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.

This study confirms the high clinical frequency of human VACV infection, even among vaccinated individuals. The infection was related to detection of IgG- or IgM-specific antibodies that correlates in most of the cases with positive PRNT. The DNAemia suggests viremia during VACV natural infections. Our data indicate that patients vaccinated against smallpox may no longer be protected.

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.

Vaccinia virus (VACV) has been associated with several bovine vaccinia outbreaks in Brazil, affecting cattle and humans. There are no available data about VACV environmental circulation or the role of wildlife in the emergence of an outbreak. Since VACV was isolated from rodents in Brazil, we investigated shedding and transmission of VACV strains in mice. The VACV excretion profile was assessed by PCR and chicken chorioallantoic membrane infection, revealing viral DNA and infectious virus in the faeces and urine of intranasally infected mice. Horizontal transmission was assessed by exposure of sentinel mice to wood shavings contaminated with excrement, to mimic a natural infection. Sentinel mice showed orthopoxvirus antibodies, and VACV DNA and infectious virus were detected in their faeces and intestines, even after six rounds of natural transmission. Together, these data suggest that murine excrement could play a relevant role in VACV spread and transmission, perhaps helping to explain how these viruses circulate between their natural hosts.

Orthopoxvirus (OPV) has been associated with worldwide exanthematic outbreaks, which have resulted in serious economic losses as well as impact on public health. Although the current classical and molecular methods are useful for the diagnosis of OPV, they are largely inaccessible to unsophisticated clinical laboratories. The major reason for the inaccessibility is that they require both virus isolation and DNA manipulation. In this report, a rapid, sensitive and low-cost semi-nested PCR method is described for the detection of OPV DNA directly from clinical specimens. A set of primers was designed to amplify the conserved OPV vgf gene. The most useful thermal and chemical conditions were selected and minimum non-inhibitory dilutions were determined. More than 100 Brazilian Vaccinia virus (VACV) field clinical specimens were tested using this semi-nested PCR in order to confirm its applicability. Cowpox virus was also detected by PCR from the ear scabs of scarified Balb/c mice. In addition, the method was highly sensitive for the detection of VACV DNA in murine blood and excreta, which are among the suggested reservoirs of OPV. Together, these data suggest that semi-nested PCR can be used for initial screening for OPV and as a routine diagnostic laboratory method.

Vaccinia virus (VACV) has been associated with several bovine vaccinia outbreaks in Brazil, causing exanthematic lesions in dairy cattle and humans. The way that VACV circulates in the environment is unknown, as is the way that this virus is transferred from wildlife to farms. Rodents are hypothetical VACV reservoirs, and murine feces has been identified as a potential source of viral shedding and transmission. In this work, we analyzed the stability of VACV infectious particles and DNA in feces from intranasally infected mice, exposed to environmental temperature and humidity, by titration assays and PCR, respectively. The results showed that VACV infectious particles were still detected at 20 days post-environmental-exposure (d.p.e.), while viral DNA was detected until 60 d.p.e. A gradual decrease in fecal viral load could be detected in all analyzed VACV strains. This work indicates long-lasting stability of VACV in murine feces and reinforces the idea that fecal matter may represent a potential source of circulating virus among rodents.

In 2010, the WHO celebrated the 30th anniversary of the smallpox eradication. Ironically, infections caused by viruses related to smallpox are being increasingly reported worldwide, including Monkeypox, Cowpox, and Vaccinia virus (VACV). Little is known about the human immunological responses elicited during acute infections caused by orthopoxviruses. We have followed VACV zoonotic outbreaks taking place in Brazil and analyzed cellular immune responses in patients acutely infected by VACV. Results indicated that these patients show a biased immune modulation when compared to noninfected controls. Amounts of B cells are low and less activated in infected patients. Although present, T CD4+ cells are also less activated when compared to noninfected individuals, and so are monocytes/macrophages. Similar results were obtained when Balb/C mice were experimentally infected with a VACV sample isolated during the zoonotic outbreaks. Taking together, the data suggest that zoonotic VACVs modulate specific immune cell compartments during an acute infection in humans.

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