In situ stress testing to identify Australian black tiger prawns (Penaeus monodon) free of gill-associated virus and Mourilyan virus.

OBJECTIVE: To identify whether black tiger prawns (Penaeus monodon) in the Weipa region of the Gulf of Carpentaria, Queensland, are free of gill-associated virus (GAV) and Mourilyan virus (MoV), which are endemic in P. monodon along the east coast of Queensland. PROCEDURE: Preliminary screening suggested that Weipa might be a source of P. monodon that are free of GAV and MoV. To assess this, more than 150 prawns captured near Weipa were maintained locally in tanks for 2 weeks and bled three times as a stressor to promote higher-level infections. The existence of GAV and MoV in lymphoid organ tissue was then determined using real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Some prawns were maintained in tanks for an additional 75 days before being tested. RESULTS: Real-time qRT-PCR did not detect GAV in any of 33 pools of RNA isolated from the 166 prawns tested. MoV was detected in five pools of RNA at extremely low viral RNA copy numbers close to the sensitivity threshold of the test. MoV was also detected at a similar low copy number in one of nine pearl-oyster mantle samples used as negative controls. CONCLUSIONS: GAV infection is either absent or, like MoV, potentially present at a very low prevalence in juvenile P. monodon inhabiting the inshore waters at Weipa. This region can thus be recommended as a good source of P. monodon certifiable as specific pathogen-free for GAV and MoV, which is desirable for domestication and selective breeding programs in Australia.

Phylogenetic relationships among rhabdoviruses inferred using the L polymerase gene.

RNA viruses of the family Rhabdoviridae include arthropod-borne agents that infect plants, fish and mammals, and also include a variety of non-vector-borne mammalian viruses. Herein is presented a molecular phylogenetic analysis, the largest undertaken to date, of 56 rhabdoviruses, including 20 viruses which are currently unassigned or assigned as tentative species within the Rhabdoviridae. Degenerate primers targeting a region of block III of the L polymerase gene were defined and used for RT-PCR amplification and sequencing. A maximum-likelihood phylogenetic analysis of a 158-residue L polymerase amino acid sequence produced an evolutionary tree containing the six recognized genera of the Rhabdoviridae and also enabled us to identify four more monophyletic groups of currently unclassified rhabdoviruses that we refer to as the 'Hart Park', 'Almpiwar', 'Le Dantec' and 'Tibrogargan' groups. The broad phylogenetic relationships among these groups and genera also indicate that the evolutionary history of rhabdoviruses was strongly influenced by mode of transmission, host species (plant, fish or mammal) and vector (orthopteran, homopteran or dipteran).

The complete genome sequence of gill-associated virus of Penaeus monodon prawns indicates a gene organisation unique among nidoviruses.

We report here a 5596 nt sequence comprising the 3'-end of the (+) ssRNA genome of gill-associated virus (GAV), an invertebrate nidovirus of Penaeus monodon prawns. The sequence extends from a subgenomic RNA start site 35 nt upstream of the 4923 nt ORF3 gene to a 3'-poly(A) tail of the 26235 nt genome of GAV. The putative 1640 amino acid (aa) ORF3 protein (MW = 182049 Da, pI = 6.62) contains 15 potential N-linked glycosylation sites, 15 potential O-linked glycosylation sites and six highly hydrophobic regions predicted to represent transmembrane (TM) domains. Three of the predicted TM domains occur in the amino-terminal 228 aa, two in the central portion, and one near the carboxy-terminus of ORF3. Only one short (83 aa) open reading frame (ORF4) was identified between ORF3 and the 3'-poly(A) tail. Completion of the genome sequence of GAV has revealed a gene organisation unique among nidoviruses in there is no discrete membrane protein gene and that the putative ORF3 spike glycoprotein gene resides downstream of a gene (ORF2) encoding a structural protein associated with nucleocapsids.

Differences in the susceptibility of some penaeid prawn species to gill-associated virus (GAV) infection.

Four species of penaeid prawn cultured in Australia (Penaeus monodon, Penaeus esculentus, Marsupenaeus japonicus and Fenneropenaeus merguiensis) were injected with a virulent preparation of gill-associated virus (GAV). P. monodon (average weight = 8.9, 13.9 and 19.2 g), P. esculentus (average weight = 19.5 g), F. merguiensis (average weight = 10.5 g), and small (average weight = 5.8 g) M. japonicus displayed overt signs of disease and mortalities which reached 82 to 100% within 23 d post-injection. Cumulative mortalities in P. esculentus and F. merguiensis were significantly lower than for P. monodon of the same size class. Medium (average weight = 13.0 g) M. japonicus also developed overt signs of disease but cumulative mortalities were not significantly higher than uninfected controls. Large (average weight = 20.3 g) M. japoncius did not display symptoms of disease and there were no significant mortalities up to 23 d post-injection.

RNA polymerase (L) gene and genome terminal sequences of ephemeroviruses bovine ephemeral fever virus and Adelaide River virus indicate a close relationship to vesiculoviruses.

The sequence of the RNA genome of bovine ephemeral fever virus (BEFV) was determined from the start of the L (polymerase) gene to the end of the untranslated 5' trailer sequence, completing the sequence of the 14900 nucleotide (nt) genome. The 6470 nt L gene encodes a single long ORF of 2144 amino acids with a deduced molecular weight of 249766 Da. The 70 nt BEFV 5' trailer region displays partial terminal complementarity with the 3' leader sequence and contains a 26 nt direct repeat of the U-rich domain of the 3' leader region. The 47 nt 5' trailer region of Adelaide River virus (ARV) displays terminal sequence similarity to the BEFV trailer and partial terminal complementarity with the ARV 3' leader sequence, but does not contain the direct repeat sequence. The BEFV L protein contains all characteristic sequence motifs of amino acid blocks I-VI, conserved among RNA polymerase proteins of single-stranded (-) RNA viruses, separated by regions of lower homology. Phylogenetic analysis using the complete BEFV L protein sequence indicated a closer relationship to vesicular stomatitis virus than to rabies virus. Sequence comparison of two conserved central domains encompassing blocks II and III and block VI of the BEFV and ARV L proteins indicated they are closely related. An extended phylogenetic analysis using the block III sequence, confirmed the relationship of these ephemeroviruses to vesiculo- and lyssaviruses and to other single-stranded (-) RNA viruses.

Gill-associated virus of Penaeus monodon prawns: an invertebrate virus with ORF1a and ORF1b genes related to arteri- and coronaviruses.

A 20089 nucleotide (nt) sequence was determined for the 5' end of the (+)-ssRNA genome of gill-associated virus (GAV), a yellow head-like virus infecting Penaeus monodon prawns. Clones were generated from a approximately 22 kb dsRNA purified from lymphoid organ total RNA of GAV-infected prawns. The region contains a single gene comprising two long overlapping open reading frames, ORF1a and ORF1b, of 4060 and 2646 amino acids, respectively. The ORFs are structurally related to the ORF1a and ORF1ab polyproteins of coronaviruses and arteriviruses. The 99 nt overlap between ORF1a and ORF1b contains a putative AAAUUUU 'slippery' sequence associated with -1 ribosomal frameshifting. A 131 nt stem-loop with the potential to form a complex pseudoknot resides 3 nt downstream of this sequence. Although different to the G/UUUAAAC frameshift sites and 'H-type' pseudoknots of nidoviruses, in vitro transcription/translation analysis demonstrated that the GAV element also facilitates read-through of the ORF1a/1b junction. As in coronaviruses, GAV ORF1a encodes a 3C-like cysteine protease domain located between two hydrophobic regions. However, its sequence suggests some structural relationship to the chymotrypsin-like serine proteases of arteriviruses. ORF1b encodes homologues of the 'SDD' polymerase, which among (+)-RNA viruses is unique to nidoviruses, as well as metal-ion-binding and helicase domains. The presence of a dsRNA replicative intermediate and ORF1a and ORF1ab polyproteins translated by a-1 frameshift suggests that GAV represents the first invertebrate member of the Order NIDOVIRALES:

Detection of Australian gill-associated virus (GAV) and lymphoid organ virus (LOV) of Penaeus monodon by RT-nested PCR.

A highly sensitive test based on reverse transcription followed by nested polymerase chain reaction (RT-nPCR) was developed to detect the Australian yellow-head-like viruses, gill-associated virus (GAV) and lymphoid organ virus (LOV) of Penaeus monodon. The RT-nPCR detected viral RNA in as little as 10 fg lymphoid organ total RNA isolated from GAV-infected P. monodon. Amplification of serial dilutions of a GAV cDNA clone showed that the nested PCR was sufficiently sensitive to detect a single genome equivalent using a DNA template. The specificity and sensitivity of the RT-nPCR was also demonstrated using experimentally infected P. (Marsupenaeus) japonicus, where GAV sequences could be amplified from lymphoid organ and haemocyte RNA as early as 6 h post infection (p.i.), and from gills by 24 h p.i. In contrast, transmission electron microscopy (TEM) identified nucleocapsids and virions in lymphoid organ cells and haemocytes from Days 3 and 6 p.i., respectively, while there was no evidence of infection in gill cells at any time. The practical application of the RT-nPCR was demonstrated by screening healthy wild-caught P. monodon broodstock. The high prevalence (>98%) of broodstock that were positive by RT-nPCR suggests that LOV is endemic in northern Queensland. In addition, results with lymphoid organ, gill and haemocyte RNA suggest that small gill biopsies may be best suited to the non-sacrificial testing of valuable broodstock. The speed and sensitivity of the RT-nPCR make it a useful adjunct to TEM for diagnosing LOV/GAV infection of P. monodon, with the additional benefit that screening of gill biopsies may facilitate selection of LOV-free broodstock.

Yellow head virus from Thailand and gill-associated virus from Australia are closely related but distinct prawn viruses.

Corresponding genomic regions of isolates of yellow head virus (YHV) from Thailand and gill-associated virus (GAV) from Australia were compared by RT-PCR and sequence analysis. PCR primers designed from sequences in the GAV ORF1b polyprotein gene amplified the corresponding 577 nucleotide region of the YHV genome. Comparison of the amplified region indicated 85.1% nucleotide and 95.8% amino acid sequence identity. YHV PCR primers designed to amplify a 135 nucleotide product previously described as a YHV diagnostic probe failed to amplify the corresponding product from GAV RNA. However, the cognate GAV sequence for this and another recently reported YHV sequence were located in an upstream region of the ORF1b gene. A comparison of these sequences indicated identities of 83.0 and 80.9% at the nucleotide level and 86.7 and 86.5% at the amino acid level, respectively. The data indicate that GAV and YHV are closely related but distinct viruses for which differential diagnostic probes can be applied.

Genome organization and transcription strategy in the complex GNS-L intergenic region of bovine ephemeral fever rhabdovirus.

A 1622 nucleotide region of the bovine ephemeral fever virus (BEFV) genome, located between the second glycoprotein (GNS) gene and the polymerase (L) gene, has been cloned and sequenced in Australian (BB7721) and Chinese (Beijing-1) isolates of the virus. In the Australian isolate, the region contains five long open reading frames (ORFs) organized into three coding regions (alpha, beta and gamma), each of which are bound by a consensus transcription initiation and transcription termination-polyadenylation-like sequences. The alpha coding region contains three long ORFs (alpha 1, alpha 2 and alpha 3). The alpha 1 ORF encodes a 10.6 kDa polypeptide which contains hydrophobic and highly basic regions characteristic of a viroporin. The alpha 2 ORF encodes a 13.7 kDa polypeptide and overlaps the alpha 3 ORF which encodes a 5.7 kDa polypeptide. The beta coding region contains a single long ORF encoding a polypeptide of 12.2 kDa. The gamma coding region, which does not occur in Adelaide River virus (ARV), contains a single long ORF encoding a polypeptide of 13.4 kDa. The Chinese isolate shares 91% nucleotide sequence identity with the Australian isolate. The organization of the alpha, beta and gamma coding regions is preserved and the sequences of the encoded polypeptides are similar to those of BB7721. The major transcription products of the region were identified in BB7721 as polycistronic alpha (alpha 1-alpha 2-alpha 3) and beta-gamma mRNAs. Sequence similarities in the BEFV alpha-beta and beta-gamma gene junctions, and the gamma-L and beta-L gene junctions of BEFV and ARV, suggest that the gamma gene may have evolved from the beta-gene by sequence duplication.

Adelaide River virus nucleoprotein gene: analysis of phylogenetic relationships of ephemeroviruses and other rhabdoviruses.

The nucleotide sequence of the Adelaide River virus (ARV) genome was determined from the 3' terminus to the end of the nucleoprotein (N) gene. The 3' leader sequence comprises 50 nucleotides and shares a common terminal trinucleotide (3' UGC-), a conserved U-rich domain and a variable AU-rich domain with other animal rhabdoviruses. The N gene comprises 1355 nucleotides from the transcription start sequence (AACAGG) to the poly(A) sequence [CATG(A)7] and encodes a polypeptide of 429 amino acids. The N protein has a calculated molecular mass of 49429 Da and a pI of 5.4 and, like the bovine ephemeral fever virus (BEFV) N protein, features a highly acidic C-terminal domain. Analysis of amino acid sequence relationships between all available rhabdovirus N proteins indicated that ARV and BEFV are closely related viruses (48.3% similarity) which share higher sequence similarity to vesiculoviruses than to lyssaviruses. Phylogenetic trees based on a multiple sequence alignment of all available rhabdovirus N protein sequences demonstrated clustering of viruses according to genome organization, host range and established taxonomic relationships.

Structural and antigenic analysis of the nucleoprotein of bovine ephemeral fever rhabdovirus.

The nucleotide sequence of the bovine ephemeral fever virus (BEFV) genome has been determined from the 3' terminus to the end of the nucleoprotein (N) gene. The 3' leader sequence comprises 50 nucleotides and shares a common terminal three nucleotides (3'-UGC-) and a downstream U-rich domain with vesicular stomatitis virus (VSV) and rabies virus. The N gene comprises 1328 nucleotides from the transcription initiation consensus sequence (AACAGG) to the conserved transcription termination-poly(A) sequence [CATG(A)7] and encodes a polypeptide of 431 amino acids with an estimated M(r) of 49,159 and a pI of 5.4. The deduced amino acid sequence of the BEFV N protein is similar to those of other mammalian rhabdoviruses and is more closely related in sequence to vesiculoviruses (VSV Indiana and New Jersey, Piry, Chandipura) than to lyssaviruses (rabies and Mokola). An almost full-length clone, 1301 bp in length, of the BEFV N gene and clones derived from 5'-terminal (559 bp) and 3'-terminal (742 bp) fragments were expressed in Escherichia coli as glutathione-S-transferase fusion proteins. A panel of 12 BEFV N protein-specific monoclonal antibodies was shown to react in immunoblots with fusion proteins containing the almost full-length N protein and the C-terminal fragment, but not the N-terminal fragment. Two of these antibodies also reacted with baculovirus-expressed rabies virus N protein. Polyclonal mouse ascitic fluids derived from BEFV, rabies virus and several other related viruses were also shown to cross-react in immunoblots with purified preparations of rabies virus and BEFV N proteins.

Complex genome organization in the GNS-L intergenic region of Adelaide River rhabdovirus.

A 2341-nucleotide region of the Adelaide River virus (ARV) genome, located immediately downstream of the second glycoprotein (GNS) gene, has been cloned and sequenced. The region contains four long open reading frames (ORFs), the last of which represents a 1088-nucleotide fragment at the start of the ARV L gene. Between the GNS and L genes are two coding regions, separated by a single nucleotide (C), and each bounded by recognized transcription initiation (AACAG) and termination/polyadenylation (CATG[A]7) sequences. The first coding region comprises 682 nucleotides and contains two long ORFs (alpha 1 and alpha 2) which are in the same reading frame but separated by two consecutive stop codons. The alpha 1 ORF encodes a 12,545-Da polypeptide which contains highly hydrophobic and highly basic domains. The alpha 2 ORF includes a potential initiation codon 18 nucleotides downstream of the tandem stop codons and encodes a polypeptide of 11,951 Da. In ARV-infected cells, the alpha region is transcribed primarily as a long 4.7-kb polycistronic mRNA containing the G, GNS, alpha 1, and alpha ORFs. Direct sequence analysis of the mRNA indicated that the tandem stop codons between the alpha 1 and alpha 2 ORFs are retained in the transcript. The second coding region contains a single long ORF (beta) comprising 493 nucleotides which encodes a polypeptide with a calculated pl of 6.614 and molecular weight of 17,102 Da. The putative beta protein is similar in size to a protein which has been reported as a minor component of virions. The beta gene is transcribed as a 0.65-kb monocistronic mRNA for which the putative transcription termination/polyadenylation signal overlaps the L gene by 22 nucleotides.

Control of bovine leukaemia virus transmission by selective culling of infected cattle on the basis of viral antigen expression in lymphocyte cultures.

The use of viral antigen expression in lymphocyte cultures to prioritize the culling of bovine leukaemia virus (BLV) infected cattle was evaluated as a means of controlling the spread of infection in heavily infected herds. Selective culling was implemented in five commercial dairy herds containing between 126 and 304 cattle with infection prevalences, based on serological testing using the agar gel immunodiffusion test, of 19.4%, 20.3%, 20.1%, 20.6% and 39%. All seropositive cattle were tested for BLV antigen expression in lymphocyte cultures, and 51% found to express detectable quantities of viral antigens. In the four herds with 19% to 21% infection prevalence, all antigen-positive animals were culled immediately. Antigen-negative animals were retained in the herds for at least 16 months. Only two new infections were recorded in these four herds after antigen-positive animals had been culled, despite the continued presence of the antigen-negative animals. In the herd with 39% infection prevalence, a rapid reduction in the incidence of infection was achieved, even though only those animals with the highest levels of antigen expression were culled initially. Experimental transmissions from seropositive cattle indicated that sheep could be infected from an antigen-positive cow with fewer than 10(3) lymphocytes, whereas more than 10(6) lymphocytes were required to transmit infection from an antigen-negative cow. Estimation of the amount of integrated BLV DNA in serial dilutions of blood from antigen-positive and antigen-negative cattle provided an explanation for the higher infectivity of antigen-positive cattle.(ABSTRACT TRUNCATED AT 250 WORDS)

The genome of bovine ephemeral fever rhabdovirus contains two related glycoprotein genes.

A 3789 nucleotide region of the bovine ephemeral fever virus (BEFV) genome, located 1.65 kb downstream of the N gene, has been cloned and sequenced. The region contains two long open reading frames (ORFs) which are bounded by putative consensus (AACAGG) and polyadenylation (CATG[A]7) sequences and are separated by an intergenic region of 53 nucleotides. Discrete mRNAs corresponding to each ORF have been identified. The first ORF encodes a polypeptide comprising 623 residues which was identified by peptide sequencing as the virion G protein. The deduced amino acid sequence of the G protein includes putative signal and transmembrane domains and five potential glycosylation sites. The second ORF encodes a polypeptide of 586 amino acids which also has characteristics of a rhabdovirus glycoprotein, including putative signal and transmembrane domains and eight potential glycosylation sites, and appears to correspond to a 90-kDa nonstructural glycoprotein (GNS) identified in BEFV-infected cells (Walker et al. [1991] J. Gen. Virol. 72, 67-74). A database search indicated that both the G and GNS proteins share significant amino acid sequence homology with other rhabdovirus G proteins and with each other. Highest homology scores for each protein were with sigma virus and vesicular stomatitis virus serotypes.

Nucleotide sequence of the genome segment encoding nonstructural protein NS1 of bluetongue virus serotype 20 from Australia.

The nucleotide sequence of the genome segment (S6) encoding the nonstructural protein NS1 of an Australian isolate of bluetongue virus serotype 20 (BTV 20) has been determined from a series of overlapping cDNA clones synthesized using two terminal 15-mer oligonucleotides as primers. The gene consists of 1769 nucleotides with an open reading frame between nucleotides 35 and 1690 encoding a protein of 552 amino acids (molecular weight 64,506 Da; net charge -2 at pH 7). Comparison of the nucleotide and deduced amino acid sequence of this genome segment with cognate segments of isolates of BTV 1 from Australia and South Africa, and BTV 10 and BTV 17 from the United States, revealed homologies of 98%, 80%, 79%, and 79%, respectively, at the nucleotide level and 98%, 90%, 89%, and 90% identity, respectively, at the amino acid level. The data indicate that the evolutionary divergence between NS1 genes of two different Australian BTV serotypes (BTV 20 and BTV 1) is less than that between isolates of the same (BTV 1) or different serotypes from different geographical locations.

Infectivity of bovine leukaemia virus infected cattle: an ELISA for detecting antigens expressed in in vitro cultured lymphocytes.

A simple ELISA is described for quantifying expression of bovine leukaemia virus (BLV) antigens in short-term cultures of peripheral blood lymphocytes (PBL) isolated from infected cattle. The PBL-ELISA demonstrated that antigen expression levels in infected cattle could vary by more than 50-fold. Inoculation of sheep with dilutions of lymphocytes from two BLV-infected cattle, differentiated in the PBL-ELISA by 50 to 100-fold, suggested that antigen expression levels were correlated with infectivity. Haematological data indicated that increased antigen expression in PBL cultures was associated with an increased number of circulating B-lymphocytes, irrespective of whether or not an animal had lymphocytosis. This supported the hypothesis that BLV-infected cattle that are PBL-ELISA positive are more infectious and may present a greater risk of transmitting the disease. The applicability of the PBL-ELISA to a field situation was assessed with 98 BLV-infected cattle from three commercial dairy herds with infection prevalences of 11%, 23% and 47%. Similar percentages (49%, 50% and 52%) of PBL-ELISA positive cattle were identified among those infected cattle available for testing in the three herds. An additional 22 infected cattle from an experimental herd were tested to assess the stability of antigen expression levels over an 8 month period. Fewer (27%) of these cattle were identified as PBL-ELISA positive and antigen expression levels were generally lower than those observed in the commercial herds. Antigen expression levels in the experimental herd remained stable over the period of the study. The potential of the PBL-ELISA to assist in BLV eradication programs by identifying those seropositive cattle with the greatest potential to transmit infection is discussed.

An enzyme-linked immunosorbent assay for detection of bovine leukaemia virus p24 antibody in cattle.

An ELISA for detecting antibody to the bovine leukaemia virus (BLV) core protein p24 is described. The test uses p24 antigen purified from concentrated cell culture supernate by lectin-affinity chromatography and gel filtration. The sensitivity and specificity of the p24-ELISA for diagnosing BLV infection relative to the gp51 agar gel immunodiffusion test, were 98.1 and 96.7%, respectively. In the event of widespread use of gp51 based vaccines, the p24-ELISA should differentiate effectively between naturally infected and vaccinated animals.

Effects of proteolytic enzymes on the infectivity, haemagglutinating activity and protein composition of bluetongue virus type 20.

The effects on virus infectivity, haemagglutinating (HA) activity and polypeptide composition of bluetongue virus type 20 (BTV 20) were determined after digestion with the proteolytic enzymes, chymotrypsin, thermolysin and trypsin. Virus infectivity increased eight to 50-fold after exposure periods which reflected the activity of the proteases. Identical maximum increases in HA activity (i.e. 4096, 1024 and 128 HAU per 0.05 ml with sheep, bovine and human erythrocytes, respectively) occurred with each of the three proteases. Peak increases in virus infectivities and HA activities occurred after similar exposure periods. Outer capsid protein VP2 was the most sensitive virus protein to proteolytic digestion, being cleaved into a number of smaller polypeptides that remained attached to the virus particle. Digestion with chymotrypsin and thermolysin yielded four common cleavage products, designated P93, P76, P54 and P25 according to their estimated molecular weight, which suggested that they shared at least three cleavage sites. VP2 cleavage products resulting from digestion with trypsin differed somewhat from those of chymotrypsin and thermolysin, although the generation of polypeptides P93, P54 and P25.5 suggested the existence of common cleavage sites for the three proteases. Possible mechanisms whereby proteolytic cleavage of VP2 may enhance the infectivity and HA activity of BTV 20 are discussed.

Cross-neutralization of genetic reassortants of bluetongue virus serotypes 20 and 21.

Genetic reassortment studies of bluetongue virus (BTV) Types 20 and 21 have revealed a reassortant genotype that was not neutralized serotype-specifically. In reciprocal neutralization tests, BTV 20 and 21 were neutralized specifically by homologous antiserum. Similarly, reassortants that possessed both outer capsid proteins (i.e., VP2 and VP5) from the same parent virus reacted with that antiserum specifically. However, two reassortants, 16(9) and 19(1), with VP2 of BTV 20 and VP5 of BTV 21 had intermediate neutralization characteristics. These reassortants were neutralized to high titres by antiserum to BTV 20 and to lower, but significant titres by antiserum to BTV 21. In addition, antiserum to BTV 20 induced 10-16-fold higher titres in plaque reduction neutralization (PRN) tests with these two reassortants compared with BTV 20 itself. Evidence of the serological cross-reactivity of Reassortants 16(9) and 19(1) was also found with respect to reductions in plaque sizes observed in the PRN tests. The average plaque sizes of these reassortants were reduced to differing extents by antiserum to BTV 20 and 21, while those formed by the parent viruses were reduced in size by homologous antiserum only. Immunoblotting analysis of the structural proteins of BTV 20 and 21 demonstrated that VP2 alone was antigenically distinct, therefore confirming its role in determining serotype specificity in virus-neutralization tests. Electrophoretic analysis revealed considerable migrational differences between VP2 and VP5 of the parent viruses, suggesting that there was some divergence in their molecular weights, intrinsic charges or structural compositions. Taken together, the data suggest that the intermediate neutralization characteristics of the reassortants that contain VP2 and VP5 from different parent viruses are due to conformational alterations in their outer capsid structure which allow antibody recognition of common neutralizing epitopes that are not exposed on BTV 20 or BTV 21.