High-yield reassortant influenza vaccine production virus has a mutation at an HLA-A 2.1-restricted CD8+ CTL epitope on the NS1 protein.
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Current influenza virus vaccines are prepared using high-yield reassortant virus strains obtained from a mixed infection of the new virus strain and a prototype high-yielding virus strain. The high-titered reassortant virus strain used as vaccine seed virus possesses the recent virus HA and NA and contains the internal genes from the high-growing prototype parent. We established a human CD8(+) cytotoxic T cell (CTL) line, 10-2C2, which recognizes an HLA-A2.1-restricted influenza A virus H1, H2, H3 cross-reactive T cell epitope on amino acids 122-130 of the NS1 protein, and unexpectedly we observed that there was decreased lysis of target cells infected with the A/Texas/36/91 (H1N1) vaccine virus strain compared to the lysis of target cells infected with the prototype A/PR/8/34 (H1N1) virus. RT-PCR results showed that the A/Texas vaccine virus strain contained a quasispecies. Approximately 50% of viral RNA of the NS1 gene had a nucleotide substitution that resulted in the N --> K amino acid change at the sixth position of the nonamer peptide. Current influenza vaccines are inactivated and do not contain the NS1 protein; however, future influenza vaccines may include live attenuated vaccines and with this mutation a live virus would fail to induce a CD8(+) CTL response to this epitope in individuals with HLA-A2.1, a very common allele, and potentially have reduced efficacy. (+info)
Sequence analysis of VP1 and VP7 genes suggests occurrence of a reassortant of G2 rotavirus responsible for an epidemic of gastroenteritis.
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G2 rotavirus was prevalent in a 1993 epidemic of acute gastroenteritis in Taiwan. In this study, the genetic relationship among G2 rotavirus strains was analysed. The VP7 genes were amplified and sequenced. Except for one strain isolated in 1981, the nucleotide sequences of the VP7 genes of most of the G2 rotaviruses were very similar (identity > 97%) and were closely related to that of a Japanese G2 reference strain, S2. The genetic relatedness of G2 rotaviruses was analysed further by RNA-RNA hybridization. The genomes of the major G2 strains of 1993 did not hybridize well with those of the G2 strains of previous seasons in RNA segments 1, 6 and 7. Partial nucleotide sequences of the VP1 gene were analysed and appeared to be similar among the major G2 strains from the same epidemic (identity > 98%), whereas the identity of the VP1 genes of the major G2 strains of the 1993 epidemic to those of previous seasons was only about 84%. Since the numbers of mutations accumulated in the VP1 and VP7 genes over a period of 10 years were comparable, the significant change in the VP1 genes of the major strains of the 1993 epidemic suggests that these G2 rotaviruses had evolved by genetic reassortment. (+info)
Detection of porcine rotavirus type G9 and of a mixture of types G1 and G5 associated with Wa-like VP4 specificity: evidence for natural human-porcine genetic reassortment.
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Rotavirus type G5 is a primarily porcine pathogen that has caused frequent and widespread diarrhea in children in Brazil and in piglets elsewhere. Initial results on the rotavirus types circulating in diarrheic piglets in Brazil disclosed a high diversity of strains with distinct G types including G1, G4, G5, and G9 and the novelty of P[8], the predominant human P specificity type. Those results add strong evidence for the emergence of new strains through natural reassortment between rotaviruses of human and porcine origins. (+info)
Derivation and biological characterization of a molecular clone of SHIV(KU-2) that causes AIDS, neurological disease, and renal disease in rhesus macaques.
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Previously, we described the derivation of a pathogenic strain of simian-human immunodeficiency virus (SHIV(KU-2)) consisting of the tat, rev, vpu, and env genes of HIV-1 (strain HXB2) in a genetic background of SIV(mac)239 that causes AIDS and productive infection of the CNS in rhesus macaques (Macca mulatta) (Raghavan et al., 1997, Brain Pathol. 7, 851-861). We report here on the characterization of a molecular clone of SHIV(KU-2), designated SHIV(KU-2MC4), that caused CD4(+) T cell loss as well as neurological and renal disease in macaques. DNA sequence analysis of selected SIV regions of SHIV(KU-2MC4) revealed 10 nucleotide changes in the LTR, whereas Gag, Vif, Vpr, Vpx, and Nef had 1, 1, 1, 2, and 13 predicted amino acid substitutions, respectively, compared to SIV(mac)239. DNA sequence analysis of HIV-1 derived regions of SHIV(KU-2MC4) revealed 2, 1, 2, and 18 predicted amino acid substitutions in the Tat, Rev, Vpu, and Env proteins, respectively, when compared to SHIV-4. Unlike the parental SHIV-4, which is not tropic for macrophages, SHIV(KU-2MC4) replicated efficiently in macrophage cultures as determined by p27 assays. However, despite the numerous changes in the Env protein and newly acquired tropism for macrophages, SHIV(KU-2MC4), like the parental SHIV-4, used CXCR4 exclusively as its coreceptor for entry into susceptible cells. Inoculation of SHIV(KU-2MC4) into two rhesus macaques resulted in severe infection in which the numbers of circulating CD4(+) T cells in the blood declined rapidly by 2 weeks postinoculation and virus producing cells in the peripheral blood mononuclear cells were identified throughout the course of infection. At the time of euthanasia (20 and 22 weeks), both macaques had lost a significant amount of weight and had no circulating CD4(+) T cells. In addition, one macaque developed intension tremors and uncoordinated movements. Virological examination of tissues at necropsy revealed active virus replication in both lymphoid and nonlymphoid tissues such as the lung and brain. Histological examination revealed that the induced immunodeficiency was associated with lymphoid depletion of the lymph nodes and spleen, opportunistic infections, lentiviral encephalitis, and severe glomerulosclerosis of the kidney. This molecular clone will serve as the basis for analyzing the molecular determinants through which SHIV(KU-2) causes severe CD4(+) T cell loss, neurological disease, and SHIV nephropathy in rhesus macaques. (+info)
Cell-mediated immune responses in cattle vaccinated with a vaccinia virus recombinant expressing the nucleocapsid protein of rinderpest virus.
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Rinderpest virus (RPV) is a member of the genus Morbillivirus in the family Paramyxoviridae which causes an acute and often fatal disease in large ruminants. To examine the immune response to the virus nucleocapsid (N) protein, a recombinant vaccinia virus expressing RPV nucleocapsid protein (rVV-RPV-N) was used to vaccinate cattle. The recombinant vaccine induced low levels of non-neutralizing anti-N antibodies. RPV-specific cell-mediated immunity induced by the recombinant was assessed by measuring both the lymphocyte proliferation and cytotoxic T-lymphocyte responses. The protective immune response was examined by challenging the vaccinated cattle with either a highly virulent (Saudi 1/81) or a mild (Kenya/eland/96) strain of the virus. The vaccinated cattle were not protected against challenge with the virulent RPV strain, except they showed a slight delay in the onset of disease when compared with the unvaccinated controls. In cattle challenged with the mild strain, apart from a transient fever, no clinical signs of rinderpest infection were seen in the vaccinated cattle. One out of two control cattle showed a similar response but the other died from classic rinderpest disease. Virus-neutralizing antibodies were induced more quickly following challenge with the mild strain in vaccinated cattle compared to the control animals. These data suggested that the cell-mediated immunity induced by rVV-RPV-N could stimulate the rapid production of neutralizing antibodies following RPV challenge but this response was not sufficient to protect against challenge with a virulent strain of the virus. Protection was seen in one of three animals challenged with a mild strain of the virus; however, a greater number of animals would need to be tested to estimate the significance of the protection afforded by the N protein. (+info)
Pathogenesis of chimeric MHV4/MHV-A59 recombinant viruses: the murine coronavirus spike protein is a major determinant of neurovirulence.
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The mouse hepatitis virus (MHV) spike glycoprotein, S, has been implicated as a major determinant of viral pathogenesis. In the absence of a full-length molecular clone, however, it has been difficult to address the role of individual viral genes in pathogenesis. By using targeted RNA recombination to introduce the S gene of MHV4, a highly neurovirulent strain, into the genome of MHV-A59, a mildly neurovirulent strain, we have been able to directly address the role of the S gene in neurovirulence. In cell culture, the recombinants containing the MHV4 S gene, S4R22 and S4R21, exhibited a small-plaque phenotype and replicated to low levels, similar to wild-type MHV4. Intracranial inoculation of C57BL/6 mice with S4R22 and S4R21 revealed a marked alteration in pathogenesis. Relative to wild-type control recombinant viruses (wtR13 and wtR9), containing the MHV-A59 S gene, the MHV4 S gene recombinants exhibited a dramatic increase in virulence and an increase in both viral antigen staining and inflammation in the central nervous system. There was not, however, an increase in the level of viral replication in the brain. These studies demonstrate that the MHV4 S gene alone is sufficient to confer a highly neurovirulent phenotype to a recombinant virus deriving the remainder of its genome from a mildly neurovirulent virus, MHV-A59. This definitively confirms previous findings, suggesting that the spike is a major determinant of pathogenesis. (+info)
Potential for evolution of California serogroup bunyaviruses by genome reassortment in Aedes albopictus.
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Aedes albopictus was introduced into the United States in used tires in 1985. Its successful colonization of the upper Midwest has potential to alter the current epidemiology of bunyaviruses that circulate in the region. It is permissive for the replication of several arboviruses, including La Crosse (LACV) and Jamestown Canyon (JCV) bunyaviruses. In this study, we demonstrate the ability of LACV and JCV to coinfect Ae. albopictus mosquitoes and to form all six possible reassortant genotypes. All reassortant viruses infect Ae. albopictus orally and can be transmitted to suckling mice. All reassortants are neurovirulent in mice. However, reassortant viruses carrying the LACV M segment in the foreign genetic background of JCV are more neuroinvasive than JCV, or any other reassortant genotype. In addition, these reassortants can replicate in gerbils and infect Ae. triseriatus, characteristics of LACV, but not JCV. Because Ae. albopictus is spreading into new geographic areas and feeds on a variety of mammals, including humans, it has the potential to transmit new, emerging bunyaviruses in nature. (+info)
Genetic reassortment of Rift Valley fever virus in nature.
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Rift Valley fever virus (RVFV), a phlebovirus of the Bunyaviridae family, is an arthropod-borne virus which emerges periodically throughout Africa, emphasizing that it poses a major threat for animal and human populations. To assess the genetic variability of RVFV, several isolates from diverse localities of Africa were investigated by means of reverse transcription-PCR followed by direct sequencing of a region of the small (S), medium (M), and large (L) genomic segments. Phylogenetic analysis showed the existence of three major lineages corresponding to geographic variants from West Africa, Egypt, and Central-East Africa. However, incongruences detected between the L, M, and S phylogenies suggested that genetic exchange via reassortment occurred between strains from different lineages. This hypothesis, depicted by parallel phylogenies, was further confirmed by statistical tests. Our findings, which strongly suggest exchanges between strains from areas of endemicity in West and East Africa, strengthen the potential existence of a sylvatic cycle in the tropical rain forest. This also emphasizes the risk of generating uncontrolled chimeric viruses by using live attenuated vaccines in areas of endemicity. (+info)