Journal of Clinical Virology 48 (2010) 69–72
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Case report
Human Vaccinia virus and Pseudocowpox virus co-infection: Clinical description and phylogenetic characterization Jônatas S. Abrahão a,1 , André T. Silva-Fernandes a,1 , Felipe L. Assis a , Maria I. Guedes a , Betânia P. Drumond b , Juliana A. Leite c , Luiz F.L. Coelho d , Filippo Turrini e , Flávio G. Fonseca a , Zélia I.P. Lobato a , Marieta Madureira a , Paulo C. Ferreira a , Cláudio A. Bonjardim a , Giliane S. Trindade a , Erna G. Kroon a,∗ a
Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil Centro de Pesquisas René Rachou-Fiocruz, Belo Horizonte, Minas Gerais, Brazil c Empresa Brasileira de Pesquisa Agropecuária, Embrapa Gado de Leite, Juiz de Fora, Minas Gerais, Brazil d Universidade Federal de Alfenas, Alfenas, Minas Gerais, Brazil e Alma Mater Studiorum – Università di Bologna, Bologna, Italy b
a r t i c l e
i n f o
Article history: Received 17 December 2009 Received in revised form 1 February 2010 Accepted 2 February 2010 Keywords: Vaccinia virus Orthopoxvirus Pseudocowpox virus Parapoxvirus Co-infection
a b s t r a c t Background: Occupational exanthematic diseases represent an important cause of public health impact and economical losses. Among the viral exanthematic diseases, two caused by poxviruses are noteworthy: the bovine vaccinia (BV), caused by the Vaccinia virus (VACV); and the milker’s nodule, in which the agent is the Pseudocowpox virus (PCPV). Both agents are zoonotic and have been associated with several cases of bovine infection. In Brazilian rural areas BV has been highly prevalent, particularly in milk herds. Farmers, milkers and their close contacts developed lesions on the hands, forearms, legs and face accompanied by several systemic symptoms. Although VACV and PCPV present with similar epidemiological and transmission patterns, no VACV and PCPV co-infection cases have to date been described. Objectives: To describe the first case of zoonotic VACV and PCVP co-infection, based on serological and molecular methods. Study design and results: In this work we report a case of a Brazilian rural worker who presented with a large severely ulcerated-pustule skin lesion, associated with fever, headache, malaise, myalgia and axillary, inguinal and cervical limphadenopathy. The worker declared occupational contact with cattle that had notable injuries on their teats. Human and bovine clinical samples were collected and submitted to serological and molecular tests. PCR and phylogenetic analysis revealed the presence of VACV DNA and PCPV DNA in the patient’s lesion. Serological tests indicated anti-VACV neutralizing antibodies and molecular assays showed the presence of VACV and PCPV DNA in the patient sera. VACV and PCPV also were detected in dairy cattle. Conclusion: Together, these results indicate a case of zoonotic VACV/PCPV co-infection. Epidemiological surveillance and appropriate medical treatment are essential for the control of both diseases, especially in the most severe cases, as described in the present study. © 2010 Elsevier B.V. All rights reserved.
1. Why this case is important Occupational exanthematic diseases have an important public health impact and can cause economic losses. Today, Vaccinia
∗ Corresponding author at: Laboratório de Vírus, Departamento de Microbiologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Av. Antônio Carlos 6627, CEP: 31270-901- Belo Horizonte, MG, Brazil. Tel.: +55 31 3409 2539; fax: +55 31 34092733. E-mail address:
[email protected] (E.G. Kroon). 1 These authors contributed equally. 1386-6532/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jcv.2010.02.001
virus (VACV) infections affecting rural workers represent a frequently reported zoonosis in Brazil.1,2 VACV is the prototype of the genus Orthopoxvirus (OPV), family Poxviridae,3 and since 1999, several VACV-isolates have been obtained from these outbreaks,1,2 also known as bovine vaccinia (BV) disease. During BV-outbreaks, infected milkers usually exhibit exanthematic-lesions on the hands and arms, apparently acquired by unprotected contact with infected dairy cattle during milking.2 BV is considered a public health problem in Brazil due to BV’s occupational character, high frequency of transmission and the improper medical treatment.2 This present report demonstrates that another zoonotic-poxvirus besides VACV circulates in Brazil and causes exanthematic disease
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in milkers and dairy cattle: the Pseudocowpoxvirus (PCPV), genus Parapoxvirus (PPV). Human infection with PCPV is commonly called milker’s nodule (MN), while the disease in dairy cattle is referred to as pseudocowpox.3 In Brazil, the data regarding PCPV are inconsistent, based only on MN clinical descriptions by local veterinaries. Although VACV and PCPV present with similar clinical profiles,3 coinfection with these viruses has never been reported. In the report, we describe, for the first time, a human VACV/PCPV co-infection. Additionally, this is the first report on the phylogenetic analysis of PCPV in Brazil.
2. Case description From June to November 2005, exanthematic outbreaks affecting humans and cattle were identified in several small counties in southeast Brazil. In general, the characteristics of these outbreaks were similar to those of BV described in the country in previous years.1,2 Our study patient was a 19-year-old man living in a rural area near Resplendor County, Minas Gerais State. The patient worked as a farmer and reported occupational contact with cows that had lesions on their teats and udders (Fig. 1B). After repeated contact with sick animals, the patient noticed the development of a single skin lesion on his right little finger that appeared as nodular swelling; the next day, he became febrile. A few days later, the nodular-lesion became a papule and then evolved into an ulcerated-pustule surrounded by a large ring of inflamed tissue (Fig. 1A). This specific transition of the disease lesion attracted our attention because the inflamed tissue area was clearly larger than that usually observed in human VACV infections in other farmers identified during the outbreaks of the previous years.1,2 The pus-
Fig. 1. Lesions caused by VACV and PCPV co-infection. (A) Exanthematic-lesion on the right little finger of our study patient, which were acquired after contact with VACV/PCPV co-infected dairy cattle. The figure highlights the large inflamed tissue area around the ulcerated-pustule. (B) Cattle teat lesions presenting several umbilicated pustules and a large and central ulcerated lesion.
tule turned into necrotic, painful-ulcers, and after a short time, the lesion developed a scab. The patient also described lymphadenopathy, headache and myalgia. An informed-consent document was signed by the patient. In order to characterize the etiological agent of this exanthematic-lesion, serological and molecular tests for the detection of OPV were performed since VACV had been detected in outbreaks in counties near Resplendor that same year. Plaque-reduction-neutralizing-tests4 revealed the presence of anti-OPV-antibodies in the patient’s serum at a titer of 1:80 neutralizing-units per milliliter at day-9 post the appearance of symptoms (p.a.s.). The serum was also submitted for quantitative real-time PCR5 and the results indicated that the patient was OPV viremic at day-9 p.a.s. (∼3 × 105 OPV genomic copies/ml). Swabs from the lesion were prepared1,2 and submitted for PCR to amplify
Fig. 2. Consensus bootstrap OPV (A) and PPV (B) phylogenetic-trees based on the nucleotide sequences of the OPV ha gene and PPV b2l gene, respectively. The trees were constructed using the maximum-parsimony method with 1000 bootstrap replicates implemented by MEGA3.1. Values >50% are shown. OPV and PPV sequences used to construct the trees were obtained from GenBank. Black dots highlight VACV-Br-Hu-1 and PCPV-Resplendor.
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the OPV-hemaglutinin gene6 (ha). As a positive control for OPV, we used the VACV-Western-Reserve strain. An amplicon of approximately 950 bp was generated, suggesting OPV infection. The amplicon was directly sequenced three times in both orientations (Mega-BACE, GE-Healthcare, UK). The sequences were aligned and compared with published OPV ha sequences from GenBank (ClustalWmethod, MEGA3.1, Phoenix, USA) and revealed strong identity with several VACV-isolates, at the nucleotide and amino acid levels. This sequence was deposited in GenBank named VACVBr-HU-1(FJ173000). A phylogenetic-tree based on the ha sequence was constructed using the maximum-parsimony method with 1000 bootstrap replicates implemented by MEGA3.1., and showed that VACV-Br-HU-1 clustered with several Brazilian-VACV-isolates obtained during BV-outbreaks (Fig. 2A). In order to investigate the causes of the exceptionally inflamed exanthematic-lesion observed in the patient and considering a previous report of OPV and PPV co-circulation,7 we decided to examine the possibility of viral co-infection. Therefore, the swab collected from the patient was tested for PPV-DNA by b2l gene PCR using the PPP1-1 and PPP-4 primers.8 Bovine swabs were also tested by PCR.5,6,8,11 As a PPV positive control, we used the Orf virusMT05.9 Surprisingly, a 590 bp amplicon was generated by the b2l PCR from the exanthematic-lesion, indicating that the milker was infected with PPV. The b2l amplicon was sequenced, as previously described, and aligned with PPV sequences from GenBank, and it showed high identity with PCPV-isolates, with the closest being PCPV-Tillquist(AY424972) and PCPV-VR634(AY453665). This sequence was deposited in GenBank and named PCPVResplendor(GU391989). A phylogenetic-tree based on the b2l gene was constructed, as described for the OPV ha tree, and showed that PCPV-Resplendor clustered with other worldwide PCPV-isolates (Fig. 2B). The presence of PCPV DNA in the patient’s blood was confirmed at day 9 p.a.s. by real-time PCR assays (∼5 × 104 PPV genomic copies/ml).8,10,11 The patient declared no immunosuppressive diseases or therapies. The VACV/PCPV co-infection was also confirmed in the dairy cattle involved in the outbreak. All of the clinical and laboratory samples were manipulated separately to avoid contamination. 3. Other similar and contrasting cases in the literature The characterization of poxviruses as agents of occupational exanthematic outbreaks in Brazilian rural areas has been extensively described.1,2 Moreover, the number of notifications of VACV outbreaks has increased in the last years, and today represents the most frequently reported zoonotic orthopoxvirus in this country. Based on electron-microscopic findings, the circulation of OPV and PPV was described in a same area in Rio de Janeiro State, however the detection of these agents was performed temporally separated by 4 months, in distinct patients.7 To date, there are no descriptions for OPV/PPV co-infection in a same patient.
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tacted showed two types of lesions on their teats (Fig. 1B). Although only the ulcerated central scab of the cow (Fig. 1B) was swabbed and analyzed (to avoid viral spread over the property due to vesicle rupture), it was possible to detect both VACV and PCPV using an OPV/PPV multiplex PCR,10 probably due to cross-contamination between the bovine lesions. The detection of VACV/PCPV DNA in the patient’s blood also supported the theory of co-infection and could be associated with the systemic manifestations. To date, other studies have described the detection of OPV or PPV-DNA in the blood related with the acute-phase of the diseases.15–17 Although other reports have described the circulation of PPV in Brazil,8,10 our study is the first to specifically describe the detection of a PCPV in Brazil. The PPV phylogenetic-tree (Fig. 2B) showed that the b2l sequence amplified from the patient lesion grouped with PCPV-isolates, excluding a hypothetical infection with Bovine papular stomatitis virus (BPSV), another zoonotic-PPV. Determining the true origin of PCPV-Resplendor is difficult, however, given that (i) PCPV-Tillquist and PCPV-VR634, the closest compared strains, were isolated many years ago and could have been spread widely to several countries before arriving in Brazil (if indeed these PCPVisolates were introduced in Brazil) and (ii) the occurrence of a milker’s nodule was previously reported in Brazil based on clinical diagnosis, but it is unknown if those samples were similar to PCPVResplendor. Additional genetic studies are necessary to determine the origin and world-distribution of PCPV-isolates. It is very difficult to predict if the disease caused by VACV and PCPV co-infection is, under all circumstances, more severe than infections with either virus alone, since could depend of the infection dose, the isolate virulence, the host immunological status, and eventual viral synergic effects. However, the VACV and PCPV epidemiology and transmission patterns are very similar.3 Manual milking and inadequate disinfection methods found in some Brazilian rural areas explain the high transmission rates during the BVand MN-outbreaks14 and probably facilitate the occurrence of viral co-infections. The frequency of poxvirus co-infections in Brazil is unknown at this time, but it is under investigation. Epidemiological surveillance and appropriate medical treatment, including the isolation of the affected properties, the administration of palliative drugs to the affected milkers, avoid the lesions debridation, etc., are essential for the control of both diseases, especially in the most severe cases, as described in the present study. Acknowledgements We thank MSc. João R. dos Santos, Angela S. Lopes, Ilda M.V. Gama, and colleagues from Laboratório de Vírus (ICB-UFMG). Financial support was provided by CNPq, CAPES, FAPEMIG and MAPA. GST, FGF, ZIPL, EGK, CAB and PCPF received fellowship from CNPq. References
4. Discussion The present work describes for the first time a case of human VACV and PCPV co-infection. Clinically, the symptoms caused by VACV and PCPV are very similar.3 Both viruses cause vesicular–pustular lesions (Fig. 1), associated with characteristic systemic symptoms.3 Recent studies have shown that the severity of poxvirus infections may be influenced by the host immunological-status, the virulence of the viral-isolate and the patient’s anti-smallpox vaccination history.12–14 Here, we describe a patient presenting with a skin lesion remarkable for a severe local inflammatory reaction, which could be related to the VACV/PCPV co-infection. Interestingly, the cattle that this patient had con-
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