ABSTRACT
An attenuated Australian strain of West Nile virus (WNV), Kunjin (KUN), shares ~98% amino acid homology with the pathogenic New York 99 NY99 strain (NY99). To investigate the viral factors involved in NY99 virulence we generated an infectious cDNA clone of the WNV NY99 4132 isolate from which virus was recovered and was shown to be indistinguishable from the parental isolate. We then introduced the regions of the NY99 non-structural (NS) proteins and/or untranslated regions (UTRs) into the KUN backbone. Chimeric KUN viruses containing NY99 5'UTR and the parts of NS coding region were more virulent in mice than parental KUN virus. Chimeric NY99 viruses, containing KUN NS2A protein with alanine 30 to proline substitution were significantly less cytopathic in cells and less virulent in mice. Our results identify the 5'UTR and NS proteins as WNV virulence determinants and confirm a role for the NS2A in WNV cytopathicity and virulence.
Subject(s)
Virulence Factors/genetics , West Nile virus/pathogenicity , Animals , Cell Line , Disease Models, Animal , Genetic Engineering , Lethal Dose 50 , Mice , Molecular Sequence Data , RNA, Viral/genetics , Recombination, Genetic , Rodent Diseases/pathology , Rodent Diseases/virology , Sequence Analysis, DNA , Survival Analysis , Viral Plaque Assay , Virulence , West Nile Fever/pathology , West Nile Fever/virology , West Nile virus/geneticsABSTRACT
Persistent gag-specific T cell immunity would be a useful component of an effective HIV vaccine. The Flavivirus Kunjin replicon was previously engineered to persistently express HIV gag and was shown to induce protective responses in mice. We evaluated Kunjin replicon virus-like-particles expressing SIVgag-pol in pigtail macaques. Kunjin-specific antibodies were induced, but no SIV-specific T cell immunity were detected. Following SIVmac251 challenge, there was no difference in SIV viremia or retention of CD4 T cells between Kunjin-SIVgag-pol vaccine immunized animals and controls. An amnestic SIV gag-specific CD8 T cell response associated with control of viremia was observed in 1 of 6 immunized animals. Refinements of this vector system and optimization of the immunization doses, routes, and schedules are required prior to clinical trials.
Subject(s)
Replicon , SAIDS Vaccines , Simian Acquired Immunodeficiency Syndrome/prevention & control , Simian Immunodeficiency Virus/immunology , Vaccines, Synthetic/immunology , West Nile virus , AIDS Vaccines , Animals , Antibodies, Viral/blood , Fusion Proteins, gag-pol/genetics , Fusion Proteins, gag-pol/immunology , Fusion Proteins, gag-pol/metabolism , Genetic Engineering , HIV-1/genetics , HIV-1/immunology , HIV-1/metabolism , Lymphocyte Activation , Macaca nemestrina , SAIDS Vaccines/administration & dosage , SAIDS Vaccines/genetics , SAIDS Vaccines/immunology , Simian Acquired Immunodeficiency Syndrome/immunology , Simian Acquired Immunodeficiency Syndrome/virology , Simian Immunodeficiency Virus/genetics , Simian Immunodeficiency Virus/metabolism , Simian Immunodeficiency Virus/pathogenicity , T-Lymphocytes/immunology , Vaccines, Synthetic/genetics , Vaccines, Synthetic/metabolism , West Nile virus/genetics , West Nile virus/immunology , West Nile virus/metabolismABSTRACT
We have recently developed a new personal sampler and demonstrated its feasibility for detection of viable airborne microorganisms including bacteria, fungi and viruses. To accelerate the time-consuming analytical procedure involving 2-5 days of biological testing, we employed a real-time PCR protocol in conjunction with the personal sampler for collection of airborne viruses. The advantage of this approach is that if the presence of a particular pathogen in the air is detected by the PCR, the remaining collecting liquid can be further analysed by more time-consuming biological methods to estimate the number of airborne infectious/live microorganisms. As sampling of bioaerosols in natural environments is likely to be associated with substantial contamination by a range of microorganisms commonly existing in an ambient air, an investigation of the specificity of detection by targeted PCR analysis is required. Here we present the results of the study on the detection of Influenza virus in the ambient air contaminated with high concentrations of bacteria and fungi using real-time PCR protocol. The combined sampling PCR detection method was found to be fully feasible for the rapid ( approximately 2.5 h) and highly specific (no cross-reactivity) identification of the labile airborne virus in the air containing elevated concentrations of other microorganisms.
