the outbreak area is characterized by small rural properties with diverse crops , pasturelands , and surrounding fragments of atlantic forest . its climate is tropical , with a relatively severe dry season , generally from april to september ( 4 ) . all dairy farms were similar , consisting of a main house with corrals and pasture fields generally with unsophisticated infrastructure . all milking was manually performed by milkers , typically without strict aseptic measures , which could have contributed to the spread of the virus among the herd and milkers . cows exhibited lesions on teats and udders that resembled the clinical features observed during other brazilian vacv outbreaks ( 1 ) . initial acute lesions were associated with a roseolar erythema with localized edema that led to the formation of vesicles . typically , a thick dark scab followed , but the formation of large areas of ulceration was also common . different stages of lesions were present , ranging from papules to vesicles , pustules , and crusts ( figure 1 ) . moreover , because of secondary infections , some cows had mastitis ( figure 1 ) . calves became infected , showing lesions on oral mucosa and muzzles ( figure 1 ) . several infected milkers reported lesions on their hands , which were apparently transmitted by unprotected contact with sick cattle ( figure 1 ) . in addition , infected persons reported severe headache , backache , lymphadenopathy , and high fever . panels 1 and 2 , ulcerative lesions on cows ' teats ; 3 , mastitis caused by bacterial secondary infection ; 4 and 5 , lesion on calves ' muzzle and oral mucosa ; 6 , lesions of dairy farm milker . for virus isolation , crusts were collected from 5 cows and 1 calf , macerated , and added to the chorioallantoic membrane of embryonated eggs ( 2 ) . the whitish pockmarks produced on chorioallantoic membranes resembled vacv pocks , differing from the red hemorrhagic ones produced by cowpox virus ( cpxv ) ( figure a1 ) . serologic cross - reactivity of antibodies to vacv  western reserve ( wr ) strain was detected in all samples , and titers of these serum samples were > 640 u / ml ( data not shown ) . transmission electron microscopy of isolates ( 6 ) showed a morphologic pattern typical of orthopoxviruses ( figure a2 ) . no a - type inclusion body ( ati ) was seen , reinforcing the conclusion that this virus was likely not a cpxv , but a vacv . viral dnas were extracted ( 6 ) and used as template for ati gene restriction fragment length polymorphism ( rflp ) analysis ( 7 ) . the ati rflp patterns of all isolates were identical to those of araatuba virus ( arav ) ( 1 ) and other vacv strains previously isolated in our laboratory ( unpub . data ) ; they were similar to those of vacv - wr and completely different from those of cpxv - brighton red ( br ) ( figure a3 ) . since all isolates showed the same ati rflp pattern , one was cloned , purified , titrated ( 1,6 ) , and named passatempo virus ( pstv ) . to better identify this etiologic agent , ha , tk , and vgf genes were amplified by polymerase chain reaction with taq polymerase ( promega , madison , wi , usa ) ( 6,8,9 ) . three clones were sequenced 3 times in both orientations by the dideoxy method , using m13 universal primers and et dynamic terminator for megabace ( ge healthcare , fairfield , ct , usa ) . the nucleotide ( nt ) sequences of ha , tk , and vgf were assembled by using the cap3 sequence assembling program ( 10 ) and deposited in genbank under accession numbers dq070848 , dq085461 , and dq085462 , respectively . the sequences and inferred amino acid sequences were aligned with those of orthopoxviruses by using the clustalw 1.6 program ( 11 ) . pstv ha gene sequence was compared to those of arav , cantagalo virus ( ctgv ) ( 1,2 ) , vacv - wr , cpxv - br , vacv instituto oswaldo cruz ( vacv - ioc ) , and vacv lister ( vacv - lst ) . vacv - ioc and vacv - lst are vaccine strains used in the brazilian smallpox eradication program ( 2,6 ) . the pstv ha gene sequences presented the same 18-nt deletion found in arav , ctgv , and vacv - ioc and shared more similarities to arav and ctgv homologous sequences . additionally , 8 amino acid substitutions were unique to pstv , arav , and ctgv . since this characteristic was not observed in the vaccine strains , an independent origin is suggested . moreover , pstv ha differs from that of arav and ctgv by 1 and 2 amino acid substitutions , respectively ( figure a4 ) . the percentage of identity between ha , tk , and vgf nucleotide sequences and inferred amino acid sequences of pstv with cpxv - br and other vacv strains are presented in the table . for the tk gene that is highly conserved among vacv , the pstv nucleotide sequence had 100% identity to arav , vacv - lst , and vacv - wr homologous sequences . additionally , pstv vgf gene had a 3-nt deletion , corresponding to nt 7,6697,671 of vacv - wr , causing the loss of 1 isoleucine in a stretch of 4 found in the arav and vacv - wr vgf sequences ( figure a4 ) . * ctgv and vacv - ioc tk and vgf nucleotide and amino acid sequences are not available in the genbank database . the alignments were used to construct phylogenetic trees by the neighbor - joining method using the tamura nei model implemented in mega3 ( 12 ) . a tk and vgf genes concatenated phylogenetic tree was constructed by placing pstv together with vacv strains ( data not shown ) . regarding ha sequences , the tree was constructed by the neighbor - joining method using the tamura - nei model of nucleotide substitutions implemented in mega3 . the tree was midpoint - rooted , 1,000 bootstrap replicates were performed , and values > 50% are shown . nucleotide sequences were obtained from genbank under accession numbers : pstv ( dq070848 ) , arav ( ay523994 ) , ctgv ( af229247 ) , vacv - wyeth ( vvz99051 ) , vacv - tiantan ( u25662 ) , vbh ( ay542799 ) , vacv - wr ( ay243312 ) , vacv - koppe ( af375122 ) , vacv - mva ( u94848 ) , vacv - ioc ( af229248 ) , vacv - lst ( af375124 ) , vacv - len ( af375123 ) , vacv - cop ( m35027 ) , bfl-3906 ( af375077 ) , vacv - malbran ( ay146624 ) , rpxv - rev ( af375118 ) , cpxv - gri90 ( cvz9904 ) , cpxv - br ( af482758 ) , ectv - mos ( af012825 ) , cmlv - cms ( ay009089 ) , cmlv - m96 ( af438165 ) , varv - bsh ( l22579 ) , varv - ind ( x69198 ) , mpxv - zre ( af380138 ) . (  ) indicates brazilian vacv isolates and (  ) indicates brazilian vaccine samples . the phylogenetic tree analysis suggested a strong phylogenetic relationship between pstv and other brazilian vacv strains . however , the vgf and ha gene analysis of pstv , arav , and ctgv indicated that genetic heterogeneity exists among these viruses , which suggests that the ha gene deletion found in pstv , arav , ctgv , and vacv - ioc could be a signature of new world or brazilian vacv strains . additionally , that rflp analysis showed a pattern identical with other brazilian strains , similar to vacv - wr and different from cpxv , suggests that a cladogenesis event may have occurred . this conclusion is feasible considering that these viruses could be circulating in the wild since smallpox vaccination or even before , going back to the colonization of south america , when cattle and other animals were brought to the new world without quarantine or inspection . the vacv variants buffalopox and rabbitpox have originated from vacv subspeciation ( 13 ) . that humans were also infected and that these persons were all milkers , phenomena that had been observed during other brazilian vacv outbreaks , points to an occupational zoonosis . although parapoxvirus infection has been placed in the category of occupational zoonosis , to our knowledge no other orthopoxviruses have been reported to cause an occupational hazard . economic losses are also a matter of concern . in addition to the reduction in milk production , extra veterinary costs are due to the usual occurrence of secondary infections on cows ' teats leading to mastitis . the reduction in milk production is a concern because brazil is a major milk exporter . therefore , the spread of these viruses could severely impact the country 's economy . in this regard , the clinical features , widespread dissemination , and epidemiology of the etiologic agent of these outbreaks must be understood . since 1963 , the growing geographic distribution of these outbreaks ( figure 3 ) indicates that these viruses may be emerging as zoonotic pathogens of cattle . this fact is especially important because a growing human population has no vaccine - derived immunity to smallpox or other orthopoxviruses . in addition , the isolation of another vacv strain strengthens the hypothesis that vacv is circulating in the new world and that these viruses seem to be endemic of this region . es , esprito santo state : esprito santo isolates in 2004 ( unpublished ) ; go , gois state : gois isolates after 2001 ( 3 ) ; mg , minas gerais state : belo horizonte virus in 1993 ( 15 ) , minas gerais isolates after 2001 ( 3 ) , passatempo virus in 2003 ; pa , par state : bean 58058 virus in 1963 ( 6 ) ; rj , rio de janeiro state : cantagalo virus in 1999 ( 2 ) ; sp , so paulo state : span232 virus in 1979 ( 14 ) , araatuba virus in 1999 ( 1 ) , so paulo isolates after 2001 ( 3 ) .
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 .