Querido et al. Parasites & Vectors 2013, 6:66 http://www.parasitesandvectors.com/content/6/1/66 RESEARCH Open Access Inoculation of Triatoma Virus (Dicistroviridae: Cripavirus) elicits a non-infective immune response in mice Jailson F B Querido1, Jon Agirre2,3, Gerardo A Marti4, Diego M A Guérin2,3,5* and Marcelo Sousa Silva1* Abstract Background: Dicistroviridae is a new family of small, non-enveloped, +ssRNA viruses pathogenic to both beneficial arthropods and insect pests. Little is known about the dicistrovirus replication mechanism or gene function, and any knowledge on these subjects comes mainly from comparisons with mammalian viruses from the Picornaviridae family. Due to its peculiar genome organization and characteristics of the per os viral transmission route, dicistroviruses make good candidates for use as biopesticides. Triatoma virus (TrV) is a pathogen of Triatoma infestans (Hemiptera: Reduviidae), one of the main vectors of the human trypanosomiasis disease called Chagas disease. TrV was postulated as a potential control agent against Chagas’ vectors. Although there is no evidence that TrV nor other dicistroviruses replicate in species outside the Insecta class, the innocuousness of these viruses in humans and animals needs to be ascertained. Methods: In this study, RT-PCR and ELISA were used to detect the infectivity of this virus in Mus musculus BALB/c mice. Results: In this study we have observed that there is no significant difference in the ratio IgG2a/IgG1 in sera from animals inoculated with TrV when compared with non-inoculated animals or mice inoculated only with non-infective TrV protein capsids. Conclusions: We conclude that, under our experimental conditions,TrVisunabletoreplicateinmice.Thisstudy constitutes the first test to evaluate the infectivity of a dicistrovirus in a vertebrate animal model. Keywords: Dicistroviridae, Triatoma virus,Chagasdisease,triatomines,Trypanosoma cruzi, Mice immune response Background Kashmir bee virus, which infects honeybees [4-6], and Dicistroviridae is a recently established family of small, Taura syndrome virus, which is pathogenic to shrimps [7]. non-enveloped viruses, containing a +ssRNA genome, and Two dicistrovirus members infect the model organism is classified under the order Picornavirales.Thisfamilycon- Drosophila melanogaster, Drosophila C virus (DCV) tains 14 members classified within two genera, Cripavirus and CrPV [8,9], and eight of them are pathogenic to in- (type species Cricket paralysis virus,CrPV),andAparavirus sect pests, as it is the case with CrPV, which infects (type species Acute bee paralysis virus,ACP)[1,2].Athird field crickets and the Olive Fruit Fly as well [7]. The genus has been recently proposed, Triatovirus (type species aforementioned CrPV is quite ubiquitous and replicates Triatoma virus, TrV) [3]. All dicistroviruses are pathogenic in many other insect species spanning five orders [10]. to arthropods, although primarily to insects, representing Although dicistroviruses are believed to be arthropod- significant threats to the health of beneficial arthropods specific, antibodies against CrPV have been detected in such as acute bee paralysis virus, Black queen cell virus, serafromapig,horse,andcattle[11].Similarly,high levels of anti-TrV antibodies were detected in chickens * Correspondence: [email protected]; [email protected] used to feed a colony of TrV-infected insects, although 2Unidad de Biofísica (UBF, CSIC-UPV/EHU), Barrio Sarriena S/N, 48940, Leioa, the authors of this work concluded that the chickens were Bizkaia, Spain 1Centre for Malaria and Tropical Diseases - Instituto de Higiene e Medicina apparently refractory to the infection with TrV [12]. Tropical - Universidade Nova de Lisboa, Lisboa, Portugal Full list of author information is available at the end of the article © 2013 Querido et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Querido et al. Parasites & Vectors 2013, 6:66 Page 2 of 6 http://www.parasitesandvectors.com/content/6/1/66 Compared to other families of picornaviruses, little is was added at a ratio of 100 mL per gram of dry material. known about dicistrovirus infection, replication mechan- PMSF (N 98.5%, purchased from Sigma) thawed at ism or gene function. Most information about cellular −20°C in isopropanol (Merck, GR for analysis) was infection and the replication cycle of dicistroviruses added up to a final concentration of 1 mM. The mixture comes from studies on DCV, and by comparison with was homogenized for 5 min using a vortex mixer and mammalian picornaviruses [1]. Many members of the then sonicated using a Sanyo MSE Soniprep 150 operat- Dicistroviridae family are considered novel candidates to ing at 10 s ON/10 s OFF for 20 pulses. After centrifuga- be used as biopesticides [1,13-17]. tion, the resulting pellet was resuspended in NMT TrV and Solenopsis invicta virus-1 are the two lone buffer and then loaded on top of a 35 mL continuous members of the Dicistroviridae family that infect insects 5%–30% sucrose gradient. The gradients were cooled in of medical importance [1]. In fact, TrV is a natural ice to avoid particle diffusion within them. The individ- enemy of Triatoma infestans (Hemiptera: Reduviidae), ual optical density of each aliquot was then measured at one of the main vectors of the trypanosomiasis disease both 260 nm and 280 nm. After plotting the absorbance called Chagas disease, a severe human illness most profile, selected aliquots were loaded onto 12.5% poly- prevalent in almost all Latin American countries [18,19]. acrylamide SDS-PAGE gels to check the purity of the To date, TrV is the only entomopathogenic virus found samples. Following gradient fractionation, equivalent in triatomines [20]. The viral particles are spherical, with samples were pooled together and dialyzed overnight a diameter of 30 nm [21], and consist of a non- against 2 L of NMT buffer. enveloped capsid that encloses a 9010 nt long viral gen- ome [22]. The capsid contains four structural proteins Infection with Triatoma virus (TrV) VP1 (29.7 kDa), VP2 (28.4 kDa), VP3 (31.8 kDa) and a Eight groups of female Mus musculus BALB/c mice (3 minor one VP0 (37.3 kDa). In addition to these four mice per group) between 5 and 8 weeks of age obtained structural proteins, this virus has a low molecular weight from Instituto de Higiene e Medicina Tropical, Lisbon, protein of 5.5 kDa that appears to be detached from the Portugal, were used in the experimental infection with capsid, lying at the particle interior and presumably in TrV. Groups 1-5 were inoculated by intraperitoneal in- contact with the genome [3,20,23]. TrV can also infect jection. Group 1 is the control group, which was inocu- natural populations of T. sordida and several experimen- lated only with PBS. Group 2 was inoculated with 3.0 μg tal populations of triatomines as well [24]. Viral replica- of empty TrV particles (capsids without genome, there- tion takes place in the midgut epithelium cells of fore non-infective) [3]. Group 3 was inoculated with 3.0 triatomines, causing many deleterious sublethal effects μg of TrV, group 4 and group 5 with 0.3 μg and 0.03 μg in T. infestans and T. patagonica colonies, such as reduc- of TrV respectively. Group 6 served as a negative control tion of the longevity, increase of the developmental time, of per os inoculation, therefore it was inoculated only a decrease in both fecundity and fertility of eggs, and even with 100 μl of PBS delivered per os intraesophageally by a cumulative mortality higher than 97% [16,25-28]. using a 1 ml tuberculin syringe and Gavage needle. As pointed out previously, the infectivity of TrV in Group 7 was inoculated with 3.0 μg of empty TrV parti- vertebrates remains unclear. Therefore, in this work we cles in 100 μl of PBS, delivered per os intraesophageally studied the infectivity of TrV in mice (Mus musculus - by using a 1 ml tuberculin syringe and Gavage needle. BALB/c mice). Our results indicate that inoculations of Group 8 was inoculated with 3.0 μg of TrV in 100 μlof both infective and non-infective TrV particles elicit the PBS, delivered per os intraesophageally by using a 1 ml same immune response, and that this insect virus is tuberculin syringe and Gavage needle. After inoculation unable to replicate in this animal model. Based on the blood and feces samples were collected at different times bicistronic character of dicistrovirus genomes, it was (3, 7, 30 and 45 days). Forty-five days after inoculation, speculated that these pathogens may not infect verte- the animals were sacrificed. brates, and this report constitutes important experimen- All experiments on animals were conducted with prior tal evidence to support that hypothesis. approval from the Animal Welfare of General Manage- ment of Veterinarians (DGV - Portugal). All manipula- Methods tions of mice satisfied the requirements of the Regulations Triatoma virus purification of Experimental Animal from the Ethics Committee of Virus purification was performed according to [3]. Dry IHMT/UNL-Portugal. feces obtained from infected insects were collected on Whatman filter papers and excised from them using a Searching for TrV in blood and feces samples standard surgery scalpel until two g of material was col- Blood samples from each mouse were collected into in- lected. A buffer composed of 10 mM NaCl, 1 mM dividual micro tubes containing 5 μl of Heparin-Sodium MgCl2 and 200 mM citric acid at pH 6 (Lysis buffer) (B. Braun, USA). The RNA was extracted according to Querido et al. Parasites & Vectors 2013, 6:66 Page 3 of 6 http://www.parasitesandvectors.com/content/6/1/66 the product manual supplied with the RNA isolation least 30% of its composition was integrated by 7 additional Kits, (Bioline, UK).