Although vesicular stomatitis virus (VSV) neurovirulence and pathogenicity in rodents have been well studied, little is known about VSV pathogenicity in non-human primates. To address this question, we measured VSV viremia, shedding, and neurovirulence in macaques. Following intranasal inoculation, macaques shed minimal recombinant VSV (rVSV) in nasal washes for 1 day post-inoculation; viremia was not detected. Following intranasal inoculation of macaques, wild type (wt) VSV, rVSV, and two rVSV-HIV vectors showed no evidence of spread to CNS tissues. However, macaques inoculated intrathalamically with wt VSV developed severe neurological disease. One of four macaques receiving rVSV developed clinical and histological signs similar to the wt group, while the remaining three macaques in this group and all of the macaques in the rVSV-HIV vector groups showed no clinical signs of disease and reduced severity of histopathology compared to the wt group. The implications of these findings for rVSV vaccine development are discussed.

Molecular surveillance of pathogens has shown the need for rapid and dependable methods for the identification of organisms of clinical and epidemiological importance. As the leading cause of community-acquired pneumonia, Streptococcus pneumoniae was used as a model organism to develop and refine a real-time fluorescence PCR assay and enhanced DNA purification method. Seventy clinical isolates of S. pneumoniae, verified by latex agglutination, were screened against 26 negative control clinical isolates employing a TaqMan assay on a thermocycler (LightCycler). The probe, constructed from the lytA gene, correctly detected all S. pneumoniae genomes without cross-reaction to negative controls. The speed and ease of this approach will make it adaptable to identification of many bacterial pathogens and provide potential for adaptation to direct detection from patient specimens.Streptococcus pneumoniae is the leading cause of community-acquired pneumonia, meningitis, and otitis media in the United States (2). While traditional antimicrobial therapy has proven an effective treatment in the past, the emergence of penicillin-and multidrug-resistant strains has resulted in an increasing number of cases of illnesses and fatalities (4, 18). Pneumococcal isolation and identification are complicated by antimicrobial suppression of growth in culture and contamination by normal flora alpha-streptococci. Detection by classical techniques, culture, and serological methods can be time-consuming and indeterminate. Sensitive and specific assays that can be completed quickly in the clinical laboratory are essential for early diagnosis and effective therapy. Molecular assays are inherently valuable because detection can be achieved with enhanced sensitivity and specificity, and detection is not diminished with nonviable organisms.Various molecular methods have been employed to assist investigations (8,11,23). These methods include restriction fragment length polymorphism (RFLP)-based protocols and fingerprinting. PCR-based assays for the detection of S. pneumoniae with primers specific to repetitive regions and genes encoding rRNA (12,14,21), pneumococcal surface adhesion A molecule (22), pneumolysin (20,27,30,37), penicillin-binding protein (5,6,7,24,31), and autolysin (10, 25, 26) have been employed with various degrees of success. Autolysin, encoded by the lytA gene, is required for S. pneumoniae pathogenesis and is a well-characterized virulence marker (1). The lytA gene has been shown to have restricted allelic variation and therefore makes an ideal target for specific identification in clinical and epidemiological studies (9, 35). Sequencing of the lytA locus and high-resolution DNA typing of S. pneumoniae demonstrated that the gene is highly conserved within the species (32, 33), and it has been shown that lytA separates S. pneumoniae from the genotypically similiar species Streptococcus mitis and S. oralis (19,34).Real-time PCR with sequence-specific primers and a fluorescent TaqMan probe allows continuous monitoring of in vitr...

Expression of Norwalk virus nonstructural polyprotein precursor in vitro resulted in rapid cotranslational cleavage at specific sites. The cleavage products were similar to those previously identified for Southampton virus, a highly related virus. We inactivated the virally encoded proteinase responsible for cleavage of the nonstructural polyprotein by mutation of the putative catalytic cysteine residue, which resulted in production of full-length polyprotein precursor. NV proteinase was expressed in Escherichia coli as a glutathione S-transferase fusion and purified by GST-affinity chromatography. Activity of the purified proteinase was demonstrated by incubation with the full-length precursor protein. trans cleavage of the nonstructural protein precursor resulted in cleavage products similar to those observed during cotranslational cleavage, however, at lesser efficiency. NV proteinase displayed sensitivities to cysteine and serine protease inhibitors similar to poliovirus 3C proteinase, suggesting that NV proteinase is a member of the viral cysteine proteinase family. We propose that the proteinase may play a regulatory role in viral replication.

The ability to express heterologous antigens from attenuated poliovirus strains suggests the potential for use as live vectored vaccines. Fullor partial-length sequences of the gene encoding rotavirus major outer capsid protein VP7 were cloned into the open reading frame of a full-length cDNA copy of poliovirus Sabin type 3. They were inserted either at the 5' end or immediately after the capsid protein coding region, at the junction between precursors P1 and P2. A protease cleavage site for 3C protease was introduced 3' to the foreign sequences to enable proteolytic processing of the antigen from the poliovirus polyprotein. Infectious viruses were generated from several of the DNA constructs, and the presence of the foreign gene sequences was confirmed by reverse transcription of the viral RNA and PCR amplification. Viruses with inserts of about 300 bases maintained the foreign sequences during passage in Vero cells. Viruses carrying larger sequences were unstable, and deletions were generated within the foreign sequences. Expression of the VP7 polypeptides was demonstrated by immunoprecipitation with specific antiserum of labeled proteins from cells infected with Sabin 3 recombinant viruses. Comparative studies of RNA synthesis showed similar kinetics for Sabin 3 and the Sabin 3/VP7 recombinants. One-step growth curves showed that production of recombinant viruses was slower than that of Sabin 3 and that the final titers were 1 to 1.5 logs lower. Accumulation of VP7-containing precursors in infected cells suggests that slow cleavage at the engineered 3C protease site may be a limiting step in the growth of these recombinant Sabin polioviruses and may influence the permissible size of foreign sequence to be inserted.