Passatempo virus, a vaccinia virus strain, Brazil

Abstract

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.

Figure 1

Lesions caused by Passatempo virus infection. 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.

Figure 2

Consensus bootstrap phylogenetic tree based on the nucleotide sequence of Orthopoxvirus ha gene. 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.

Figure 3

Brazilian states where vaccinia viruses were isolated. ES, Espírito Santo State: Espírito Santo isolates in 2004 (unpublished); GO, Goiás State: Goiás isolates after 2001 (3); MG, Minas Gerais State: Belo Horizonte virus in 1993 (15), Minas Gerais isolates after 2001 (3), Passatempo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, Pará State: BeAn 58058 virus in 1963 (6); RJ, Rio de Janeiro State: Cantagalo virus in 1999 (2); SP , São Paulo State: SPAn232 virus in 1979 (14), Araçatuba virus in 1999 (1), São Paulo isolates after 2001 (3).

Figure A1

Pocks morphology in chorioallantoic membranes. 1 and 2, whitish and opaque pocks could be observed after an incubation period of 72 hours with the Passatempo and vaccinia virus Western Reserve (WR), respectively, on chorioallantoic membrane of 10-day-old embryonated pathogen-free eggs; 3, cowpox virus Brighton Red (BR) formed red hemorrhagic lesions.

Figure A2

Electron micrographs of cells infected with Passatempo virus. Vero cells were infected with Passatempo virus at a multiplicity of infection of 5 and fixed by OsO4, 24 hours after inoculation. 1, typical orthopoxvirus "brick-shaped" morphologic pattern, with biconcave core and outer membranes were observed in cell-associated enveloped virus (CEV); 2, intracelullar mature virions (IMV) were visualized in the cytoplasm of infected cells within virosomes with the presence of spheric immature virus (IV). No A-type inclusion body (ATI) was visualized in infected cells. N, nucleus. Bars, 500 nm.

Figure A3

Restriction fragment length polymorphism analysis of the ati gene of viral isolates. ati fragments were amplified by polymerase chain reaction by using primers based on the cowpox virus ati gene nucleotide sequence, followed by XbaI digestion. The amplified DNA was fractionated by electrophoresis on a 1% agarose gel and stained with ethidium bromide. Lanes: 1, vaccinia virus WR; 2, isolate-cow1; 3, isolate-cow2; 4, isolate-cow3; 5, isolate-cow4; 6, isolate-cow5; 7, isolate-calf; 8, cowpox virus BR.

Figure A4

Alignment of Passatempo virus (PSTV) hemagluttinin antigen (HA) and vaccinia grown factor (VGF) amino acid inferred sequences with other orthopoxvirus sequences. 1, HA sequence of PSTV (DQ070848) was compared to the region corresponding to amino acids 6 to 309 of vaccinia virus (VACV-WR, AY243312) HA sequence and to HA sequences of Araçatuba virus (ARAV, AY523994); Cantagalo virus (CTGV, AF229247); vaccinia virus IOC (VACV-IOC, AF229248); vaccinia virus Lister (VAVC-LST, AF375124) and cowpox virus Brighton Red (BR) (CPXV-BR, AF482758). 2– The VGF sequence of PSTV (DQ085462) was compared to the region corresponding to amino acids 19 to 135 of vaccinia virus WR (VACV-WR, AY243312) and to the same region of Araçatuba virus (ARAV, AF503170), vaccinia virus Lister (VACV-LST, AY678276); and cowpox virus BR (CPXV-BR, AF482758). The sequences were obtained from GenBank and aligned by using the default parameters of CLUSTAL W and hand-edited to enhance biologically significant regions. The amino acid positions are shown according to the VACV-WR HA and VGF protein sequences. (.) indicates identity of amino acids, (-) indicates deletions, and (*) indicates the PSTV amino acid substitutions. The box shows the conserved deletion observed in the HA sequence of the Brazilian VACVs. Amino acids substitution found amongst PSTV and other Brazilian VACVs are shown in gray boxes.