Aracatuba Virus: A Vaccinialike Virus Associated With Infection in Humans and Cattle

Giliane de Souza Trindade, Flávio Guimarães da Fonseca, João Trindade Marques, Maurício Lacerda Nogueira, Luiz Claudio Nogueira Mendes, Alexandre Secorun Borges, Juliana Regina Peiró, Edviges Maristela Pituco, Cláudio Antônio Bonjardim, Paulo César Peregrino Ferreira, Erna Geessien Kroon

Disclosures

Emerging Infectious Diseases. 2003;9(2) 

In This Article

Results

After Araçatuba virus was isolated in bovine fetal kidney cell monolayers, the samples were viewed by transmission electronic microscopy. Typical brick-shaped poxvirus forms were observed, measuring about 260 x 360 nm, with a superficial structure formed by tubules on long irregularly arranged filaments (data not shown). Samples were also added to embryonated chicken eggs so pock formations could be visualized on chorioallantoic membranes. White, nonhemorrhagic pocks were found (data not shown).

Orthopoxvirus Genus and Nucleotide Sequence Analysis

PCR amplification of TK, VGF, and HA genes generated 528-, 381-, and 960-bp fragments, respectively. Amplicons were cloned into pGEMT vector and sequenced in both orientations. When compared to nucleotide sequences available in the GenBank databases using the BLASTN program, the TK and VGF genes from Araçatuba virus were highly similar to homologous genes of VACV-WR. Optimal alignment showed similarity rates of up to 99.5% between Araçatuba virus and VACV-WR genes and minimal differences from nucleic acid substitutions. The coding region of HA gene was analyzed by alignment with similar sequences of VACV-WR and Cantagalo virus deposited in GenBank (accession nos. AF229247 and AF482758.1). The Araçatuba virus HA nucleotide sequence contained a signature deletion identical to a deletion detected in the sequence of Cantagalo virus (Figure 2A). This feature, absent in the sequence of most VACV strains, was used to correlate Araçatuba virus with VACV strain Istituto Ozwaldo Cruz (IOC), which was used as vaccine in some regions of Brazil during the smallpox eradication campaign.[14] Using the nucleotide sequences from Araçatuba virus and other poxviruses, we constructed evolutionary trees with the Treecon program and placed Araçatuba virus isolate in the same cluster as other VACV strains (Figure 2B and 2C).

(A) Nucleotide sequence of the Araçatuba virus hemagglutinin (HA).

(A) Nucleotide sequence of the Araçatuba virus hemagglutinin (HA).

(A) Nucleotide sequence of the Araçatuba virus hemagglutinin (HA).

Although the formation of typical A-type inclusions is restricted to cells infected with cowpox virus, ectromelia virus, and raccoonpox virus,[2] the sequence coding the N-terminus of the protein is highly conserved in many viruses, including CPXV, VACV, variola virus, camelpox virus, and ectromelia virus. These conserved sequences flank variable regions containing different size deletions, which may generate different size fragments after PCR amplification. The specificity of this assay is enhanced by the use of restriction enzymes, Xba I or Bgl II, allowing the detection of mutations at the restriction sites for these enzymes. We amplified the ATI gene from Araçatuba virus, VACV-WR, and CPXV for comparison. As described, the VACV-WR ATI amplicon generated 3 fragments after digestion with Xba I[26] (Figure 3). The larger fragment has approximately 900 bp, and the shorter fragments migrate closely, between the 300-bp and 400-bp markers. The profile obtained after digestion of Araçatuba virus ATI amplicon was similar to that of VACV-WR (Figure 3). The main difference, however, is that the larger fragment generated after XbaI digestion of the Araçatuba virus ATI amplicon is smaller than the VACV-WR fragments. These differences in size are also detected when nondigested ATI amplicons from Araçatuba virus and VACV are compared. Nevertheless, the pattern obtained for Araçatuba virus is completely different from the CPXV ATI pattern (Figure 3).

Detection and restriction fragment length polymorphism taxonomic analysis of the Araçatuba virus ATI gene.

Detection and restriction fragment length polymorphism taxonomic analysis of the Araçatuba virus ATI gene.

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