Genomic and phylogenetic characterization of viruses included in the Manzanilla and Oropouche species complexes of the genus Orthobunyavirus, family Bunyaviridae.

A thorough characterization of the genetic diversity of viruses present in vector and vertebrate host populations is essential for the early detection of and response to emerging pathogenic viruses, yet genetic characterization of many important viral groups remains incomplete. The Simbu serogroup of the genus Orthobunyavirus, family Bunyaviridae, is an example. The Simbu serogroup currently consists of a highly diverse group of related arboviruses that infect both humans and economically important livestock species. Here, we report complete genome sequences for 11 viruses within this group, with a focus on the large and poorly characterized Manzanilla and Oropouche species complexes. Phylogenetic and pairwise divergence analyses indicated the presence of high levels of genetic diversity within these two species complexes, on a par with that seen among the five other species complexes in the Simbu serogroup. Based on previously reported divergence thresholds between species, the data suggested that these two complexes should actually be divided into at least five species. Together these five species formed a distinct phylogenetic clade apart from the rest of the Simbu serogroup. Pairwise sequence divergences among viruses of this clade and viruses in other Simbu serogroup species complexes were similar to levels of divergence among the other orthobunyavirus serogroups. The genetic data also suggested relatively high levels of natural reassortment, with three potential reassortment events present, including two well-supported events involving viruses known to infect humans.

Key genomic changes necessary for an in vivo lethal mouse marburgvirus variant selection process.

Marburgvirus (MARV) infections are generally lethal in humans and nonhuman primates but require in vivo lethal mouse variant selection by the serial transfer (passage) of the nonlethal virus into naïve mice to propagate a lethal infection. The passage of progenitor (wild-type) MARV or Ravn virus (RAVV) from infected scid BALB/c mouse liver homogenates into immunocompetent BALB/c mice results in the selection of lethal mouse viruses from within the quasispecies sufficient to establish lethality in immunocompetent mice. Genomic analysis in conjunction with the passage history of each mutation detailed the altered primary and secondary structures of the viral genomic RNA throughout the process. Key findings included the following: (i) a VP40:D184N mutation previously identified in the lethal guinea pig MARV genome was the first mutation to occur during the passage of both the MARV and RAVV variants; (ii) there was biased hypermutagenesis in the RAVV variant genome; (iii) there were two identical mutations in lethal mouse MARV and RAVV variants, VP40:Y19H in the PPPY motif and VP40:D184N in a loop structure between the two VP40 domains; (iv) the passage of wild-type MARV and RAVV in mice resulted in the selection of viral variants from among the quasispecies with different genotypes than those of the wild-type viruses; and (v) a lethal mouse RAVV variant had different tissue tropisms distinct from those of its wild-type virus. These studies provide insights into how marburgviruses manipulate the host for enzymes, metabolites, translation regulators, and effectors of the innate immune response to serve as potential viral countermeasures.

Development and characterization of a mouse model for Marburg hemorrhagic fever.

The lack of a mouse model has hampered an understanding of the pathogenesis and immunity of Marburg hemorrhagic fever (MHF), the disease caused by marburgvirus (MARV), and has created a bottleneck in the development of antiviral therapeutics. Primary isolates of the filoviruses, i.e., ebolavirus (EBOV) and MARV, are not lethal to immunocompetent adult mice. Previously, pathological, virologic, and immunologic evaluation of a mouse-adapted EBOV, developed by sequential passages in suckling mice, identified many similarities between this model and EBOV infections in nonhuman primates. We recently demonstrated that serially passaging virus recovered from the liver homogenates of MARV-infected immunodeficient (SCID) mice was highly successful in reducing the time to death in these mice from 50 to 70 days to 7 to 10 days after challenge with the isolate MARV-Ci67, -Musoke, or -Ravn. In this study, we extended our findings to show that further sequential passages of MARV-Ravn in immunocompetent mice caused the MARV to kill BALB/c mice. Serial sampling studies to characterize the pathology of mouse-adapted MARV-Ravn revealed that this model is similar to the guinea pig and nonhuman primate MHF models. Infection of BALB/c mice with mouse-adapted MARV-Ravn caused uncontrolled viremia and high viral titers in the liver, spleen, lymph node, and other organs; profound lymphopenia; destruction of lymphocytes within the spleen and lymph nodes; and marked liver damage and thrombocytopenia. Sequencing the mouse-adapted MARV-Ravn strain revealed differences in 16 predicted amino acids from the progenitor virus, although the exact changes required for adaptation are unclear at this time. This mouse-adapted MARV strain can now be used to develop and evaluate novel vaccines and therapeutics and may also help to provide a better understanding of the virulence factors associated with MARV.

Reidentification of Ebola Virus E718 and ME as Ebola Virus/H.sapiens-tc/COD/1976/Yambuku-Ecran.

Ebola virus (EBOV) was discovered in 1976 around Yambuku, Zaire. A lack of nomenclature standards resulted in a variety of designations for each isolate, leading to confusion in the literature and databases. We sequenced the genome of isolate E718/ME/Ecran and unified the various designations under Ebola virus/H.sapiens-tc/COD/1976/Yambuku-Ecran.

Genomic differences between guinea pig lethal and nonlethal Marburg virus variants.

The complete genome sequences of 2 closely related plaque-derived variants of Marburg virus (MARV) species Lake Victoria marburgvirus, strain Musoke, indicate only a few regions of the RNA genome as underlying the differences between the 2 viruses. One variant is >90% lethal for guinea pigs and the other much less virulent, when guinea pigs are challenged with 1000 pfu of virus. Only 4 mutations that result in amino acid changes were identified, 1 in viral matrix protein VP40 and 3 in L, the RNA-dependent RNA polymerase. In addition, 6 differences were identified in noncoding regions of transcribed mRNA, and 1 silent codon change was identified in the L gene. Interestingly, the amino acid mutation identified in VP40 occurs in a nonconserved loop structure between 2 domains that are homologues only among MARV species. The L gene mutations were equally intriguing, clustering near a highly conserved motif in viral RNA-dependent RNA polymerases.

Venezuelan equine encephalitis replicon immunization overcomes intrinsic tolerance and elicits effective anti-tumor immunity to the 'self' tumor-associated antigen, neu in a rat mammary tumor model.

Many tumor-associated antigens (TAAs) represent 'self' antigens and as such, are subject to the constraints of immunologic tolerance. There are significant barriers to eliciting anti-tumor immune responses of sufficient magnitude. We have taken advantage of a Venezuelan equine encephalitis-derived alphavirus replicon vector system with documented in vivo tropism for immune system dendritic cells. We have overcome the intrinsic tolerance to the 'self' TAA rat neu and elicited an effective anti-tumor immune response using this alphavirus replicon vector system and a designed target antigen in a rigorous rat mammary tumor model. We have demonstrated the capacity to generate 50% protection in tumor challenge experiments (p = 0.004) and we have confirmed the establishment of immunologic memory by both second tumor challenge and Winn Assay (p = 0.009). Minor antibody responses were identified and supported the establishment of T helper type 1 (Th1) anti-tumor immune responses by isotype. Animals surviving in excess of 300 days with established effective anti-tumor immunity showed no signs of autoimmune phenomena. Together these experiments support the establishment of T lymphocyte dependent, Th1-biased anti-tumor immune responses to a non-mutated 'self' TAA in an aggressive tumor model. Importantly, this tumor model is subject to the constraints of immunologic tolerance present in animals with normal developmental, temporal, and anatomical expression of a non-mutated TAA. These data support the continued development and potential clinical application of this alphaviral replicon vector system and the use of appropriately designed target antigen sequences for anti-tumor immunotherapy.