Brazilian vaccinia viruses and their origins

Abstract

Although the World Health Organization (WHO) declared global smallpox eradicated in 1980, concerns over emergent poxvirus infections have increased. Most poxvirus infections are zoonotic; exploring their genetic diversity will illuminate the genetic and evolutionary aspects of poxvirus infections, ecology, and epidemiology. In recent decades, several strains of the orthopoxvirus vaccinia virus (VACV) have been isolated throughout Brazil, including genetically distinct isolates within the same outbreak. To further investigate the diversity and origins of these viruses, we analyzed molecular data from 8 Brazilian VACV isolates and compared several genes involved in virus structure and pathogenicity. Genetic variation among isolates suggests that ancestral Brazilian VACVs existed before the beginning of the WHO smallpox eradication vaccination campaigns and that these viruses continue to circulate.

Figure 1

Timeline of events regarding the introduction and circulation of orthopoxviruses in Brazil (2,3). Double slashes indicate a gap in the timeline.

Figure 2

A) Strict consensus of the 6 most parsimonious trees derived from Orthopoxvirus species on the basis of B19R, E3L, and A56R sequences and rooted with ectromelia virus from Moscow (ECTV-MOS). Bootstrap values based on 100 bootstrap replicates of 10 random addition replicates each are shown at each node where the value was >50%. Black rectangles represent the 18-nt deletion shared by group 1 Brazilian vaccinia virus (BRZ-VACV) and VACV-Institute Oswaldo Cruz (IOC) and where it maps on the tree. Open rectangles represent the 3 digestion profiles (P1, P2, and P3) of the rpo132-ATI-p4c-A27L region assigned at each appropriate node. Blue, branches of group 1; red, branches of group 2; orange, isolation of VACV-Guarani P1 (GP1) and VACV-GP2 at adjacent farms in the same area. Colors correspond to information presented in panel B. B) Map of Brazil showing states and ecological biomes. Collection sites are represented by numbers that refer to Brazilian isolates and their respective years of isolation. Map used with permission from Instituto Brasileiro de Geografia e Estatistica. GO, Goiás; SP, São Paulo; MG, Minas Gerais. See Table 2 for other definitions.

Figure 3

A) Partial amino acid alignment of A56R vaccinia virus Copenhagen (VACV-COP). Sequences were retrieved from GenBank and aligned using the BioEdit program (www.mbio.ncsu.edu/bioedit/bioedit.html). VACV-COP (M35027, 161908-161957) is used as the reference sequence; International Union of Pure and Applied Chemistry symbols, amino acids; dots, residues identical to the reference sequence; dashes, gaps created by the alignment; gold box surrounds the 18-nt deletion once proposed as a Brazilian (BRZ)-VACV molecular identifier. RPXV-UTR, rabbitpox virus-Utrecht; HSPV, horsepox virus; MVA, modified vaccinia Ankara; LIVP, Liverpool; BFL, buffalopox; BAV, BeAn 58058 virus; SAV, SpAn232 virus; VBH, Belo Horizonte virus; GP, Guarani virus; ARAV, Araçatuba virus; CTGV, Cantagalo virus; PSTV, Passatempo virus; IOC, Institute Oswaldo Cruz. B) Depiction of the rpo132-ATI-p4c-A27 region in Cowpox virus Brighton Red (CPXV-BR) and VACV strains. Positions of primers RNApol, ATI-low, ATI-up, and P4c1 are illustrated. These primers have been used for the molecular diagnosis of Brazilian (BRZ)-VACV (5,26). The 300-bp fragment amplified by primers RNApol and P4c1 from GP1V and VBH was sequenced by Trindade et al. (9,13). Sequence outside this region is unknown. ATI-f is used to designate coding regions that resemble fragments of the wild-type (CPXV-BR) ATI gene. The gap in VACV-COP lies within the A26L gene. *This gene is often referred to as ATI due to its original description. However, the coding region is actually a fusion of the C-terminus of ATI and the N-terminus of P4c where the sequence in between has been deleted. The gene is currently identified by the Poxviridae Bioinformatics Resource (http://athena.bioc.uvic.ca/database.php?db=poxviridae) as belonging to the P4c precursor family. †Western Reserve is presumed similar in structure to SpAn232 virus, GP2V, ARAV, and PSTV due to similar restriction fragment length polymorphism results (6,7,11,13).