Journal of Medical Virology 82:692–699 (2010) Rapid Detection of Orthopoxvirus by Semi-Nested PCR Directly From Clinical Specimens: A Useful Alternative for Routine Laboratories Joˆ natas Santos Abraha˜ o, Betaˆ nia Paiva Drumond, Giliane de Souza Trindade, Andre´ Tavares da Silva-Fernandes, Jaqueline Maria Siqueira Ferreira, Pedro Augusto Alves, Rafael Kroon Campos, Larissa Siqueira, Cla´ udio Antoˆ nio Bonjardim, Paulo Ce´sar Peregrino Ferreira, and Erna Geessien Kroon* Laborato´rio de Vı´rus, Departamento de Microbiologia, Instituto de Cieˆncias Biolo´gicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil Orthopoxvirus (OPV) has been associated with OPVs have been reported previously to infect humans. worldwide exanthematic outbreaks, which have The first of these is Variola virus (VARV), which is resulted in serious economic losses as well as the etiological agent of smallpox, which had been impact on public health. Although the current eradicated. The other three are zoonotic species, includ- classical and molecular methods are useful for ing Monkeypox virus (MPXV), Cowpox virus (CPXV), the diagnosis of OPV, they are largely inacces- and Vaccinia virus (VACV). These species have been sible to unsophisticated clinical laboratories. The associated with outbreaks of infection in Africa, major reason for the inaccessibility is that they Europe, South America and Asia [Heymann et al., require both virus isolation and DNA manipula- 1998; Haenssle et al., 2006; Trindade et al., 2006; Singh tion. In this report, a rapid, sensitive and low-cost et al., 2007]. The incidence of zoonotic OPV infections semi-nested PCR method is described for the has been increasing in recent years [reviewed by detection of OPV DNA directly from clinical speci- Ferreira et al., 2008]. These findings can be explained mens. A set of primers was designed to amplify the in part by the suspension of smallpox vaccination in the conserved OPV vgf gene. The most useful early 1980s. Cessation of vaccination has also led to the thermal and chemical conditions were selected emergence of an unimmunized generation against OPVs and minimum non-inhibitory dilutions were deter- [Kulesh et al., 2004]. mined. More than 100 Brazilian Vaccinia virus The diagnosis of OPV infections involves clinical, (VACV) field clinical specimens were tested using serological, virological, microscopic and molecular this semi-nested PCR in order to confirm its techniques [Kulesh et al., 2004; Lobato et al., 2005; applicability. Cowpox virus was also detected by Trindade et al., 2006; Saijo et al., 2008; Vestergaard PCR from the ear scabs of scarified Balb/c mice. In et al., 2008; Strenger et al., 2009]. Zoonotic OPV has the addition, the method was highly sensitive for potential to cause either local or disseminated vesicular- the detection of VACV DNA in murine blood pustular lesions, which are associated with fever, and excreta, which are among the suggested lymphadenopathy, malaise and acute muscle pain reservoirs of OPV. Together, these data suggest [Fenner et al., 1989]. The infection induces a robust that semi-nested PCR can be used for initial humoral immune response. Due to this response, anti- screening for OPV and as a routine diagnostic OPV antibodies can be detected by ELISA, immuno- laboratory method. J. Med. Virol. 82:692–699, fluorescence or neutralization tests in sera several days 2010. ß 2010 Wiley-Liss, Inc. after the initial infection [Pelkonen et al., 2003; Karem KEY WORDS: viral diagnosis; vaccinia out- breaks; poxvirus; Orthopoxvi- Grant sponsor: CNPq CAPES FAPEMIG. rus *Correspondence to: Erna Geessien Kroon, Laborato´rio de Vı´rus, Departamento de Microbiologia, Instituto de Cieˆncias Biolo´gicas, Universidade Federal de Minas Gerais, Av. Antoˆnio Carlos, 6627, CEP: 31270-901, Belo Horizonte, MG, Brazil. INTRODUCTION E-mail: [email protected] Accepted 13 July 2009 Orthopoxviruses (OPVs) are large, enveloped, linear DOI 10.1002/jmv.21617 double-stranded DNA viruses. They are classified as a Published online in Wiley InterScience genus within the Poxviridae family [Moss, 2007]. Four (www.interscience.wiley.com) ß 2009 WILEY-LISS, INC. Direct Detection of Orhopoxvirus by vgf Semi-Nested PCR 693 et al., 2005; Lederman et al., 2008]. The epidermal gen, Carlsbad, CA) supplemented with 5% fetal calf lesions typically present high titers of virions, and serum (FCS), 25 mg/ml fungizone (Amphotericin B, viruses can be isolated from vesicular secretions or by Crista´lia, Brazil), 500 U/ml penicillin, and 50 mg/ml inoculation of permissive cell lines or the chorioallantoic gentamicin (Schering-Plough, Rio de Janeiro, Brazil). membrane (CAM) of embryonated hen’s eggs [Sarkar VACV strain Western Reserve (VACV-WR) and CPXV et al., 1974; Leite et al., 2005; Singh et al., 2006; Kurth strain Brighton Red (CPXV-BR), kindly provided by Dr. et al., 2008]. The viral particles obtained from the lesions C. Jungwirth (Universitat Wurzburg, Germany), were are then visualized by electron microscopy. This grown in Vero cells (ATCC number CCL81) and purified method is laborious due to required additional specimen subsequently using a sucrose gradient as described manipulation, including fixing, staining and labeling previously [Joklik, 1962]. VACV-WR was then used for [reviewed by Trindade et al., 2007]. Molecular and the PCR standardization, minimum non-inhibitory immunological techniques, including real-time PCR dilutions (MNIDs) and sensitivity assays. [Putkuri et al., 2009], nested and semi-nested PCR [Sa´nchez-Seco et al., 2006; Damaso et al., 2007], Murine Sample Collection PCR-RFLP [Meyer et al., 1997], and Western blot assays Clinical specimens were collected from Mus musculus [Fedorko et al., 2005] have been applied widely in OPV Balb/c mice to assess the semi-nested PCR of vgf research. The use of real-time PCR for the detection of sensitivity and MNID. Four male 4-week-old Balb/c VACV directly from lesions without DNA or virus mice were held in cages with access to filtered water and manipulation was described by de Souza Trindade autoclaved industrial food. To confirm the absence of et al. [2008]. However, the other mentioned previously OPV, all animals were tested previously by the neutra- processes require viral isolation and amplification. lization assay [Abdalrhman et al., 2006]. To mimic In addition, some described recently methods have a epidermal lesions of OPV, scabs were obtained by restricted application because they were designed to scarification in phosphate buffered saline (PBS) of detect only specific strains [Damaso et al., 2007; Saijo Balb/c mice ears as described previously [Tscharke and et al., 2008; Singh et al., 2008] or have become out of date Smith, 1999]. Feces and urine were collected with due to novel polymorphisms in new OPV isolates [Meyer microcentrifuge tubes positioned directly below the et al., 1997; Leite et al., 2007]. anus or penis of the mice. Blood was also collected in Although the methods referred to above are employed microcentrifuge tubes with EDTA after the mice were in research centers, they are often not useful in killed. All samples were then stored at À708C. unsophisticated routine clinical laboratories. OPV dia- Feces and scabs were homogenized in PBS (0.1 g gnosis in such laboratories, based on detection of anti- clinical specimen/0.9 ml PBS). The samples were then OPV antibodies by ELISA, cannot differentiate anti- centrifuged at 2,000g for 3 min, and the supernatants bodies associated with acute infection from antibodies were then collected. Pools of blood, urine and feces resulting from prior vaccination [Jahrling et al., 2007]. supernatants were spiked subsequently with VACV-WR This is a concern in OPV diagnosis because patients to assess the sensitivity of vgf semi-nested PCR and that vaccinated against smallpox patients can be susceptible of MNID. to other OPV infections [Silva-Fernandes et al., 2009]. Attempting to reduce both, the cost and time for OPV Collection and Preparation of Human and diagnosis, a highly sensitive conventional semi-nested Bovine BV Clinical Samples PCR technique was developed to amplify the highly conserved OPV virus growth factor (vgf) gene directly Vesicle contents and dried scabs from cattle from dried scabs and vesicular contents. This study udders and the hands of the milking personnel were discusses a simple and rapid technique, which has collected during BV outbreaks (Table II). This collection facilitated the diagnosis of more than 100 Brazilian was accomplished using 1-ml insulin syringes with cases of VACV (BV) infection during the last 2 years. In 0.45 mm  13 mm needles and cotton swabs or a pair of addition, the potential for further use of this method as forceps. Collected samples were transported under cold an epidemiological tool was assessed under laboratory conditions to the laboratory and stored at À708C until conditions. The method was able to detect OPV DNA processing. Vesicular liquid swabs were added to 200 ml directly from rodent specimens, which are the believed of PBS and centrifuged at 2,000g for 3 min. Scabs to be reservoirs for OPV. The vgf semi-nested PCR were macerated in PBS (0.1 g scab/0.9 ml PBS) using method does not require viral isolation or DNA manip- a homogenizer (Politron, Littau, Switzerland) and ulation and therefore can be useful for unsophisticated clarified by centrifugation at 2,000g for 3 min. Two routine clinical diagnostic laboratories as a rapid microliters of each supernatant was used in the vgf screening tool for OPV. semi-nested PCR. Several expected PCR products were sequenced directly (ET Dynamic Termnator for MATERIALS AND METHODS MegaBACE—GE Healthcare, Fairfield, NJ) and com- pared with available GenBank sequences using the Cells and Viruses online BLAST software (http://www.ncbi.nlm.nih.gov/ Vero cells were propagated at 378C in Eagle’s- blast). In parallel, two microliters of the same clarified minimum essential medium (MEM; GibcoBRL, Invitro- BV field samples were added to 198 ml of PBS and J.