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.

BackgroundDespite 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 VACV's 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 FindingsIn 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/SignificanceThese 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?

Outbreaks of bovine vaccinia disease caused by circulation of Vaccinia virus (VACV) strains have been a common occurrence in Brazil in the recent years, being an important emergent zoonosis. During a single outbreak that took place in 2001, two genetically different VACV strains were isolated and named Guarani P1 virus (GP1V) and Guarani P2 virus (GP2V). Molecular diagnosis was done through restriction fragment length polymorphism (RFLP) of ati gene (A26L) and by sequence analysis of a group of five VACV genes including the C11R, J2R, A56R, B18R, and E3L genes. These findings confirmed the co-circulation of two different Vaccinia virus strains during the same outbreak, raising important questions about the origin, emergence, and circulation of VACV strains in Brazil.

Brazilian Vaccinia virus had been isolated from sentinel mice, rodents and recently from humans, cows and calves during outbreaks on dairy farms in several rural areas in Brazil, leading to high economic and social impact. Some phylogenetic studies have demonstrated the existence of two different populations of Brazilian Vaccinia virus strains circulating in nature, but little is known about their biological characteristics. Therefore, our goal was to study the virulence pattern of seven Brazilian Vaccinia virus strains. Infected BALB/c mice were monitored for morbidity, mortality and viral replication in organs as trachea, lungs, heart, kidneys, liver, brain and spleen. Based on the virulence potential, the Brazilian Vaccinia virus strains were grouped into two groups. One group contained GP1V, VBH, SAV and BAV which caused disease and death in infected mice and the second one included ARAV, GP2V and PSTV which did not cause any clinical signals or death in infected BALB/c mice. The subdivision of Brazilian Vaccinia virus strains into two groups is in agreement with previous genetic studies. Those data reinforce the existence of different populations circulating in Brazil regarding the genetic and virulence characteristics.

To detect orthopoxvirus in the Brazilian Amazon, we conducted a serosurvey of 344 wild animals. Neutralizing antibodies against orthopoxvirus were detected by plaque-reduction neutralizing tests in 84 serum samples. Amplicons from 6 monkey samples were sequenced. These amplicons identified vaccinia virus genetically similar to strains from bovine vaccinia outbreaks in Brazil.

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. CASE REPORTWe examined the clinical and epidemiological aspects of one case of zoonotic human infection during a bovine vaccinia virus outbreak and characterized the causative agent as a vaccinia virus (VACV) strain.In August 2000, vesicular and exanthemous disease outbreaks affecting dairy cattle and milkers were observed on several farms in Minas Gerais, the most important milk-producing State in Brazil. At one farm, a smallpox unvaccinated 19-year-old male dairy farm milker (Patient one) became sick and developed many lesions after being in contact with cows presenting lesions on their udders. These lesions started as itchy points on the hands, followed by the appearance of local edema and, after 3 days, the patient reported fever, headache, exhaustion, and vesicles on his hands. After a period of 3 or 4 days, these vesicles evolved to ulcerated lesions mainly characterized as umbilicated pustules surrounded by inflammatory tissue and painful ulcers on the hands and fingers (Fig. 1A and B). At this time, the milker reported the presence of peripheral erythema and lymphadenitis, with enlarged lymph nodes and a secondary bacterial infection on the lesions. Despite having the clinical symptoms of systemic illness, the patient did not cease working. Only on day 10 was the patient admitted at the local public hospital and received treatment based on fever and pain relievers and topical antibiotics due to the secondary bacterial infection. A serum sample and a biopsy from the necrotic black scab were collected and sent to the laboratory in order to establish the etiology of the lesions. Antibodies to orthopoxvirus were detected in the patient's serum sample by a serum neutralization assay. The biopsy sample was then ground, homogenized, and inoculated onto a chorioallantoic membrane of embryonated chicken eggs where, after 72 h, white and opaque classical pock formations were observed, suggesting a VACV infection (3,6,9). The "pocks" were used for virus propagation in Vero cells, where a typical cytopathic effect was also observed. The isolated virus showed typical poxvirus brickshaped particles when visualized by atomic force microscopy (AFM) (5) (Fig. 1C and D).In addition, for the molecular characterization of the isolated poxvirus, the thymidine kinase (tk), vaccinia virus growth factor (vgf), and hemagglutinin (ha) genes were amplified, the amplicons were cloned into the pGEM-T vector (pGEM-T Easy Vector Systems; Promega Corp., Madison, WI), and three clones for each gene were sequenced (3,6,9). tk and vgf are conserved genes in the Orthopoxvirus genus and have previously been used as taxonomic tools to study ungrouped poxvirus (3,9). When the tk and vgf nucleotide...

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