Flávio Guimarães da Fonseca

Araçatuba Virus: A Vaccinialike Virus Associated with Infection in Humans and Cattle

We describe a vaccinialike virus, Araçatuba virus, associated with a cowpoxlike outbreak in a dairy herd and a related case of human infection. Diagnosis was based on virus growth characteristics, electron microscopy, and molecular biology techniques. Molecular characterization of the virus was done by using polymerase chain reaction amplification, cloning, and DNA sequencing of conserved orthopoxvirus genes such as the vaccinia growth factor (VGF), thymidine kinase (TK), and hemagglutinin. We used VGF-homologous and TK gene nucleotide sequences to construct a phylogenetic tree for comparison with other poxviruses. Gene sequences showed 99% homology with vaccinia virus genes and were clustered together with the isolated virus in the phylogenetic tree. Araçatuba virus is very similar to Cantagalo virus, showing the same signature deletion in the gene. Araçatuba virus could be a novel vaccinialike virus or could represent the spread of Cantagalo virus.

Passatempo Virus, a Vaccinia Virus Strain, Brazil

Passatempo virus was isolated during a zoonotic outbreak. Biologic features and molecular characterization of hemagglutinin, thymidine kinase, and vaccinia growth factor genes suggested a vaccinia virus infection, which strengthens the idea of the reemergence and circulation of vaccinia virus in Brazil. Molecular polymorphisms indicated that Passatempo virus is a different isolate.

One More Piece in the VACV Ecological Puzzle: Could Peridomestic Rodents Be the Link between Wildlife and Bovine Vaccinia Outbreaks in Brazil?

Background Despite the fact that smallpox eradication was declared by the World Health Organization (WHO) in 1980, other poxviruses have emerged and re-emerged, with significant public health and economic impacts. Vaccinia virus (VACV), a poxvirus used during the WHO smallpox vaccination campaign, has been involved in zoonotic infections in Brazilian rural areas (Bovine Vaccinia outbreaks – BV), affecting dairy cattle and milkers. Little is known about VACVs natural hosts and its epidemiological and ecological characteristics. Although VACV was isolated and/or serologically detected in Brazilian wild animals, the link between wildlife and farms has not yet been elucidated. Methodology/Principal Findings In this study, we describe for the first time, to our knowledge, the isolation of a VACV (Mariana virus - MARV) from a mouse during a BV outbreak. Genetic data, in association with biological assays, showed that this isolate was the same etiological agent causing exanthematic lesions observed in the cattle and human inhabitants of a particular BV-affected area. Phylogenetic analysis grouped MARV with other VACV isolated during BV outbreaks. Conclusion/Significance These data provide new biological and epidemiological information on VACV and lead to an interesting question: could peridomestic rodents be the link between wildlife and BV outbreaks?

Characterization of the Vaccinia Virus H3L Envelope Protein: Topology and Posttranslational Membrane Insertion via the C-Terminal Hydrophobic Tail

ABSTRACT The vaccinia virus H3L open reading frame encodes a 324-amino-acid immunodominant membrane component of virus particles. Biochemical and microscopic studies demonstrated that the H3L protein was expressed late in infection, accumulated in the cytoplasmic viral factory regions, and associated primarily with amorphous material near immature virions and with intracellular virion membranes. Localization of the H3L protein on the surfaces of viral particles and anchorage via the hydrophobic tail were consistent with its extraction by NP-40 in the absence of reducing agents, its trypsin sensitivity, its reactivity with a membrane-impermeable biotinylation reagent, and its immunogold labeling with an antibody to a peptide comprising amino acids 247 to 259. The H3L protein, synthesized in a coupled in vitro transcription/translation system, was tightly anchored to membranes as determined by resistance to Na2CO3 (pH 11) extraction and cytoplasmically oriented as shown by sensitivity to proteinase K digestion. Further studies demonstrated that membrane insertion of the H3L protein occurred posttranslationally and that the C-terminal hydrophobic domain was necessary and sufficient for this to occur. These data indicated that the H3L protein is a member of the C-terminal anchor family and supported a model in which it is synthesized on free ribosomes and inserts into the membranes of viral particles during their maturation.

Morphological and molecular characterization of the poxvirus BeAn 58058

SummaryBeAn 58058 virus (BAV) was isolated from an Oryzomis rodent in Brazil. BAV was shown to be antigenically related to another poxvirus also isolated in Brazil, the Cotia virus, but it remained ungrouped. Electron microscopy revealed that BAV has a typical poxvirus morphology. The Hind III DNA profile of BAV genome was similar with that of VV WR and Lister, but some differences in the profile were detected. We have also detected the presence of genes homologous to vaccinia virus (VV WR) genes in the genome of BAV. Genes related to vaccinia thymidine kinase (TK) gene and vaccinia growth factor (VGF) gene were found. The patterns of TK and VGF mRNA transcripts described for vaccinia virus infected cells were observed in BAV infected cells. Nucleotide sequence of BAV VGF homologous gene was similar to VV WR VGF sequences. This similarity was further seen when cross-hybridization of total genomes of BAV and VV was done. Polypeptide synthesis of BAV and vaccinia in infected cells also showed similar profiles. The genetic data was used to construct a phylogenetic tree where BAV and VV were placed at the same cluster. Based on our findings we propose that BAV is a vaccinia virus variant.

Characterization of a vaccinia-like virus isolated in a Brazilian forest

The SPAn232 virus (SPAnv) was isolated from sentinel mice in the forest of Cotia, São Paulo, Brazil. It was grouped originally as a Cotia virus (CV) sample due to serological cross-reaction with the latter. However, SPAnv presented genetic characteristics that differed from CV and indicated that SPAnv is a member of the vaccinia virus (VV) subgroup. SPAnv showed a HindIII-digested DNA pattern similar to those of the WR and Lister strains of VV. Also, SPAnv presented genes homologous to the vaccinia growth factor, thymidine kinase and A-type inclusion (ATI) genes from VV. RFLP analysis of the SPAnv ATI homologous gene indicated that the virus belongs to the VV group. Nucleotide sequences from SPAnv genes showed up to 99% similarity with the same genes from VV. Such a relationship was confirmed visually through the drawing of phylogenetic trees. The results point out the occurrence of a VV strain that is possibly in active circulation in the forests of Southeast Brazil.

Zoonotic Brazilian Vaccinia virus: From field to therapy

Vaccinia virus (VACV), the prototype species of the Orthopoxvirus (OPV) genus, causes an occupational zoonotic disease in Brazil that is primarily associated with the handling of infected dairy cattle. Cattle and human outbreaks have been described in southeastern Brazil since 1999 and have now occurred in almost half of the territory. Phylogenetic studies have shown high levels of polymorphisms among isolated VACVs, which indicate the existence of at least two genetically divergent clades; this has also been proven in virulence assays in a mouse model system. In humans, VACV infection is characterized by skin lesions, primarily on the hands, accompanied by systemic symptoms such as fever, myalgia, headache and lymphadenopathy. In this review, we will discuss the virological, epidemiological, ecological and clinical aspects of VACV infection, its diagnosis and compounds that potentially could be used for the treatment of severe cases.

Zoonotic Vaccinia Virus Infection in Brazil: Clinical Description and Implications for Health Professionals

ABSTRACT Bovine vaccinia virus outbreaks have been occurring in different regions of Brazil. We report here the time course of natural human infection by vaccinia virus and describe important clinical and epidemiological aspects of this zoonotic infection. The diagnosis of vaccinia virus infection was based on clinical, serological, and molecular procedures.

Brazilian Vaccinia virus strains are genetically divergent and differ from the Lister vaccine strain.

Vaccinia virus is responsible for an important zoonotic disease affecting dairy cattle and humans in Brazil, but little is known about the origin, epidemiology and evolution of these Brazilian Vaccinia virus strains. In this work, seven Brazilian Vaccinia virus strains and the Lister-derived Brazilian vaccine strain, named Lister-Butantan, were compared based on the sequences of ten host range and virulence related genes. Comparison of Brazilian Vaccinia virus strains with Lister-Butantan revealed several differences. Phylogenetic analyses confirmed the existence of genetically distinct Brazilian Vaccinia virus groups and has not thus far demonstrated a close relationship between Brazilian strains and Lister-Butantan. In this study, the BeAn58058 and SPAn232 strains were grouped together with the Belo Horizonte and Guarani P1 strains. Additionally, genetic polymorphisms in host range and virulence genes as well as differences in the deduced amino acid sequences were detected among Brazilian Vaccinia virus. This genetic diversity may result in a plethora of different biological properties presented by Brazilian Vaccinia virus, including differences in adaptation to the host as well as pathogenic properties. Furthermore, co-circulation of these divergent strains could increase the possibility of recombination events in nature, leading to the formation of new variants with unpredictable pathogenic potential.

Effects of Deletion or Stringent Repression of the H3L Envelope Gene on Vaccinia Virus Replication

ABSTRACT The C-terminal membrane anchor protein encoded by the H3L open reading frame of vaccinia virus is located on the surfaces of intracellular mature virions. To investigate the role of the H3L protein, we constructed deletion (vH3Δ) and inducible (vH3i) null mutants. The H3L protein was not detected in lysates of cells infected with vH3Δ or vH3i in the absence of inducer. Under these conditions, plaques were small and round instead of large and comet shaped, indicative of decreased virus replication or cell-to-cell spread. The mutant phenotype was correlated with reduced yields of infectious intra- and extracellular virus in one-step growth experiments. The defect in vH3i replication could not be attributed to a role of the H3L protein in virus binding, internalization, or any event prior to late gene expression. Electron microscopic examination of cells infected with vH3Δ or vH3i in the absence of inducer revealed that virion assembly was impaired, resulting in a high ratio of immature to mature virus forms with an accumulation of crescent membranes adjacent to granular material and DNA crystalloids. The absence of the H3L protein did not impair the membrane localization of virion surface proteins encoded by the A27L, D8L, and L1R genes. The wrapping of virions and actin tail formation were not specifically blocked, but there was an apparent defect in low-pH-mediated syncytium formation that could be attributed to decreased virus particle production. The phenotypes of the H3L deletion and repression mutants were identical to each other but differed from those produced by null mutations of genes encoding other vaccinia virus membrane components.