Neutralizing antibody responses to bluetongue and epizootic hemorrhagic disease virus serotypes in beef cattle

Blood samples were obtained from sentinel beef cattle at monthly intervals, and the sera were tested for antibodies, using a bluetongue virus (BTV) immunodiffusion test (IDT) and virus-neutralization test (VNT), for 5 BTV serotypes (2, 10, 11, 13, and 17) and 2 epizootic hemorrhagic disease virus (EHDV) serotypes (1 and 2). The cattle tested were transported from Tennessee to Texas in 1984 and 1985. All cattle were seronegative by the BTV IDT at the initial bleeding in Texas in 1984 and 1985. In 1984, 16 of 40 (40%) cattle seroconverted as assessed by results of the BTV IDT. In the 16 seropositive cattle in 1984, neutralizing antibodies were detected to BTV serotypes 10 (n = 7), 11 (n = 3), and 17 (n = 11), and EHDV serotypes 1 (n = 1) and 2 (n = 7). In 1984, no cattle seroconverted to BTV-2 or BTV-13. In 1985, 10 of 36 (27.8%) cattle seroconverted as assessed by results of the IDT. Of the 10 seropositive cattle in 1985, neutralizing antibodies were detected to BTV serotypes 10 (n = 10), 11 (n = 10), 13 (n = 7), and 17 (n = 5), and EHDV serotypes 1 (n = 1) and 2 (n = 7). In 1985, no cattle seroconverted to BTV-2. Clinical diseases attributable to BTV or EHDV was not detected in these cattle in 1984 or 1985.     

Recombinant cDNA probe from bluetongue virus genome segment 10 for identification of bluetongue virus

Genome segment 10 of bluetongue virus (BTV) serotype 11 UC8 strain was cloned and subsequently hybridized to viral double-stranded RNA extracted from 90 field isolates of BTV serotypes 10, 11, 13, and 17; the prototype strains of BTV 2, 10, 11, 13, and 17; the prototype strain epizootic hemorrhagic disease virus (EHDV) serotype 1; and 4 field isolates of EHDV serotype 2. The 90 field isolates were obtained from different counties in California, Louisiana, and Idaho during the years 1979, 1980, and 1981. The cloned genetic probe hybridized with all the BTV samples tested, showing different degrees of cross-hybridization at the stringency conditions used in this study. This indicated that BTV genome segment 10 has conserved nucleotide sequences among the BTV serotypes 2, 10, 11, 13, and 17. No cross-hybridization signals were detected between the cloned genome segment 10 of BTV 11 UC8 strain and the prototype strain of EHDV serotype 1 and the field isolates of serotype 2. This probe recognized a wide variety of BTV isolates.     

Serological studies of Australian and Papua New Guinean cattle and Australian sheep for the presence of antibodies against bluetongue group viruses

Following isolation of a virus (CSIRO19) from insects in Australia and its identification as bluetongue virus serotype 20 (BTV20), a nationwide survey of antibodies in cattle and sheep sera was undertaken. Initial studies using the serum neutralization (SN) test showed that the distribution of BTV20 antibodies in cattle was confined to the northern part of Australia. Group-reactive antibody tests (agar gel diffusion precipitin, AGDP, and complement-fixation, CF) showed group-reactive cattle sera south of the BTV20 zone (northern Australia), and southwards from Queensland to New South Wales. Very few group-reactive sheep sera (45 out of 16213) were found and these were of doubtful epidemiological significance. Some of these BTV group-reactive, BTV20-negative, sera were tested in SN tests against BTV1 to 17 and Ibaraki (IBA) virus. The results indicated that BTV1, or a closely related orbivirus, was active in cattle in Queensland, northern Western Australia, and New South Wales, and that antibody to BTV15 was present in some of the cattle sera in northern Western Australia and the Northern Territory. Antibody to IBA virus was present in some cattle sera in Queensland, northern Western Australia and New South Wales. SN antibody titres ?60 were also found to a number of other BTV serotypes in cattle sera in northern Western Australia and Queensland (principally, BTV2 and BTV7). Low level reactions were commonly observed against these and a number of other BTV serotypes, often in the same serum samples. Further, 22% of the group-reactive cattle sera did not react with any of the viruses in the SN tests. Such results were difficult to interpret in terms of known Australian BTV or BTV-related isolates.     

Recovery of dual serotypes of bluetongue virus from infected sheep and cattle

Dual serotypes of bluetongue virus (BTV) were recovered from field-collected samples of sheep and cattle blood. Two sheep, each infected with both BTV serotypes 10 and 17, were found in a flock with bluetongue disease associated with these two serotypes. One sheep infected with BTV serotypes 11 and 17 was found in a second flock; it was the only viremic sheep detected and was clinically ill. Dual serotype infections of one beef and two dairy cattle were found in three geographically separate herds; mixtures recovered were of BTV serotypes 10 and 17 and serotypes 11 and 17. Clinical signs of illness were absent in the cattle in two herds, but severe conjuctivitis was seen in several cows in a third herd, including the cow with a dual serotype infection (BTV 11 and 17). Two of the cattle with dual infections had no serological evidence of BTV as determined by the agar gel precipitin test; serum was not available from the other cow with a dual serotype infection. The significance of dual infections and immune tolerance are discussed.     

Serogrouping of United States and some African serotypes of bluetongue virus using RT-PCR

The diagnostic potential of RT-PCR for detection of bluetongue virus (BTV) ribonucleic acid (RNA) sequence in cell culture and tissue samples from infected ruminants from United States, Sudan, South Africa and Senegal, was evaluated. The non structural protein 1 (NS1) gene of North American BTV serotype 11 was targeted for PCR amplification. The United States BTV serotypes 2, 10, 11, 13 and 17 and the Sudanese BTV serotypes 1, 2, 4 and 16 and BTV serotype 4 from South Africa and BTV serotype 2 from Senegal were studied. RNAs from all BTV field isolates used in this study, propagated in cell cultures, were detected by the described RT-PCR-based assay. The first specific 790bp BTV PCR products were amplified using a pair of outer primers (BTV1 and BTV2). Specificity of the PCR products was confirmed by a nested amplification of a 520bp PCR product using a pair of internal (nested) primers (BTV3 and BTV4). The BTV PCR products were visualized on ethidium bromide-stained agarose gels. Amplification products were not detected when the RT-PCR-based assay was applied to RNAs from closely related orbiviruses including, epizootic hemorrhagic disease virus (EHDV) prototypes serotypes 1, 2, 4; RNA from Sudanese isolate of palyam orbiviruses serogroup and total nucleic acid extracts from uninfected Vero cells. Application of the nested BTV RT-PCR to clinical samples resulted in amplification of BTV RNA from blood and serum samples from goats experimentally infected with BTV4 and from naturally infected sheep, goats, cattle and deer. The results of this study indicated that this RT-PCR assay could be applied for rapid detection of BTV, in cell culture and clinical samples from susceptible ruminants during an outbreak of the disease, in the United States and African.     

Bluetongue and epizootic hemorrhagic disease in ruminants in Georgia: survey by serotest and virologic isolation

The frequencies of precipitating antibodies to bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV) in domestic ruminants and white-tailed deer (WTD) in Georgia were 36% and 32%, respectively (n = 2,200). The frequencies of seropositivity to BTV and EHDV were high among cattle (47% and 42%, respectively [n = 1,068]) and less so in WTD (36% and 34% [n = 414]). The frequencies among sheep were 34% for BTV and 29% for EHDV (n = 286), whereas among goats, seropositivity was 8% for BTV and 7% for EHDV (n = 433). Serum samples from northeastern Georgia (1 of the 4 regions in the survey) had the highest frequency of precipitating antibodies for BTV (45%) and EHDV (38%). The lowest frequency was in southeastern Georgia, with 29% seropositivity for BTV and 24% seropositivity for EHDV. Of the 175 farms or herds in the serosurvey, 70% included animals that had BTV-precipitating antibodies, and 67% included animals which had EHDV-precipitating antibodies. Seventeen viral isolates were obtained from individual animals on 9 different farms. Fifteen of the isolates were BTV--8 from cattle, 4 from sheep, and 3 from WTD; 8 of them were serotype 11, and 7 were serotype 17. Viral isolates from each of 2 WTD were identified as EHDV serotype 1 and serotype 2. Of the total 17 isolates, 11 were from clinically healthy ruminants, and 6 were from animals with clinical signs of BT or EHD. Five of the viral isolates originated from northeastern Georgia, 7 from the northwestern region, and 5 from the southwestern region; none was obtained from specimens from the southeastern region.     

Analysis of genetic variation of epizootic hemorrhagic disease virus and bluetongue virus field isolates by coelectrophoresis of their double-stranded RNA.

Thirty-two bovine field isolates of bluetongue virus (BTV), 6 field isolates of epizootic hemorrhagic disease virus (EHDV) from deer, 4 BTV prototype serotypes (10, 11, 13, and 17), and 2 EHDV prototype serotypes (1 and 2) were coelectrophoresed, using polyacrylamide gels. Field isolates were obtained from various regions of the United States. Analysis of polyacrylamide gels and scattered plots generated for comparison of migration patterns for different isolates within each serotype of BTV revealed wide variation among the individual segments. The BTV serotypes 10 and 11 had more variation, compared with BTV serotypes 13 and 17, especially for migration of genome segment 5. A definitive correlation was not seen between the double-stranded RNA migration profiles on polyacrylamide gel electrophoresis, geographic origin, herd of origin, or year of collection. One BTV field isolate contained more than 1 electropherotype, with 2 bands at the segment-7 position, and it was further characterized as BTV serotype 11. Segments 2 and 5 of EHDV isolates were more variable in their migration than were the other gene segments. Generally, migration profiles for EHDV double-stranded RNA were more variable, compared with those of BTV isolates. Although a correlation was found between migration profiles and serotype of 2 isolates of EHDV, a study of additional EHDV isolates is required before the diversity of electrophoretic patterns of EHDV can be determined.     

Epizootiologic study of bluetongue: virologic and serologic results

Heparinized blood and serum samples were obtained from 1,295 ruminants in herds or flocks with bluetongue virus (BTV) infection in 4 western states. Submissions were from herds or flocks with clinical bluetongue (BT), as well as from animals on premises with no history of BT disease. Insects, including Culicoides variipennis, were collected in areas enzootic for BT disease. Viral isolations were in 10-day-old embryonating chicken eggs that were then adapted to Vero cells for serotyping. Sera were tested from group-specific antibody to BTV by the micro agar gel precipitin (AGP) test. Viral isolations were from cattle (81), sheep (122), goats (9), antelope (2), and C varipennis (5). There were 7 isolates of serotype 120, 114 of serotype 11, 42 of serotype 13, and 56 of serotype 17. In herds or flocks from which BTV was isolated, 51% of cattle, 56% of sheep, 21% of goats, and 52% of antelope had AGP antibodies. Virus was isolated from 43% of the cattle and 23% of the sheep that had no demonstrable evidence of AGP antibodies. Viral isolations were seasonal, occurring from August until December. Approximately 30% of the herds or flocks from which virus was isolated had more than one serotype of virus causing infection.     

Serological studies of two additional Australian blue-tongue virus isolates CSIRO 154 and CSIRO 156

Serological surveys revealed that some cattle in northern Australia possessed bluetongue virus (BTV) group-reactive (agar gel diffusion precipitin, AGDP, and complement-fixing, CF) antibodies, but not serum neutralizing (SN) antibodies, to BTV20, a new type previously found in Australia. Attempts were made during 1979 to isolate viruses causing these reactions. There was one isolate of a virus (CSIRO 154) and eight isolates of another virus (CSIRO 156) made from the blood of healthy cattle in the Northern Territory. These viruses could not be distinguished from BTV20 by AGDP, CF or fluorescent-abtibody tests and hence were designated members of the bluetongue serogroup. Serotyping was carried out using the plaque-inhibition and plaque-reduction SN tests. CSIRO 156 virus could not be distinguished from BTV1 by any of the SN tests and it was concluded that it was an Australian isolate of the BTV1 serotype. CSIRO 154 virus was found to be related to, but not identical with, BTV6. It is probably not one of the known 20 BTV serotypes and may represent a new BTV serotype. None of the three Australian BTV isolates is known to cause clinical disease in sheep or cattle under natural conditions, and biochemical comparisons with the African BTV serotypes may show differences not revealed by these serological studies.     

Evaluation of bluetongue virus diagnostic tests in free-ranging bighorn sheep

Five bluetongue virus (BTV) diagnostic tests were evaluated for use in free-ranging bighorn sheep. We sampled one bighorn sheep population four times between 1989 and 1995. The tests evaluated included virus isolation (VI), polymerase-chain reaction (PCR), serum neutralization (SN), agar-gel immunodiffusion (AGID), and competitive enzyme-linked immunosorbent assay (c-ELISA). The c-ELISA, AGID and SN tests had high levels of agreement in determining serogroup exposure in bighorn sheep. We used maximum-likelihood algorithms to estimate the parameters of each diagnostic test used. Although the c-ELISA and AGID had high sensitivity and specificity, the SN had perfect specificity but lower apparent sensitivity. Due to the potential of cross-reactions among multiple serotypes, results of the SN must be interpreted with caution when assessing serotype exposure in an area where multiple serotypes are endemic. The PCR assay delineated convalescent antibody titers from more-recent infections, and consequently, was pivotal in distinguishing a different exposure pattern between the bighorn sheep and cattle in an adjacent herd. Based on an increasing seroprevalence (50% to 100%), BTV circulated through this bighorn sheep population between 1989 and 1993. This increase in seroprevalence coincided with a bighorn die-off due to BTV infection in June, 1991. An adjacent cattle herd was sampled in 1995 for comparison. The bighorn sheep and adjacent cattle had different patterns of exposure to BTV between 1994 and 1995. There was no evidence that BTV circulated through the bighorn sheep population from 1994 to 1995. In 1995, seroprevalence to BTV decreased to 72%, none of yearling bighorn was seropositive, and all of the 39 bighorn sheep were PCR-negative. In contrast, all adult cattle were seropositive to BTV by c-ELISA and SN, and 4 of the calves were seropositive; 11 of the 24 cattle were PCR-positive, including all five calves. Overall, the pattern of temporal herd immunity in the bighorn sheep appeared to follow a classic epidemic curve, with the appearance and subsequent disappearance of herd immunity coinciding with the 1991 die-off in this population. As low levels of herd immunity and high proportions of susceptible animals are key factors in the development of epidemics, this population of bighorn sheep may be at increased risk for a BTV epidemic in the future.     

Recombinant DNA probe for serotype-specific identification of bluetongue virus 17

The double-stranded RNA genome from 117 field isolates of bluetongue virus (BTV) serotypes 10, 11, 13, and 17 was blotted onto nitrocellulose paper and hybridized with a radioactively labeled cloned copy of DNA genome segment 2 of BTV-17. Viral RNA from BTV prototype strains 2, 10, 11, 13, and 17 were used as controls. The probe hybridized only with the viral RNA from prototype BTV-17 virus and field isolates of BTV-17. There was no cross hybridization with field isolates of BTV serotypes 10, 11, and 13. A complementary DNA probe developed from genes coding for BTV serotype specificity was effectively used in a slot-blot hybridization system for efficiently characterizing the viral serotype.     

Variation amongst the neutralizing epitopes of bluetongue viruses isolated in the United States in 1979-1981.

Neutralizing epitopes present on field isolates of bluetongue virus (BTV) serotypes 10, 11, 13 and 17 were evaluated with a panel of polyclonal and neutralizing monoclonal antibodies (MAbs). A total of 91 field isolates were evaluated, including 15 isolates of BTV-10, 29 isolates of BTV-11, 26 isolates of BTV-13, and 21 isolates of BTV-17. The viruses were isolated from cattle, goats, sheep, elk and deer in Idaho, Louisiana, Nebraska and, predominantly, California, in the years 1979, 1980 and 1981. The isolates were analyzed and compared using a panel of neutralizing MAbs which included five MAbs raised against BTV-2, seven against BTV-10, five against BTV-13, and six against BTV-17. Neutralization patterns obtained with the MAb panel and individual field isolates were compared to those obtained with prototype viruses of each serotype. All field isolates were neutralized by at least some of the MAbs raised against the prototype virus of the same serotype. All field isolates of BTV-10 were neutralized by the seven MAbs raised to BTV-10, whereas the field isolates of BTV-11, BTV-13 and BTV-17 were not consistently neutralized by all of the MAbs raised against the prototype virus of the same serotype. Variation in neutralizing epitopes recognized by the MAb panel was most pronounced amongst the field isolates of BTV-17. A one-way cross neutralization was evident between BTV-10 and BTV-17 as all field isolates of BTV-17 were neutralized by four of the MAbs raised against BTV-10. In contrast, no BTV-10 isolates were neutralized by the MAbs raised against BTV-17. Differences in the MAb neutralization patterns of field isolates of BTV-11, BTV-13 and BTV-17 suggest that the immunogenic domain responsible for their neutralization is plastic, such that individual epitopes within the domain may vary in their significance to the neutralization of different viruses, even of the same serotype. The apparent conservation of neutralizing epitopes on field isolates of BTV-10 suggests that the field isolates may be derived from the modified-live vaccine strain of BTV-10.     

Antigenic relationship of Ibaraki,bluetongue, and epizootic Hemorrhagic disease viruses

Ibaraki virus, which causes a bluetongue-like disease of cattle in Japan, was compared antigenically with the four serotypes of bluetongue virus (BTV) found in the U.S. and with the two serotypes of epizootic hemorrhagic disease virus (EHDV). No antigenic relationship was found between Ibaraki virus and BTV serotypes 10, 11, 13, and 17 in tests for group or serotype-specific antigens. However, Ibaraki virus and EHDV were related antigenically. The agar gel precipitin and indirect fluorescent antibody tests for group antigens showed two-way cross relationships between Ibaraki virus and EHDV serotypes 1 and 2. The more restrictive serotype-specific neutralization test revealed that antigenic relatedness was stronger between Ibaraki virus and the serotype 2 (Alberta strain) of EHDV than between Ibaraki virus and the serotype 1 (New Jersey strain) of EHDV.     

Temporal appearance, geographic distribution, and species of origin of bluetongue virus serotypes in the United States.

Beginning in 1973, all available laboratory and field strains of bluetongue virus (BTV) from the United States were serotyped. Of the viral strains serotyped, 27 were collected from 1953 through 1972; 173 were collected from 1973 through 1977. Although 20 BTV serotypes have been found worldwide, only BTV serotypes 10, 11, 13, and 17 have been found in the United States. Since 1973, serotypes 11 and 17 have been the prevalent serotypes. Samples were collected over a 24-year period in the United States and represent a wide geographic area and diverse host sources (sheep, cattle, wild ruminants, and insect vectors). The collection was not a statistical sampling.     

Regional seroprevalence of bluetongue virus in cattle in Illinois and western Indiana

OBJECTIVE: To estimate seroprevalence of bluetongue virus (BTV) and the geographic distribution of seropositive cattle herds in Illinois and western Indiana. SAMPLE POPULATION: 10,585 serum samples obtained from cattle in 60 herds during 3 transmission seasons (2000 through 2002). PROCEDURES: In a longitudinal study, serum samples were tested for BTV antibodies by use of a competitive ELISA. Four geographic zones were created by use of mean minimum January temperature. A multivariable mixed-effects logistic regression model with a random effect for herd was used to estimate seropositive risk for zone, age of cattle, herd type, and transmission season. RESULTS: Overall, BTV antibodies were detected in 156 (1.5%) samples. Estimated seroprevalence in 2000, 2001, and 2002 was 1.49%, 0.97%, and 2.18%, respectively. Risk of being seropositive for BTV was associated with geographic zone and age. Seroprevalence increased progressively from northern to southern zones, with no evidence of BTV infection in the northernmost zone. In the southernmost zone, annual seroprevalence ranged from 8.65% to 11.00%. Adult cattle were 2.35 times as likely as juvenile cattle to be seropositive. CONCLUSIONS AND CLINICAL RELEVANCE: Overall seroprevalence was lower than has been reported for Illinois cattle. Bluetongue virus antibodies were distributed heterogeneously in this region. Only in the southernmost zone was seroprevalence consistently > 2%. Regionalization of BTV risk based on state borders does not account for such variability. Serologic data could be combined with landscape, climate, and vector data to develop predictive models of BTV risk within transitional regions of the United States.     

Bluetongue and epizootic hemorrhagic disease in white-tailed deer from Oklahoma: serologic evaluation and virus isolation

Blood samples were collected from 194 white-tailed deer from 27 locations in Oklahoma from 1977 through 1984. Sixty-eight (35%) of the deer had antibody against bluetongue virus (BTV) and 78 (40%) had antibody against epizootic hemorrhagic disease virus. Seropositive deer were detected in each of the 4 geographic quadrants of the state. Virus isolation was attempted in 40 deer from the northeast quadrant of Oklahoma (1983 through 1984); BTV was isolated from 11 deer, but epizootic hemorrhagic disease virus was not isolated. The isolation of BTV serotype 11 from these deer from 1983 through 1984 coincided with reported isolations of this serotype in other ruminants in Oklahoma during this time.     

Bluetongue and epizootic hemorrhagic disease virus isolation from vertebrate and invertebrate hosts at a common geographic site

One serotype of bluetongue virus (BTV) and two serotypes of epizootic hemorrhagic disease virus (EHDV) were isolated from vertebrate and invertebrate hosts on a farm in Colorado. The isolations were from blood samples collected a week apart from a dairy heifer with stomatitis and laminitis; EHDV serotypes 1 and 2 were isolated from the first blood sample, and BTV serotype 13 and EHDV serotype 1 were isolated from the second. Antibodies to EHDV and BTV were detected in the serum from this heifer. Both EHDV serotypes and BTV serotype 13 were isolated from pools of female biting gnats (Culicoides variipennis) that had not had a recent blood meal. The BTV insect isolate was biologically transmitted by female gnats from an infected donor sheep to a recipient host sheep. Culicoides variipennis was the predominant insect collected during three nights of light trap captures at the farm.     

Isolation and Identification of Arbovirus from the Sentinel Herds in Jinghong City,Yunnan Province,China in 2019

The purpose of this study was to understand the prevalence of arboviruses in Jinghong city,Yunnan province.Three sentinel cattle were set up in Menghan township of Jinghong city in 2019,blood samples were collected regularly for isolation and identification of arboviruses,a total of 7 virus isolates were obtained.Viral nucleic acid were identification by RT-PCR,and the results showed that two strains of epidemic hemorrhagic fever virus (EHDV) with serotypes of 6 and 7,two strains of bluetongue virus (BTV) with serotypes 4 and 5,D’Aguilar virus (DAV) serotype in one strain of PALV and two unidentified circoviruses were isolated,respectively.The ORF region of virus Seg-2 and Seg-3 sequences was compared and analyzed,all the 7 regional strains of the virus were Eastern,which was most closely related to strains in Japan,Australia and India.The blood and serum of three sentinel animals were tested by viral nucleic acid and serum neutralization test,which proved that all three animals were infected with the corresponding virus.When animals were infected with the virus,specific antibodies in the serum rise rapidly,it reached peaks 3 to 4 weeks later and could remain at this level for a long time.However,the content of viral nucleic acid in the blood decreased rapidly after reaching the peak from 2 to 4 weeks.The isolation,sequence characteristics and infection characteristics of Jinghong arboviruses in animals were reported in this study,the results provided data support for further understanding of local bovine arboviruses.At the same time,7 strains of virus were isolated from 3 cattle,suggesting that there might be more arboviruses in the area.     

2019年云南省景洪市哨兵动物多种虫媒病毒的分离鉴定

本研究旨在了解云南省景洪市虫媒病毒的流行情况。2019年在景洪市勐罕镇设立了3头哨兵动物牛,定期采血进行虫媒病毒的分离与鉴定,共获得7株病毒分离物。经病毒核酸的RT-PCR鉴定,分离到2株血清型分别为6型和7型流行性出血热病毒（epizootic haemorrhagic disease virus,EHDV）,2株血清型分别为4型和5型的蓝舌病病毒（bluetongue virus,BTV）,1株帕利亚血清群病毒（palyam serogroup virus,PALV）中的D’Aguilar virus（DAV）血清型病毒和2株未鉴定出的环状病毒。经病毒Seg-2、Seg-3序列ORF区的比对和进化分析显示,7株病毒的地域型均为Eastern型,与日本、澳大利亚和印度毒株具有最近的亲缘关系。3头哨兵动物的血液和血清,经病毒核酸及血清中和试验检测,证明3头动物均被相应的病毒感染。动物感染病毒后,血清中的特异性抗体迅速上升,3~4周后达最高点并能够在该水平维持较长时间,而血液中病毒核酸含量2~4周到达最高点后则呈迅速下降趋势。本研究报道了景洪虫媒病毒的分离、毒株序列特征以及在动物上的感染特性,研究结果为进一步了解当地的牛虫媒病毒提供数据支撑,同时3头牛分离获得7株病毒,提示当地可能还存在更多种类的虫媒病毒。