Studies on overwintering of bluetongue viruses in insects.
(49/353)
Bluetongue viruses (BTVs) are economically important arboviruses that affect sheep and cattle. The overwintering mechanism of BTVs in temperate climates has eluded researchers for many years. Many arboviruses overwinter in their invertebrate vectors. To test the hypothesis that BTVs overwinter in their vertically infected insect vectors, Culicoides sonorensis larvae were collected from long-term study sites in northern Colorado, USA, and assayed for the presence of BTV RNA by nested RT-PCR. Sequences from BTV RNA segment 7 were detected in 30 % (17/56) of pools composed of larvae and pupae collected in 1998 and in 10 % (31/319) of pools composed of adults reared from larvae collected in 1996. BTV was not isolated from the insects. Additionally, Culicoides cell-culture lines derived from material collected at one of the sites, or derived from insect samples collected during a BTV outbreak, contained BTV RNA segment 7. In contrast, segment 2 RNA was detected at half the rate of segment 7 RNA in the field-collected larvae and was only detected in the Culicoides cell lines with one of two primer sets. These data suggest that BTVs could overwinter in the insect vector and that there is reduced expression of the outer capsid genes during persistent infection. (+info)
Phosphorylation of bluetongue virus nonstructural protein 2 is essential for formation of viral inclusion bodies.
(50/353)
In bluetongue virus (BTV)-infected cells, large cytoplasmic aggregates are formed, termed viral inclusion bodies (VIBs), which are believed to be the sites of viral replication and morphogenesis. The BTV nonstructural protein NS2 is the major component of VIBs. NS2 undergoes intracellular phosphorylation and possesses a strong single-stranded RNA binding activity. By changing phosphorylated amino acids to alanines and aspartates, we have mapped the phosphorylated sites of NS2 to two serine residues at positions 249 and 259. Since both of these serines are within the context of protein kinase CK2 recognition signals, we have further examined if CK2 is involved in NS2 phosphorylation by both intracellular colocalization and an in vitro phosphorylation assay. In addition, we have utilized the NS2 mutants to determine the role of phosphorylation on NS2 activities. The data obtained demonstrate that NS2 phosphorylation is not necessary either for its RNA binding properties or for its ability to interact with the viral polymerase VP1. However, phosphorylated NS2 exhibited VIB formation while unmodified NS2 failed to assemble as VIBs although smaller oligomeric forms of NS2 were readily formed. Our data reveal that NS2 phosphorylation controls VIBs formation consistent with a model in which NS2 provides the matrix for viral assembly. (+info)
Assembly and intracellular localization of the bluetongue virus core protein VP3.
(51/353)
The bluetongue virus (BTV) core protein VP3 plays a crucial role in the virion assembly and replication process. Although the structure of the protein is well characterized, much less is known about the intracellular processing and localization of the protein in the infected host cell. In BTV-infected cells, newly synthesized viral core particles accumulate in specific locations within the host cell in structures known as virus inclusion bodies (VIBs), which are composed predominantly of the nonstructural protein NS2. However, core protein location in the absence of VIBs remains unclear. In this study, we examined VP3 location and degradation both in the absence of any other viral protein and in the presence of NS2 or the VP3 natural associate protein, VP7. To enable real-time tracking and processing of VP3 within the host cell, a fully functional enhanced green fluorescent protein (EGFP)-VP3 chimera was synthesized, and distribution of the fusion protein was monitored in different cell types using specific markers and inhibitors. In the absence of other BTV proteins, EGFP-VP3 exhibited distinct cytoplasmic focus formation. Further evidence suggested that EGFP-VP3 was targeted to the proteasome of the host cells but was dispersed throughout the cytoplasm when MG132, a specific proteasome inhibitor, was added. However, the distribution of the chimeric EGFP-VP3 protein was altered dramatically when the protein was expressed in the presence of the BTV core protein VP7, a normal partner of VP3 during BTV assembly. Interaction of EGFP-VP3 and VP7 and subsequent assembly of core-like particles was further examined by visualizing fluorescent particles and was confirmed by biochemical analysis and by electron microscopy. These data indicated the correct assembly of EGFP-VP3 subcores, suggesting that core formation could be monitored in real time. When EGFP-VP3 was expressed in BTV-infected BSR cells, the protein was not associated with proteasomes but instead was distributed within the BTV inclusion bodies, where it colocalized with NS2. These findings expand our knowledge about VP3 localization and its fate within the host cell and illustrate the assembly capability of a VP3 molecule with a large amino-terminal extension. This also opens up the possibility of application as a delivery system. (+info)
Induction of human immunodeficiency virus type 1-specific T cells by a bluetongue virus tubule-vectored vaccine prime-recombinant modified virus Ankara boost regimen.
(52/353)
In the absence of strategies for reliable induction of antibodies broadly neutralizing human immunodeficiency virus type 1 (HIV-1), vaccine efforts have shifted toward the induction of cell-mediated immunity. Here we describe the construction and immunogenicity of novel T-cell vaccine NS1.HIVA, which delivers the HIV-1 clade A consensus-derived immunogen HIVA on the surface of tubular structures spontaneously formed by protein NS1 of bluetongue virus. We demonstrated that NS1 tubules can accommodate a protein as large as 527 amino acids without losing their self-assembly capability. When injected into BALB/c mice by several routes, chimeric NS1.HIVA tubules induced HIV-1-specific major histocompatibility complex class I-restricted T cells. These could be boosted by modified virus Ankara expressing the same immunogen and generate a memory capable of gamma interferon (IFN-gamma) production, proliferation, and lysis of sensitized target cells. Induced memory T cells readily produced IFN-gamma 230 days postimmunization, and upon a surrogate virus challenge, NS1.HIVA vaccine alone decreased the vaccinia virus vv.HIVA load in ovaries by 2 orders of magnitude 280 days after immunization. Thus, because of its T-cell immunogenicity and antigenic simplicity, the NS1 delivery system could serve as a priming agent for heterologous prime-boost vaccination regimens. Its usefulness in primates, including humans, remains to be determined. (+info)
Nonstructural protein 3 of bluetongue virus assists virus release by recruiting ESCRT-I protein Tsg101.
(53/353)
The release of Bluetongue virus (BTV) and other members of the Orbivirus genus from infected host cells occurs predominantly by cell lysis, and in some cases, by budding from the plasma membrane. Two nonstructural proteins, NS3 and NS3A, have been implicated in this process. Here we show that both proteins bind to human Tsg101 and its ortholog from Drosophila melanogaster with similar strengths in vitro. This interaction is mediated by a conserved PSAP motif in NS3 and appears to play a role in virus release. The depletion of Tsg101 with small interfering RNA inhibits the release of BTV and African horse sickness virus, a related orbivirus, from HeLa cells up to fivefold and threefold, respectively. Like most other viral proteins which recruit Tsg101, NS3 also harbors a PPXY late-domain motif that allows NS3 to bind NEDD4-like ubiquitin ligases in vitro. However, the late-domain motifs in NS3 do not function as effectively in facilitating the release of mini Gag virus-like particles from 293T cells as the late domains from human immunodeficiency virus type 1, human T-cell leukemia virus, and Ebola virus. A mutagenesis study showed that the arginine residue in the PPRY motif is responsible for the low activity of the NS3 late-domain motifs. Our data suggest that the BTV late-domain motifs either recruit an antagonist that interferes with budding or fail to recruit an agonist which is different from NEDD4. (+info)
Detection of bluetongue virus RNA by in situ hybridization: comparison with virus isolation and antigen detection.
(54/353)
An in situ nucleic acid hybridization (ISH) technique was developed to detect bluetongue virus (BTV) RNA in cell culture. The sensitivity of the ISH technique was compared with virus isolation (VI) and antigen detection, using an indirect fluorescent-antibody (IFA) or an enzyme immunocytoassay (EICA) technique, for detection of 5 BTV serotypes indigenous to the United States. The VI was the most sensitive technique, detecting BTV early after infection of the cells. The IFA and EICA were of similar sensitivity; BTV antigen could be detected shortly after demonstration of virus by isolation. The sensitivity of ISH for detection of BTV-17 was equivalent to that of antigen detection. The ISH was not as sensitive as VI or antigen detection when assaying for the other BTV serotypes. (+info)
The complete sequence of the group-specific antigen, VP7, of African horsesickness disease virus serotype 4 reveals a close relationship to bluetongue virus.
(55/353)
The complete sequence of the S7 RNA that codes for the major group-specific coat protein. VP7, of African horsesickness virus serotype 4 (AHSV-4) was determined from cDNA analyses and found to be 1179 nucleotides in length. One single open reading frame of 353 codons was observed defining a protein of Mr 38,107 with a net charge of -1.5 at neutral pH. Comparison of the AHSV-4 VP7 sequence with that of bluetongue virus serotype 10 revealed an overall similarity of 44%, with the amino- and carboxy-terminal regions exhibiting the greatest levels of homology. In addition, potential secondary structures of the terminal sequences of the S7 RNA segments of AHSV-4 and BTV serotypes 10, 13 and 17 are presented. (+info)
Improvements in the modified direct complement fixation test and its application in the detection of bluetongue antibodies in cattle and sheep sera.
(56/353)
In this study, improvements were made in the technique and the preparation of the antigen. It was possible to perform three extractions and elutions resulting in a soluble reactive preparation from each batch of infected mouse brain. This led to an appreciable increase in the yield of highly reactive antigen. The presence of bluetongue antibodies was not detected in 13,210 sheep sera. Of the 13,486 bovine sera tested, only three questionable reactions were obtained. It was possible to determine that two of these animals were imported. Various isolation methods, including transmission trials to susceptible sheep followed by serological tests on the sheep sera, failed to confirm the infection in the three reactors. (+info)