Cooperativity between the 3' untranslated region microRNA binding sites is critical for the virulence of eastern equine encephalitis virus.

Eastern equine encephalitis virus (EEEV), a mosquito-borne RNA virus, is one of the most acutely virulent viruses endemic to the Americas, causing between 30% and 70% mortality in symptomatic human cases. A major factor in the virulence of EEEV is the presence of four binding sites for the hematopoietic cell-specific microRNA, miR-142-3p, in the 3' untranslated region (3' UTR) of the virus. Three of the sites are "canonical" with all 7 seed sequence residues complimentary to miR-142-3p while one is "non-canonical" and has a seed sequence mismatch. Interaction of the EEEV genome with miR-142-3p limits virus replication in myeloid cells and suppresses the systemic innate immune response, greatly exacerbating EEEV neurovirulence. The presence of the miRNA binding sequences is also required for efficient EEEV replication in mosquitoes and, therefore, essential for transmission of the virus. In the current studies, we have examined the role of each binding site by point mutagenesis of the seed sequences in all combinations of sites followed by infection of mammalian myeloid cells, mosquito cells and mice. The resulting data indicate that both canonical and non-canonical sites contribute to cell infection and animal virulence, however, surprisingly, all sites are rapidly deleted from EEEV genomes shortly after infection of myeloid cells or mice. Finally, we show that the virulence of a related encephalitis virus, western equine encephalitis virus, is also dependent upon miR-142-3p binding sites.

The transmission dynamic of Madariaga Virus by bayesian phylogenetic analysis: Molecular surveillance of an emergent pathogen.

Madariaga Virus (MADV) is an emergent Alphavirus of the eastern equine encephalitis virus (EEEV) strain complex causing epizootic epidemics. In this study the genetic diversity and the transmission dynamics of Madariaga virus has been investigated by Bayesian phylogenetics and phylodynamic analysis. A database of 32 sequences of MADV group structural polyprotein were downloaded from GenBank, aligned manually edited by Bioedit Software. ModelTest v. 3.7 was used to select the simplest evolutionary model that adequately fitted the sequence data. Neighbor-joining tree was generated using MEGA7. The phylogenetic signal of the dataset was tested by the likelihood mapping analysis. The Bayesian phylogenetic tree was built using BEAST. Selective pressure analysis revealed one positive selection site. The phylogenetic trees showed two main clusters. In particular, Lineage II showed an epizootic infection in monkeys and Lineage III, including 2 main clusters (IIIa and IIIB), revealing an epizootic infection in humans in Haiti and an epizootic infection in humans in Venezuela during the 2016, respectively. The Bayesian maximum clade credibility tree and the time of the most common recent ancestor estimates, showed that the root of the tree dated back to the year 346 with the probable origin in Brazil. Gene flow analysis revealed viral exchanges between different neighbor countries of South America. In conclusion, Bayesian phylogenetic and phylodynamic represent useful tools to follow the transmission dynamic of emergent pathogens to prevent new epidemics spreading worldwide.

Natural variation in the heparan sulfate binding domain of the eastern equine encephalitis virus E2 glycoprotein alters interactions with cell surfaces and virulence in mice.

Recently, we compared amino acid sequences of the E2 glycoprotein of natural North American eastern equine encephalitis virus (NA-EEEV) isolates and demonstrated that naturally circulating viruses interact with heparan sulfate (HS) and that this interaction contributes to the extreme neurovirulence of EEEV (C. L. Gardner, G. D. Ebel, K. D. Ryman, and W. B. Klimstra, Proc. Natl. Acad. Sci. U. S. A., 108:16026-16031, 2011). In the current study, we have examined the contribution to HS binding of each of three lysine residues in the E2 71-to-77 region that comprise the primary HS binding site of wild-type (WT) NA-EEEV viruses. We also report that the original sequence comparison identified five virus isolates, each with one of three amino acid differences in the E2 71-to-77 region, including mutations in residues critical for HS binding by the WT virus. The natural variant viruses, which possessed either a mutation from lysine to glutamine at E2 71, a mutation from lysine to threonine at E2 71, or a mutation from threonine to lysine at E2 72, exhibited altered interactions with heparan sulfate and cell surfaces and altered virulence in a mouse model of EEEV disease. An electrostatic map of the EEEV E1/E2 heterotrimer based upon the recent Chikungunya virus crystal structure (J. E. Voss, M. C. Vaney, S. Duquerroy, C. Vonrhein, C. Girard-Blanc, E. Crublet, A. Thompson, G. Bricogne, and F. A. Rey, Nature, 468:709-712, 2010) showed the HS binding site to be at the apical surface of E2, with variants affecting the electrochemical nature of the binding site. Together, these results suggest that natural variation in the EEEV HS binding domain may arise during EEEV sylvatic cycles and that this variation may influence receptor interaction and the severity of EEEV disease.

Evolutionary patterns of eastern equine encephalitis virus in North versus South America suggest ecological differences and taxonomic revision.

The eastern equine encephalitis (EEE) complex consists of four distinct genetic lineages: one that circulates in North America (NA EEEV) and the Caribbean and three that circulate in Central and South America (SA EEEV). Differences in their geographic, pathogenic, and epidemiologic profiles prompted evaluation of their genetic diversity and evolutionary histories. The structural polyprotein open reading frames of all available SA EEEV and recent NA EEEV isolates were sequenced and used in evolutionary and phylogenetic analyses. The nucleotide substitution rate per year for SA EEEV (1.2 x 10(-4)) was lower and more consistent than that for NA EEEV (2.7 x 10(-4)), which exhibited considerable rate variation among constituent clades. Estimates of time since divergence varied widely depending upon the sequences used, with NA and SA EEEV diverging ca. 922 to 4,856 years ago and the two main SA EEEV lineages diverging ca. 577 to 2,927 years ago. The single, monophyletic NA EEEV lineage exhibited mainly temporally associated relationships and was highly conserved throughout its geographic range. In contrast, SA EEEV comprised three divergent lineages, two consisting of highly conserved geographic groupings that completely lacked temporal associations. A phylogenetic comparison of SA EEEV and Venezuelan equine encephalitis viruses (VEEV) demonstrated similar genetic and evolutionary patterns, consistent with the well-documented use of mammalian reservoir hosts by VEEV. Our results emphasize the evolutionary and genetic divergences between members of the NA and SA EEEV lineages, consistent with major differences in pathogenicity and ecology, and propose that NA and SA EEEV be reclassified as distinct species in the EEE complex.

Structural and nonstructural protein genome regions of eastern equine encephalitis virus are determinants of interferon sensitivity and murine virulence.

Eastern equine encephalitis virus (EEEV) causes sporadic epidemics of human and equine disease in North America, but South American strains have seldom been associated with human neurologic disease or mortality, despite serological evidence of infection. In mice, most North American and South American strains of EEEV produce neurologic disease that resembles that associated with human and equine infections. We identified a South American strain that is unable to replicate efficiently in the brain or cause fatal disease in mice yet produces 10-fold higher viremia than virulent EEEV strains. The avirulent South American strain was also sensitive to human interferon (IFN)-alpha, -beta, and -gamma, like most South American strains, in contrast to North American strains that were highly resistant. To identify genes associated with IFN sensitivity and virulence, infectious cDNA clones of a virulent North American strain and the avirulent South American strain were constructed. Two reciprocal chimeric viruses containing swapped structural and nonstructural protein gene regions of the North American and South American strains were also constructed and found to replicate efficiently in vitro. Both chimeras produced fatal disease in mice, similar to that caused by the virulent North American strain. Both chimeric viruses also exhibited intermediate sensitivity to human IFN-alpha, -beta, and -gamma compared to that of the North American and South American strains. Virulence 50% lethal dose assays and serial sacrifice experiments further demonstrated that both structural and nonstructural proteins are important contributors to neurovirulence and viral tissue tropism. Together, the results of this study emphasize the complex and important influences of structural and nonstructural protein gene regions on EEEV virulence.

The Old World and New World alphaviruses use different virus-specific proteins for induction of transcriptional shutoff.

Alphaviruses are widely distributed throughout the world. During the last few thousand years, the New World viruses, including Venezuelan equine encephalitis virus (VEEV) and eastern equine encephalitis virus (EEEV), evolved separately from those of the Old World, i.e., Sindbis virus (SINV) and Semliki Forest virus (SFV). Nevertheless, the results of our study indicate that both groups have developed the same characteristic: their replication efficiently interferes with cellular transcription and the cell response to virus replication. Transcriptional shutoff caused by at least two of the Old World alphaviruses, SINV and SFV, which belong to different serological complexes, depends on nsP2, but not on the capsid protein, functioning. Our data suggest that the New World alphaviruses VEEV and EEEV developed an alternative mechanism of transcription inhibition that is mainly determined by their capsid protein, but not by the nsP2. The ability of the VEEV capsid to inhibit cellular transcription appears to be controlled by the amino-terminal fragment of the protein, but not by its protease activity or by the positively charged RNA-binding domain. These data provide new insights into alphavirus evolution and present a plausible explanation for the particular recombination events that led to the formation of western equine encephalitis virus (WEEV) from SINV- and EEEV-like ancestors. The recombination allowed WEEV to acquire capsid protein functioning in transcription inhibition from EEEV-like virus. Identification of the new functions in the New World alphavirus-derived capsids opens an opportunity for developing new, safer alphavirus-based gene expression systems and designing new types of attenuated vaccine strains of VEEV and EEEV.

Arboviruses Pathogenic for man in Brazil

Os mais importantes aspectos clinicicos e ecoepidemiologicos e aspectos preventivos acerca das arboviroses associadas com doenca humana no Brasil sao discutidos.Trinta e seis arbovirus dentre os tipos presentemente isolados no Pais tem sido incriminados como causadores de doenca humana. Destes, cinco sao importantes em termos de saude publica pois estao associados com epidemias , sao os virus Dengue (DEN), Mayaro(MAY), Oropouche (ORO), Rocio (ROC) e Febre amarela (FA). DEN e ORO estao associados com doenca humana epidemica em areas urbanas enquanto MAY, ROC e FA especialmente em areas rurais. Basicamente, o virus ORO determina um quadro febril algumas vezes acmpanhado por meningite asseptica. MAY e DEN sao responsaveis por quadros exantematicos, sendo que DEN, nos ultimos anos tem sido associado com quadros de febre hemorragica, o que sabidamente e o mecanismos pelo qual o virus FA determinaa sua apresentacao clinica classica e o ROC esta associado com graves quadros de encefalite. Trinta e um outros arbovirus tem sido associados com doenca febril benigna em poucos e esporadicos casos. Afora DEN e os Arenavirus Flexal e Sabia ( nao sao arbovirus), todos os arbovirus envolvidos com doenca humana na AmazoniaBrasileira, sao mantidos em natureza atraves de um ciclo silvestre desenvolvido na floresta, onde diversas especies de insetos hematofagos e vertebrados silvestres atuam como vetores e hospedeiros, respectivamente.O virus DEN tem um ciclo urbano em que o mosquito Aedes aegypti e o vetor e o homem atua como hospedeiro. Os arenavirus sao transmitidos diretamente ao homen atraves de excretas de roedores que sao seus principais hospedeiros.Excetuando os cinco virus associados com epidemias que causam um grande impacto socio-economico, inclusive levando a morte, casos verificados com FA, DEN e ROC, o verdadeiro papel dessesvirus como agentes sistematicos de doencas humanas e ainda puco conhecido. Novos estudos sao necessarios para esclarecer aspectos ainda obscuros acerca da epidemiologia da maioria desses arbovirus

Glycoproteins E2 of the Venezuelan and eastern equine encephalomyelitis viruses contain multiple cross-reactive epitopes.

Enzyme immunoassay (EIA) with sixty types of monoclonal antibodies (MAbs) was used to study cross-reactive epitopes on the attenuated and virulent strains of the Eastern equine encephalomyelitis (EEE) and Venezuelan equine encephalomyelitis (VEE) viruses. All three structural proteins of the EEE and VEE viruses were demonstrated to have both cross-reactive and specific antigenic determinants. The glycoprotein E1 of EEE and VEE viruses possesses three cross-reactive epitopes for binding to MAbs. The glycoprotein E2 has a cluster of epitopes for 20 cross-reacting MAbs produced to EEE and VEE viruses. Cross-reactive epitopes were localised within five different sites of glycoprotein E2 of VEE virus and within four sites of that of the EEE virus. There are no cross-neutralising MAbs to the VEE and EEE viruses. Only one type of the protective Mabs was able to cross-protect mice against lethal infection by the virulent strains of the VEE and EEE viruses. Eight MAbs blocked the hemagglutination activity (HA) of both viruses. Antigenic alterations of neutralising and protective sites were revealed for all attenuated strains of the VEE and EEE viruses. Comparative studies of the E2 proteins amino acid sequences show that the antigenic modifications observed with the attenuated strains of the VEE virus may be caused by multiple amino acid changes in positions 7, 62, 120, 192 and 209-213. The escape-variants of the VEE virus obtained with cross-reactive MAbs 7D1, 2D4 and 7A6 have mutations of the E2 protein at positions 59, 212-213 and 232, respectively. Amino acid sequences in these regions of the VEE and EEE viruses are not homologous. These observations indicate that cross-reactive MAbs are capable of recognising discontinuous epitopes on the E2 glycoprotein.

[Damaging action of Venezuelan equine and Eastern equine encephalomyelitis viruses on the chromosome apparatus of mouse bone marrow cells]. / Povrezhdaiushchee deistvie virusov venesuél'skogo i vostochnogo éntsefalomielitov loshadei na khromosomnyi apparat kletok kostnogo mozga myshi.

Cytogenetical study of bone marrow cells of mice infected with pathogenic and attenuated strains of Venezuelan (VEE) and Eastern (EEE) equine encephalomyelitis viruses was carried out to elucidate the pattern of changes of the chromosomal apparatus in the infected animals, and differences in the effect of strains with different degree of pathogenicity on the cell during mitosis. It was shown that inoculation of mice with pathogenic and attenuated VEE and EEE virus strains led to the appearance in the bone marrow of a larger number of aberrant cells. Both VEE virus strain induced a significant increase both of the total number of aberrant cells and of the cells with true aberrations. The pathogenic and attenuated EEE virus strains also caused a marked increase in the number of aberrant cells, but while the number of true aberrant cells is significant for the pathogenic strain, the attenuated strain causes an insignificant change in this parameter.

Correlation between virus-cell receptor properties of alphaviruses in vitro and virulence in vivo.

Virulent and avirulent clones of Venezuelan, Western, and Eastern equine encephalitis viruses were examined for their in vitro attachment characteristics to the surface of cultured cell monolayers. These attachment characteristics were correlated with in vivo plasma clearance rates and virulence. For the clones investigated, avirulence correlated in vitro with attachment pH optima close to physiologic pH and in vivo with a rapid clearance from plasma. Conversely, virulent clones had lower in vitro attachment pH optima and low plasma clearances in vivo.

[Interferonogenic and antiviral activity of the tobacco mosaic virus, tilorone and sodium nucleinate]. / Interferonogennaia i protivovirusnaia aktivnost' virusa tabachnoi mozaiki, tilorona i nukleinata natriia

Production of endogenic interferon in animals in responce to administration of tobaco mozaic virus, tilorone and sodium nucleinate was shown. Dependence of interferon production on the type of the inductor and the route of its administration was studied. Absolute innocuiuty of the tobaco mozaic virus for monkeys (macaco-resus) and mice, as well as the absence of any side effects in humans treated with it perorally was shown. The tobaco mozaic virus, tilorone and sodium nucleinate used perorally in treatment of experimental infections of mice caused by the viruses of East and West encephalomyelitis, influenza and tick encephalitis had a pronounced protective effect.