replication, IBAV VP6 is likely to accumulate in some parts of infected cells to assemble efficiently into the primary replication complex (subcore).

Serologic, trace element, and fecal parasite survey of free-ranging, female mule deer (Odocoileus hemionus) in eastern Washington, USA.

Blood and fecal samples collected from 97 free-ranging mule deer (Odocoileus hemionus), from four distinct herds during the spring of 2000 or 2001 in eastern Washington, US, were tested for exposure to selected pathogens, concentrations of trace elements, and presence of parasites in feces. Antibodies were detected to the following: Leptospira interrogans serovar Bratislava (4%), Leptospira interrogans serovar Canicola (1%), Leptospira interrogans serovar Grippotyphosa (13%), Bovine viral diarrhea virus 1 (57%), Bovine respiratory syncytial virus (71%), Infectious bovine rhinotracheitis virus (51%), Bovine parainfluenza virus 3 (61%), Bluetongue virus (25%), and Epizootic hemorrhagic disease virus (25%); 3 of 63 (5%) samples had antibody to Neospora spp. All samples tested for antibody to Brucella abortus and L. interrogans serovar Icterohaemorrhagiae, L. interrogans serovar Pomona, and L. interrogans serovar Hardjo samples were negative. Trace element concentrations from 97 sera were deficient for selenium (17%), copper (19%), iron (34%), calcium (3%), and phosphorus (2%) compared with thresholds established for domestic livestock. Parasites detected in 97 fecal samples included dorsal-spined larvae (probably Parelaphostrongylus sp.) (40%), abomasal nematode eggs (1%), Capillaria sp. eggs (1%), Nematodirus sp. eggs (26%), Moniezia sp. eggs (1%), and Eimeria sp. (2%).

Testicular lesions and antler abnormalities in Colorado, USA mule deer (Odocoileus hemionus): a possible role for epizootic hemorrhagic disease virus.

Antler abnormalities of deer and other cervids often result from testicular lesions and decreased levels of testosterone, inhibiting normal cycles of antler growth. Affected males have antlers with retained velvet, numerous short, misshapen points ("cactus bucks"), and failure to shed these abnormal antlers annually. In Colorado, US, we observed a high occurrence of "cactus bucks" in mule deer (Odocoileus hemionus) populations after management efforts to increase the number of mature male deer in the state. Affected males consistently had antibody to epizootic hemorrhagic disease virus serotype 2 (EHDV-2), and examination of the testes of these animals demonstrated nonspecific end-stage lesions of chronic inflammation, fibrosis, and mineralization. To examine more acute stages of testicular lesions, and to screen for EHDV specifically within the testes, we sampled 16 male mule deer from affected herds, but with essentially normal antlers (n = 14) or retained velvet only (n = 2). Testicular and epididymal lesions identified from these samples included necrotizing vasculitis (n = 2), hemorrhage (n = 6), edema (n = 2), seminiferous tubular necrosis (n = 5), orchitis (n = 5), epididymitis (n = 10), hypospermia (n = 6), and end-stage lesions of seminiferous tubular loss (n = 2), fibrosis (n = 2), and mineralization (n = 2). Each of the 16 cases was blindly scored on the basis of number of histologic lesions, with a median score of two. Five of seven (71%) testes that were PCR positive for EHDV had lesion scores above the median, whereas none of the nine (0%) EHDV PCR-negative testes had lesion scores above the median, suggesting an association between testicular lesions and detection of EHDV RNA in the testes (P = 0.003). Although the role of EHDV infection remains unconfirmed, the association between testicular and epididymal lesions and presence of EHDV RNA in the affected tissues suggests that cactus buck antlers may be a sequela of EHDV infection.

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).

Analysis of murine B-cell epitopes on bluetongue virus 12 nonstructural protein 1.

The bluetongue virus (BTV) NS1 protein is one of the major proteins synthesized during BTV infection and is responsible for the generation of virus-specific tubules. Although some functional and structural studies on the BTV NS1 protein have been reported, there have been no reports describing the linear B-cell epitopes recognized by humoral immune responses published to date. In this study, 25 BTV12 NS1-reactive monoclonal antibodies (MAbs) and polyclonal antisera (polyclonal antibodies, PAbs) were generated and analyzed. We identified 14 linear NS1 epitopes recognized by the PAbs and MAbs using NS1-derived peptides in an enzyme-linked immunosorbent assay. Moreover, we predicted 23 linear B-cell epitopes using the ABCpred online server which employs an artificial neural network. Analysis of the predicted and identified epitopes of NS1 demonstrated the feasibility of B-cell epitope prediction. Sequence alignments indicated that the epitopes recognized by MAbs are highly conserved among BTV serotypes, but not among the other members of the genus Orbivirus, such as the African horse sickness virus (AHSV), epizootic hemorrhagic disease virus (EHDV), and Chuzan disease virus (CV). Importantly, we identified specific MAbs that recognized all BTV serotypes tested as well as MAbs that recognized only BTV12, suggesting that these NS1-specific MAbs could serve as a basis for BTV diagnostic approaches. The generation and identification of NS1 protein epitopes will provide the foundation for further studies about the function and structure of NS1 and novel epitope-based vaccines.

Identification of bluetongue virus and epizootic hemorrhagic disease virus serotypes in French Guiana in 2011 and 2012

In French Guiana, the sero- and viro-prevalence of Bluetongue virus (BTV) is high but the circulating serotypes remain unknown. No data are available regarding the prevalence of Epizootic hemorrhagic disease (EHD). This study was conducted to assess the prevalence and to identify the circulating serotypes of these two Orbiviruses in this region (BTV and EHDV). Blood samples were collected in main livestock areas, from 122 young cattle between June and August 2011, to perform virological (PCR and viral isolation) and serological (ELISA) analyses. Moreover, samples from sheep and goat showing BTV-like clinical signs and from newly imported animals were analyzed using the same assays. Results confirmed an important viral circulation, with viro- and seroprevalence of 85% and 84% and 60% and 40% for BTV and EHDV, respectively. Ten Orbivirus serotypes were identified (BTV-1, 2, 6, 10, 12, 13, 17 and 24, EHDV-1 and 6). The circulation of many serotypes in intertropical America and in the Caribbean region underlines the need to establish measures to monitor and control animal movements.

Characterization of epizootic hemorrhagic disease virus from a bovine with clinical disease with high nucleotide sequence identity to white-tailed deer isolates.

Epizootic hemorrhagic disease virus (EHDV) was isolated from a pregnant cow in Indiana, USA, exhibiting excessive salivation, pyrexia and abortion. VP2, VP5, and VP7 sequences of the isolated bovine EHDV showed 97.7, 97.4, and 97.9 % identity to a serotype 2 reference virus. Bovine EHDV was closely related (>99.9 %) to white tailed deer (WTD) EHDV collected from Iowa in 2013 and showed less than 2.1 % divergence from EHDV collected from WTD across the USA in 2013. The high degree of sequence identity between bovine and WTD EHDV isolates demonstrates that similar viruses concurrently circulate in both species and suggests possible further incursions into bovines.

Molecular Typing of Epizootic Hemorrhagic Disease Virus Serotypes by One-Step Multiplex RT-PCR

Epizootic hemorrhagic disease virus (EHDV) causes a highly infectious noncontagious hemorrhagic disease in wild and captive deer (Cervidae) populations in the US. Although rapid and accurate identification of the disease is important, identification of the serotype is equally important for understanding the epidemiology of the disease in white-tailed deer (Odocoileus virginianus) populations. We developed a one-step multiplex reverse transcriptase PCR assay for rapid differentiation and identification of EHDV serotypes 1, 2, and 6 in cell culture and clinical samples by targeting the viral gene segment 2 (L2) that encodes for the structural protein VP2. From 2009 to 2012, 427 clinical samples including tissue and blood (in ethylenediaminetetraacetic acid) from white-tailed deer, found EHDV positive by real-time PCR, were used to evaluate this subtyping assay. Eighteen percent of the positive samples tested were EHDV-1, 59% were EHDV-2, and 21% were EHDV-6; 2% of the samples were positive for more than one subtype, indicating mixed infection. This assay provides a rapid, sensitive, specific diagnostic tool for differentiation and identification of EHDV serotypes in field samples and virus isolates.

Epizootic haemorrhagic disease virus in Reunion Island: Evidence for the circulation of a new serotype and associated risk factors

Bluetongue virus (BTV) and epizootic haemorrhagic disease virus (EHDV) are members of the Orbivirus genus of the Reoviridae family transmitted between ruminants by the bites of Culicoides midges. BTV went undetected in Reunion Island between its first documented emergence in 1979 and two other serious outbreaks with both BTV-3 and EHDV-6 in 2003, and both EHDV-6 and BTV-2 in 2009. In these outbreaks, infected animals developed symptoms including hyperthermia, anorexia, congestion, prostration and nasal discharge.Samples were collected in 2011 to assess the prevalence of BT and EHD in ruminants native to Reunion Island by serological analysis. A cross-sectional study was undertaken on 67 farms, including a total of 276 cattle, 142 sheep and 71 goats. The prevalence rates of BT and EHD were 58% (95% CI [54.03–62.94]) and 38% (95% CI [33.85–42.63], respectively. Two further suspected outbreaks were confirmed to involve EHDV and BTV/EHDV. A new circulating EHDV serotype 1 of unknown origin was isolated. Our results confirm that the prevalence of both BT and EHD is high and that both are likely currently circulating. A high risk of BTV and EHDV infections was associated with the introduction of ruminants from neighbouring farms without quarantine, the presence of organic and other waste on the farm, and treatment against ectoparasites and insects.

Tibet Orbivirus, a Novel Orbivirus Species Isolated from Anopheles maculatus Mosquitoes in Tibet, China

BackgroundThe genus Orbivirus includes a number of important pathogenic viruses, including Bluetongue virus (BTV), African horse sickness virus (AHSV), and Epizootic hemorrhagic disease virus (EHDV). In this study we describe the isolation and characterization of an Orbivirus strain isolated from Anopheles maculatus mosquitoes collected in Tibet, China.Methods and ResultsInitial viral screening identified a viral strain (XZ0906) that caused significant cytopathic effect (CPE) in BHK-21 cells, including rounding, cell rupture, and floating. Although CPE was not observed in insect cells (C6/36), these cells supported viral replication. Polyacrylamide gel analysis revealed a genome consisting of 10 segments of double-stranded RNA (dsRNA), with a distribution pattern of 3-3-3-1. 454 high throughput sequencing of culture supernatant was used for viral identification. Complete genome sequencing was performed by Sanger sequencing in combination with 5′-RACE and 3′-RACE. Sequence analysis demonstrated that all 5′- and 3′- untranslated regions (UTRs) for each of the 10 genome segments contained a series of six highly conserved nucleotides. In addition, homology analysis and phylogenetic analysis based on amino acid sequence was completed, and all results show that virus XZ0906 was not a member of any known species or serotype of Orbivirus, indicating it to be a new species within the genus Orbivirus.ConclusionsThe isolated Orbivirus strain was designated Tibet Orbivirus, TIBOV to denote the location from which it was isolated. TIBOV is a novel orbivirus species which is isolated from Anopheles maculatus mosquitoes collected in Tibet, China.

The host specific NS3 glycosylation pattern reflects the virulence of Ibaraki virus in different hosts

The non-structural protein NS3 was investigated in Ibaraki virus (IBAV), an epizootic hemorrhagic disease virus, serotype 2. Degree of NS3 glycosylation, cytopathic effect, and virus release efficiency were compared between mammalian and insect cells. The molecular weight of synthesized NS3 was compared in Western blot analysis following the removal of the glycochain by PNGase F treatment and revealed that glycosylation of NS3 occurred only in mammalian cells. Also, it was revealed that the amount of infectious IBAV in the extracellular fraction continued to increase for insect cells even after 60h post infection without disrupting cells. These results suggested that glycosylation of NS3 controls pathogenicity of IBAV in host cells to protect vector insects by altering the release pathway of assembled progeny viruses.

Whole genome analysis of epizootic hemorrhagic disease virus identified limited genome constellations and preferential reassortment

Epizootic hemorrhagic disease virus (EHDV) is a Culicoides transmitted orbivirus that causes haemorrhagic disease in wild and domestic ruminants. A collection of 44 EHDV isolated from 2008 to 2012 was fully sequenced and analysed phylogenetically. Serotype 2 viruses were the dominant serotype all years except 2012 when serotype 6 viruses represented 63 % of the isolates. High genetic similarity (>94 % identity) between serotype 1 and 2 virus VP1, VP3, VP4, VP6, NS1, NS2 and NS3 segments prevented identification of reassortment events for these segments. Additionally, there was little genetic diversity (>96 % identity) within serotypes for VP2, VP5 and VP7. Preferential reassortment within the homologous serotype was observed for VP2, VP5 and VP7 segments for type 1 and type 2 viruses. In contrast, type 6 viruses were all reassortants containing VP2 and VP5 derived from an exotic type 6 with the remaining segments most similar to type 2 viruses. These results suggest that reassortment between type 1 and type 2 viruses requires conservation of the VP2, VP5 and VP7 segment constellation while type 6 viruses only require VP2 and VP5 and are restricted to type 2-lineage VP7. As type 6 VP2 and VP5 segments were exclusively identified in viruses with type 2-derived VP7, these results suggest functional complementation between type 2 and type 6 VP7 proteins.