Taxonomic Structure of the Family
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Birnaviridae |
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Virions are about 60 nm in diameter, single-shelled, non-enveloped icosahedrons (Fig. 1). Electron micrographs of frozen, unstained virions indicate that the capsid structure is based on the geometry of a skewed T = 13 icosahedral lattice of the right-handed type. Computer image processing at 20 Å resolution produces a model of the virion with 260 trimeric subunits. The inner layer consists of 200 trimeric subunits of VP3 with VP4 residues at the inner 5-fold axes.
Physicochemical and Physical Properties
Virion Mr is about 55 106, S20w is 435S; buoyant density in CsCl is 1.33 g/cm3. Defective virions with interfering activity have been demonstrated to band at 1.30 g/cm3. Viruses are stable at pH 3-9, resistant to heat (60°C, 1 hr), ether and 1% SDS at 20°C, pH 7.5 for 30 min.
Virions contain two segments (A, B) of dsRNA which constitute between 9-10% of the particle by weight. The size of the larger segment A for Infectious pancreatic necrosis virus (IPNV) ranges from 2,962 bp (IPNV strain Sp) to 3,092 bp (IPNV, strain Jasper) and 3,104 bp (IPNV, strains N1 and DRT). The size of segment B for IPNV ranges from 2,731 (strain DRT) to 2,784 bp (IPNV, strain Jasper). For Infectious bursal disease virus (IBDV), segment A ranges in size from 3,063 bp (IBDV, strain Farragher) to 3,261 bp (IBDV, strain P2). Segment B for IBDV ranges in size from 2,715 bp (IBDV isolate UK661) to 2,922 bp (IBDV, strain QC-2). The Drosophila X virus (DXV) was found to have an A segment containing 3,360 bp. Despite the differences in the sizes of the A segments among members of the same genus, the coding region of the genome produces a polyprotein of 972 aa residues for IPNV, 1,012 aa residues for IBDV, and 1,032 aa residues for DXV. Similarly, the IPNV virion polymerases have a reported coding region for 844-845 aa residues; for IBDV it is 879 aa residues and there is a report of an unusually large polymerase of 891 aa for IBDV strain QC-2. Both genome segments contain a 94 103 5 genome-linked protein (VPg). There are no poly (A) tracts at the 3 ends of the RNA segments. The defective particles banding at 1.30 g/cm3 appear to have a truncated A segment.
Virions contain five polypeptides: VP1 (94 103) which is the RNA-dependent RNA polymerase as well as the VPg; pre-VP2 (62 103) and VP2 (54 103), the major capsid polypeptides and type specific antigens; VP3 (30 103), an internal capsid protein and group specific antigen. A nonstructural protein, NS, for IPNV is the virus-coded protease that autocatalytically cleaves the polyprotein to produce VP2, NS, and VP3. A viral protein, which has been designated as VP4 (29 103) for IBDV despite reports that VP4 is not a component of the virion, may also be a nonstructural protein. More than likely, VP4 is also a virus-encoded protease similar to the NS of IPNV, but its proteolytic activity has not been determined. An additional nonstructural polypeptide that is positively charged has been designated as VP5 for IBDV (16.5 103) or the 17 103 protein for IPNV. This polypeptide is encoded by segment A in an ORF preceding the segment A encoded polyprotein. The 16.5 103 protein has been shown to be nonessential for IBDV replication. A second ORF encoding an arginine-rich protein has also been identified in genome segment A for DXV. Although VP1 can guanylylate itself, it has no guanylyl transferase activity and the viral mRNA made in the cell retains its 5 VPg. There is no evidence for 5’ capping of any of the viral mRNAs.
None present.
No N-linked glycosylation of any of the virion proteins of IPNV or IBDV has been detected. There is a report of O-linked glycosylation for VP2 of IPNV.
Genome Organization and Replication
The genome organization of segment A of the three different birnavirus genomes, is illustrated in Figure 2. The arrangements are similar although there is some variation in the sizes of the individual proteins. For IPNV, genome segment A contains two ORFs: ORF1 encoding a 17 103 protein and ORF2 encoding a large 106 103 polyprotein in an overlapping reading frame. The cleavage sites for the autocatalytic protease have not been fully determined for both IBDV and DXV. However, the putative cleavage sites are shown in Figure 2 where two basic dipeptides RR and KR are located at the indicated sites for IBDV. The N-terminal amino acid sequence defining the cleavage site between preVP2 and VP4 for DXV has been determined. In this case, an S-A dipeptide (aa residues 500 and 501) was identified by N-terminal amino acid sequencing. This result assumes that VP4 is not trimmed after cleavage by cellular amino peptidase(s). For IPNV, the region where cleavage occurs is shown as hatched areas since the exact NS protease cleavage sites have not been determined. DXV contains an ORF1 encoding a 27 103 protein (237 aa residues) located between the VP4 and VP3 encoded sequences. Genome segment B contains one large 94 103 product (Fig. 3, ORF3).
A single cycle of replication takes about 18-22 hr for IPNV and 6-8 hr for IBDV. For IBDV, virus-binding proteins at the surface of various types of chicken cells have been determined. After entry into the host cell, the virion RNA-dependent RNA polymerase becomes activated and produces two genome length (24S) mRNA molecules from each of the 14S dsRNA genome segments. These mRNAs are not capped; rather, nascent mRNAs have a VPg attached to their 5 ends and they lack 3 poly(A) tracts. Replicative intermediates have been identified in infected cells. Virus RNA is transcribed by a semi-conservative strand displacement mechanism in vitro; however, re-initiation of RNA synthesis in vitro has not been observed. There is no information on minus strand RNA synthesis. The two mRNAs can be detected in infected cells by 3-4 hr post-infection and are synthesized in the same relative proportions throughout the replicative cycle (i.e., about twice as many A as B mRNA molecules). Virus-specific polypeptides can be detected at 4-5 hr post-infection and are present in the same relative proportions to each other until the end of the replication cycle. There are no specifically early or late proteins. The segment A mRNA is translated to a 106 103 polyprotein which contains (5 to 3) the pre-VP2, NS and VP3 polypeptides (Fig. 3). It has been speculated that the NS protease co-translationally cleaves the polyprotein to generate the three polypeptides. Pre-VP2 is later processed by a slow maturation cleavage to produce VP2. This cleavage is incomplete since both pre-VP2 and VP2 are found in purified virus, although VP2 predominates. The polyprotein has been detected in in vitro translation systems and antisera have detected some unprocessed polyprotein in IPNV-infected cells. The NS protease is unique and no homologous protease has been identified in extensive computer generated similarity searches. The active site of the protease has been mapped to the carboxy end of NS. The exact cleavage sites on the polyprotein are not known. The product of the 16.5-17 103 ORF has been detected in both IBDV and IPNV infected cells.
The mRNA from segment B is translated to a 94 103 polypeptide which is the viral RNA-dependent RNA polymerase (VP1, Fig. 3). It is found in virions both in a “free”, and genome-linked form (VPg).
Virus particles assemble and accumulate in the cytoplasm. For IBDV, the VP4 accumulates in the nucleus as well as in the cytoplasm of infected cells. The mechanism of virus release is unknown. In tissue culture about half of the progeny virions remains cell-associated. In vitro, defective interfering particles are formed depending on the multiplicity of infection.
The major capsid protein VP2 is the type-specific antigen and contains the virus neutralizing epitopes. Anti-VP3 antibodies do not neutralize virus infectivity. There is no serological cross-reaction between the fish, avian and insect birnaviruses.
The natural hosts of IPNV are salmonid fish, although the virus has also been isolated from other fresh-water and marine fishes, as well as from bivalve mollusks. The virus is transmitted both vertically and horizontally. There are no known vectors. The geographic distribution is world-wide. IPNV can cause epizootics resulting in high mortality in hatchery-reared salmonid fries and fingerlings. The virus causes necrotic lesions in the pancreas and is also found, without lesions, in other organs such as kidney, gonad, intestine, brain etc. Infected adult fish become life-long carriers without exhibiting overt signs of infection.
The natural hosts of IBDV are chickens and turkeys. Rarely, IBDV has been isolated from ducks and other domestic fowl. The mode of transmission is horizontal. There are no known vectors. IBDV has a world-wide distribution. The virus destroys the bursa of Fabricius of young chicks causing B lymphocyte deficiency. Mortality occurs between 3 to 6 weeks of age and is associated with inflammation of the bursa of Fabricius, formation of immune complexes, depletion of complement and clotting abnormalities.
Drosophila melanogaster populations are the natural host of DXV. The mode of transmission is horizontal and there are no known vectors. The geographic distribution is unknown. Infected fruitflies become sensitive to CO2. The target organs and histopathology are not known. DXV has also been isolated from populations of Culicoides spp.
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