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

Methods

Five adult Girolanda cows from a herd of 40 animals were sent to the Veterinary Teaching Hospital at Unesp-Araçatuba, São Paulo State, Brazil; they had painful lesions on their teats, which interfered with milking. Lesions initially appeared on 2 cows and spread quickly to 35 animals, as well as the milker's hands (Figure 1). Starting as a red focal area, the lesions developed quickly into a wound that healed with difficulty. No such episode had previously occurred on that farm. The cows had these symptoms for approximately 8 days before being taken to the veterinary surgeon. During the clinical examination, lesions in different stages were recognized; in most of the cows, nodular ulcerative wounds of 2-6 mm in diameter were predominant. Lesions were localized only on teats and udder, and many of them had dark, raw crusts. The teats had increased local temperature and were sensitive to touch. Because of the pain, cows avoided their suckling calves. At the farm, the only manual milker was also affected. The milker had approximately 10 lesions on both hands and arms, but he did not initially accept any medical help and did not consent to examination. Because asepsic measures were not carried out, contact between the cows' teats and the milker's hands during milking probably enhanced the rapid spread of virus within the herd. Oral vesicles were not observed on calves' muzzles or on buccal mucosae. Sterile samples of the vesicles and crusts were collected and sent to the Laboratório de Viroses de Bovídeos, Instituto Biológico, São Paulo for analysis. The animals were isolated from the herd, and teat lesions were treated with glycerine and a topical antibiotic, while the milker received medication at a nearby hospital. Three months after onset of infection, the remaining lesions on the cows were in an advanced healing process; however, all affected cows produced substantially less milk.

Lesions from suspected Araçatuba virus on hand of dairy farm worker (milker) (A) and teats of cow (B).

Lesions from suspected Araçatuba virus on hand of dairy farm worker (milker) (A) and teats of cow (B).

The material collected was prepared in 20% suspension of Eagle minimal essential medium (MEM) with 1% antibiotic to isolate the virus by inoculations in bovine fetal kidney cell monolayers at the Instituto Biológico, São Paulo. Samples that showed cytopathic effects were analyzed by transmission electronic microscopy. Material isolated from bovine fetal kidney cell monolayers was spread on the chorioallantoic membrane of embryonated chicken eggs and incubated at 37°C for 72 h.[21]

VACV, WR strain, was obtained from the National Institute for Medical Research (Mill Hill, London, U.K.) and CPXV, Brighton strain, was provided by Dr. C. Jungwirth, WÜrzburg, Germany. Viruses were propagated in Vero cells and purified in a sucrose gradient as described.[22] Vero cells were propagated at 37°C in MEM, supplemented with 5% fetal calf serum. Vero cells were also used for viral titration.[23]

The primers based on the TK and VGF nucleotide sequence of VACV-WR were produced as described by Fonseca et al..[6] The purified Araçatuba virus genome was used as a template, and temperatures of 45°C were used for annealing. Amplified fragments were cloned into the pGEMT vector (pGEM-T Easy Vector Systems, Promega Corp., Madison, WI). The portion of the HA coding sequence was amplified by using primers EACP1 and EACP2 as described by Roop et al.,[24] and the approximately 900-bp fragment was produced and cloned into the pGEMT vector.

A PCR-based method for rapid screening and taxonomic differentiation is currently used to explicate Orthopoxvirus taxonomy.[25,26] The assay uses primers designed from the ATI gene sequence from CPXV. We performed PCR with the primer pair ATI-up-1 5'AATACAAGGAGGATCT3' and ATI-low-1 5'CTTAACTTTTTCTTTCTC3'. After the amplification reactions were carried out, the amplicons were digested with XbaI at 37°C for 3 h, as described.[26]

The PCR-amplified TK, VGF, and HA fragments of Araçatuba virus, cloned into the pGEMT plasmids, were sequenced in both orientations by the dideoxy-chain termination method[27] by using M13 universal primers (fmol DNA Sequencing System; Promega Corp.) and [a32 P]dCTP for oligonucleotide labeling. Sequences were analyzed by using the BLASTN and BLASTX programs.[28] The DNA sequences of the Araçatuba virus, TK, and VGF genes were deposited in GenBank (accession nos. AF 503169 and AF503170). A phylogenetic tree was constructed by using the Treecon program with the Araçatuba virus-TK and Araçatuba virus-VGF nucleotide sequences.[29]

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