Short Communication

Genetic Characterization of Avian Rotavirus Groups F and G in Brazilian Farms

Ligia Neves Scuarcialupi, Laila Andreia Rodrigues Beserra, Julia Rosas Hochheim, Rodrigo Martins Soares, Fabio Gregori*

Department of Preventive Veterinary Medicine and Animal Health, School of Veterinary Medicine and Animal Science, University of São Paulo. Av. Professor Dr. Orlando Marques de Paiva, 87, CEP 05508-270, São Paulo, SP, Brazil.

Received | August 29, 2017; Accepted | October 12, 2017; Published | April 25, 2018

*Correspondence | Fabio Gregori, Department of Preventive Veterinary Medicine and Animal Health, School of Veterinary Medicine and Animal Science, University of São Paulo. Av. Professor Dr. Orlando Marques de Paiva, 87, CEP 05508-270, São Paulo, SP, Brazil; E-mail: [email protected]

DOI | http://dx.doi.org/10.17582/journal.hv/2018/5.2.12.16

Citation | Scuarcialupi,, L.N., Beserra, L.A.R., Hochheim, J.R., Soares, R.M. and Gregori, F. 2018. Genetic characterization of avian rotavirus groups F and G in Brazilian farms. Hosts and Viruses, 5(2): 12-16.

Keywords: Rotavirus, Avian, Group F, Group G, PCR

Introduction

Rotaviruses (RVs) are one the causes of enteritis and diarrhea in avian species, resulting in negative impact on egg and poultry production (Islam et al., 2010; Dhama et al., 2015).

Belonging to Reoviridae Family, RVs are triple-shelled non-enveloped particles with an icosahedral morphology. Their genome consists of 11 double-stranded RNA segments, encoding one or two proteins each, resulting in six structural proteins (viral proteins VP1-VP4, VP6 and VP7) and six nonstructural proteins (NSP1-NSP6). They are classified in eight major groups named A to H according to the antigenic properties and nucleotide sequences of the VP6 internal capsid viral protein (Dhama et al., 2015; Mihalov-Kovács et al., 2015), and two new group candidates (I and J) have been proposed more recently (Bányai et al., 2017; Phan et al., 2017).

Rotavirus F (RVF) and Rotavirus G (RVG) have only been detected in avian species so far (Kindler et al., 2013; Dhama et al., 2015). Data on the occurrence of these RV groups in Brazil are scarce, despite the relevance of the avian industry in this country and the significant economic losses that this infection may result.

In this study, we report the occurrence of RVF and RVG in avian farms, based on the amplification of the VP6 gene, broadening the knowledge about the circulation of these viruses in different Brazilian regions.

Materials and Methods

Samples

A total of 97 intestinal contents was used in this study, collected between the years 2008 and 2015. Farms were located in ten different Brazilian states (Ceará, Goiás, Mato Grosso, Mato Grosso do Sul, Minas Gerais, Paraná, Pernambuco, Santa Catarina, São Paulo, Rio Grande do Sul), including broilers (43.29%; 42/97), layers (41.23%; 40/97), and breeders (15.46%; 15/97). There is no information on clinical signs, nor the age of sampled animals.

Reverse transcriptase-PCR (RT-PCR)

Samples were prepared as 50% (w/v) suspensions in diethylpyrocarbonate-treated water and centrifuged at 12,000 g for 15 min at 4ºC; the resulting supernatants were used in the RNA extraction. Total RNA was extracted using TRIzol reagent (Invitrogen™), 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 targeting a fragment of VP6 gene followed by nucleotide sequencing of the detected amplicons, using a group F primer pair (forward primer VP6AVF10 5’-AAGTCAATCAGTCGCAATG-3’ and reverse primer VP6AVF891 5’-GGTCTAACTATTCGTAGCCTAA-3’) generating a 903-bp fragment (Beserra and Gregori, 2014). For RVG detection, a PCR primer pair was designed (forward primer VP6AVG26 5’-CAGCATGGATCTCATCGA-3’ and reverse primer VP6AVG1095 5’-TCACGAACGAATCTTGAGT-3’), targeting the VP6 gene, based on the nucleotide sequence of avian RVG strain 03V0567 (accession number HQ403604) (Johne et al., 2011), generating a fragment of 1070-bp.

For both reactions, 5µL of cDNA was added to the PCR mix, which consisted of 1X PCR buffer (Invitrogen™), 0.2mM of each DNTP, 0.5mM of each primer, 2mM MgCl2, 1.25U Taq DNA polymerase (Invitrogen™), and ultrapure water up to 25 µL, and submitted to the following thermocycling conditions: initial denaturation at 94ºC for 3 min; 30 amplification cycles (94ºC for 45 sec, 50ºC for 30 sec, and 72ºC for 45 sec); with a final extension at 72ºC for 10 min. The products were resolved in a 1.5% agarose gel stained with 3µL of SYBR Safe (Invitrogen™).

DNA sequencing

VP6 PCR amplicons were purified with EXOSAP-it (USB Products®) reagent, submitted to bidirectional DNA sequencing using BigDye 3.1 (Applied Biosystems™), and resolved in an ABI-3500 Genetic Analyzer (Applied Biosystems™), according to the manufacturer’s instructions. The nucleotide sequences from each sample were aligned with other rotavirus groups retrieved from GenBank using CLUSTAL/W v. 2.1 (Larkin et al., 2007). A phylogenetic tree was generated with the neighbor-joining distance algorithm and the maximum composite likelihood substitution model with 1000 bootstrap replicates using MEGA v.7.0.26 (Kumar et al., 2016) from a 615-nucleotide-long VP6 partial fragment.

Accession numbers

The RVF sequences were deposited in GenBank under the accession numbers MF361095 to MF361099, and RVG under the accession number MF361100.

Results

Frequency of rotavirus F ang G

Through the RT-PCR and sequencing reactions, there were 5 RVF positive samples (5.15%; 5/97), from Paraná state (n=2/broiler); Mato Grosso do Sul state (n=1/layer); Rio Grande do Sul state (n=1/breeder) and Goiás state (n=1/breeder). Regarding RVG, this study detected only one positive sample (1.03%; 1/97) in a layer hen from Mato Grosso do Sul state. Moreover, this sample presented coinfection with RVF.

Phylogenetic tree

The phylogenetic tree confirmed the PCR results, and the 5 RVF sequenced samples clustered together with rotavirus F representatives. Regarding RVG, the sample detected herein was more related to HK18 (accession number KC876014) RVG strain isolated from pigeon, than the previously described in broilers from Brazilian Amazon. The tree topology could segregate all RV (A, B, C, D, F, G, H and I) clades, supported by high bootstrap values, as shown in Figure 1.

Discussion

It was found a relatively low frequency of RVs, with values of 5.15% for RVF and 1.03% for RVG, among different Brazilian States and types of avian farming. These results are similar to those found in Brazilian Amazon, where incidences of 9.4% (8/85) and 10.6% (9/85) were shown for RVF and RVG, respectively, in broiler chickens (Mascarenhas et al., 2016). Similarly, RVF presented a frequency of 9.43% (5/53) in broilers farms from two different Brazilian States (Beserra and Gregori, 2014), which were distant from Amazon region. A low frequency of RVF and RVG were already described in Germany and United Kingdom (Otto et al., 2012).

One of the samples detected in this study demonstrated a coinfection between RVF and RVG. Double rotaviral infections have been detected previously (Mcnulty, 2008, Otto et al., 2012; Beserra and Gregori, 2014) and they may lead to the generation of reassortants. These findings show the importance of performing a diagnosis covering different avian rotavirus groups, otherwise the infections may be underdiagnosed and, consequently, fewer measures could be taken to control the disease.

Even though only partial VP6 gene sequences have been used, the phylogenetic tree depicted a distinction among the rotavirus groups, supported by high bootstrap values (greater than 90%). Figure 1 shows a clade including RVF, RVA, RVD and RVC, and another one with RVB, RVG, RVH and RVI, in agreement with previous studies (Johne et al., 2011; Kindler et al., 2013; Beserra and Gregori, 2014; Mascarenhas et al., 2016).

Considering previous descriptions of RVF and data generated in this study, this agent was diagnosed in all Brazilian geographical regions, and hence a wide distribution in poultry farms, but with relatively low genetic variability (Figure 1).

The positive sample detected in Mato Grosso do Sul is the first RVG description in this state and probably has a different origin from those previously detected in Amazon region (Mascarenhas et al., 2016), given the tree topology, with two different clades (Figure 1). Interestingly, RVG sample was closely related to a pigeon strain detected in Hong-Kong (Phan et al., 2013), forming a separated clade. Additional sequencing of remaining genes may elucidate this finding and provide useful data on epidemiological aspects of this reservoir to the avian industry, once cross-species transmission may occur when different species are mixed (Pauly et al., 2017).

Prophylactic measures that promote biosafety should be strengthened, since there is no vaccine commercially available and there is a limited knowledge about the RV pathogenicity in birds.

Author’s Contributions

All the authors contributed equally and approved the manuscript for submission

Conflict of interest

The authors declare that there is no conflict of interests regarding the publication of this article

References

• • Bányai, K., Kemenesi, G., Budinski, I., Földes, F., Zana, B., Marton, S., Varga-Kugler, R., Oldal, M., Kurucz, K., Jakab, F. Candidate new rotavirus species in Schreiber’s bats, Serbia. Infection, Genetics and Evolution, 2017; 48: 19-26. https://doi.org/10.1016/j.meegid.2016.12.002
• • Beserra, L.A.R., Gregori, F. Description of rotavirus F in broilers from Brazilian poultry farms. Avian Diseases, 2014; 58(3):458-61. https://doi.org/10.1637/10747-121613-ResNote.1
• • Dhama, K., Saminathan, M., Karthik, K., Tiwari, R., Shabbir, M.Z., Kumar, N., Malik, Y.S., Singh, R.K. Avian rotavirus enteritis – an updated review. Veterinary Quarterly, 2015; 35(3):142-58. https://doi.org/10.1080/01652176.2015.1046014
• • Islam, M.S., Alam, M.M., Ahmed, M.U., Saifuzzaman, A.B.M., Kobayashi, N., Kayesh, M.E.H., Uddin, M.S. Molecular epidemiologic study on rotavirus infection in human and birds in association with gastroenteritis. Bangladesh Journal of Veterinary Medicine, 2010; 7(1): 233-7. http://dx.doi.org/10.3329/bjvm.v7i1.5066
• • Johne, R., Otto, P., Roth, B., Löhren, U., Belnap, D., Reetz, J., Trojnar, E. Sequence analysis of the VP6-encoding genome segment of avian group F and G rotaviruses. Virology Journal, 2011; 412 (2):384-91. https://doi.org/10.1016/j.virol.2011.01.031
• • Kindler, E., Trojnar, E., Heckel, G., Otto, P.H., Johne, R. Analysis of rotavirus species diversity and evolution including the newly determined full-length genome sequences of rotavirus F and G. Infection, Genetics and Evolution, 2013; 14:58-67. https://doi.org/10.1016/j.meegid.2012.11.015
• • Kumar, S., Stecher, G., Tamura, K. MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets. Molecular Biology and Evolution, 2016; 33(7):1870-4. https://doi.org/10.1093/molbev/msw054
• • Larkin, M.A., Blackshields, G., Brown, N.P., Chenna, R., McGettigan, P.A., McWilliam, H., Valentin, F., Wallace, I.M., Wilm, A., Lopez, R., Thompson, J.D., Gibson, T.J., Higgins, D.G. Clustal W and Clustal X version 2.0. Bioinformatics, 2007; 23(21):2947-8. https://doi.org/10.1093/bioinformatics/btm404
• • Mascarenhas, J.D., Bezerra, D.A., Silva, R.R., Silva, M.J., Júnior, E.C., Soares, L.S. Detection of the VP6 gene of group F and G rotaviruses in broiler chicken fecal samples from the Amazon region of Brazil. Archives of Virology, 2016; 161(8):2263-8. https://doi.org/10.1007/s00705-016-2882-z
• • Mcnulty, M. S. 2008: Rotavirus infections. In: Saif, Y. M.(ed), Diseases of poultry: 12th edn., pp. 338-350. Iowa State University Press, Ames, IA.
• • Mihalov-Kovács, E., Gellért, A., Marton, S., Farkas, S.L., Fehér, E., Oldal, M., Jakab F., Martella, V., Bányai, K. Candidate new rotavirus species in sheltered dogs, Hungary. Emerging Infectious Diseases, 2015; 21(4):660-3. https://doi.org/10.3201/eid2104.141370
• • Nirwati, H., Hakim, M.S., Aminah, S., Dwija, I.B.N.P., Pan, Q., Aman, A.T. Identification of rotavirus strains causing diarrhoea in children under five years of age in Yogyakarta, Indonesia. Malaysian Journal of Medical Sciences, 2017; 24(2):68-77. https://doi.org/10.21315/mjms2017.24.2.9
• • Otto, P.H., Ahmed, M.U., Hotzel, H., Machnowska, P., Reetz, J., Roth, B., Trojnar, E., Johne, R. Detection of avian rotaviruses of groups A, D, F and G in diseased chickens and turkeys from Europe and Bangladesh. Veterinary Microbiology, 2012; 156(1-2):8-15. https://doi.org/10.1016/j.vetmic.2011.10.001
• • Pauly, M., Oni, O.O., Sausy, A., Owoade, A.A., Adeyefa, C.A.O., Muller, C.P., Hübschen, J.M., Snoeck, C.J. Molecular epidemiology of avian rotaviruses group A and D shed by different bird species in Nigeria. Virology Journal, 2017; 14:111. https://doi.org/10.1186/s12985-017-0778-5
• • Phan, T.G., Leutenegger, C.M., Chan, R., Delwart, E. Rotavirus I in feces of a cat with diarrhea. Virus Genes. 2017; 53(3):487-490. https://doi.org/10.1007/s11262-017-1440-4
• • Phan, T.G., Vo, N.P., Boros, Á., Pankovics, P., Reuter, G., Li, O.T., Wang, C., Deng, X., Poon, L.L., Delwart, E. The viruses of wild pigeon droppings. PLoS One, 2013; 8(9):e72787. https://doi.org/10.1371/journal.pone.0072787