Preparation and characterization of new challenge stocks of SIVmac32H J5 following rapid serial passage of virus in vivo.

Background: A new challenge stock of the simian immunodeficiency virus SIVmacJ5 has been produced following passage in vivo.

Methods: SIVmacJ5 3/92 (J5M), was passaged serially through cynomolgus macaques (Macaca fascicularis) by intravenous inoculation of infected spleen cells isolated and prepared 14 days post-infection. Two challenge stocks, SIVmacJ5 S61MLN and SIVmacJ5 S62spl, were prepared by culture of lymphoid tissue ex vivo.

Immunological responses and viral modulatory effects of vaccination with recombinant modified vaccinia virus Ankara (rMVA) expressing structural and regulatory transgenes of simian immunodeficiency virus (SIVmac32H/J5M).

Background: The immunogenicity and protective efficacy of recombinant modified vaccinia virus Ankara (rMVA) vectors expressing structural (gag/pol, env) and regulatory (tat, rev, nef) genes of SIVmac251/32H-J5 (rMVA-J5) were assessed.

Methods: Immunization with rMVA constructs (2.5 x 10(7) IU) 32, 20 and 8 weeks pre-challenge was compared with 32 and 20 weeks but with a final boost 8 weeks pre-challenge with 2 x 10(6) fixed-inactivated HSC-F4 cells infected with SIVmac32H.

Vaccination with live attenuated simian immunodeficiency virus for 21 days protects against superinfection.

The identification of mechanisms that prevent infection with human immunodeficiency virus (HIV) or simian immunodeficiency virus (SIV) would facilitate the development of an effective AIDS vaccine. In time-course experiments, protection against detectable superinfection with homologous wild-type SIV was achieved within 21 days of inoculation with live attenuated SIV, prior to the development of detectable anti-SIV humoral immunity. Partial protection against superinfection was achieved within 10 days of inoculation with live attenuated SIV, prior to the development of detectable anti-SIV humoral and cellular immunity.

Mechanisms of protection induced by attenuated simian immunodeficiency virus.

To determine whether attenuated simian immunodeficiency virus (SIV) vaccines confer protection against superinfection via secondary cellular immune responses, we searched for markers of immune activation following rechallenge. Productive infection with either attenuated SIVmacC8 or wild-type SIVmacJ5 resulted in a transient increase in T-lymphocyte CD25 and Mafa-DR expression. A pronounced increase in the frequency of FAS+ CD8+ lymphocytes was observed following SIVmacJ5 infection only.

Specific proliferative T cell responses and antibodies elicited by vaccination with simian immunodeficiency virus Nef do not confer protection against virus challenge.

The efficacy of immunizing with a combination of simian immunodeficiency virus (SIV) Nef vaccines was evaluated. Four vaccinates received three intradermal immunizations with recombinant vaccinia virus that expressed SIV Nef, followed by three intramuscular immunizations with rDNA also expressing SIV Nef. Finally, the four vaccinates received two subcutaneous boosts with recombinant SIV Nef protein.

Characteristics of a pathogenic molecular clone of an end-stage serum-derived variant of simian immunodeficiency virus (SIV(F359)).

End-stage simian immunodeficiency virus (SIV) isolates are suggested to be the most fit of the evolved virulent variants that precipitate the progression to AIDS. To determine if there were common characteristics of end-stage variants which emerge from accelerated cases of AIDS, a molecular clone was derived directly from serum following in vivo selection of a highly virulent SIV isolate obtained by serial end-stage passage in rhesus monkeys (Macaca mulatta). This dominant variant caused a marked cytopathic effect and replicated to very high levels in activated but not resting peripheral blood lymphocytes.

Mechanisms of protection induced by live attenuated simian immunodeficiency virus: III. Viral interference and the role of CD8+ T-cells and beta-chemokines in the inhibition of virus infection of PBMCs in vitro.

In this study, we investigated whether a type of retroviral interference might be one mechanism that mediates the powerful protection induced by live attenuated SIVC8. Our results show that retroviral interference could be demonstrated between SIV and SHIV-HXBc2 in human T-cell lines chronically infected with either SIVC8 or SIVJ5. Lymphocytes from macaques infected with live attenuated SIVC8 were significantly less sensitive (P < 0.

Cloning and sequencing of cynomolgus macaque CCR3, GPR15, and STRL33: potential coreceptors for HIV type 1, HIV type 2, and SIV.

The characterization of several seven-transmembrane G protein-coupled receptors, which function as coreceptors for HIV-1, HIV-2, and/or SIV, has opened up a whole new area of AIDS research. Animal models that have played a central role in the understanding of lentivirus pathogenesis and the design of novel vaccine strategies may also be invaluable in studying the role of these secondary receptors in infection and disease progression. However, since it is known that minor species-specific sequence changes in CCR3 and STRL33 affect their ability to act as coreceptors for HIV-1, HIV-2, and/or SIV, it is important to ascertain whether the relevant receptors function as expected in the animal model of choice.

HIV-1 kills renal tubular epithelial cells in vitro by triggering an apoptotic pathway involving caspase activation and Fas upregulation.

HIV-infected patients suffer several renal syndromes, which can progress rapidly from renal insufficiency to end-stage renal disease. Histologically, HIV-induced nephropathy is characterized by prominent tubulopathy with apoptosis of tubular cells. Clinical and experimental evidence suggests that renal injury may be directly related to virus infection.

Bluetongue virus serotypes 1 and 3 infection in Poll Dorset sheep.

Objective: To study the clinical signs following bluetongue virus serotypes 1 and 3 infection in Poll Dorset sheep.

Design: A clinical and pathological study.

Procedure: Twenty Poll Dorset sheep were inoculated with bluetongue virus serotypes 1 or 3, each inoculum having a different passage history.

VP7 from African horse sickness virus serotype 9 protects mice against a lethal, heterologous serotype challenge.

An established mouse model system was used to evaluate the effectiveness of the major outer core protein VP7 of African horse sickness virus (AHSV) serotype 9 as a subunit vaccine. Balb C mice were immunised with VP7 crystals purified from AHSV infected BHK cells. In groups of mice, each of which was immunised with > or = 1.

African horsesickness virus VP7 sub-unit vaccine protects mice against a lethal, heterologous serotype challenge.

An established mouse model was used to evaluate the effectiveness of the major outer core protein of African horsesickness virus (AHSV), VP7, as a subunit vaccine. Adult female BALB/c mice were immunized with VP7 crystals purified from BHK cells infected with AHSV serotype 9 (AHSV-9), using three inoculations in Freund's adjuvant. Eighty to one hundred per cent of the immunized mice were protected against a heterologous challenge with a known lethal dose of AHSV-7.

Expression of the major core structural protein (VP7) of bluetongue virus, by a recombinant capripox virus, provides partial protection of sheep against a virulent heterotypic bluetongue virus challenge.

A recombinant capripox virus was constructed containing a cDNA copy of genome segment 7 of bluetongue virus (BTV) serotype 1 from South Africa (BTV 1SA), which expressed high levels of the major BTV core protein VP7 in infected lamb testis (LT) cells. Sheep vaccinated with this recombinant virus developed antibodies to VP7 (detected by ELISA) but no neutralizing antibodies to either the homologous or heterologous BTV serotype, prior to challenge (BTV 1 or BTV 3, respectively). Following challenge with a virulent heterotypic strain of BTV (BTV3 SA), all of the animals developed clinical signs of disease, indicating that they were infected and that the challenge virus did replicate.

Proteolytic cleavage of VP2, an outer capsid protein of African horse sickness virus, by species-specific serum proteases enhances infectivity in Culicoides.

Purified African horse sickness virus (AHSV) was fed, as part of a blood meal, to adult females from a susceptible colony of Culicoides variipennis, established in the insectories at the Institute for Animal Health, Pirbright Laboratory, UK. The meal consisted of heparinized blood obtained from ovine, bovine, equine (horse and donkey) or canine sources spiked with AHSV serotype 9 (AHSV9). The infectivity levels observed for C.

Application of the polymerase chain reaction to the detection of African horse sickness viruses.

The development of a coupled reverse transcriptase-polymerase chain reaction assay (RT-PCR) is described for the detection of African horse sickness virus (AHSV) double-stranded RNA. Genome segments 7 and 10 were chosen as target templates for primers selected for use in the RT-PCR. Using these AHSV-specific primers all 9 serotypes were detectable.

Complete nucleotide sequence of RNA segment 3 of bluetongue virus serotype 2 (Ona-A). Phylogenetic analyses reveal the probable origin and relationship with other orbiviruses.

The nucleotide sequence of the RNA segment 3 of bluetongue virus (BTV) serotype 2 (Ona-A) from North America was determined to be 2772 nucleotides containing a single large open reading frame of 2703 nucleotides (901 amino acid). The predicted VP3 protein exhibited general physiochemical properties (including hydropathy profiles) which were very similar to those previously deduced for other BTV VP3 proteins. Partial genome segment 3 sequences, obtained by polymerase chain reaction (PCR) sequencing, of BTV isolates from the Caribbean were compared to those from North America, South Africa, India, Indonesia, Malaysia and Australia, as well as other orbiviruses, to determine the phylogenetic relationships amongst them.

The use of African horse sickness virus VP7 antigen, synthesised in bacteria, and anti-VP7 monoclonal antibodies in a competitive ELISA.

A full-length cDNA clone of genome segment 7 of African Horse Sickness Virus, serotype 9 (AHSV9) was obtained using the PCR technique. The clone was sequenced and found to be 98.27% homologous to the previously published sequence of the equivalent cDNA clone from AHSV4 at the nucleotide level and to exhibit 99.

Sequence of genome segment 9 of bluetongue virus (serotype 1, South Africa) and expression analysis demonstrating that different forms of VP6 are derived from initiation of protein synthesis at two distinct sites.

Bluetongue virus (BTV) VP6 is often resolved into two closely migrating bands by SDS-PAGE (VP6 and VP6a). RNA segment 9 of BTV-serotype 1 South Africa (encoding VP6) has been cloned as cDNA, and the complete sequence has been determined. Expression of this clone both in vitro and in tissue culture produced the same polypeptide doublet as seen previously in extracts from BTV-infected cells.

The complete sequence of genome segment 8 of bluetongue virus, serotype 1, which encodes the nonstructural protein, NS2.

Bluetongue virus has a ten-segment double-stranded RNA genome, of which segment 8 encodes a nonstructural protein NS2. This protein is the only bluetongue viral protein to be phosphorylated and also has the ability to bind single-stranded RNA. At present, the function of NS2 is unknown and in order to analyse its characteristics in more detail, it was first necessary to obtain a full-length cDNA clone of the genome segment.

Development of the polymerase chain reaction for the detection of bluetongue virus in tissue samples.

Total genomic dsRNA, extracted from purified core particles of bluetongue virus serotype 1 from South Africa (BTV1SA), was used as template to optimise a polymerase chain reaction (PCR) for the detection of bluetongue virus RNA. Pairs of oligonucleotides complementary to the 3' termini of eight of the ten genome segments were tested. Those representing the 5' termini of genome segment 7 gave the best amplification results producing a single DNA band with the same mobility during agarose gel electrophoresis as genome segment 7.

Identification of a bluetongue virus serotype 1-specific ovine helper T-cell determinant in outer capsid protein VP2.

Ovine T-cell lines (including one clone [101A]), which are specific for Bluetongue virus serotype 1 (BTV1), have been established and characterized. Although these T-cell lines react with different isolates of BTV1 (including those from South Africa, Australia, Nigeria, and Cameroon), they do not react with heterologous BTV serotypes. Antigen specificity of these T-cells was studied using purified virus particles, infectious subviral particles (ISVP) and cores, or using individual BTV structural proteins that were either isolated by SDS-PAGE or expressed by recombinant strains of vaccinia virus.