Occurrence and Characterization of Rotavirus A in Broilers, Layers, and Broiler Breeders from Brazilian Poultry Farms Author(s): L. A. R. Beserra, B. R. P. Barbosa, N. T. C. G. Bernardes, P. E. Brandão, and F. Gregori Source: Avian Diseases, 58(1):153-157. 2014. Published By: American Association of Avian Pathologists DOI: http://dx.doi.org/10.1637/10626-080513-ResNote.1 URL: http://www.bioone.org/doi/full/10.1637/10626-080513-ResNote.1
BioOne (www.bioone.org) is a nonprofit, online aggregation of core research in the biological, ecological, and environmental sciences. BioOne provides a sustainable online platform for over 170 journals and books published by nonprofit societies, associations, museums, institutions, and presses. Your use of this PDF, the BioOne Web site, and all posted and associated content indicates your acceptance of BioOne’s Terms of Use, available at www.bioone.org/page/terms_of_use. Usage of BioOne content is strictly limited to personal, educational, and non-commercial use. Commercial inquiries or rights and permissions requests should be directed to the individual publisher as copyright holder.
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AVIAN DISEASES 58:153–157, 2014
Research Note— Occurrence and Characterization of Rotavirus A in Broilers, Layers, and Broiler Breeders from Brazilian Poultry Farms L. A. R. Beserra,A B. R. P. Barbosa, N. T. C. G. Bernardes, P. E. Branda˜o, and F. Gregori Department of Preventive Veterinary Medicine and Animal Health, College of Veterinary Medicine, University of Sa˜o Paulo, Av. Professor Dr. Orlando Marques de Paiva, 87, 05508-270 Sa˜o Paulo, Brazil Received 5 August 2013; Accepted 30 September 2013; Published ahead of print 1 October 2013 SUMMARY. Rotaviruses are a major cause of diarrhea in humans and animals, including several mammalian and avian species. Using different PCR protocols, we report the occurrence of rotavirus A in 21 (53.84%; 21/39) from 39 fecal pool samples of broilers, layers, and broiler breeders from Brazilian avian farms. We typed the G5, G8, G11, G19, and P[31] genotypes. RESUMEN. Nota de Investigacio´n—Presentacio´n y caracterizacio´n de rotavirus A en pollos de engorde, gallinas de postura y en reproductores pesados. Los rotavirus son una causa importante de diarrea en los seres humanos y animales, incluyendo varias especies de mamı´feros y aves. Mediante el uso de diferentes protocolos de PCR, se reporta la presentacio´n de rotavirus A en 21 (53.84%, 21/39) de 39 muestras fecales agrupadas de pollos de engorde, ponedoras, reproductoras pesados de granjas avı´colas brasilen˜as. Se detectaron los genotipos G5, G8, G11, G19 y P [31]. Key words: rotavirus, avian, group A, genotypes, PCR Abbreviations: bp 5 base pair; NSP 5 nonstructural protein; RT 5 reverse transcriptase; VP 5 viral protein
Rotaviruses are a major cause of diarrhea in humans and several animal species (3,5). Among avian species, rotavirus has been isolated in chickens, turkeys, pigeons, ducks, and pheasants, with or without symptoms (16). Rotaviruses belong to the family Reoviridae, subfamily Sedoreovirinae, and genus Rotavirus (11) and have a nonenveloped viral capsid containing a genome of 11 double-stranded RNA segments that encode six structural viral proteins (VP1–VP4, VP6, and VP7) and six nonstructural proteins (NSP1–NSP6) (6). On the basis of antibody reactivity or genetic sequencing of VP6, rotaviruses have been classified into seven groups: group A to group G (6,17); however, in 2012, Matthijnssens et al. proposed the existence of a novel group H that was originally designated as ADRV-N (new adult diarrhea rotavirus) (15). According to the antigenic and genetic characteristics of VP7 and VP4, which are important markers for vaccine development and interspecies transmission studies (10,18), group A rotaviruses are classified in genotypes G (glycoprotein) and P (protease-sensitive), respectively (14). Rotaviruses are classified into at least 29 G and 37 P genotypes (13,23). In Brazil, there have been reports of avian rotavirus occurrence since 1989 (1), with frequency ranging from 8.5% (32/378 samples) in broilers (20) to 45.3% (58/128 samples) from intestinal contents of layers and broilers by polyacrylamide gel electrophoresis (24), but no data on group characterization were provided. Later, group A rotavirus was detected in six fecal samples from ostriches (19) and a typical bovine rotavirus genotype G6P(1) found on commercial turkey farms (2). In addition, in broilers, group D rotavirus was previously detected in the northern Brazilian region by a specific reverse transcriptase–polymerase chain reaction (RT-PCR) assay, with 53% of occurrence (16/30 samples) (4).
Here we report the occurrence and characterization of genotypes G and P of group A rotavirus in the feces of broilers, layers, and broiler breeders from Brazilian poultry farms. MATERIALS AND METHODS Samples. A total of 39 pools of intestinal contents were used in this study. Each pool consisted of enteric contents of three to five birds within a single batch, collected between 2008 and 2012. Samples were collected across five Brazilian states (Sa˜o Paulo, Parana´, Santa Catarina, Rio Grande do Sul, and Goia´s) and included broilers (61.53%, 24/39), layers (23.07%, 9/39), and broiler breeders (15.38%, 6/39), without symptoms of diarrhea. This study was approved by the Committee on Ethics for Animal Trials of the School of Veterinary Medicine, University of Sa˜o Paulo, under protocol number 2236/2010. RT-PCR for genotyping VP7 and VP4. Samples were prepared as 40% (v/v) suspensions in diethylpyrocarbonate-treated water and clarified at 12,000 3 g for 15 min at 4 C; the resultant supernatants Table 1. VP7 nucleotide sequences of group A avian rotavirus, recovered from GenBank, used for designing the primers, according to accession number, genotype, and host. Accession no.
Genotype
Host
FN393054 EU486973 S58166 AB080737 AB080738 JQ085407 EU486977 EU486975 X56784 FJ169861 D82979
G23 G22 G17 G7 G19 G19 G19 G19 G7 G19 G18
Pheasant Turkey Turkey Turkey Chicken Chicken Chicken Chicken Chicken Chicken Pigeon
A
Corresponding author. E-mail: [email protected]
153
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Fig. 1. Nucleotide neighbor-joining distance tree (maximum composite likelihood substitution model) for the partial VP7 rotavirus gene showing the known genotypes. Strains detected in the present study are preceded by black triangles. The numbers at each node are bootstrap values greater than 70% from 1000 replicates. The bar represents the number of substitutions per site. were used for RT-PCR. Total RNA was extracted using TRIzolH reagent (Invitrogen, Carlsbad, CA), and cDNA was synthesized using random primers (Invitrogen) and M-MLV Reverse Transcriptase (Invitrogen) as described by the manufacturer. Rotavirus genotyping based on VP7 (G type) was carried out with a nested multiplex PCR described by Gouvea et al. (7). The negative samples were further screened/genotyped by PCR followed by nucleotide sequencing of the generated amplicons, using
two consensual primers (forward primer VP7AVEFW 59-TGTATAGTACTGARTGTACTATCCTT-39 and reverse primer VP7ANYRW 59-TGCCACCAYYTYTTCC-39) designed in this study, which were used to amplify an 863-base pair (bp) fragment. The PCR conditions were as follows: initial denaturation at 94 C/3 min, followed by 30 amplification cycles (94 C/45 s, 50 C/30 s, and 72 C/45 s), and a final extension at 72 C/10 min. For primer design, VP7 nucleotide sequences
Occurrence of rotavirus A in Brazil
155
Fig. 2. Nucleotide neighbor-joining distance tree (maximum composite likelihood substitution model) for the partial VP4 rotavirus gene showing the known genotypes. Strains detected in the present study are preceded by black triangles. The numbers at each node are bootstrap values greater than 70% from 1000 replicates. The bar represents the number of substitutions per site.
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Table 2. Positive samples, according to ID, genotype (VP7 and/or VP4), avian farm, and Brazilian state. The samples with ‘‘P’’ were positive for VP4, but their genotypes were not defined. Positive samples ID
Genotype (VP7)
Genotype (VP4)
1 3 4 7 12 15 28 41 46 53 55 57 64 65 76 77 79 80 84 103 106 Total (%): 21/39 (53.84)
— — — G19 G5 G8 — G8 — G11 G8 — — G19 G8 G8 G8 — — — — 10/21 (47.61)
P[31] P P P[31] — — P — P[31] — — P P P[31] — — — P P P[31] P[31] 13/21 (61.90)
of group A avian rotaviruses were imported from GenBank (Table 1), aligned with Clustal W software version 2.1 (12), and selected from conserved areas, using the software Bioedit (9). The biochemical features—melting temperature (Tm), hairpins, dimers, and repeats— were calculated with Netprimer software (PREMIER Biosoft, Palo Alto, CA) and submitted to BLAST in order to ensure the rotavirus specificity. The VP4 genotyping was carried out on the basis of the nucleotide sequencing of the first round of PCR amplicons, by using the primers CON3 and CON2 (8), as follows: cDNA was added to the PCR mix with 13 PCR Buffer (Invitrogen), 0.2 mM of each DNTP, 0.5 mM of each primer (CON3 and CON2), 2 mM MgCl2, 1.25 U of Taq DNA Polymerase (Invitrogen), and 12.625 ml of ultrapure water, in a 25 ml final reaction volume and submitted to initial denaturation at 94 C/1.5 min; followed by 30 amplification cycles (94 C/1 min, 40 C/ 2 min, and 72 C/1 min); and a final extension at 72 C/10 min. The products of the PCR were resolved on a 1.5% agarose gel stained with 0.5 mg/ml ethidium bromide, generating an 876-bp fragment. DNA sequencing. Amplicons of VP7 and VP4 were purified with EXOSAP-it (USBH) reagent, submitted to bidirectional DNA sequencing with BigDye 3.1 (Applied Biosystems, Foster City, CA), and resolved in an ABI-3500 Genetic Analyser (Applied Biosystems), according to the respective manufacturer’s instructions. The sequences from each sample were aligned with homologous sequences representing different rotavirus genotypes retrieved from GenBank with CLUSTAL/ W 2.1 (12); then a phylogenetic tree was generated with the neighborjoining distance algorithm and the maximum composite likelihood model with 1000 bootstrap replicates using MEGA 5.1 (21). Deduced amino acid identities of the generated sequences were calculated with Bioedit 7.1.3.0 (9) software.
Avian farm
Layer Broiler Broiler Broiler Layer Broiler Layer Layer Broiler Broiler Broiler Broiler Broiler Broiler Layer Layer Layer Layer Broiler Broiler Broiler
Breeder
Breeder Breeder Breeder
State
Parana´ Parana´ Parana´ Parana´ Rio Grande do Sul Goia´s Parana´ Parana´ Parana´ Sa˜o Paulo Rio Grande do Sul Rio Grande do Sul Sa˜o Paulo Parana´ Sa˜o Paulo Sa˜o Paulo Sa˜o Paulo Sa˜o Paulo Sa˜o Paulo Sa˜o Paulo Sa˜o Paulo
nucleotide sequencing data (Fig. 1). Regarding VP4 genotyping, a total of 13 samples (13/39; 33.33%) presented a specific first-round amplification band on an agarose gel (8); from these, only 6 (15.38%; 6/39) could be defined as P[31] (Fig. 2). Two samples (ID 7 and 65) (Table 2) were positive in both PCRs, VP7 and VP4, with primers designed herein and those described by Gouvea et al. (8), respectively. This way, was defined simultaneously the G19 and P[31] genotypes in these 2 samples. Nucleotide and deduced amino acid identities and phylogenetic tree. Comparison of the VP7 gene’s nucleotide and predicted amino acid sequences obtained from our study to those already deposited in GenBank revealed that they are more closely related to the avian rotavirus AvRV2 strain (accession number JQ085407): 93.7% in terms of nucleotides and 97.7% with amino acids. With regard to the samples of the VP4 gene, they shared maximum identities with the avian rotavirus Ch06V0661 strain (accession number EU486962): 89.3% in terms of nucleotides and 94.5% with amino acids. The phylogenetic tree depicted that the sequences of the VP7 and VP4 genes clustered with the G19 and P[31] genotypes, respectively (Figs. 1 and 2). Accession numbers. The nucleotide sequences of VP7 and VP4 genes of group A rotavirus from this study have been deposited in GenBank under the accession numbers KC962122 and KC962123 for VP7 and KC962114 to KC962121 for VP4. DISCUSSION
RESULTS
Frequency of rotavirus occurrence and genotypes. Using the different aforementioned PCR protocols and generating a specific amplicon, we detected 21 rotavirus samples (53.84%; 21/39) overall. Out of 39 pools of fecal samples tested, we characterized the G5 (2.56%; 1/39), G8 (15.38; 6/39), and G11 (2.56%; 1/39) genotypes by nested multiplex PCR (7). Additionally, another 2 (5.12%; 2/39) samples were further detected by PCR with primers designed herein, and their genotype was G19, as defined by genetic analysis from
The primers described by Govea et al. (7) amplify the G5, G6, G8, G10 and G11 genotypes. The G5, G8, and G11 detected in this study have been identified in bovine (G8) and porcine (G5 and G11) strains, while the genotypes described in avian species are G7, G17, and G22 in turkeys, G18 in pigeons, G19 in chickens, and G23 in pheasants (13), which may suggest interspecies transmission. In fact, interspecies transmission of rotavirus has been described in Brazil from avian samples presenting a typical bovine rotavirus G6P(1) genotype from turkey fecal samples (2) as well as the combination of porcine and bovine rotavirus P(1)P(7), G6P(1), and G10P(1) genotypes
157
Occurrence of rotavirus A in Brazil
found in fecal samples of ostriches (19). More recently, the G9 genotype, generally found in humans, was detected in pigs (22). The VP7 partial nucleotide sequences (accession numbers KC912122 and KC912123) generated with primers designed herein, showed 93.7% nucleotide identity and 96.6% amino acid identity between them. These samples were collected in the same broiler farm of Parana´ State, Brazil, but in different years, which suggests the circulation of the different strains in the region throughout the time. The phylogenetic tree of VP7 nucleotide sequences (Fig. 1) showed that the strains segregated with G19 genotype, inside a subcluster having only avian strain representatives. The phylogenetic tree of VP4 nucleotide sequences (Fig. 2) showed that all Brazilian samples clustered together in P[31], indicating that it belonged to a clade composed mostly of avian samples, except the strain 993/83 (accession number D16352), which came from calves with diarrhea, in Germany (5). In both VP7 and VP4 phylogenetic trees, the topology was maintained by genotype segregation, and notably, the avian clades were supported by high bootstrap values. Here we describe the combination of G19 and P[31] genotypes in two samples (accession numbers KC962122/KC962118 and KC962123/KC962119), and to our knowledge, this is the first report of the G19P[31] genotype from broilers in Brazil. This same genotype combination has also been described in the sample Ch661G1 (accessions numbers EU486962 and EU486977 for VP7 and VP4, respectively) from chickens without symptoms in Germany (18). Despite these reports, the current information on the pathogenicity of avian rotavirus genotypes is limited. In conclusion, group A rotavirus can be found in commercial poultry farms in Brazil, where they exist as common avian genotypes; however, others have also been described in mammalian hosts. REFERENCES 1. Alfieri, A. F., A. A. Alfieiri, M. Resende, and J. S. Resende. Detection and propagation of avian enteric reovirus in chicken. Arq. Bras. Med. Vet. Zootec. 41:493–501. 1989. 2. Asano, K. M., F. Gregori, S. P. Souza, D. Rotava, R. N. Oliveira, L. Y. Villarreal, L. J. Richtzenhain, and P. E. Branda˜o. Bovine rotavirus in turkeys with enteritis. Avian Dis. 55:697–700. 2011. 3. Bergeland, M. E., J. P. Mcadaragh, and J. Stotz. Rotaviral enteritis in turkey poults. In: Proc. 26th Western Poultry Disease Conference. University of California, Davis. pp. 129–130. 1977. 4. Bezerra, D. A., R. R. Silva, J. H. Kaiano, R. V. Silvestre, D. S. Oliveira, A. C. Linhares, Y. B. Gabbay, and J. D. Mascarenhas. Detection of avian group D rotavirus using the polymerase chain reaction for the VP6 gene. J. Virol. Methods 185:189–192. 2012. 5. Brussow, H., O. Nakagomi, G. Gerna, and W. Eichhorn. Isolation of an avian like group A rotavirus from a calf with diarrhea. J. Clin. Microbiol. 30:67–73. 1992. 6. Estes, M. K., and A. Z. Kapikian. Rotaviruses. In: Fields virology, 5th ed. D. M. Knipe, and P. M. Howley, eds. Lippincott, Williams and Wilkins, Philadelphia. pp. 1917–1974. 2007. 7. Gouvea, V., N. Santos, and M. C. Timenetsky. Identification of bovine and porcine rotavirus G types by PCR. J. Clin. Microbiol. 32:1338–1340. 1994. 8. Gouvea, V., N. Santos, and M. C. Timenetsky. VP4 typing of bovine and porcine group A rotaviruses by PCR. J. Clin. Microbiol. 32:1333–1337. 1994.
9. Hall, T. A., and Bioedit. A user-friendly biological sequence alignment editor and analysis program for windows 95/98/nt. Nucleic Acids. Symp. Series 41:95–98. 1999. 10. Hoshino, Y., and A. Z. Kapikian. Rotavirus serotypes: classification and importance in epidemiology, immunity, and vaccine development. J. Health Pop. Nutr. 18:5–14. 2000. 11. King, A. M. Q., M. J. Adams, E. B. Carstens, and E. J. Lefkowitz, eds. Virus taxonomy: classification and nomenclature of viruses, 9th ed. Elsevier Academic Press, San Diego. 2012. 12. Larkin, M. A., G. Blackshields, N. P. Brown, R. Chenna, P. A. Mcgettigan, H. Mcwilliam, F. Valentin, I. M. Wallace, A. Wilm, R. Lopez, J. D. Thompson, T. J. Gibson, and D. G. Higgins. Clustal W and Clustal X version 2.1. Bioinformatics 23:2947–2948. 2007. 13. Matthijnssens, J., M. Ciarlet, S. M. McDonald, H. Attoui, K. Ba´nyai, J. R. Brister, J. Buesa, M. D. Esona, and M. K. Estes. Uniformity of rotavirus strain nomenclature proposed by the Rotavirus Classification Working Group (RCWG). Arch. Virol. 156:1397–1413. 2011. 14. Matthijnssens, J., M. Ciarlet, M. Rahman, H. Attoui, K. Ba´nyai, M. K. Estes, J. R. Gentsch, M. Iturriza-Go´mara, C. D. Kirkwood, V. Martella, P. P. Mertens, O. Nakagomi, J. T. Patton, F. M. Ruggeri, L. J. Saif, N. Santos, A. Steyer, K. Taniguchi, U. Desselberger, and M. Van Ranst. Recommendations for the classification of group A rotaviruses using all 11 genomic RNA segments. Arch. Virol. 153:1621–1629. 2008. 15. Matthijnssens, J., P. Otto, M. Ciarlet, U. Desselberger, M. Van Ranst, and R. Johne. VP6 sequence-based cut-off values as a criterion for rotavirus species demarcation. Arch. Virol. 157:1177–1182. 2012. 16. McNulty, M. S. Rotavirus infections. In: Diseases of poultry, 11th ed. Y. M. Saif, H. J. Barnes, J. R. Glisson, A. M. Fadly, L. R. McDougald, and D. E. Swayne, eds. Iowa State University Press, Ames, Iowa. pp. 308–317. 2003. 17. Ramig, R. F., M. Ciarlet, P. P. C. Mertens, and T. S Dermody. Genus Rotavirus. In: Virus taxonomy. Eighth report of the ICTV. C. M. Fauquet, M. A. Mayo, J. Maniloff, U. Desselberger, and L. A. Ball, eds. Elsevier, Amsterdam, the Netherlands. pp. 484–496. 2005. 18. Schumann, T., H. Hotzel, P. Otto, and R. Johne. Evidence of interspecies transmission and reassortment among avian group A rotaviruses. Virology 386:334–343. 2009. 19. Silva, L. C., A. A. Sanche, F. Gregori, P. E. Branda˜o, A. A. Alfieri, S. A. Headley, and J. A. Jerez. First description of group A rotavirus from fecal samples of ostriches (Struthio camelus). Res. Vet. Sci. 93:1066–1069. 2012. 20. Tamehiro, C. Y., A. F. Alfieri, K. C. Medici, and A. A. Alfieri. Segmented double-stranded genomic RNA viruses in fecal samples from broiler chicken. Braz. J. Microbiol. 34:349–353. 2003. 21. Tamura, K., D. Peterson, N. Peterson, G. Stecher, M. Neu, and S. Kumar. Mega5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol. Biol. Evol. 28:2731–2739. 2011. 22. Tonietti, P. O., A. S. Hora, F. D. F. Silva, V. L. A. Ruiz, and F. Gregori. Phylogenetic analyses of the VP4 and VP7 genes of porcine group A rotaviruses in Sa˜o Paulo State, Brazil: first identification of G5P[23] in piglets. J. Clin. Microbiol. 51(8):2750–2753. 2013. 23. Trojnar, E., J. Sachsenro¨der, S. Twardziok, R. Jochen, P. H. Otto, and R. Johne. Identification of an avian group A rotavirus containing a novel VP4 gene with a close relationship to those of mammalian rotaviruses. J. Gen. Virol. 94:136–142. 2013. 24. Villarreal, L. Y. B., G. Uliana, C. Valenzuela, J. L. V. Chaco´n, A. B. S. Saidenberg, A. A. Sanches, P. E. Branda˜o, J. A. Jerez, and A. J. P. Ferreira. Rotavirus detection and isolation from chickens with or without symptoms. Braz. J. Poult. Sci. 8:187–191. 2006.
ACKNOWLEDGMENT We are grateful to Fundac¸a˜o de Amparo a` Pesquisa de Sa˜o Paulo for financial support for this study (Grant No. 2011/10244-9).
BioOne (www.bioone.org) is a nonprofit, online aggregation of core research in the biological, ecological, and environmental sciences. BioOne provides a sustainable online platform for over 170 journals and books published by nonprofit societies, associations, museums, institutions, and presses. Your use of this PDF, the BioOne Web site, and all posted and associated content indicates your acceptance of BioOne’s Terms of Use, available at www.bioone.org/page/terms_of_use. Usage of BioOne content is strictly limited to personal, educational, and non-commercial use. Commercial inquiries or rights and permissions requests should be directed to the individual publisher as copyright holder.
BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors, nonprofit publishers, academic institutions, research libraries, and research funders in the common goal of maximizing access to critical research.
AVIAN DISEASES 58:153–157, 2014
Research Note— Occurrence and Characterization of Rotavirus A in Broilers, Layers, and Broiler Breeders from Brazilian Poultry Farms L. A. R. Beserra,A B. R. P. Barbosa, N. T. C. G. Bernardes, P. E. Branda˜o, and F. Gregori Department of Preventive Veterinary Medicine and Animal Health, College of Veterinary Medicine, University of Sa˜o Paulo, Av. Professor Dr. Orlando Marques de Paiva, 87, 05508-270 Sa˜o Paulo, Brazil Received 5 August 2013; Accepted 30 September 2013; Published ahead of print 1 October 2013 SUMMARY. Rotaviruses are a major cause of diarrhea in humans and animals, including several mammalian and avian species. Using different PCR protocols, we report the occurrence of rotavirus A in 21 (53.84%; 21/39) from 39 fecal pool samples of broilers, layers, and broiler breeders from Brazilian avian farms. We typed the G5, G8, G11, G19, and P[31] genotypes. RESUMEN. Nota de Investigacio´n—Presentacio´n y caracterizacio´n de rotavirus A en pollos de engorde, gallinas de postura y en reproductores pesados. Los rotavirus son una causa importante de diarrea en los seres humanos y animales, incluyendo varias especies de mamı´feros y aves. Mediante el uso de diferentes protocolos de PCR, se reporta la presentacio´n de rotavirus A en 21 (53.84%, 21/39) de 39 muestras fecales agrupadas de pollos de engorde, ponedoras, reproductoras pesados de granjas avı´colas brasilen˜as. Se detectaron los genotipos G5, G8, G11, G19 y P [31]. Key words: rotavirus, avian, group A, genotypes, PCR Abbreviations: bp 5 base pair; NSP 5 nonstructural protein; RT 5 reverse transcriptase; VP 5 viral protein
Rotaviruses are a major cause of diarrhea in humans and several animal species (3,5). Among avian species, rotavirus has been isolated in chickens, turkeys, pigeons, ducks, and pheasants, with or without symptoms (16). Rotaviruses belong to the family Reoviridae, subfamily Sedoreovirinae, and genus Rotavirus (11) and have a nonenveloped viral capsid containing a genome of 11 double-stranded RNA segments that encode six structural viral proteins (VP1–VP4, VP6, and VP7) and six nonstructural proteins (NSP1–NSP6) (6). On the basis of antibody reactivity or genetic sequencing of VP6, rotaviruses have been classified into seven groups: group A to group G (6,17); however, in 2012, Matthijnssens et al. proposed the existence of a novel group H that was originally designated as ADRV-N (new adult diarrhea rotavirus) (15). According to the antigenic and genetic characteristics of VP7 and VP4, which are important markers for vaccine development and interspecies transmission studies (10,18), group A rotaviruses are classified in genotypes G (glycoprotein) and P (protease-sensitive), respectively (14). Rotaviruses are classified into at least 29 G and 37 P genotypes (13,23). In Brazil, there have been reports of avian rotavirus occurrence since 1989 (1), with frequency ranging from 8.5% (32/378 samples) in broilers (20) to 45.3% (58/128 samples) from intestinal contents of layers and broilers by polyacrylamide gel electrophoresis (24), but no data on group characterization were provided. Later, group A rotavirus was detected in six fecal samples from ostriches (19) and a typical bovine rotavirus genotype G6P(1) found on commercial turkey farms (2). In addition, in broilers, group D rotavirus was previously detected in the northern Brazilian region by a specific reverse transcriptase–polymerase chain reaction (RT-PCR) assay, with 53% of occurrence (16/30 samples) (4).
Here we report the occurrence and characterization of genotypes G and P of group A rotavirus in the feces of broilers, layers, and broiler breeders from Brazilian poultry farms. MATERIALS AND METHODS Samples. A total of 39 pools of intestinal contents were used in this study. Each pool consisted of enteric contents of three to five birds within a single batch, collected between 2008 and 2012. Samples were collected across five Brazilian states (Sa˜o Paulo, Parana´, Santa Catarina, Rio Grande do Sul, and Goia´s) and included broilers (61.53%, 24/39), layers (23.07%, 9/39), and broiler breeders (15.38%, 6/39), without symptoms of diarrhea. This study was approved by the Committee on Ethics for Animal Trials of the School of Veterinary Medicine, University of Sa˜o Paulo, under protocol number 2236/2010. RT-PCR for genotyping VP7 and VP4. Samples were prepared as 40% (v/v) suspensions in diethylpyrocarbonate-treated water and clarified at 12,000 3 g for 15 min at 4 C; the resultant supernatants Table 1. VP7 nucleotide sequences of group A avian rotavirus, recovered from GenBank, used for designing the primers, according to accession number, genotype, and host. Accession no.
Genotype
Host
FN393054 EU486973 S58166 AB080737 AB080738 JQ085407 EU486977 EU486975 X56784 FJ169861 D82979
G23 G22 G17 G7 G19 G19 G19 G19 G7 G19 G18
Pheasant Turkey Turkey Turkey Chicken Chicken Chicken Chicken Chicken Chicken Pigeon
A
Corresponding author. E-mail: [email protected]
153
154
L. A. R. Beserra et al.
Fig. 1. Nucleotide neighbor-joining distance tree (maximum composite likelihood substitution model) for the partial VP7 rotavirus gene showing the known genotypes. Strains detected in the present study are preceded by black triangles. The numbers at each node are bootstrap values greater than 70% from 1000 replicates. The bar represents the number of substitutions per site. were used for RT-PCR. Total RNA was extracted using TRIzolH reagent (Invitrogen, Carlsbad, CA), and cDNA was synthesized using random primers (Invitrogen) and M-MLV Reverse Transcriptase (Invitrogen) as described by the manufacturer. Rotavirus genotyping based on VP7 (G type) was carried out with a nested multiplex PCR described by Gouvea et al. (7). The negative samples were further screened/genotyped by PCR followed by nucleotide sequencing of the generated amplicons, using
two consensual primers (forward primer VP7AVEFW 59-TGTATAGTACTGARTGTACTATCCTT-39 and reverse primer VP7ANYRW 59-TGCCACCAYYTYTTCC-39) designed in this study, which were used to amplify an 863-base pair (bp) fragment. The PCR conditions were as follows: initial denaturation at 94 C/3 min, followed by 30 amplification cycles (94 C/45 s, 50 C/30 s, and 72 C/45 s), and a final extension at 72 C/10 min. For primer design, VP7 nucleotide sequences
Occurrence of rotavirus A in Brazil
155
Fig. 2. Nucleotide neighbor-joining distance tree (maximum composite likelihood substitution model) for the partial VP4 rotavirus gene showing the known genotypes. Strains detected in the present study are preceded by black triangles. The numbers at each node are bootstrap values greater than 70% from 1000 replicates. The bar represents the number of substitutions per site.
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Table 2. Positive samples, according to ID, genotype (VP7 and/or VP4), avian farm, and Brazilian state. The samples with ‘‘P’’ were positive for VP4, but their genotypes were not defined. Positive samples ID
Genotype (VP7)
Genotype (VP4)
1 3 4 7 12 15 28 41 46 53 55 57 64 65 76 77 79 80 84 103 106 Total (%): 21/39 (53.84)
— — — G19 G5 G8 — G8 — G11 G8 — — G19 G8 G8 G8 — — — — 10/21 (47.61)
P[31] P P P[31] — — P — P[31] — — P P P[31] — — — P P P[31] P[31] 13/21 (61.90)
of group A avian rotaviruses were imported from GenBank (Table 1), aligned with Clustal W software version 2.1 (12), and selected from conserved areas, using the software Bioedit (9). The biochemical features—melting temperature (Tm), hairpins, dimers, and repeats— were calculated with Netprimer software (PREMIER Biosoft, Palo Alto, CA) and submitted to BLAST in order to ensure the rotavirus specificity. The VP4 genotyping was carried out on the basis of the nucleotide sequencing of the first round of PCR amplicons, by using the primers CON3 and CON2 (8), as follows: cDNA was added to the PCR mix with 13 PCR Buffer (Invitrogen), 0.2 mM of each DNTP, 0.5 mM of each primer (CON3 and CON2), 2 mM MgCl2, 1.25 U of Taq DNA Polymerase (Invitrogen), and 12.625 ml of ultrapure water, in a 25 ml final reaction volume and submitted to initial denaturation at 94 C/1.5 min; followed by 30 amplification cycles (94 C/1 min, 40 C/ 2 min, and 72 C/1 min); and a final extension at 72 C/10 min. The products of the PCR were resolved on a 1.5% agarose gel stained with 0.5 mg/ml ethidium bromide, generating an 876-bp fragment. DNA sequencing. Amplicons of VP7 and VP4 were purified with EXOSAP-it (USBH) reagent, submitted to bidirectional DNA sequencing with BigDye 3.1 (Applied Biosystems, Foster City, CA), and resolved in an ABI-3500 Genetic Analyser (Applied Biosystems), according to the respective manufacturer’s instructions. The sequences from each sample were aligned with homologous sequences representing different rotavirus genotypes retrieved from GenBank with CLUSTAL/ W 2.1 (12); then a phylogenetic tree was generated with the neighborjoining distance algorithm and the maximum composite likelihood model with 1000 bootstrap replicates using MEGA 5.1 (21). Deduced amino acid identities of the generated sequences were calculated with Bioedit 7.1.3.0 (9) software.
Avian farm
Layer Broiler Broiler Broiler Layer Broiler Layer Layer Broiler Broiler Broiler Broiler Broiler Broiler Layer Layer Layer Layer Broiler Broiler Broiler
Breeder
Breeder Breeder Breeder
State
Parana´ Parana´ Parana´ Parana´ Rio Grande do Sul Goia´s Parana´ Parana´ Parana´ Sa˜o Paulo Rio Grande do Sul Rio Grande do Sul Sa˜o Paulo Parana´ Sa˜o Paulo Sa˜o Paulo Sa˜o Paulo Sa˜o Paulo Sa˜o Paulo Sa˜o Paulo Sa˜o Paulo
nucleotide sequencing data (Fig. 1). Regarding VP4 genotyping, a total of 13 samples (13/39; 33.33%) presented a specific first-round amplification band on an agarose gel (8); from these, only 6 (15.38%; 6/39) could be defined as P[31] (Fig. 2). Two samples (ID 7 and 65) (Table 2) were positive in both PCRs, VP7 and VP4, with primers designed herein and those described by Gouvea et al. (8), respectively. This way, was defined simultaneously the G19 and P[31] genotypes in these 2 samples. Nucleotide and deduced amino acid identities and phylogenetic tree. Comparison of the VP7 gene’s nucleotide and predicted amino acid sequences obtained from our study to those already deposited in GenBank revealed that they are more closely related to the avian rotavirus AvRV2 strain (accession number JQ085407): 93.7% in terms of nucleotides and 97.7% with amino acids. With regard to the samples of the VP4 gene, they shared maximum identities with the avian rotavirus Ch06V0661 strain (accession number EU486962): 89.3% in terms of nucleotides and 94.5% with amino acids. The phylogenetic tree depicted that the sequences of the VP7 and VP4 genes clustered with the G19 and P[31] genotypes, respectively (Figs. 1 and 2). Accession numbers. The nucleotide sequences of VP7 and VP4 genes of group A rotavirus from this study have been deposited in GenBank under the accession numbers KC962122 and KC962123 for VP7 and KC962114 to KC962121 for VP4. DISCUSSION
RESULTS
Frequency of rotavirus occurrence and genotypes. Using the different aforementioned PCR protocols and generating a specific amplicon, we detected 21 rotavirus samples (53.84%; 21/39) overall. Out of 39 pools of fecal samples tested, we characterized the G5 (2.56%; 1/39), G8 (15.38; 6/39), and G11 (2.56%; 1/39) genotypes by nested multiplex PCR (7). Additionally, another 2 (5.12%; 2/39) samples were further detected by PCR with primers designed herein, and their genotype was G19, as defined by genetic analysis from
The primers described by Govea et al. (7) amplify the G5, G6, G8, G10 and G11 genotypes. The G5, G8, and G11 detected in this study have been identified in bovine (G8) and porcine (G5 and G11) strains, while the genotypes described in avian species are G7, G17, and G22 in turkeys, G18 in pigeons, G19 in chickens, and G23 in pheasants (13), which may suggest interspecies transmission. In fact, interspecies transmission of rotavirus has been described in Brazil from avian samples presenting a typical bovine rotavirus G6P(1) genotype from turkey fecal samples (2) as well as the combination of porcine and bovine rotavirus P(1)P(7), G6P(1), and G10P(1) genotypes
157
Occurrence of rotavirus A in Brazil
found in fecal samples of ostriches (19). More recently, the G9 genotype, generally found in humans, was detected in pigs (22). The VP7 partial nucleotide sequences (accession numbers KC912122 and KC912123) generated with primers designed herein, showed 93.7% nucleotide identity and 96.6% amino acid identity between them. These samples were collected in the same broiler farm of Parana´ State, Brazil, but in different years, which suggests the circulation of the different strains in the region throughout the time. The phylogenetic tree of VP7 nucleotide sequences (Fig. 1) showed that the strains segregated with G19 genotype, inside a subcluster having only avian strain representatives. The phylogenetic tree of VP4 nucleotide sequences (Fig. 2) showed that all Brazilian samples clustered together in P[31], indicating that it belonged to a clade composed mostly of avian samples, except the strain 993/83 (accession number D16352), which came from calves with diarrhea, in Germany (5). In both VP7 and VP4 phylogenetic trees, the topology was maintained by genotype segregation, and notably, the avian clades were supported by high bootstrap values. Here we describe the combination of G19 and P[31] genotypes in two samples (accession numbers KC962122/KC962118 and KC962123/KC962119), and to our knowledge, this is the first report of the G19P[31] genotype from broilers in Brazil. This same genotype combination has also been described in the sample Ch661G1 (accessions numbers EU486962 and EU486977 for VP7 and VP4, respectively) from chickens without symptoms in Germany (18). Despite these reports, the current information on the pathogenicity of avian rotavirus genotypes is limited. In conclusion, group A rotavirus can be found in commercial poultry farms in Brazil, where they exist as common avian genotypes; however, others have also been described in mammalian hosts. REFERENCES 1. Alfieri, A. F., A. A. Alfieiri, M. Resende, and J. S. Resende. Detection and propagation of avian enteric reovirus in chicken. Arq. Bras. Med. Vet. Zootec. 41:493–501. 1989. 2. Asano, K. M., F. Gregori, S. P. Souza, D. Rotava, R. N. Oliveira, L. Y. Villarreal, L. J. Richtzenhain, and P. E. Branda˜o. Bovine rotavirus in turkeys with enteritis. Avian Dis. 55:697–700. 2011. 3. Bergeland, M. E., J. P. Mcadaragh, and J. Stotz. Rotaviral enteritis in turkey poults. In: Proc. 26th Western Poultry Disease Conference. University of California, Davis. pp. 129–130. 1977. 4. Bezerra, D. A., R. R. Silva, J. H. Kaiano, R. V. Silvestre, D. S. Oliveira, A. C. Linhares, Y. B. Gabbay, and J. D. Mascarenhas. Detection of avian group D rotavirus using the polymerase chain reaction for the VP6 gene. J. Virol. Methods 185:189–192. 2012. 5. Brussow, H., O. Nakagomi, G. Gerna, and W. Eichhorn. Isolation of an avian like group A rotavirus from a calf with diarrhea. J. Clin. Microbiol. 30:67–73. 1992. 6. Estes, M. K., and A. Z. Kapikian. Rotaviruses. In: Fields virology, 5th ed. D. M. Knipe, and P. M. Howley, eds. Lippincott, Williams and Wilkins, Philadelphia. pp. 1917–1974. 2007. 7. Gouvea, V., N. Santos, and M. C. Timenetsky. Identification of bovine and porcine rotavirus G types by PCR. J. Clin. Microbiol. 32:1338–1340. 1994. 8. Gouvea, V., N. Santos, and M. C. Timenetsky. VP4 typing of bovine and porcine group A rotaviruses by PCR. J. Clin. Microbiol. 32:1333–1337. 1994.
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ACKNOWLEDGMENT We are grateful to Fundac¸a˜o de Amparo a` Pesquisa de Sa˜o Paulo for financial support for this study (Grant No. 2011/10244-9).
