Description of Rotavirus F in Broilers from Brazilian Poultry Farms Author(s): L. A. R. Beserra and F. Gregori Source: Avian Diseases, 58(3):458-461. 2014. Published By: American Association of Avian Pathologists DOI: http://dx.doi.org/10.1637/10747-121613-ResNote.1 URL: http://www.bioone.org/doi/full/10.1637/10747-121613-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.
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 58:458–461, 2014
Research Note— Description of Rotavirus F in Broilers from Brazilian Poultry Farms L. A. R. BeserraA 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, SP, Brazil Received 16 December 2013; Accepted 11 March 2014; Published ahead of print 12 March 2014 SUMMARY. Rotaviruses are segmented double-stranded RNA viruses that cause gastroenteritis in mammals and birds. Here we describe the first partial nucleotide sequences of the structural protein VP6 from the genomes of group F rotaviruses that were detected in 5 out of 53 fecal samples (9.43%) from healthy broilers from Brazilian poultry farms based on reverse-transcriptase–PCR with primers designed for this study. The findings support the development of molecular detection systems, which can be used for the assessment of the distribution of rotavirus F in birds, their potential involvement in diseases, and their impact on poultry health. RESUMEN. Nota de Investigacio´n—Descripcio´n de un rotavirus F en pollos de granjas avı´colas brasilen˜as. Los rotavirus son virus de genoma de ARN segmentado y de doble cadena que causan gastroenteritis en los mamı´feros y en las aves. Aquı´ se describen las primeras secuencias de nucleo´tidos parciales de la proteı´na estructural VP6 de los genomas de rotavirus del grupo F que se detectaron en 5 de 53 muestras fecales (9.43%) en pollos de granjas avı´colas de Brasil, mediante transcriptasa reversa y PCR con iniciadores disen˜ados en este trabajo. Los resultados apoyan el desarrollo de sistemas de deteccio´n molecular, que pueden ser utilizados para la evaluacio´n de la distribucio´n de rotavirus F en las aves, su papel potencial en enfermedades y su impacto en la salud de las aves comerciales. Key words: rotavirus, avian, group F, PCR Abbreviations: NSP 5 nonstructural protein; RT 5 reverse transcriptase; VP 5 viral protein
Rotaviruses, the major etiologic agents of acute viral gastroenteritis in young animals and children worldwide, are members of the Reoviridae family, and their genome is enclosed in three concentric layers comprising 11 segments of double-stranded RNA that encode six structural proteins (VP1–VP4, VP6, and VP7) and six nonstructural proteins (NSP1–NPS5/6) (1,2,13). Based on their antigenic properties and/or nucleotide sequencing of VP6, eight groups (groups A–H) of rotaviruses have been described so far (7). In avian species, rotavirus groups A, D, F, and G have so far been detected (8,9,16). Rotavirus groups F and G have been identified from the intestinal contents of broilers and turkeys, both with or without outward symptoms by polyacrylamide gel electrophoresis and more recently by PCR and the analysis of nucleotide sequences (4,10,11,12,15). Data on rotavirus F genome sequences are scarce and to date, only one rotavirus F complete sequence is available at GenBank (5). Here, we report the occurrence of group F rotavirus in the feces of broilers from Brazilian poultry farms from eight different states. We used RT-PCR–based amplification of the VP6 gene, followed by nucleotide sequencing reaction of the amplicons. To the best of our knowledge, this is the first report of rotavirus F in Brazil. MATERIALS AND METHODS Samples. A total of 53 pooled intestinal contents were used in this study. Each pool consisted of the enteric contents of three to five birds within a single batch, collected between the years 2008 and 2012. More than one pool was obtained from each farm; farms were located in eight different Brazilian states (Ceara´, Bahia, Espı´rito Santo, Sa˜o Paulo, Parana´, Santa Catarina, Rio Grande do Sul, and Goia´s) and included broilers A
Corresponding author. E-mail: [email protected]
(50.94%, 27/53), broiler breeders (24.30%, 15/53), layers (15.09%, 8/ 53), and grandparent farms (5.66%, 3/53); none of these birds had symptoms of diarrhea. The birds’ ages varied from 40 days to 11 mo. Reverse transcriptase–PCR(RT-PCR). Samples were prepared as 40% (v/v) suspensions in diethylpyrocarbonate-treated water and centrifuged at 12,000 3 g for 15 min at 4 C; the resulting supernatants were used in the RNA extraction. 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. The samples were screened by PCR based on VP6 gene sequences followed by nucleotide sequencing of the detected amplicons, using a group F primer pair (forward primer VP6AVF10 59AAGTCAATCAGTCGCAATG-39 and reverse primer VP6AVF891 59AAGTCAATCAGTCGCAATG-39) that were designed in this study. For primer design, the VP6 complete nucleotide sequence of group F avian rotaviruses (accession number HQ403603) was imported from GenBank, and areas were selected based on the biochemical features (nucleotide positions 10 to 28 for forward primer and 891 to 912 for reverse primer within VP6 sequence HQ403603), using the software Bioedit 7.1.3.0 (3). The biochemical features—melting temperature, hairpins, dimers, and repeats—were calculated with Netprimer software (2013 Premier, Biosoft, Palo Alto, CA) These primers were used to amplify a 881-bp fragment 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 (VP6AVF10 and VP6AVF891), 2 mM MgCl2, 1.25 U 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 for 1.5 min. The PCR cycle consisted of initial denaturation at 94 C for 3 min, followed by 30 amplification cycles (94 C for 45 sec, 50 C for 30 sec, and 72 C for 45 sec); with a final extension at 72 C for 10 min. The products of the PCR were resolved on a 1.5% agarose gel stained with 0.5 mg/ml ethidium bromide. DNA sequencing. PCR amplicons of VP6 were purified with EXOSAP-it (USB Products, Affymetrix, Cleveland, OH) reagent, submitted to bidirectional DNA sequencing using BigDye 3.1 (Applied Biosystems, Foster City, CA), and resolved in an ABI-3500 Genetic Analyzer (Applied Biosystems), according to the manufacturer’s
458
Rotavirus F in broilers from Brazilian poultry farms
459
Fig. 1. Nucleotide neighbor-joining distance tree (maximum composite likelihood substitution model) for the partial VP6 rotavirus gene (698 nucleotides) showing the known groups (A–H). Strains detected in the present study are preceded by black triangles. The numbers at each node are bootstrap values greater than 80% from 1000 replicates. The bar represents the number of substitutions per site. instructions. The nucleotide sequences from each sample were aligned with other rotavirus groups retrieved from GenBank with CLUSTAL/W 2.1 (6). A nucleotide sequence phylogenetic tree, based on rotavirus reference sequences available in GenBank, was generated with the neighbor-joining distance algorithm and the maximum composite likelihood model with 1000 bootstrap replicates using MEGA 5.2.2 (14), from a 698-nucleotide-long VP6 partial fragment. Deduced amino acid identities of the generated sequences were calculated with Bioedit 7.1.3.0 (3) software.
RESULTS
Frequency of rotavirus F. From the 53 pools of fecal samples tested, we detected rotavirus group F samples by PCR in five samples
(9.43%). All of the positive samples were detected in broilers (40 days of age) from the Brazilian states of Sa˜o Paulo (20%, 1/5) and Parana´ (80%, 4/5). Two samples from Parana´ State were also positive for avian rotavirus A (based on VP7 and VP4 genes, of which G[19] and P31 genotypes were defined, respectively; data not shown). Accession numbers. The nucleotide sequences of VP6 group F rotavirus from this study were deposited in GenBank under the accession numbers KF926653 to KF926657. Nucleotide and deduced amino acid identities. Comparison of the VP6 gene 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 F 03V0568 strain (accession number HQ403603), with a maximum nucleotide identity of 88.4% and 96.7% if translated into amino acids.
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Fig. 2. Comparison of amino acid sequences of the VP6 gene of avian rotavirus F obtained from our study and HQ403603 (retrieved from GenBank).
Phylogenetic tree. The phylogenetic tree of the VP6 nucleotide sequences demonstrates that the five Brazilian rotavirus strains segregate with the rotavirus F representative, while the topology was maintained, with the remaining groups (A, B, C, D, E, G, and H) distinct from each other and supported by high bootstrap values, as shown in Fig. 1. DISCUSSION
In contrast to the other avian rotaviruses, A and D, the incidence of rotaviruses F and G is relatively low and their involvement in the disease is still unclear (11). In fact, here we detected only five positives in 53 samples without symptoms of diarrhea. Two samples were positive for avian rotaviruses A and F in the same broiler farm in Parana´ State, Brazil, thereby indicating that both groups could simultaneously infect apparently healthy birds. All positives samples were sequenced and the VP6 partial nucleotide sequences (accession numbers KF926653 to KF926657) generated with primers designed herein showed nucleotide identity ranging from 94.6% to 99.6% and amino acid identity ranging from 99.2% to 100%. These samples were obtained from Sa˜o Paulo and Parana´ states, Brazil. A high degree of variability was observed between the sequences we detected and the avian rotavirus F 03V0568 strain retrieved from GenBank (Fig. 2). The phylogenetic tree of the VP6 nucleotide sequences (Fig. 1) showed that all of the Brazilian samples consistently clustered together in rotavirus F inside a subcluster together with rotavirus groups A, C, and D. In fact, it is evident that the highest percentages of sequence identity occur with rotavirus groups A, C, and D, ranging from 46.9% to 52.3% on the nucleotide level (4). The phylogenetic analyses of the sequences identified two major clades formed by rotavirus A/C/D/F and rotavirus B/G/H, which may indicate divergent evolution of these groups (4). Within the clades, further relationships between the species are evident. For example, from the genome-wide tree, differentiation between rotavirus A and the other species within the same clade is evident.
Subsequently, the more closely related rotavirus species D and F seem to have both been separated from rotavirus C (5). The coinfection between rotaviruses A and F in this study, in spite of birds not presenting diarrhea, needs further epidemiologic investigation in order to evaluate the implications of these findings, considering that rotaviruses A, D, F, and G are capable of replication within the same host species and that double infections have been detected previously (8,11). In conclusion, rotavirus F can be found in Brazil in healthy birds from commercial poultry farms. The genome sequences generated herein may serve as a basis for the development of molecular detection systems, which can be used for the assessment of the distribution of rotavirus F in birds, its potential involvement in diseases, and its impact on poultry health. Further investigations are needed to determine the etiologic role of the virus detected in order to answer these questions.
REFERENCES 1. Attoui, H., P. P. C. Mertens, J. Becnel, S. Belaganahalli, M. Bergoin, C. P. Brussaard, J. D. Chappell, M. Ciarlet, M. del Vas, T. S. Dermody, P. R. Dormitzer, R. Duncan, Q. Fang, R. Graham, K. M. Guglielmi, R. M. Harding, B. Hillman, A. Makkay, C. Marzachi, J. Matthijnssens, R. G. Milne, F. M. Jaafar, H. Mori, A. A. Noordeloos, T. Omura, J. T. Patton, S. Rao, M. Maan, D. Stoltz, N. Suzuki, N. M. Upadhyaya, C. Wei, and H. Zhou. Family: Reoviridae. In: Virus taxonomy: ninth report of the ICTV. A. M. Q. King, M. J. Adams, E. B. Carstens, and E. J. Lefkowitz, eds. Elsevier Academic Press, Amsterdam. pp. 541–637. 2012. 2. 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. 3. 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. 4. Johne, R., P. Otto, B. Roth, U. Lo¨hren, D. Belnap, J. Reetz, and E. Trojnar. Sequence analysis of the VP6-encoding genome segment of avian group F and G rotaviruses. Virology 412:384–391. 2011.
Rotavirus F in broilers from Brazilian poultry farms
5. Kindler, E., E. Trojnar, G. Heckel, P. H. Otto, and R. Johne. Analysis of rotavirus species diversity and evolution including the newly determined full-length genome sequences of rotavirus F and G. Infect. Genet. Evol. 14:58–67. 2013. 6. 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. 7. 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. 8. McNulty, M. S. Rotavirus infections. In: Diseases of poultry, 12th ed. Y. M. Saif, ed. Iowa State University Press, Ames, IA. pp. 338–350. 2008. 9. McNulty, M. S., G. M. Allan, D. Todd, J. B. McFerran, E. R. McKillop, D. S. Collins, and R. M. McCracken. Isolation of rotaviruses from turkeys and chickens: demonstration of distinct serotypes and RNA electropherotypes. Avian Pathol. 9:363–375. 1980. 10. McNulty, M. S., D. Todd, G. M. Allan, J. B. McFerran, and J. A. Greene. Epidemiology of rotavirus infection in broiler chickens: recognition of four serogroups. Arch. Virol. 81:113–121. 1984.
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11. Otto, P., M. U. Ahmed, H. Hotzel, P. Machnowska, J. Reetz, B. Roth, E. Trojnar, and R. Johne. Detection of avian rotaviruses of groups A, D, F and G in diseased chickens and turkeys from Europe and Bangladesh. Vet. Microbiol. 156:8–15. 2012. 12. Otto, P., E. M. Liebler-Tenorio, M. Elschner, J. Reetz, U. Lo¨hren, and R. Diller. Detection of rotaviruses and intestinal lesions in broiler chicks from flocks with runting and stunting syndrome (RSS). Avian Dis. 50:411–418. 2006. 13. Saif, Y. M. 2008. Viral enteric infections. Introduction. In: Diseases of poultry, 12th ed. Y. M. Saif, ed. Iowa State University Press, Ames, IA. p. 329. 2008. 14. Tamura, K., D. Peterson, N. Peterson, G. Stecher, M. Neu, and S. Kumar. Mega 5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol. Biol. Evol. 28:2731–2739. 2011. 15. Theil, K. W., L. J. Saif, P. D. Moorhead, and R. E. Whitmoyer. Porcine rotavirus-like virus (group B rotavirus): characterization and pathogenicity for gnotobiotic pigs. J. Clin. Microbiol. 21:340–345. 1985. 16. 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.
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 58:458–461, 2014
Research Note— Description of Rotavirus F in Broilers from Brazilian Poultry Farms L. A. R. BeserraA 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, SP, Brazil Received 16 December 2013; Accepted 11 March 2014; Published ahead of print 12 March 2014 SUMMARY. Rotaviruses are segmented double-stranded RNA viruses that cause gastroenteritis in mammals and birds. Here we describe the first partial nucleotide sequences of the structural protein VP6 from the genomes of group F rotaviruses that were detected in 5 out of 53 fecal samples (9.43%) from healthy broilers from Brazilian poultry farms based on reverse-transcriptase–PCR with primers designed for this study. The findings support the development of molecular detection systems, which can be used for the assessment of the distribution of rotavirus F in birds, their potential involvement in diseases, and their impact on poultry health. RESUMEN. Nota de Investigacio´n—Descripcio´n de un rotavirus F en pollos de granjas avı´colas brasilen˜as. Los rotavirus son virus de genoma de ARN segmentado y de doble cadena que causan gastroenteritis en los mamı´feros y en las aves. Aquı´ se describen las primeras secuencias de nucleo´tidos parciales de la proteı´na estructural VP6 de los genomas de rotavirus del grupo F que se detectaron en 5 de 53 muestras fecales (9.43%) en pollos de granjas avı´colas de Brasil, mediante transcriptasa reversa y PCR con iniciadores disen˜ados en este trabajo. Los resultados apoyan el desarrollo de sistemas de deteccio´n molecular, que pueden ser utilizados para la evaluacio´n de la distribucio´n de rotavirus F en las aves, su papel potencial en enfermedades y su impacto en la salud de las aves comerciales. Key words: rotavirus, avian, group F, PCR Abbreviations: NSP 5 nonstructural protein; RT 5 reverse transcriptase; VP 5 viral protein
Rotaviruses, the major etiologic agents of acute viral gastroenteritis in young animals and children worldwide, are members of the Reoviridae family, and their genome is enclosed in three concentric layers comprising 11 segments of double-stranded RNA that encode six structural proteins (VP1–VP4, VP6, and VP7) and six nonstructural proteins (NSP1–NPS5/6) (1,2,13). Based on their antigenic properties and/or nucleotide sequencing of VP6, eight groups (groups A–H) of rotaviruses have been described so far (7). In avian species, rotavirus groups A, D, F, and G have so far been detected (8,9,16). Rotavirus groups F and G have been identified from the intestinal contents of broilers and turkeys, both with or without outward symptoms by polyacrylamide gel electrophoresis and more recently by PCR and the analysis of nucleotide sequences (4,10,11,12,15). Data on rotavirus F genome sequences are scarce and to date, only one rotavirus F complete sequence is available at GenBank (5). Here, we report the occurrence of group F rotavirus in the feces of broilers from Brazilian poultry farms from eight different states. We used RT-PCR–based amplification of the VP6 gene, followed by nucleotide sequencing reaction of the amplicons. To the best of our knowledge, this is the first report of rotavirus F in Brazil. MATERIALS AND METHODS Samples. A total of 53 pooled intestinal contents were used in this study. Each pool consisted of the enteric contents of three to five birds within a single batch, collected between the years 2008 and 2012. More than one pool was obtained from each farm; farms were located in eight different Brazilian states (Ceara´, Bahia, Espı´rito Santo, Sa˜o Paulo, Parana´, Santa Catarina, Rio Grande do Sul, and Goia´s) and included broilers A
Corresponding author. E-mail: [email protected]
(50.94%, 27/53), broiler breeders (24.30%, 15/53), layers (15.09%, 8/ 53), and grandparent farms (5.66%, 3/53); none of these birds had symptoms of diarrhea. The birds’ ages varied from 40 days to 11 mo. Reverse transcriptase–PCR(RT-PCR). Samples were prepared as 40% (v/v) suspensions in diethylpyrocarbonate-treated water and centrifuged at 12,000 3 g for 15 min at 4 C; the resulting supernatants were used in the RNA extraction. 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. The samples were screened by PCR based on VP6 gene sequences followed by nucleotide sequencing of the detected amplicons, using a group F primer pair (forward primer VP6AVF10 59AAGTCAATCAGTCGCAATG-39 and reverse primer VP6AVF891 59AAGTCAATCAGTCGCAATG-39) that were designed in this study. For primer design, the VP6 complete nucleotide sequence of group F avian rotaviruses (accession number HQ403603) was imported from GenBank, and areas were selected based on the biochemical features (nucleotide positions 10 to 28 for forward primer and 891 to 912 for reverse primer within VP6 sequence HQ403603), using the software Bioedit 7.1.3.0 (3). The biochemical features—melting temperature, hairpins, dimers, and repeats—were calculated with Netprimer software (2013 Premier, Biosoft, Palo Alto, CA) These primers were used to amplify a 881-bp fragment 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 (VP6AVF10 and VP6AVF891), 2 mM MgCl2, 1.25 U 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 for 1.5 min. The PCR cycle consisted of initial denaturation at 94 C for 3 min, followed by 30 amplification cycles (94 C for 45 sec, 50 C for 30 sec, and 72 C for 45 sec); with a final extension at 72 C for 10 min. The products of the PCR were resolved on a 1.5% agarose gel stained with 0.5 mg/ml ethidium bromide. DNA sequencing. PCR amplicons of VP6 were purified with EXOSAP-it (USB Products, Affymetrix, Cleveland, OH) reagent, submitted to bidirectional DNA sequencing using BigDye 3.1 (Applied Biosystems, Foster City, CA), and resolved in an ABI-3500 Genetic Analyzer (Applied Biosystems), according to the manufacturer’s
458
Rotavirus F in broilers from Brazilian poultry farms
459
Fig. 1. Nucleotide neighbor-joining distance tree (maximum composite likelihood substitution model) for the partial VP6 rotavirus gene (698 nucleotides) showing the known groups (A–H). Strains detected in the present study are preceded by black triangles. The numbers at each node are bootstrap values greater than 80% from 1000 replicates. The bar represents the number of substitutions per site. instructions. The nucleotide sequences from each sample were aligned with other rotavirus groups retrieved from GenBank with CLUSTAL/W 2.1 (6). A nucleotide sequence phylogenetic tree, based on rotavirus reference sequences available in GenBank, was generated with the neighbor-joining distance algorithm and the maximum composite likelihood model with 1000 bootstrap replicates using MEGA 5.2.2 (14), from a 698-nucleotide-long VP6 partial fragment. Deduced amino acid identities of the generated sequences were calculated with Bioedit 7.1.3.0 (3) software.
RESULTS
Frequency of rotavirus F. From the 53 pools of fecal samples tested, we detected rotavirus group F samples by PCR in five samples
(9.43%). All of the positive samples were detected in broilers (40 days of age) from the Brazilian states of Sa˜o Paulo (20%, 1/5) and Parana´ (80%, 4/5). Two samples from Parana´ State were also positive for avian rotavirus A (based on VP7 and VP4 genes, of which G[19] and P31 genotypes were defined, respectively; data not shown). Accession numbers. The nucleotide sequences of VP6 group F rotavirus from this study were deposited in GenBank under the accession numbers KF926653 to KF926657. Nucleotide and deduced amino acid identities. Comparison of the VP6 gene 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 F 03V0568 strain (accession number HQ403603), with a maximum nucleotide identity of 88.4% and 96.7% if translated into amino acids.
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Fig. 2. Comparison of amino acid sequences of the VP6 gene of avian rotavirus F obtained from our study and HQ403603 (retrieved from GenBank).
Phylogenetic tree. The phylogenetic tree of the VP6 nucleotide sequences demonstrates that the five Brazilian rotavirus strains segregate with the rotavirus F representative, while the topology was maintained, with the remaining groups (A, B, C, D, E, G, and H) distinct from each other and supported by high bootstrap values, as shown in Fig. 1. DISCUSSION
In contrast to the other avian rotaviruses, A and D, the incidence of rotaviruses F and G is relatively low and their involvement in the disease is still unclear (11). In fact, here we detected only five positives in 53 samples without symptoms of diarrhea. Two samples were positive for avian rotaviruses A and F in the same broiler farm in Parana´ State, Brazil, thereby indicating that both groups could simultaneously infect apparently healthy birds. All positives samples were sequenced and the VP6 partial nucleotide sequences (accession numbers KF926653 to KF926657) generated with primers designed herein showed nucleotide identity ranging from 94.6% to 99.6% and amino acid identity ranging from 99.2% to 100%. These samples were obtained from Sa˜o Paulo and Parana´ states, Brazil. A high degree of variability was observed between the sequences we detected and the avian rotavirus F 03V0568 strain retrieved from GenBank (Fig. 2). The phylogenetic tree of the VP6 nucleotide sequences (Fig. 1) showed that all of the Brazilian samples consistently clustered together in rotavirus F inside a subcluster together with rotavirus groups A, C, and D. In fact, it is evident that the highest percentages of sequence identity occur with rotavirus groups A, C, and D, ranging from 46.9% to 52.3% on the nucleotide level (4). The phylogenetic analyses of the sequences identified two major clades formed by rotavirus A/C/D/F and rotavirus B/G/H, which may indicate divergent evolution of these groups (4). Within the clades, further relationships between the species are evident. For example, from the genome-wide tree, differentiation between rotavirus A and the other species within the same clade is evident.
Subsequently, the more closely related rotavirus species D and F seem to have both been separated from rotavirus C (5). The coinfection between rotaviruses A and F in this study, in spite of birds not presenting diarrhea, needs further epidemiologic investigation in order to evaluate the implications of these findings, considering that rotaviruses A, D, F, and G are capable of replication within the same host species and that double infections have been detected previously (8,11). In conclusion, rotavirus F can be found in Brazil in healthy birds from commercial poultry farms. The genome sequences generated herein may serve as a basis for the development of molecular detection systems, which can be used for the assessment of the distribution of rotavirus F in birds, its potential involvement in diseases, and its impact on poultry health. Further investigations are needed to determine the etiologic role of the virus detected in order to answer these questions.
REFERENCES 1. Attoui, H., P. P. C. Mertens, J. Becnel, S. Belaganahalli, M. Bergoin, C. P. Brussaard, J. D. Chappell, M. Ciarlet, M. del Vas, T. S. Dermody, P. R. Dormitzer, R. Duncan, Q. Fang, R. Graham, K. M. Guglielmi, R. M. Harding, B. Hillman, A. Makkay, C. Marzachi, J. Matthijnssens, R. G. Milne, F. M. Jaafar, H. Mori, A. A. Noordeloos, T. Omura, J. T. Patton, S. Rao, M. Maan, D. Stoltz, N. Suzuki, N. M. Upadhyaya, C. Wei, and H. Zhou. Family: Reoviridae. In: Virus taxonomy: ninth report of the ICTV. A. M. Q. King, M. J. Adams, E. B. Carstens, and E. J. Lefkowitz, eds. Elsevier Academic Press, Amsterdam. pp. 541–637. 2012. 2. 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. 3. 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. 4. Johne, R., P. Otto, B. Roth, U. Lo¨hren, D. Belnap, J. Reetz, and E. Trojnar. Sequence analysis of the VP6-encoding genome segment of avian group F and G rotaviruses. Virology 412:384–391. 2011.
Rotavirus F in broilers from Brazilian poultry farms
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