Journal of Virological Methods 185 (2012) 189192

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Journal of Virological Methods

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Detection of avian group D rotavirus using the polymerase chain reaction for the VP6 gene
Delana Andreza Melo Bezerra a , Ren Ribeiro da Silva b , Jane Haruko Lima Kaiano a , Rodrigo Vellasco Duarte Silvestre a , Darleise de Souza Oliveira a , Alexandre C. Linhares a , Yvone Benchimol Gabbay a , Joana DArc Pereira Mascarenhas a,
a b

Sec o de Virologia, Instituto Evandro Chagas, Secretaria de Vigilncia em Sade, Ministrio da Sade, Rodovia BR 316KM 07, S/N, Levilndia, 67.030-000, Ananindeua, Par, Brazil Laboratrio Nacional Agropecurio (LANAGRO), Ministrio da Agricultura, Avenida Almirante Barroso, 1234, Marco, 66.093-020, Belm, Par, Brazil

a b s t r a c t
Article history: Received 3 December 2011 Received in revised form 6 July 2012 Accepted 11 July 2012 Available online xxx Keywords: Group D rotaviruses Avian VP6 gene RT-PCR

Group D rotaviruses (RVs-D) have been documented in birds and, while they may be common in these animals, few molecular studies are available for this specic group. In this study, specic primers for the gene that encodes for the RVs-D VP6 protein were designed and used in a reverse transcription polymerase chain reaction (RT-PCR). Thirty pools of samples were tested by polyacrylamide gel electrophoresis (PAGE) yielding a 30% (9/30) positivity. These pools were subjected subsequently to RT-PCR, with a 53% (16/30) positivity rate. The sensitivity of the PCR assay was demonstrated up to a dilution of 5 104 ng/L (0.5 pg/L) of the cloned VP6 gene. The four samples were sequenced and showed 90.891.1% similarity with regards to the RVs-D VP6 gene. To assess for specicity our RT-PCR was applied to nine samples known to contain enteric viral agents other than group D rotaviruses including picobirnavirus, rotavirus group A, and reovirus with negative results. Overall, the data conrm the specicity of the primers used for detecting the RVs-D by RT-PCR, suggesting that this assay can be used for diagnostic purposes. Published by Elsevier B.V.

1. Introduction The infectious diseases of the gastrointestinal tract that affect birds have a worldwide distribution and a variety of etiologies et al., 2009). (Reynolds et al., 1987; Yegani and Korve, 2008; Lojkic Among the viral agents, rotaviruses are the most common viruses in birds and are found both in birds with enteropathic infections and in healthy birds (Tamehiro et al., 2003; Villarreal, 2006; Pantin-Jackwood et al., 2008). The rotaviruses belong to the family Reoviridae, and are non-enveloped icosahedral particles that contain a genome of 11 segments of double-stranded RNA, each of which encodes at least one viral protein (Ramig et al., 2005). Rotaviruses are classied into seven groups (AG) based on the antigenic sites located on the VP6 protein of the internal capsid (Estes and Kapikian, 2007). The rotaviruses that infect birds belong to groups A, D, F, and G (Otto et al., 2006, 2011; Trojnar et al., 2010; Johne et al., 2011). The RVs-D have been documented in birds, but a few molecular studies are available for this specic group because techniques used for detection rely mainly on the use of polyacrylamide gel

electrophoresis (PAGE). As more sensitive molecular methods are not available, it is likely that the true frequency of RVs-D in birds is underestimated. Therefore there is a need for developing more sensitive techniques in order to improve our knowledge of the molecular diversity of RVs-D infecting birds. The aim of this study was to develop a method to detect RVs-D in birds using on RT-PCR assay based on the amplication of the VP6 gene. 2. Materials and methods 2.1. Fecal specimens The samples (n = 30) analyzed in this study were represented by pools of fecal samples obtained from broiler chickens of the genus Gallus in 12 farms located in four cities in Par State, Northern Brazilian Amazon. 2.2. RNA-PAGE The viral RNA was extracted from 20% fecal suspensions using silica powder glass, as described by Boom et al. (1990). After extraction these samples were tested by PAGE and RT-PCR. PAGE was performed according to the method described by Pereira et al. (1983).

Corresponding author. Tel.: +55 91 3214 2016; fax: +55 91 3214 2005. E-mail address: joanamascarenhas@iec.pa.gov.br (J.D.P. Mascarenhas). 0166-0934/$ see front matter. Published by Elsevier B.V. http://dx.doi.org/10.1016/j.jviromet.2012.07.017

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Fig. 1. (a) PAGE migration prole of the group D rotaviruses (lanes 14). (b) RVs-D as detected by RT-PCR: lane 1, 123-bp DNA marker from Invitrogen; lanes 25, amplication of the 742-bp fragment of the VP6-coding gene; and lane 6, the avian rotavirus group A sample (negative control).

2.3. RT-PCR Based on the sequences reported by Trojnar et al. (2010), specic primers were designed targeting at the gene coding for the VP6 protein of the RVs-D, using the Primer BLAST program available at NCBI. The forward primer used in this study was RD6F (5 -GGAGGCGCTGTCTTCAATTGCG-3 ) and the reverse primer was RD6R (5 -TGGCCAATAGTGTGTGGCAGCT-3 ), which were used to amplify a 742-bp fragment. The RT-PCR for detection of RVs-D samples was carried out in two steps. To obtain the cDNA, 3 L of the extracted dsRNA was added to the pair of primers followed by denaturation during 5 min at 95 C and chilling on ice thereafter. The rst step was reverse transcription carried out using a nal volume of 25 L including 16.5 L of H2 O free of RNase and DNase, 2.5 L of 10 buffer (Invitrogen), 1 L of dNTPs (10 mM, Invitrogen), 0.75 L of MgCl2 (50 mM, Invitrogen), 0.25 L of RT (SuperScriptTM II, 20U, Invitrogen), and 0.5 L of each primer (20 mM, Invitrogen). The reaction was incubated at 42 C for 1 h. The second step was represented by PCR performed by adding 25 L of PCR reagents to the cDNA sample for a nal reaction volume of 50 L. The PCR reagents included 18.5 L of H2 O free of RNase and DNase, 2.5 L of 10 First-Stand Buffer, 3 L of dNTPs (10 mM), 0.75 L of MgCl2 (50 mM,), 0.25 L of Taq DNA polymerase (2.5 U/L, Invitrogen), with the following cycling conditions: 93 C for 3 min, 35 cycles of 93 C for 1 min, 55 C for 1 min, and 72 C for 1 min, with a nal incubation at 68 C for 7 min. The PCR products were analyzed by gel electrophoresis on a 1.5% agarose gel with TBE buffer, and the gel was stained with SYBR Safe DNA gel stain (Invitrogen). Visualization of the 742-bp band was conducted using a GEL DOC 1000 instrument. To determine the sensitivity of the method, a PCR was performed including 10-fold serial dilutions of cleansed recombinant colonies containing the fragment of VP6 of the RVs-D, with initial concentration of 50 ng/L and nal concentration of 5 105 ng/L. 2.4. Cloning and sequencing Four samples from different farms and municipalities in the Belm area were selected to perform cloning and sequencing. The fragments amplied by RT-PCR were cloned using the Invitrogen TOPO TA Cloning Kit, according to the manufacturers protocol. Approximately 5 L of the plasmid preparations containing the inserted amplicons were used to transform competent E. coli DH5

cells by heat shock, and the transformed cells were plated on Luria-Bertani medium (LB) plates containing ampicillin (100 g/mL) plus IPTG (0.1 mM) and X-Gal (20 g/mL). The recombinant colonies were identied based on the white color of the colonies and then transferred to liquid LB medium containing ampicillin (100 g/mL) and incubated at 37 C for 12 h at 250 rpm. The presence of recombinant colonies containing the target fragment was conrmed by PCR using the primers RD6F and RD6R. These PCR products were puried for nucleotide sequencing using the QIAquick@ PCR Purication Kit, following the protocol provided by the manufacturer. The nucleotide sequences were determined by sequencing with the Big Dye Terminator Kit (Applied Biosystems) using the same primers as those used for RT-PCR. Sequencing was conducted on an ABI PRISM 3100 automatic sequencer (Applied Biosystems).

2.5. Sequence analysis The phylogenetic dendrogram was constructed by comparing rotavirus sequences from various groups, including group A (from humans and animals), group D (chicken/05V0049/DEU/2005 [U733448]), group F (chicken/03V0568/DEU/2003 [HQ403603]), and group G (chicken/03V0567/DEU/2003 [HQ403604]). The analysis included the entire 742-bp sequence amplied by PCR, which corresponded to nucleotides 3-744 of the coding region of the VP6 gene, using the group D prototype sequence as a reference (Trojnar et al., 2010). The sequences obtained for the VP6 gene were aligned and edited using BioEdit (v.7.0.5.3), and the dendrogram was generated by MEGA 5 (v.5.05) using the neighbor-joining method and Kimura 2-parameter model (Kimura, 1980); the reliability was tested by a nonparametric bootstrap analysis with 2000 replicates.

2.6. Statistical analysis Statistical analysis was performed using the software EpiInfo 3.3.2. (http://www.cdc.gov/epiinfo) and BioEstat 5.0 (Ayres et al., 2007). The screening test was performed to assess the sensitivity of the results obtained by RT-PCR compared with the PAGE and the kappa test was used to assess the reproducibility of the RT-PCR. p values 0.05 were regarded as statistically signicant.

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3.2. Sequencing The sequences of three cDNA clones were obtained from four samples from different municipalities in Belm area, corresponding to the coding sequence for the VP6 protein, were identied and analyzed, and sequence analysis conrmed the RT-PCR results with a 99% bootstrap value for group D rotaviruses (Fig. 3). Compared with the RVs-D prototype, samples ch27/PA, ch37/PA, ch69/PA, and ch105A/PA showed a similarity of 90.891.1%, at the nucleotide level, to the RVs-D prototype sequence and 98.599.5% nucleotide similarity between each of these four samples. In contrast, low nucleotide similarities were obtained when comparison was made between groups A (52.956.8%), F (53.554%), and G (42.943.1%). The sequences for samples ch27/PA, ch37/PA, ch69/PA, and ch105A/PA are available in GenBank (Accession nos. JQ065735, JQ065734, JQ065736, and JN703463).
Fig. 2. Assessment of RT-PCR sensitivity for RVs-D detection. Lanes 17, 10-fold serial dilutions of cleansed recombinant colonies containing the VP6 fragment of RVs-D, with initial concentration of 50 ng/L (lane 1). The lowest dilution yielding virus detection was 105 (0.5 pg; lane 6); lane 8, 123-bp DNA marker from Invitrogen.

4. Discussion The molecular diversity of rotavirus group A in birds has been well established (Elschner et al., 2005; Pantin-Jackwood et al., 2008; Schuman et al., 2009; Trojnar et al., 2009; Jindal et al., 2010; Ursu et al., 2011). However, groups D, F and G have only been described recently (Trojnar et al., 2010; Johne et al., 2011). In this study, specic primers (RD6F and RD6R) were constructed to amplify the RVs-D VP6 protein gene based on its gene sequence. These primers demonstrated good specicity, as they only amplied the samples that were positive for group D rotaviruses and not those samples positive for other enteric viruses such as group A rotaviruses, picobirnaviruses and reoviruses. Worldwide, studies on the occurrence of RVs-D have been carried out based essentially on the use of PAGE. In Brazil, distinct electrophoretic proles of rotaviruses have been reported in fecal samples of Brazilian birds using PAGE, but these studies did not classify rotaviruses into groups (Tamehiro et al., 2003). Using the same technique, studies in Germany (Otto et al., 2006), India (Savita et al., 2008), and Bangladesh (Islam et al., 2009) classied rotaviruses into groups and found that RVs-D were found most commonly. However, detection of RVs-D using PAGE is problematic, since this method is likely to underestimate the true frequency of this group of rotaviruses due to its lower sensitivity as compared to other molecular methods such as RT-PCR. In addition, group D rotaviruses cannot be propagated in MA104 cell culture systems (Devitt and Reynolds, 1993). Samples with lower viral titers that are not detected by PAGE need a more sensitive detection method, such as RT-PCR.

3. Results 3.1. Detection of RVs-D by PAGE and RT-PCR Of a total of 30 pools of fecal samples examined by PAGE, nine (30%) were positive, displaying typical avian migration patterns (5:2:2:2). These results were consistent with those related to RVs-D (Fig. 1a). The same 30 pools were subjected subsequently to RT-PCR assay to VP6 gene showing that 16 samples (53%) were positive for RVs-D, each generating a fragment of 742 bp as shown in Fig. 1b. The primers used did not yield positive results in nine (control) samples known to be RVs-D negative, although positive for other enteric viruses, such as rotavirus group A (one sample), picobirnavirus (two samples), reovirus (two samples), rotavirus group A and picobirnavirus (one sample), reovirus and picobirnavirus (three samples). The sensitivity of the method was demonstrated by amplication following serial 10-fold dilutions up to 105 with a limit of detection which was equivalent to 5 104 ng/L (0.5 pg/L) (Fig. 2). The comparison of the results obtained by PAGE (9/30) and RT-PCR (16/30) suggested good reproducibility (kappa = 0.5, p = 0.0004) and in comparing the two techniques, the screening test had a sensitivity of 100% for RT-PCR and the sensitivity of the PAGE was of 56.3%.

77 JQ065734 RVD/Chicken-wt/BRA/37/2009/GxP[x]
99 99 78

JQ065735 RVD/Chicken-wt/BRA/27/2008/GxP[x] JQ065736 RVD/Chicken-wt/BRA/69/2009/GxP[x] JN703463 RVD/Chicken-wt/BRA/105A/2010/GxP[x] GU733448 RVD/Chicken-wt/DEU/05V0049/2005/GXP[x] HQ403603 RVF/Chicken-wt/DEU/03V0568/2003/GXP[x] FJ169858 RVA/Chicken-tc/DEU/02V0002G3/2002/G19P[30]

92 100 88

EF554130 RVA/Human-tc/ITA/PA169/1988/G6P[14]
*DQ119822 RVA/Pig-xx/China DQ870496 RVA/Cow-tc/USA/NCDV/1967/G6P[1] K02254 RVA/Cow-tc/FRA/RF/1982/G6P[1] HQ403604 RVG/Chicken-wt/03V0567/DEU/2003/GXP[x]

0.1

Fig. 3. Dendrogram based on the partial (742 bp) nucleotide sequence of the VP6 gene. The numbers adjacent to the nodes represent the percentage of bootstrap support for the cluster. Bootstrap values lower than 70% are not shown. The strains analyzed in this study are shown in bold type. *No complete information was obtained for this strain.

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D.A.M. Bezerra et al. / Journal of Virological Methods 185 (2012) 189192 Elschner, M., Hotzel, H., Reetz, J., Diller, R., Otto, P., 2005. Isolation, identication and characterization of group A from a chicken: the inner capsid protein sequence shows only a distant phylogenetic relationship to most other avian group A rotaviruses. Journal of Veterinary Medicine 52, 211213. Estes, M.K., Kapikian, A.Z., 2007. Rotaviruses. In: Kniepe, D.M., Howley, P.M., Grifn, D.E., Martin, M.A., Roizman, B., Straus, S.E. (Eds.), Fields Virology. , 5th ed. Lippincott, Williams and Wilkins, Philadelphia, pp. 19171974. Islam, M.S., Alam, M.M., Ahmed, M.U., Saifuzzaman, A.B.M., Kobayashi, N., Kayesh, M.E.H., Udin, M.S., 2009. Molecular epidemiologic study on rotavirus infection in human and birds in association with gastroenteritis. Bangladesh Journal of Veterinary Medicine 7, 233237. Jindal, N., Patnayak, D.P., Chander, Y., Ziegler, A.F., Goyal, S.M., 2010. Detection and molecular characterization of enteric viruses in breeder turkeys. Avian Pathology 39, 5361. Johne, R., Otto, P., Roth, B., Lhren, U., Belnap, D., Reetz, J., Trojnar, E., 2011. Sequence analysis of the VP6-encoding genome segment of avian group F and G rotaviruses. Virology 442, 384439. Kimura, M., 1980. A simple method for estimating evolutionary rate of base substitution through comparative studies of nucleotide sequences. Journal of Molecular Evolution 16, 111120. I., Bidin, M., Bidin, Z., Mikec, M., 2009. Viral agents associated with Lojkic, poult enteritis in croatian commercial turkey ocks. Acta Veterinaria 79, 9198. Otto, P., Liebler-Tenorio, E.M., Elschner, M., Reetz, J., Lohren, U., Dille, R., 2006. Detection of rotaviruses and intestinal lesions in broiler chicks from ocks with runting and stunting syndrome (RSS). Avian Diseases 50, 411418. Otto, P.H., Ahmed, M.U., Hotzel, H., Machnowska, P., Reetz, J., Roth, B., Trojnar, E., Johne, R., 2011. Detection of avian rotaviruses of groups A, D, F and G in diseased chickens and turkeys from Europe and Bangladesh. Veterinary Microbiology 156, 815. Pantin-Jackwood, M.J., Day, J.M., Jackwood, M.W., Spackman, E., 2008. Enteric viruses detected by molecular methods in commercial chicken and turkey ocks in the United States between 2005 and 2006. Avian Diseases 52, 235244. Pereira, H.G., Azeredo, R.S., Leite, J.P., Barth, O.M., Sutmoller, F., De Farias, V., Vidal, M.N., 1983. Comparison of polyacrilamide gel electrophoresis (PAGE), immuno-electron microscopy (IEM) and enzyme immunoassay (EIA) for the rapid diagnosis of rotavirus infection in children. Memrias do Instituto Oswaldo Cruz 78, 483490. Ramig, R.F., Ciarlet, M., Mertens, P.P.C., Dermody, T.S., 2005. Genus rotavirus. In: Fauquet, C.M., Mayo, M.A., Maniloff, J., Desselberger, U., Ball, L.A. (Eds.), Virus Taxonomy. Eighth report of the ICTV. Elsevier Academic Press, Amsterdam, Netherlands, pp. 484496. Reynolds, D.L., Saif, Y.M., Theil, K.W., 1987. A survey of enteric viruses of turkey poults. Avian Diseases 31, 8998. Savita. Kusumakar, A.L., Malik, Y.P.S., Minakshi Prasad, G., 2008. Detection and characterization of group A and D avian rotaviruses in India. Indian Journal of Biotechnology 7, 554556. Schuman, T., Hotzel, H., Otto, P., Johne, R., 2009. Evidence of interspecies transmission and reassortment among avian group A rotaviruses. Virology 386, 334343. Tamehiro, C.Y., Aleri, A.F., Medici, C., Aeri, A.A., 2003. Segmented double-stranded genomic RNA viruses in fecal samples from broiler chicken. Brazilian Journal of Microbiology 34, 344348. Trojnar, E., Otto, P., Johne, R., 2009. The rst complete genome sequence of a chicken group A rotavirus indicates independent evolution of mammalian and avian strains. Virology 386, 325333. Trojnar, E., Otto, P., Roth, B., Reetz, J., Johne, E., 2010. The genome segments of a group D rotavirus possess group A-like conserved termini but encode group-specic proteins. Journal of Virology 84, 1025410265. Ursu, K., Papp, H., KIsfali, P., Rig, D., Melegh, B., Martella, V., Bnyai, K., 2011. Monitoring of group A rotaviruses in wild-living birds in Hungary. Avian Diseases 55, 123127. Villarreal, L.Y.B., Uliana, G., Valenzuela, C., Chcon, J.L.V., Saindenberg, A.B.S., Sanches, A.A., Brando, P.E., Jerez, J.A., Ferreira, A.J.P., 2006. Rotavirus detection and isolation from chickens with or without symptoms. Brazilian Journal of Poultry Science 3, 187191. Yegani, M., Korve, D.R., 2008. Factors affecting intestinal health in poultry. Poultry Science 87, 20522063.

Based on the description of the complete sequence of the RVs-D by Trojnar et al. (2010), it was possible to develop new techniques for the molecular detection of RVs-D, such as RT-PCR and real-time PCR (Otto et al., 2011). The present analysis was based essentially on the use of PAGE and RT-PCR for detection of RVs-D and showed that PAGE has a low sensitivity to these rotaviruses, since only 30% of the samples had typical avian RNA proles, whereas RT-PCR had a 53% positivity rate. These results demonstrate that 23% (7/30) of samples could not be diagnosed by PAGE, suggesting the potential advantage of RTPCR. Of note, studies on rotavirus group A have shown divergence between the results obtained by PAGE and RT-PCR (Otto et al., 2006). To our knowledge, the use of RT-PCR has only been reported to detect RVs-D in one study (Otto et al., 2011). However, a direct comparison in terms of sensitivity between our data and those from the latter authors seems difcult: while we were able to detect RVsD by PCR up to 5 104 ng/L of the cloned product, Otto et al. (2011) reported having detected RVs-D strains up to the limit of 102 dilution of the RVs-D strains. The RT-PCR technique proposed by us enables the detection of more samples, providing representative data on the prevalence of pathogenic RVs-D in broiler chickens. This will improve the knowledge on the infection by this rotavirus group. 5. Conclusions The results using RT-PCR suggests that it is highly sensitive because amplication could be observed up to a 5 104 ng/L dilution of the cloned product. This method appears to be more sensitive than PAGE for detection of RVs-D and did not amplify RVs-D-negative samples containing other enteric viral agents and this suggests a high specicity. Acknowledgements This study was partially supported by a grant from Par State Secretary of Science and Technology, contract no. 067/2008 (SEDECT/FAPESPA/PA). Delana A.M. Bezerra and Jane H.L. Kaiano received a Grant fellowship from the Brazilian National Council for the Development of Science and Technology (CNPq). We gratefully acknowledge the valuable technical support provided by Mrs. Euzeni Maria de Ftima Menezes. References
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