Galileu Barbosa Costa

Spread of Vaccinia Virus to Cattle Herds, Argentina, 2011

To the Editor: Since 1999, several zoonotic outbreaks of vaccinia virus (VACV) infection have been reported in cattle and humans in rural areas of Brazil. The infections have caused exanthematous lesions on cows and persons who milk them, and thus are detrimental to the milk industry and public health services (1,2). In Brazil during the last decade, VACV outbreaks have been detected from the north to the extreme south of the country (1–4). Because Brazil shares extensive boundaries with other South American countries, humans and cattle on dairy and beef-producing farms in those countries may be at risk of exposure to VACV. To determine if VACV has spread from Brazil to Argentina, we investigated the presence of VACV in serum samples from cattle in Argentina. During 2011, we obtained serum samples from 100 animals (50 dairy and 50 beef cattle) on farms in Cordoba, Corrientes, Entre Rios, and Santa Fe Provinces in Argentina (Technical Appendix, panel A). No VACV cases had been reported in humans or cattle in these provinces. However, Corrientes Province borders the Brazilian state of Rio Grande do Sul, where VACVs (Pelotas 1 and Pelotas 2 viruses) were isolated during an outbreak affecting horses in 2008 (2). To determine the presence of neutralizing antibodies in the serum samples, we used an orthopoxvirus 70% plaque-reduction neutralization test as described (4). On the basis of previous studies that detected viral DNA in serum samples (4–6), we used real-time PCR to amplify the highly conserved orthopoxvirus vaccinia growth factor (vgf) gene DNA (P.A. Alves, unpub. methods). To amplify the hemagglutinin (HA) gene DNA from the serum samples, we used real-time PCR with primers as described by de Souza Trindade et al. 2008 (7). The HA PCR products were directly sequenced in both orientations by using specific primers and capillary electrophoresis (Genetic Analyzer 3130; Applied Biosystems, Grand Island, NY, USA). We used ClustalW (http://www.clustal.org) and MEGA4 software (http://megasoftware.net/) to align nucleotide sequences and construct a phylogenetic tree (neighbor-joining method, 1,000 bootstraps) from the obtained HA fragment. Of the 50 dairy cattle samples, 4 (8.0%) had neutralizing antibodies against orthopoxvirus; of these, 3 (75.0%) had titers of 100 neutralizing units (NU)/mL, and 1 (25.0%) had a titer of 400 NU/mL. Of the 50 beef cattle, 8 (16.0%) had antibodies to orthopoxvirus, 1 (12.5%) of which had a titer of 800 NU/mL. Most of the positive samples were from cattle in Corrientes and Entre Rios Provinces (Table). Table Diagnosis of Orthopoxvirus infection in beef and dairy cattle during a study of the spread of vaccinia virus to cattle herds, Argentina, 2011* Of the 100 serum samples, 5 (3 from beef and 2 from dairy cattle) were positive for vgf by real-time PCR. HA DNA was amplified from 2 of the 3 vgf PCR–positive beef cattle samples; plaque-reduction neutralization test results were also positive for the 2 samples (Table). Alignment of the HA fragment nucleotide sequence of the isolates from Argentina showed that the sequence was highly similar to that of the homologous gene of VACV isolates from Brazil. Furthermore, the sequences showed a signature deletion that is also present in the sequences of VACV isolates from Brazil. Compared with sequences for other VACV isolates, those from Argentina had 2 polymorphisms (Technical Appendix, panel C). The HA sequences from the isolates from Argentina demonstrated 100% identity among themselves and exhibited higher identity with group 1 (98.2% identity) versus group 2 (93.6% identity) isolates from Brazil (Technical Appendix, panel D). In the phylogenetic tree based on the HA nucleotide sequences (Technical Appendix, panel B), the VACVs from Argentina clustered with several group 1 VACVs detected during outbreaks in Brazil. Although no outbreaks of exanthematous VACV infection have been described in cattle or humans in Argentina, we detected neutralizing antibodies against orthopoxvirus and detected VACV DNA in serum samples from cattle in the country. Most of the seropositive samples were from cattle in Entre Rios Province, which shares a border with Uruguay, and Corrientes Province, which shares a border with Rio Grande do Sul State in Brazil, where Pelotas VACVs have been isolated (2). We believe that the seropositive cattle in this study may have been exposed to VACV, the only orthopoxvirus known to be circulating in South America (1–4,8–10). Despite veterinary surveillance efforts of border control organizations, VACV control may be hampered by the circulation of infected rural workers and the misdiagnosis of VACV infection; misdiagnoses occur because VACV lesions resemble those of other exanthematous diseases. Moreover, peridomestic rodents have been hypothesized to act as VACV hosts, and could facilitate the spread of VACV in border areas (10). In addition, we could not rule out the circulation of autochthonous VACV in Argentina, but this is a less likely explanation. Our findings suggest that cattle herds in areas of Argentina near the border with Brazil may be exposed to VACV from Brazil and, thus, may be at risk for VACV infection. Further research is needed to determine the risk factors for VACV infection and to assess the circulation of VACV in South America Technical Appendix: Map of Argentina, indicating locations where blood samples were collected from cattle and results of phylogenetic analysis of vaccine virus isolated from the samples. Click here to view.(611K, pdf)

Serologic and Molecular Evidence of Vaccinia Virus Circulation among Small Mammals from Different Biomes, Brazil

Vaccinia virus (VACV) is a zoonotic agent that causes a disease called bovine vaccinia, which is detected mainly in milking cattle and humans in close contact with these animals. Even though many aspects of VACV infection have been described, much is still unknown about its circulation in the environment and its natural hosts/reservoirs. To investigate the presence of Orthopoxvirus antibodies or VACV DNA, we captured small rodents and marsupials in 3 areas of Minas Gerais state, Brazil, and tested their samples in a laboratory. A total of 336 animals were tested; positivity ranged from 18.1% to 25.5% in the 3 studied regions located in different biomes, including the Atlantic Forest and the Cerrado. Analysis of nucleotide sequences indicated co-circulation of VACV groups I and II. Our findings reinforce the possible role played by rodents and marsupials in VACV maintenance and its transmission chain.

Natural Vaccinia Virus Infection: Diagnosis, Isolation, and Characterization

Natural infections of Vaccinia virus (VACV)—the prototype species of the Orthopoxvirus genus, from the family Poxviridae and subfamily Chordopoxvirinae—cause an occupational emergent zoonotic disease that is primarily associated with the handling of infected dairy cattle. In humans, VACV infection is characterized by skin lesions, primarily on the hands, and accompanied by systemic symptoms such as fever, myalgia, headache, and lymphadenopathy. The diagnosis of VACV is usually performed according to the methods described for other orthopoxviruses. This unit describes the methods utilized to obtain clinical samples, the serological and molecular techniques used for diagnosis, and the isolation methods and techniques used for molecular and biological characterization of the viruses.

Detection of Vaccinia Virus in Urban Domestic Cats, Brazil

We investigated possible vaccinia virus (VACV) in urban house cats in Brazil. Serum samples from 6 cats were positive for VACV by PCR, indicating likely VACV circulation among house cats in urban areas of Brazil. This finding highlights the importance of epidemiologic surveillance to avoid outbreaks among urban human populations.

Neutralizing antibodies associated with exposure factors to Orthopoxvirus in laboratory workers

Extensive use of Vaccinia virus (VACV) in research has led to associated accidental human exposure in laboratories worldwide. In spite of the social and economic relevance of Bovine Vaccinia outbreaks in Brazil, national data concerning laboratory workers handling these infectious agents are relatively scarce. Therefore, a serological survey was conducted in a Brazilian laboratory to evaluate staff exposure to orthopoxviruses (OPVs). Information concerning direct work with OPVs, vaccination status and laboratory accidents was collected and correlated to serology results. This study presents an opportunity for discussion of routine procedures involving OPVs in laboratories and their intrinsic risks. Aspects of the live attenuated smallpox vaccine are also discussed.

Vaccinia Virus Natural Infections in Brazil: The Good, the Bad, and the Ugly

The orthopoxviruses (OPV) comprise several emerging viruses with great importance to human and veterinary medicine, including vaccinia virus (VACV), which causes outbreaks of bovine vaccinia (BV) in South America. Historically, VACV is the most comprehensively studied virus, however, its origin and natural hosts remain unknown. VACV was the primary component of the smallpox vaccine, largely used during the smallpox eradication campaign. After smallpox was declared eradicated, the vaccination that conferred immunity to OPV was discontinued, favoring a new contingent of susceptible individuals to OPV. VACV infections occur naturally after direct contact with infected dairy cattle, in recently vaccinated individuals, or through alternative routes of exposure. In Brazil, VACV outbreaks are frequently reported in rural areas, affecting mainly farm animals and humans. Recent studies have shown the role of wildlife in the VACV transmission chain, exploring the role of wild rodents as reservoirs that facilitate VACV spread throughout rural areas. Furthermore, VACV circulation in urban environments and the significance of this with respect to public health, have also been explored. In this review, we discuss the history, epidemiological, ecological and clinical aspects of natural VACV infections in Brazil, also highlighting alternative routes of VACV transmission, the factors involved in susceptibility to infection, and the natural history of the disease in humans and animals, and the potential for dissemination to urban environments.

Detection of mimivirus genome and neutralizing antibodies in humans from Brazil

In recent years, giant viruses belonging to the family Mimiviridae have been proposed to be infectious agents in humans. In this work we provide evidence of mimivirus genome and neutralizing antibodies detection in humans.

Evaluating anti-Orthopoxvirus antibodies in individuals from Brazilian rural areas prior to the bovine vaccinia era

Vaccinia virus naturally circulates in Brazil and is the causative agent of a zoonotic disease known as bovine vaccinia (BV). We retrospectively evaluated two populations from the Amazon and Southeast Regions. BV outbreaks had not been reported in these regions before sample collection. Neutralising antibodies were found in 13 individuals (n = 132) with titres ranging from 100 ≥ 6,400 neutralising units/mL. Univariate analysis identified age and vaccination as statistically significant risk factors in individuals from the Southeast Region. The absence of detectable antibodies in vaccinated individuals raises questions about the protection of smallpox vaccine years after vaccination and reinforces the need for surveillance of Orthopoxvirus in Brazilian populations without evidence of previous outbreaks.

Cross-sectional study involving healthcare professionals in a Vaccinia virus endemic area

Orthopoxviruses (OPV) are emerging viruses with great importance in human and veterinary medicine, such as Vaccinia virus (VACV), which causes outbreaks of bovine vaccinia (BV) in South America. The clinical aspects of BV are similar to other vesicular infections, complicating the clinical diagnosis. This cross-sectional study evaluated the knowledge of Healthcare Professionals about BV and revealed their unpreparedness about BV in a VACV hyper-endemic area in Brazil, highlighting the public health issues associated with VACV infections. This study presents an opportunity to discuss the importance of vaccination for healthcare professionals who work in areas of VACV circulation and brings an educational measure on VACV infections for health professionals around the world.

Absence of vaccinia virus detection in a remote region of the Northern Amazon forests, 2005-2015.

Vaccinia virus (VACV) circulates in Brazil and other South America countries and is responsible for a zoonotic disease that usually affects dairy cattle and humans, causing economic losses and impacting animal and human health. Furthermore, it has been detected in wild areas in the Brazilian Amazon. To better understand the natural history of VACV, we investigated its circulation in wildlife from French Guiana, a remote region in the Northern Amazon forest. ELISA and plaque reduction neutralization tests were performed to detect anti-orthopoxvirus antibodies. Real-time and standard PCR targeting C11R, A56R and A26L were applied to detect VACV DNA in serum, saliva and tissue samples. No evidence of VACV infection was found in any of the samples tested. These findings provide additional information on the VACV epidemiological puzzle. The virus could nevertheless be circulating at low levels that were not detected in areas where no humans or cattle are present.