Search

Birnaviridae is a family of viruses (birnaviruses) which consists of four genera, members of which cause diseases in fish, birds, mollusks, and insects. The genome of birnaviruses encodes the highly immunogenic VP2 capsid protein. In order to demonstrate that the VP2 protein can be exploited as a diagnostic antigen for birnaviruses, we developed a lateral flow assay based on the surface-exposed VP2 protrusion domain of a representative birnavirus, infectious bursal disease virus (IBDV) of serotype 1 which causes the highly devastating infectious bursal disease in chickens. The biophysical characterization of the purified domain reveals that the domain predominantly consists of β-sheets, exists in a trimeric form, and remains folded at high temperatures, making it suitable for diagnostic purposes. Owing to its highly immunogenic nature and excellent biophysical properties, we employed the VP2 protrusion domain in a gold nanoparticle-based lateral flow assay for rapid detection of anti-IBDV antibodies in serum samples of infected chickens. Our results indicate that the domain binds anti-IBDV antibodies with high specificity during laboratory testing and on-site testing. The lateral flow assay reported here yields comparable results in a qualitative manner as obtained through a commercial enzyme-linked immunosorbent assay (ELISA). As VP2 is a common capsid protein of birnaviruses, the lateral flow assay can be generalized for other birnaviruses, and members of Tetraviridae and Nodaviridae families which contain homologous VP2 capsid proteins. Electronic supplementary material The online version of this article (10.1007/s00253-020-10458-6) contains supplementary material, which is available to authorized users.

Infectious bursal disease is a widely spread threatening contagious viral infection of chickens that induces major damages to the Bursa of Fabricius and leads to severe immunosuppression in young birds causing significant economic losses for poultry farming. The etiological agent is the infectious bursal disease virus (IBDV), a non-enveloped virus belonging the family of Birnaviridae. At present, the treatment against the spread of this virus is represented by vaccination schedules mainly based on inactivated or live-attenuated viruses. However, these conventional vaccines present several drawbacks such as insufficient protection against very virulent strains and the impossibility to differentiate vaccinated animals from infected ones. To overcome these limitations, in the last years, several studies have explored the potentiality of recombinant subunit vaccines to provide an effective protection against IBDV infection. In this review, we will give an overview of these novel types of vaccines with special emphasis on current state-of-the-art in the use of plants as “biofactories” (plant molecular farming). In fact, plants have been thoroughly and successfully characterized as heterologous expression systems for the production of recombinant proteins for different applications showing several advantages compared with traditional expression systems (Escherichia coli, yeasts and insect cells) such as absence of animal pathogens in the production process, improved product quality and safety, reduction of manufacturing costs, and simplified scale-up.

Bipolaris maydis is the causal agent of corn southern leaf blight. Here, we report a novel double-stranded RNA (dsRNA) mycovirus designated Bipolaris maydis botybirnavirus 1 (BmBRV1) from B. maydis strain JZ11 in Jingzhou, Hubei province of China. BmBRV1 has a genome consisting of two dsRNAs (dsRNA1 and dsRNA2) with a size of 6435 and 5987 bp, respectively, each of which contains a single open reading frame (ORF). The two polyproteins encoded by dsRNA1 and dsRNA2 share the highest amino acid identities of 81.8 and 75.3%, respectively, with the RdRp and coat protein of Sclerotinia sclerotiorum botybirnavirus 1 (SsBRV1), a tentative species of the genus Botybirnavirus. Phylogenetic analysis based on the amino acid sequences of RdRp indicated that BmBRV1 belongs to a distinct species of the newly proposed family Botybirnaviridae.

The VP5 protein of infectious bursal disease virus (IBDV) was shown in previous reports to be involved in the cytopathogenicity of IBDV. Here, using a rescued VP5-deficient IBDV infectious clone, it was demonstrated that a lack of VP5 expression significantly hinders the release of viral progeny from infected cells but does not block intracellular virus production. Monoclonal VP5-expressing Vero cells did not exhibit induction of cell death. Using VP5-specific mAbs generated in our laboratory as a tool, it was shown by flow cytometry analysis that VP5 was detectable on the surface of IBDV-infected and monoclonal VP5-expressing Vero cells and bursal cells in IBDV-infected chickens. Taken together, these data suggest that the VP5 protein is involved in regulation of the release of intracellular IBDV virions and may be used as a cell-surface marker for detecting IBDV-infected cells in FCM analysis. This study contributes to the further characterization of the VP5 protein, which will allow a better understanding of the mechanism of IBDV pathogenicity.

Infectious bursal disease virus (IBDV) is a double-stranded RNA virus in the Birnaviridae family. Four pathotypes, attenuated, virulent, antigenic variant, and very virulent, have been identified. In this study, we examined the phylogenetic relationship of 25 field isolates that were collected from a single farm during 1989-2008. A sequence analysis of PCR amplified 714 bp VP2 region showed that all the samples were derived from very virulent infectious bursal disease virus (vvIBDV) and were more closely related to the vvIBDV isolate UK661. From 1999, the isolate XA1999 had amino acids I228 and T394. XA2000, XA2001, XA2002, and XA2003-09 had amino acids E279 and T394. From 2004 to 2008, the isolates had amino acids H320, I349, S375, and R381 while the UK661 virus had T228, D279, Q320, V349, P375, K381, and A394. Such mutations do not change key amino acid residues in the domains which are essential for its virulence. It suggests that a virulent IBDV strain could maintain its virulence for a long period in the same chicken farm and the strain is highly stable under normal environments.

In this paper, the infectivity and propagation of two attenuated infectious bursal disease virus (IBDV) strains in DT40 cells were investigated. The results showed that both of the tested strains, TAD and HN(3), directly infect and proliferate in DT40 cells, requiring no adaptive passages. Unexpectedly, IBDV can be rapidly propagated and continuously harvested at high titers for a long time, accompanied by the rapid growth of host cells and showing no increase in pathogenicity. Our results provide further support to suggest that DT40 cells can be used as an ideal model for studying IBDV pathogenesis. Additionally, the DT40 cell line could also serve as a potential system for commercial IBDV vaccine preparation.

Four putative aquabirnaviruses, based on morphology, nucleic acid type and partial RNA-dependent RNA polymerase gene (VP1) sequence, isolated from three tropical freshwater fish species were not neutralised by antisera against type members of the Aquabirnavirus genus serogroups A, B or C. Antisera produced against two of the isolates neutralised the homologous and heterologous isolates, but not any type member of Aquabirnavirus serogroups A, B or C. The serological comparisons suggest that the four isolates should be regarded as members of a fourth Aquabirnavirus serogroup, D.

A birnavirus strain, Paralichthys olivaceus birnavirus (POBV), was isolated and characterized from cultured flounder in China, and its complete genomic sequence was subsequently determined. The virus could induce cytopathic effects (CPE) in four of seven fish cell lines and was resistant to chloroform, 5-iodo-2'-deoxyuridine, acid and alkaline pH, and heat treatment. Purified virus particles had a typical icosahedral shape, with a diameter of approximately 55-60 nm. The genomic segments A and B of POBV were 3,091 and 2,780 bp in length and shared many of the features of the members of the family Birnaviridae. Segment A contained two partially overlapping ORFs encoding a polyprotein, pVP2-VP4-VP3, and a nonstructural protein, VP5, while segment B had only one ORF encoding for the VP1, a viral RNA-dependent RNA polymerase (RdRp). This is the first report about a birnavirus strain from a new non-salmonid host in China and its complete genome sequence.

Infectious bursal disease virus (IBDV) is a bi-segmented, dsRNA virus of the Birnaviridae family. The nonstructural protein VP5 has been reported to be associated with virus-induced cell apoptosis and pathogenicity, but its role in viral replication has not been unequivocally identified. Based on a PCR introduced mutagenesis strategy, the 33 bp of 96–129 bp located between ORF A1 and ORF A2 of genomic segment A of IBDV strain HZ2 were deleted, and an Nhe I (GcTaGc) site was inserted at 96–102 bp simultaneously. The mutated segment A was ligated into pCI, resulting in pCI-ANhe3. A chimeric and deficient IBDV strain, named strain ANhe3, was recovered from chicken embryo fibroblast (CEF) cells by co-transfection with pCI-ANhe3 and pCI-mB, derived from the genomic segment B strain HZ2. The indirect fluorescent assay identified that strain ANhe3 could replicate on CEF cells without expression of VP5. Further examination showed that the pathogenesis of strain ANhe3 replicating on SPF chicken embryos was attenuated compared to strain HZ2. This paper provides a new rapid rescue strategy for gene-deleted virus. This strategy lays a basis for gene-deleted vaccine of IBDV.

A phage-displayed single chain variable fragment (scFv) antibody library was constructed from the immune spleen cells of chickens immunized with very virulent infectious bursal disease virus (vvIBDV) strain CS89. A library consisting of around 9.2 x 10(7) clones was subjected to 3 rounds of panning against captured CS89 virus. Analysis of individual clones by nucleotide sequencing revealed at least 22 unique scFv antibodies binding to vvIBDV in ELISA. Testing of the scFv antibody panel in ELISA against classical, variant or vaccine strains and a wide variety of vvIBDV isolates from the UK, China, France, Belgium, Africa, Brazil, Indonesia and the Netherlands identified one antibody, termed chicken recombinant antibody 88 (CRAb 88) that was specific for vvIBDV. CRAb 88 was capable of recognizing all vvIBDV strains tested regardless of their country of origin and showed no reactivity with classical, variant or vaccine strains, lending support to the use of this scFv as a powerful diagnostic tool for the differentiation of vvIBDV strains. Immunoprecipitation studies revealed that CRAb 88 was directed towards a highly conformational epitope located within the major neutralizing protein VP2. Sequence analysis of the hypervariable region of VP2 of the IBDV strains tested indicate that Ile(256) and Ile(294) may play roles in binding of CRAb 88. This is the first reagent of its type capable of positively distinguishing vvIBDV from other IBDV strains.

The rapid genomic characterization of infectious bursal disease virus (IBDV) requires determining which partial nucleotide (nt) sequences derived from IBDV segments A or B would produce phylogenetic information as significant as sequencing the whole corresponding segments. Long nt coding sequences of 27 IBDV segments A (aa 20-991) and 21 segments B (aa 7-stop codon) were retrieved from databanks and used to compute reference phylogenetic trees using Neighbor Joining (NJ) and Parsimony (P): clusters appearing in the NJ and P reference trees with a bootstrap value greater than 80% were considered as significant (Whole Segment Clusters, WSC). The sequences were then cut into overlapping regions. These were used to compute phylogenetic trees which were compared with reference ones. Of the partial sequences, the VP2 gene best represented IBDV segment A (10 out of 13 WSC were conserved), and the 5' two thirds of segment B best represented segment B (5 to 6 conserved WSC out of 6). Implementation of the Plato programme finally demonstrated that the region encoding VP2 variable domain (vVP2, segment A) is the only region of IBDV genome with a significantly different evolution rate, which result is consistent with vVP2 being subjected to a high selection pressure.

Infectious bursal disease virus (IBDV) causes lymphocytolysis and immunosuppression in infected poultry. The IBDV genome encodes a polyprotein VP243 that is post-translationally cleaved by the VP4 protease into the two structural proteins pVP2 and VP3. The objective of the present study was to determine if IBDV polyprotein induced suppression of bursal B lymphocyte growth and their capacity for proliferation. Bursal B cells were examined both for chickens infected with IBDV and for chickens orally inoculated with a DNA construct expressing IBDV VP243 polyprotein. Bursae were collected at 0, 12, 24 and 48 hours after inoculation. Proliferation of bursal B cells (purified AvBu1(+) cells) in response to concanavalin A mitogenic stimulation was significantly suppressed by infection at 1 day old with either the classical STC or variant E strains of IBDV. Oral administration of DNA constructs expressing the IBDV VP243 polyprotein from either the classical STC or variant E strains in the pCR3.1 vector resulted in persistent, moderate levels of construct in the bursa until at least 48 hours after inoculation. The VP243 DNA construct similarly induced suppression of proliferation for bursal lymphocytes independently of the virus infection. Expression of VP243 polyprotein in transiently transfected DT40 B lymphocyte culture also suppressed cell growth and proliferative responses to mitogen stimulation. Polyprotein expression did not affect cell viability and suppression of proliferation probably occurred by means of cell cycle arrest. The expression of the mature viral proteins VP2, VP4 or VP3 did not change the rate of cell proliferation or response of B cell cultures to mitogen. The results suggested that IBDV polyprotein is a mediator of immunosuppression.

Summary. Infectious bursal disease virus (IBDV) is the agent of an immune-depressive disease affecting the poultry industry worldwide. Infection of IBDV leads to expression of five mature virus-encoded proteins. Proteolytic processing of the virus-encoded polyprotein generates VP3 which coats the inner surface of the IBDV capsid. In this report, we describe the characterization of the RNA-binding activity of VP3. For these studies, the VP3 coding region was fused to a histidine tag and expressed in insect cells using a recombinant baculovirus. The histidine-tagged VP3 was affinity-purified and used to study its ability to bind RNA molecules using three complementary methods: (i) Northwestern blotting; (ii) binding of VP3 protein-RNA complexes to nitrocellulose membranes; and (iii) electrophoretic mobility shift assays. The results demonstrated that VP3 efficiently bound ssRNA and dsRNA. Under the experimental conditions used in this study, the formation of VP3-RNA complexes did not depend upon the presence of specific RNA sequences. A series of histidine-tagged VP3 deletion mutants spanning the whole VP3 coding region were generated. The use of these mutants revealed that the VP3 RNA-binding domain layed in a highly conserved 69 aa stretch close to the N-terminus of the protein.

An Indonesian very virulent (vv) strain of infectious bursal disease virus (IBDV), designated Tasik94, was characterised both in vivo and at the molecular level. Inoculation of Tasik94 into 5-week-old specific-pathogen-free (SPF) chickens resulted in 100% morbidity and 45% mortality. The complete nucleotide and predicted amino acid sequences of genomic segments A and B were determined. Across each of the three deduced open reading frames (ORFs), Tasik94 shared the greatest nucleotide homology to Dutch vv strain D6948. Phylogenetic analyses were performed using 15 full-length polyprotein sequences and a total of 105 VP2 hypervariable region sequences from geographically and pathogenically diverse strains. In each case, Tasik94 grouped closely with vv strains, particularly those from Europe. The deduced VP1, VP2, VP3, VP4 and VP5 protein sequences of Tasik94 were aligned with those from published strains and putative virulence determinants were identified in VP2, VP3 and VP4. Alignment of additional protein sequences across the VP2 hypervariable region confirmed that residues Ile[242], Ile[256] and Ile[294] were highly-conserved amongst vv strains, and may account for their enhanced virulence.

Sera from adult and chick king penguins were examined for antibodies to infectious bursal disease, influenza A and Newcastle disease viruses. This study was completed by a 1-year survey of clinical signs of disease in the colony. About 4% and 1% of the birds were positive for serotypes 1 and 2, respectively, of infectious bursal disease virus. In 1996, two coughing peaks occurred in adults, during autumn and spring, and a peak of conjunctivitis occurred at the end of the winter. Although this showed the presence of infectious agents, it was not possible to be precise about whether the detected viruses were responsible for the clinical signs, or to assess if the infectious agents were native or introduced.

Summary. A full-length cDNA clone of the segment B of the very virulent infectious bursal disease virus (IBDV) strain BD 3/99 was constructed and the full-length nucleotide sequence was established. The nucleotide sequence encoding VP1, an RNA-dependent RNA polymerase, of BD 3/99 was aligned with that of 17 other IBDV strains including six very virulent, three classical virulent, five classical attenuated, one antigenic variant and two serotype 2 strains. The VP1 genes of all very virulent strains were 97.5% to 99.8% identical. With the exception of an atypical Australian strain, 002-73, all of the classical virulent or attenuated and antigenic variant strains were also 97.5% to 100% identical. Serotype 2 strains showed only 4–6% divergence from serotype 1 classical virulent or attenuated strains; in contrast, however, the very virulent strains were 10.5% to 12.5% divergent from the classical virulent or attenuated strains as well as serotype 2 strains. Analysis of the deduced amino acid sequence of VP1 revealed 17 common, including 8 unique amino acid substitutions in the very virulent strains. In the phylogenetic tree the very virulent strains formed a distinct cluster and all other strains including classical virulent, attenuated and antigenic variant strains and even serotype 2 strains were grouped together. It to is suggested that the VP1 of very virulent IBDV is phylogenetically distinct from that of all other IBDV strains and probably originated from a hitherto unidentified source.

Isolates of infectious bursal disease virus (IBDV) were obtained from domestic poultry in New Zealand in 1997 and 1998. An in-vivo pathogenicity study carried out in specific pathogen free (SPF) chickens demonstrated the low virulence of one of the virus isolates. The nucleotide sequences of the hypervariable region of the VP2 gene of two isolates were determined and compared with published sequences of strains from other countries. The deduced amino acid sequence of the two New Zealand IBDV isolates showed 100% identity with each other, suggesting that little genetic drift had occurred. Phylogenetic analysis showed that the New Zealand isolates were more closely related to two attenuated IBDV strains (Cu1 and PBG98) than to classical (STC and 52/70), very virulent (DV86), variant (variant E) or Australian (002-73) strains. The results support the hypothesis that an attenuated strain of the virus was inadvertently introduced into the NZ poultry population in 1993.

The 2362 base pair sequence of the larger of the two double stranded RNA genome segments of a rabbit strain of picobirnavirus (PBV) has a major open reading frame (ORF) of 591 amino acids and two smaller ORFs of 55 and 155 amino acids. A clone of the segment did not hybridise with other viral bisegmented ds RNAs from faecal samples. There is no relationship in sequence or organisation between this PBV sequence and the bisegmented dsRNAs found associated with Cryptosporidium parvum. This suggests that there are at least two distinct classes of bisegmented dsRNA viruses or viral-like agents in faeces.

Rainbow trout gonad cells (RTG-2) display a dramatic cytopathic effect and lysis following productive infection by infectious pancreatic necrosis virus (IPNV). In this study viruses were efficiently released into the growth medium together with low amounts of the monomeric free form of the structural protein VP3, heterodimers of VP2-VP3, aggregates of pVP2 and viral RNA associated with VP3. Ribonucleoprotein complexes of RNA-VP3 contained RNA equivalent to at the most 25% of full length viral genomes. Infectivity of material released into the growth medium late in infection was only associated with fully assembled viruses and isolated subviral RNA-VP3 complexes were not infectious. Upon purification of IPNV, viral hexa- and pentagonal particles of approx. 15 nm diameter were occasionally co-purified with the virus and then appeared in large quantities. Similar particle-like structures were seen as substructures of purified viruses that were treated with and partially disintegrated by CsCl of virus isodensity concentration.

A flow cytometric virus binding assay that directly visualizes the binding of infectious bursal disease virus (IBDV) to its target cells was established. The chicken B lymphoblastoid cell line, LSCC-BK3, which is permissive for IBDV infection, bound high levels of the virus. Another B lymphoblastoid cell line, LSCC-1104-B1, bound low levels of the virus, although it was nonpermissive. No virus binding was detected in nonpermissive T lymphoblastoid cell lines. In the binding assay to heterogeneous cell populations of chicken lymphocytes, IBDV (a highly virulent OKYM strain) bound to 94% cells in the lymphocytes prepared from the bursa of Fabricius, 37% cells in those prepared from the spleen, 3% cells in those prepared from the thymus, and 21% cells in those prepared from the blood. Most of the cells, which bound the virus, were surface immunoglobulin M (SIgM)-positive, but a small number of them were SIgM-negative. Additionally, the binding of IBDV to the LSCC-BK3 cells was affected by treatment of the cells with proteases and N-glycosylation inhibitors. These findings may indicate that the IBDV host range is mainly controlled by the presence of a virus receptor composed of N-glycosylated protein associated with the subtle differentiation stage of B-lymphocytes represented mostly by SIgM-bearing cells.

Summary The right hand end Nde I fragment 3 (90.8–100 map units) of the fowl adenovirus serotype 10 (FAV-10) was characterised so as to allow the location of an insertion site for recombinant vector construction. Infectious bursal disease virus (IBDV) VP2 gene from the Australian classical strain 002/73, under the control of the FAV-10 major late promoter/leader sequence (MLP/LS) was inserted into a unique Not I site that was generated at 99.5 map units. This recombinant virus was produced without deletion of any portion of the FAV-10 genome. When administered to specific pathogen free (SPF) chickens intravenously, intraperitoneally, subcutaneously or intramuscularly, it was shown that the FAV-10/VP2 recombinant induced a serum VP2 antibody response and protected chickens against challenge with IBDV V877, an intermediate virulent classical strain. Birds were not protected when the recombinant was delivered via the conjunctival sac.

The VP1 encoded by genomic segment B of birnaviruses is generally known to exist as a genome-linked protein (VPg) and as a "free" polypeptide of 90 kDa in virus particles. The guanylylation activity associated with infectious bursal disease virus (IBDV) was demonstrated by incubating purified virus in presence of [alpha 32P] GTP; optimum activity in the 90 kDa form of VP1 was seen in low salt concentration in the presence of 4 mM magnesium ions over a wide range of incubation temperatures. The IBDV VP1 was shown to lack guanyl transferase activity. Northwestern (RNA-protein) blot analysis of purified virus using a radiolabelled cDNA probe consisting of 3' and 5' ends of genomic segment B indicated that both forms of virion-associated VP1 contained viral RNA sequences of which those linked to VPg corresponded to the two genome segments and those linked to the 90 kDa VP1 were probably a short oligonucleotide of the terminal viral RNA sequences.

Bivalve molluscs can bioaccumulate, in their tissues, well known viruses that infect humans and higher animals. However, since 1969 there have been reports, based largely on ultrastructural studies, that have indicated there are also viruses which infect these molluscs. Lack of bivalve molluscan cell lines has limited the isolation of these viruses, although some viruses isolated in fish cell lines are claimed to be infective for bivalves. Distinctive morphogenesis and characteristics of iridoviruses have been reported in larval and adult oysters in association with infections of epithelial and haemocytic tissues. Herpes viruses have been reported in the American oyster, Pacific oyster, and European flat oyster from three continents. The herpes viral agent in Pacific oysters has been experimentally transmitted and requires temperatures of 25–26°C for a productive infection and clinical disease. Papova-like viruses, picorna-like, and other small virus-like particles have been reported in several bivalve species. A leukemia-like disease of many bivalve species, collectively termed disseminated neoplasias, is of unconfirmed aetiology, but may be related to retroviral infections. Representatives of the Reoviridae and Birnaviridae have been isolated by using fish cell lines. One definitive study concludes that at least one example is not infective for bivalves while other studies claim molluscan infectivity. Phages have been reported in three pathogenic agents infecting bivalves. Advancement in the field of molluscan virology will require increased application of physical isolation methods, refinement of primary cell culture methods, use of molecular diagnostic tools, and the development of continuous molluscan cell lines.

Terminal sequences of the bi-segmented dsRNA genome of 3 different strains of infectious bursal disease virus (IBDV) were analyzed by the rapid amplification of cDNA 5' ends (5'RACE) procedure. Both segments are 85% homologous in a 32-nucleotide sequence comprising the 5' end, whereas the 3' end has a conserved pentamer. Comparison to published terminal sequences of other IBDV strains revealed high conservation between the two segments but more serotype-specific nucleotide changes (5 on segment A and 3 on segment B) in the 5' noncoding region compared to the 3' noncoding region (none on segment A and 1 on segment B).

A picobirnavirus with an atypical genome profile was detected by polyacrylamide gel electrophoresis (PAGE) in 37% (20/54) of human faecal samples also containing oocysts of Cryptosporidium typical of C. parvum. This virus shares many of the characteristics of the previously described picobirnaviruses, but has a significantly smaller genome (1.75 and 1.55 Kbp).

Eleven of thirteen infectious bursal disease viruses (IBDV) were shown to be similar following polymerase chain reaction (PCR) amplification of two regions (293 bp and 651 bp cDNA fragments) in genomic segment B and restriction enzyme analysis of the PCR products. Two IBDV strains differed from the eleven viruses when examined by BstEII digestion of the 293 bp PCR product, but all thirteen viruses were similar using the 651 bp cDNA fragment and digestion with AvaI. Because segment B encodes replicating enzyme(s), the results suggest that its sequences may be highly conserved among IBDV isolates.

Quantitative counter-immunoelectrophoresis was standardized to detect antibodies to the avian infectious bursal disease virus. This technique correlated well with the conventional quantitative agar gel precipitation test in estimating antibodies to IBDV. The use of blood dried on filter paper as an alternative to serum is discussed. QCIE is simple, easy to perform and faster than QAGP.

Two fowlpox virus recombinants were constructed which expressed the host-protective antigen, VP 2, of infectious bursal disease virus (IBDV). Recombinant FPV-VP 2.4.3 contained the gene for the VP 2-VP 4-VP 3 polyprotein under the control of the vaccinia virus late promoter P.L 11 inserted within the thymidine kinase (TK) gene of FPV. In infected chicken embryo skin (CES) cells VP 2 and VP 3 proteins were correctly processed from the polyprotein precursor molecule. Recombinant FPV-VP 2 contained only the VP 2 encoding region under the control of the fowlpox early/late promoter P.E/L inserted immediately downstream of theTK gene. The expression level of VP 2 from FPV-VP 2 was approximately 5 times higher than from FPV-VP 2.4.3. Wing web inoculation of birds resulted in the development of typical fowlpox lesions and the development of antibodies to FPV with either of the recombinants, but only birds vaccinated with FPV-VP 2 developed antibodies to IBDV. When challenged with IBDV (strain 002-73), a significant level of protection was provided by FPV-VP 2 vaccination, although the level was lower than the protection provided by an oil adjuvanted inactivated whole IBDV vaccine. Birds vaccinated with FPV-VP 2.4.3 were not protected from infection as assessed by ELISA for the presence of IBD virus in bursae.

Using neutralizing monoclonal antibodies, three categories of escape mutants were selected from a stock of wild-type infectious bursal disease virus (IBDV). Additional mutants were found, where alterations coexisted in two or three of these epitopes. Although each group of mutants had a distinct reaction pattern with neutralizing monoclonal antibodies, all types of mutants were neutralized by convalescent chicken sera to the same extent. In spite of the lack of homogeneity in these antigenic sites located on IBDV structural polypeptide VP2, all neutralizing monoclonal antibodies reacted with epitopes in extracts prepared from the bursa of Fabricius from animals that had died during recent outbreaks of infectious bursal disease in the F.R.G. and Africa. Since binding to VP2 of the escape mutants, demonstrable by immunoprecipitation, correlated with the neutralizing capacity of these antibodies, a combined immunoprecipitation-immunoblotting technique was established as equivalent for a neutralization assay. The results of our experiments indicate that IBDV did not undergo a major antigenic variation in these two areas of Europe and Africa. The significance of protein conformation for the interaction of VP2 with neutralizing antibodies was underlined by the finding that renatured VP2 was capable of binding neutralizing antibodies; the antibodies induced in animals by immunization with this protein, however, were not neutralizing.

The coding sequences of VP2 from a virulent strain, 52/70, of infectious bursal disease virus (IBDV) were excised from a cDNA clone and inserted into a fowlpox plasmid insertion vector. The resulting plasmid, pIBD 1, was used to construct a recombinant fowlpox virus, fpIBD 1, which expressed VP 2 as a beta-galactosidase fusion protein. Chickens vaccinated with fpIBD 1 at 1 and 14 days of age, were challenged at 28 days with either IBDV strain 52/70 or the highly virulent strain CS 89. These chickens were protected against mortality, but not against damage to the bursa of Fabricius. The protection achieved by the use of fpIBD 1 shows that VP 2 is a host protective antigen.

Approximately 40 viruses are known from marine sponges; turbellarian and monogenetic flatworms; cephalopod, bivalve, and gastropod mollusks; nereid polychaetes; and isopod and decapod crustaceans. Most of the viruses can be tentatively assigned to the Herpesviridae, Baculoviridae, Iridoviridae, Adenoviridae, Papovaviridae, Reoviridae, “Birnaviridae”, Bunyaviridae, Rhabdoviridae, and Picornaviridae. Viruslike particles found in oysters might be representatives of the Togaviridae and Retroviridae. Enveloped single-stranded RNA viruses from crustaceans have developmental and morphological characteristics intermediate between families, and some show evidence of relationships to the Paramyxoviridae as well as the Bunyaviridae or Rhabdoviridae. Certain small viruses of shrimp cannot be assigned, even tentatively, to a particular family. Some viruses cause disease in wild and captive hosts, others are associated with disease states but may not be primary instigators, and many occur in apparently normal animals. The frequency of viral disease in natural populations of marine invertebrates is unknown. Several viruses that cause disease in captive animals, with or without experimental intervention, have also been found in diseased wild hosts, including herpeslike viruses of crabs and oysters, iridovirus of octopus, and reolike and bunyalike viruses of crabs. Iridolike viruses have been implicated in massive mortalities of cultured oysters. Baculoviruses, and IHHN virus, which is of uncertain affinities, cause economically damaging diseases in cultured penaeid shrimp. Double or multiple viral infection is common in crabs. For example, a reolike virus and associated rhabdolike virus act synergistically to cause paralytic and fatal disease inCallinectes sapidus. Information on host range, most susceptible stage, and viral latency is available only for viruses of shrimp. One baculovirus attacks five species of New World penaeid shrimp. IHHN virus infects three species ofPenaeus and causes catastrophic mortalities inP. stylirostris, but usually exhibits only inapparent infection inP. vannamei. Some shrimp viruses apparently are latent in larvae, causing disease only when shrimp have reached the postlarval or juvenile stages. Others are equally or more pathogenic in larvae. Studies of shrimp viruses and iridovirus-associated disease in cultured oysters point up the need for rapid and accurate diagnostic methods. Until appropriate cell cultures from marine invertebrates are devised, the viral identifications necessary for understanding of epizootiology, rapid containment of epizootics in cultured animals, and decisions regarding introductions of exotic species will be difficult or impossible.