Avian rotaviruses (RVs) are important etiologic agents of gastroenteritis in birds. In general, avian RVs are understudied; consequently, there is a paucity of information regarding these viruses. Therefore, the characterization of these viral species is highly relevant because more robust information on genetic, epidemiologic, and evolutionary characteristics can clarify the importance of these infections, and inform efficient prevention and control measures. In this study, we describe partial genome characterizations of two avian RV species, RVF and RVG, detected in asymptomatic poultry flocks in Brazil. Complete or partial sequences of at least one of the genomic segments encoding VP1, VP2, VP4, VP6, VP7, NSP1, NSP4, NSP4, or NSP5 of 23 RVF and 3 RVG strains were obtained, and demonstrated that multiple variants of both RVF and RVG circulate among Brazilian poultry. In this study, new and important information regarding the genomic characteristics of RVF and RVG is described. In addition, the circulation of these viruses in the study region and the genetic variability of the strains detected are demonstrated. Thus, the data generated in this work should help in understanding the genetics and ecology of these viruses. Nonetheless, the availability of a greater number of sequences is necessary to advance the understanding of the evolution and zoonotic potential of these viruses.
Keywords: rotavirus; RVF; RVG; chickens; genome sequencing
Rotaviruses (RVs) are members of the Rotavirus genus in the Reoviridae family and are divided into 12 species A–L [[
The RV genome is composed of 11 segments of double-stranded RNA (dsRNA) encoding six structural proteins, VP1–VP4, VP6 and VP7, and five or six nonstructural proteins, NSP1–NSP5 and NSP6, depending on the viral strain [[
Avian RV infections were first described in 1977 in the United States, when virions resembling those of mammalian strains were visualized by electronic microscopy in the intestinal contents of turkeys with enteritis [[
The evolution of RVs has been elucidated by analysis of the genetic repertoire of circulating strains, primarily by using classical Sanger sequencing. Consequently, several mechanisms, alone or in combination, that have been identified lead to RV evolution and diversity. Point mutations are among the main sources of diversity among these viruses [[
As established by the Rotavirus Classification Working Group (RCWG), the classification of RVA is based on the analysis of the 11 genomic RNA segments. Cutoff values have been defined for identifying the genotype of each segment [[
RVF and RVG were originally detected in chickens [[
Fecal samples included in this study were from a collection housed at the Laboratory of Respiratory, Enteric and Ocular Viruses (LAVIREO) at the Federal University of Rio de Janeiro. Samples in this collection were obtained from asymptomatic chickens in the southeastern Brazilian cities of São José do Vale do Rio Preto (May 2013), and Bom Jardim (October 2018), in the state of Rio de Janeiro, and Marechal Floriano (October 2018), in the state of Espírito Santo (Figure 1), and were analyzed previously for RVF and RVG by RT-PCR [[
Stool suspensions were prepared in 10% (w/v) phosphate-buffered saline (pH 7.2) and then centrifuged at 2500× g for 5 min; viral dsRNA was extracted from 300 μL of the supernatant using the guanidine isothiocyanate-phenol-chloroform method [[
Complete or partial sequences of at least one of the genomic segments encoding VP1, VP2, VP4, VP6, VP7, NSP1, NSP2, NSP4, or NSP5 of 23 RVF and 3 RVG strains were obtained (Appendix A Table A3).
Complete or partial sequences of the open reading frame (ORF) of gene segment 1, encoding VP1, were obtained from three strains detected in Bom Jardim, Rio de Janeiro. A comparison of the complete sequence of the BJ12 strain, and reference strains from Germany and the Republic of Korea showed nucleotide identities of 88.7% and 89.7%, respectively, and amino acid identities of 96% and 97%, respectively. Partial sequences were obtained for strains BJ1 and BJ7 from Bom Jardim. To facilitate the analysis, all sequences were edited to the length of 1115 bp, which permitted the analysis of the three strains from Rio de Janeiro and reference strains from Germany and South Korea, and other RVF strains detected previously in Brazil (Figure 2). The comparison of the partial sequences of the three strains detected in Bom Jardim showed 100% identity and that they were closer to the Brazilian RS-15-4S-2 strain (90.5% identity), detected in the state of Rio Grande do Sul, southern Brazil, than to the strains from Germany (03V0568; 88.4%) and Republic of Korea (D62; 89.7%).
Partial sequences of gene segment 2, encoding VP2 of three strains, BJ1, BJ7, and BJ12, were obtained. For phylogenetic analysis, the sequences were edited to the length of 1200 bp (Figure 2). The strains detected in this study had 99.4 to 100% identity among themselves, and 88.7% to 90.7% nucleotide identity with German and Republic of Korean reference strains, respectively.
Complete sequences of the ORFs of gene segment 3, which encodes VP4, of two strains, BJ1 and BJ7, were obtained. Phylogenetic analysis showed high nucleotide (99.9%) and amino acid (100%) identities. Comparison with reference strains showed that the strains from Bom Jardim were closer to the German strain 03V0568, with percentage identities of nucleotides and amino acids of 96.1% and 96.8%, respectively. Identities with the Republic of Korean strain (D62) were 78.1% for nucleotides and 83.7% for amino acids. Interestingly, another Brazilian strain from Rio Grande do Sul, RS-BR-14-4S-3, was closer to the German and Republic of Korea strains than to the strains from Bom Jardim.
The partial sequences of BJ1, BJ7, and BJ12 strains were analyzed by adjusting all sequence lengths to 1130 bp (Figure 2). Phylogenetic analyses showed that the strains detected in this study had 99.9 to 100% identity with each other, and were more closely related to the reference strains from Germany than to those from South Korea and Brazil.
Complete sequences of the OFR of gene segment 6, encoding VP6, were obtained for the BJ1, BJ7, and BJ12 strains. Phylogenetic analysis showed that the strains detected in this study were identical. Comparison with German (03V0568) and Swiss (PB56-SII36) reference strains showed nucleotide identities of 88.1–85.8% and amino acid identities of 95.7–92.1%, respectively, with RVF from Bom Jardim. On the other hand, the comparison with a Brazilian strain detected in the state of Rio Grande do Sul showed 91.9% nucleotide and 99.7% amino acid identities.
In addition, we obtained the partial VP6 sequences of 18 RVF strains from both Bom Jardim in the state of Rio de Janeiro and Marechal Floriano in the state of Espírito Santo. Sequences were adjusted to the size of 280 bp (Figure 2). Phylogenetic analysis showed that our strains presented greater genetic proximity to RVFs previously detected in Brazil, although they form a separate clade than those detected in Germany (03V0568), the Republic of Korea (D62), and Switzerland (PB56-SI36). Interestingly, the BRA57 strain, detected in the state of Pará, northern Brazil, grouped separately with a partridge strain (956_1) detected in Italy.
Partial sequences of gene segment 9, which encodes VP7, were obtained for strains BJ1, BJ7, and BJ12. Sequences were adjusted to 398 bp, which covered the same ORF regions of all samples (Figure 2). Phylogenetic analysis showed that the strains detected in this study had 99.2 to 100% identity. Comparison with German (03V0568), Chinese (361BR-K141_94516), and Republic of Korean (D62) strains showed identities of 75.9% to 80.4%.
The complete sequence of segment 5, which encodes NSP1, was obtained for BJ12. A partial sequence was determined for BJ1. Sequence lengths were adjusted to 997 bp to include both strains (Figure 2). Phylogenetic analyzes showed that the BJ strains presented 99.7% nucleotide identity and were closer to the Brazilian strain from Rio Grande do Sul, BR-15-4S-5 (91.5–91.9%) than RVF strains from China (361BR-K141_94516), Germany (03V0568), and South Korea (D62) (85.6–86.9%).
Sequence of the complete ORF of gene segment 8, encoding the NSP2 of the BJ1 strain, and partial sequences of the BJ7 and BJ12 strains were obtained. The sequence analysis of the BJ1 ORF disclosed an identity of 88.6% and 92.8% of nucleotides and amino acids, respectively, with the German reference strain (03V0568), and 90.3% and 95% with two Brazilian strains (RS-BR-15-4S-7 and RS-BR-15-5R) detected in Rio Grande do Sul. When the length of the sequences was adjusted to 594 bp (Figure 2), phylogenetic analysis showed that they grouped in a single clade close to, but distinct from, other strains detected in southern Brazil. This could suggest the independent evolution of the RVF strains circulating in the country. However, the analysis of the complete ORFs of a larger number of samples is necessary to confirm this hypothesis.
Complete sequences of the ORF of gene segment 11, which encodes NSP4, were obtained for six strains, BJ1, BJ7, BJ12, and BJ37 from Rio de Janeiro, and MF104 and MF110 from Espírito Santo (Figure 2). Phylogenetic analyses showed that the strains detected in this study presented high nucleotide and amino acid identity. Comparison with reference strains from Germany (03V0568), China (361bR-k141_94516), and the Republic of Korea (D62) showed that the Brazilian strains had nucleotide identities of 88.4–88.6% and amino acid identities of 88.9–91.8%. Unfortunately, genetic information regarding NSP4 of other Brazilian RVF strains is not available.
The complete ORF of gene segment 10, encoding NSP5, was obtained for five RVF strains: BJ1, BJ7, BJ10, BJ11, and BJ12. Phylogenetic analyses showed that the BJ strains had 91.2–100% nucleotide identity and 94.5–100% amino acid identity among themselves. Comparison with reference strains from Germany, China, and the Republic of Korea showed that the strains from Rio de Janeiro showed nucleotide identities of 86.7% to 89.5%, respectively, and amino acid identities of 89.8% and 94.5%, respectively. Adjusting the length of the sequences to 400 bp enabled the analysis of eight more strains: BJ14, BJ30, BJ31, BJ32, BJ37, BJ39, BJ41, and BJ51 detected in Bom Jardim, and MF06 detected in Marechal Floriano. The dendrogram shows that the Brazilian strains were closely related but not identical (Figure 2).
Complete or partial sequences of the sixth segment encoding VP6 of three RVG strains detected in Marechal Floriano were obtained: MF48 (complete ORF), MF40, and MF41 (partial ORFs). When comparing the complete ORF sequence of the MF48 strain with the reference strains that are available on GenBank, we observed that the strain from Brazil is genetically closer to chicken strains from South Africa (MRC-DPRU1679) and Germany (03V0568), with 89% and 89.3% nucleotide identity, and 96.9 and 97.7% amino acid identity, respectively. It has 80% and 89% nucleotide and amino acid identity, respectively, with the US turkey strains (Minnesota-1 and Minnesota-2) and is phylogenetically distant from the Hong Kong pigeon strain (HK18) (Figure 3). When we adjusted the sequence length to 642 bp, we were able to include the MF40 and MF41 strains in the analysis (Figure 3). The dendrogram showed that the strains detected in Brazil were genetically similar (94.5 to 96.9% identity) yet grouped differently to form three sub-clades: the strains from Espírito Santo (MF40, MF41 and MF48), detected in 2018, grouped in one clade and the strains from Rio Grande do Sul, detected in 2009, grouped in two different clades. These data suggest that the Brazilian strains have a common ancestor and possibly evolved independently of each other, leading to the observation of different variants circulating in our country. Partial sequences from a partridge strain (956-2) from Italy and a gull strain (H01-10385) from the Netherlands were also included in this analysis. The phylogenetic analysis showed a separation between strains of different host species, except for the partridge strain that grouped with chicken samples. These findings suggest (i) that different VP6 genotypes circulate among RVG strains and (ii) that these genotypes may be associated with different hosts (chickens, turkeys, and pigeons).
A partial sequence of the eighth gene segment that encodes NSP2 was obtained for the MF48 RVG strain (Figure 2) and showed 89.6–90.1% identity with the RVG strains detected in chickens, 84.7% with the pigeon strain, and 80.4% with turkey strains.
Three complete ORFs of gene segment 11, encoding the NSP5, from strains MF40, MF41, and MF48 were obtained. Phylogenetic analysis showed that, similar to the findings of VP6, the Brazilian strains were genetically closer to chicken strains and once again separated into different clades according to host species (Figure 2).
RVs are the most prevalent etiologic agents of diarrhea among diverse hosts that include mammals and birds [[
In this study, we obtained the complete ORF sequences of the genome segments that encode VP6, NSP2, NSP4, and NSP5 of RVF strains circulating in two southeastern states of Brazil: Rio de Janeiro and Espírito Santos. When compared to the sequences of reference strains, they presented nucleotide identity values for VP6, NSP2, and NSP4 greater than 85%, the suggested cutoff value for RVA. The application of the same criteria to RVF would suggest that these belong to the same genotype. However, it is important to point out that the Brazilian strains grouped in a distinct clade in the dendrograms, suggesting their independent evolution.
On the other hand, the percentages of nucleotide identity observed for NSP5 could suggest the circulation of different genotypes, as they are below 91%, which is the cutoff value for this gene in the case of RVA. Furthermore, analysis of partial NSP5 sequences showed that although the strains in this study clustered closely, they are not all in the same clade. This difference may have arisen through the typical mechanisms of RV evolution, primarily through point mutations. The MF06 strain originating from Espírito Santo presented similarity with the strains from Rio de Janeiro; however, its distribution in the dendrogram shows segregation in a specific clade, suggesting the circulation of separate variants in different Brazilian states.
When analyzing partial sequences, we observed that for VP1, using a fragment that corresponded to 54% of the ORF, the Brazilian strains and the reference strains shared >83% identity, a cutoff value stipulated to differentiate the VP1 genotypes from RVA. For genes encoding VP2, VP4, VP7, and NSP1, the sequences corresponded to <50% of the ORF (20%, 20%, 45%, and 40%, respectively). In the case of genes encoding VP2 and NSP1, the identity between all strains was well above a cutoff value of 84%, suggesting a single genotype. However, for VP4, the nucleotide identity values and the topology of the dendrogram suggest two distinct genotypes: one represented by the strain from Germany and the strains from Rio de Janeiro, and a second represented by the strain from South Korea and the strain from Rio Grande do Sul. Interestingly, these data suggest a distinct origin of the strains from Rio de Janeiro and Rio Grande do Sul. In the case of VP7, the results suggest three different genotypes represented by the Brazilian strains, the strains from Germany and China, and the strain from South Korea.
VP4 and VP7 are responsible for cell entry and the induction of neutralizing antibodies and, therefore, represent the main targets for vaccine development [[
Overall, phylogenetic analyzes of all sequenced RVF genome segments demonstrated that Brazilian strains, although possibly having a common ancestor, evolved independently, leading to different variants circulating in the country.
As for RVG strains, three genes, VP6, NSP2, and NSP5, were sequenced. Analysis of the complete and partial ORFs of VP6 suggests the existence of different genotypes and possibly the segregation of these genotypes according to host species. Furthermore, the circulation of different variants in Rio de Janeiro and Rio Grande do Sul was also demonstrated. This evidence was reinforced by the analysis of NSP2 and NSP5.
The RCWG proposal for RVA defines the cutoff values for the genetic classification of individual genes. However, for the application of these criteria, the RCWG imposes conditions for the identification of genotypes. Therefore, no genotype should be assigned based on less than 500 nucleotides or less than 50% of the ORF sequence. As the percentage cutoff values for each of the 11 genomic segments were calculated based on complete ORFs, applying these cutoff percentages to a partial ORF sequence can lead to erroneous conclusions. Only under certain circumstances, when all three of the following restrictions are met, can a partial gene sequence be used to assign an already established genotype to a given RVA gene: (
Birds are recognized as important reservoirs of viruses that can threaten human health, such as the influenza A virus, which infects both birds and mammals, enabling the reassortment of genomic segments and changes in tropism and transmission efficiency [[
In this study, new and important information regarding the genomic characteristics of RVF and RVG was described, despite the small number of characterized strains. These difficulties can be partially explained by the samples used for amplification of the viral genomes, which came from asymptomatic chickens and therefore contained low viral loads, as well as the limited number of available RVF and RVG sequences, which confounded the selection of more efficient primers. However, despite the limitations of this study, the circulation of RVF and RVG variants among poultry was clearly demonstrated. Nonetheless, the availability of a greater number of sequences is necessary for a better understanding of the evolution and zoonotic potential of these viruses.
MAP: Figure 1 Map of Brazil showing the study region (circle; ES = Espírito Santo and RJ = Rio de Janeiro), located in the southeast of the country (A). Maps of the locations where fecal samples were obtained from birds analyzed in this study: (B) map of the state of Rio de Janeiro showing the cities of São José do Vale do Rio Preto in the East Region, and Bom Jardim in Bom Jardim in the Center Region of the state; (C) map of the state of Espírito Santo showing the city of Marechal Floriano in the Southwest Region of the state.
Graph: Figure 2 Dendrograms constructed from complete or partial sequences of the VP1, VP2, VP4, VP6, VP7, NSP1, NSP2, NSP4, and NSP5-encoding genome segments of RVF strains. Distances were corrected using the Kimura 2-parameter model. The dendrogram was constructed using the maximum likelihood method. Statistical support was provided by bootstrapping 1000 pseudoreplicates. Bootstrap values above 70% are given as branch nodes. GenBank reference strain accession numbers are shown next to the strain identification. RVF strains detected in this study are indicated by black triangles.
Graph: viruses-15-01089-g002b.tif
Graph: Figure 3 Dendrograms constructed from complete or partial sequences of the VP6, NSP2, and NSP5-encoding genome segments of RVG strains. Distances were corrected using the Kimura 2-parameter model, and the dendrogram was constructed using the maximum likelihood method. Statistical support was provided by bootstrapping 1000 pseudoreplicates. Bootstrap values above 70% are given as branch nodes. GenBank reference strain accession numbers are shown next to the strain identification. RVG strains detected in this study are indicated by black triangles.
Conceptualization, N.S.; Formal analysis, M.S.P. and J.B.L.D.; Funding acquisition, N.S.; Investigation, M.S.P.; Methodology, M.S.P., J.B.L.D., M.P.P., C.E.P.F.T. and G.S.M.; Project administration, N.S.; Resources, N.S.; Supervision, N.S.; Validation, N.S.; Writing—original draft, M.S.P. and J.B.L.D.; Writing—review & editing, M.P.P., C.E.P.F.T., G.S.M. and N.S. All authors have read and agreed to the published version of the manuscript.
This study was approved by the Animal Research Ethics Committee of the Federal University of Rio de Janeiro, Rio de Janeiro, Brazil (protocol 151/18).
Not applicable.
The nucleotide sequences of the 2014–2018 viruses obtained in this study were submitted to the GenBank database under accession numbers OL688642, OL688643, OL688646, OL688659-OL688661, and OQ627756-OQ627800.
The authors declare no conflict of interest.
Table A1 Primers used for sequencing the RVF and RVG strains.
Primer Primer Sequence 5′ → 3′ Gene Position † RFVP1.F1 ATTAATTGCGTACGATGGGGA VP1 5–25 RFVP1.F2 GGATCCTGCAATATCTACATC 429–449 RFVP1.F3 GCAAATGTWTATTCATGGTC 991–1010 RFVP1.F4 TGCTTAAAGATGAYGTTATTA 1145–1165 RFVP1.F5 GGTAGAAGRGATGTACCAGGT 1372–1392 RFVP1.R1 TTAATYATTTTTGATGAATA 835–854 RFVP1.R2 ACTTCCAAATTTTATTTCC 1242–1260 RFVP1.R3 GCTAAATTTGCAATAGAATTA 1807–1827 RFVP1.R4 TAACRTTCATAAGTGCATAAG 1985–2005 RFVP1.R5 CCTGATATYTTAACTGATGTC 2229–2249 RFVP1.R6 GATARTAAATCATAAACATATG 2984–3005 RFVP1.R7 ATTATTGCAGCTTACTCTTGG 3270–3290 RFVP2.F1 GGCTATTATTGGCAGGATGT VP2 1–20 RFVP2.F2 GCAGARATGAGRCATAGAGT 764–783 RFVP2.F3 GGGAAACATTAACGTTAACT 1041–1060 RFVP2.F4 GTAATGAATCAGCAATATGC 1772–1791 RFVP2.R1 CAGATTATAAGGAACATATC 978–997 RFVP2.R2 CTAACTATATTAGCTTCAGC 788–807 RFVP2.R3 AGAGCTAATACAAACATTCC 1307–1326 RFVP2.R4 GATGCATCAGGATATACAGC 1862–1881 RFVP2.R5 GGAGATAATACTCCTCCAGC 2741–2760 RFVP4.F1 GATGGCTTCTCGCTTTTGGG VP4 11–30 RFVP4.F2 AAATGCGGTAGTTTTGGAATAG 352–372 RFVP4.F3 GGTTATCAATTTTCAACGTCT 787–809 RFVP4.F4 GTAGGAAGTATAACTCCATA 1104–1123 RFVP4.F5 ATCAAGRTGGCAGAAAARTT 1500–1519 RFVP4.R1 CATATACCATGTTTCTGAAT 1010–1029 RFVP4.R2 AATCCAGCTTGTGACACGAC 690–709 RFVP4.R3 AGGATAATATGGAGTTATACT 1110–1130 RFVP4.R4 TTGAAT TCCTGTCGTAATTGG 1480–1500 RFVP4.R5 TATGCTATATTAGCATCCTTA 1980–2000 RFVP4.R6 ATACTGTTTCTCGCTCAAAGT 2250–2270 RFVP6.F1 GGCTTATAAAAGTCAATCAG VP6 1–20 RFVP6.F2 AAGTCAATCAGTCGCAATGG 10–29 RFVP6.F3 CGTACAGAACCAGGTCAAATGTG 620–642 RFVP6.F4 GCCAGTGCCAATATCTGATGC 667–687 F5.RVF ATGTGAAACTGAAATGTGTCTTGAATC 310–336 F6.RVF TACAAAAAGTTAGAGTTAGAACAGCTTA 447–474 RFVP6.F7 GGTCTACTTAATGATCAARTAC 730–737 RFVP6.F8 CAATACAAGTTGATACTGATGC 690–710 RFVP6.R1 CCTGCYACATCATCCATAGC 539–558 R2.RVF TTGATCATTAAGTAGACCAGAYGCATC 707–733 RFVP6.R3 AGCTGTTCTAACTCTAACTT 453–472 RFVP6.R4 CAGTGATACTATCGGAATAAACCA 1241–1264 RFVP6.R5 CACAGTTGCCCGGCCAAACG 1268–1287 RFVP6.R6 GCATATTATCTTGTCTAGAT 1150–1169 RFVP7.F1 CGAACAGCCTCCATCAGCTCGTGT VP7 17–40 RFVP7.F2 CCTGTAATTCAGGATGTTTGCTG 57–79 RFVP7.F2 GTARCCTGTAATTCAGGATGTT 53–74 RFVP7.F3 GAAGTAAATGAGTTATTTCAAT 355–376 RFVP7.F4 TCGACTGATGTGAAAACATATG 601–622 RFVP7.F4 TCAACTGATGCTACTACATATG 601–622 RFVP7.F5 TATAGAATGAATATTACTGG 673–692 RFVP7.R1 GGTCATAATGTTGTTCGCAAC 970–990 RFVP7.R2 CAACGTTAATGATTATTTATC 953–973 RFVP7.R3 GAGGAATAATCTGGTCCAAC 733–752 RFVP7.R4 GATAAGTCATCTGGTCCATG 412–431 RFVP7.R4 GATAAATCACTCGGCCCAT 413–431 RFNSP1.F1 GTG TGC CGA TTC AGA GAT GG NSP1 23–42 RFNSP1.F2 GGT ATG AGT GTA GTW CCA GC 711–730 RFNSP1.F3 CAA TTY CGT GAT TGG AA 1176–1192 RFNSP1.R1 CCG TGT GCG ATG CTG AAT CGG 1745–1765 RFNSP1.R2 GTA TAC TCA CAA TCT TAT CAT 792–812 RFNSP1.R3 ACT AAT CAR TCA CCT CTT AT 1401–1420 RFNSP2.F1 TTRTTTTTGATATAGAGCAGT NSP2 10–30 RFNSP2.F3 GGTCCGGCAAAATCCTGCCTGCCG 28–51 RFNSP2.F4 GAATGATGCAGAAGATAGAC 412–431 RFNSP2.F5 GAGTATAAAATTACATTCAA 608–627 RFNSP2.R1 GGTCGTAGTATGATATAGAT 1049–1068 RFNSP2.R2 TTGCTTTAATAGCTTTRAGAG 786–806 RFNSP2.R3 GTACTTATTCATATATTCAATA 529–550 RFNSP2.R4 GTTTCAGGATTAAGTTATTGGG 1019–1040 RFNSP4.F1 CCTCATCTTAGTTATACGTAC NSP4 11–31 RFNSP4.F2 CCCTCAGTGGTTTTGACAAG 31–50 RFNSP4.R1 GGTCATAACTCATCCGTTAG 659–678 RFNSP4.R2 GCTGATCGCTGCACTCTGG 641–659 RFNSP4.R3 GGTACAGTATGTTATACGC 607–621 RFNSP4.R4 ACCACTAGCATCTTCAGTTC 406–425 RFNSP5.F1 GAGCATGGATCTTGATATAGAC NSP5 20–42 RFNSP5.F2 ATGGAATCTGTAAATAAT 303–320 RFNSP5.F3 TCAGTTAAGTCATCAAATTC 357–376 RFNSP5.R1 CATGATTATAGATCGGATATAAGC 656–679 RFNSP5.R2 GCTGAGTAATGCTTTGCACTGC 411–432 RGVP6.F1 GGAAAGAAATCTCCAACCTAG VP6 1–21 RGVP6.F2 GCTAAGCTCGAACCTCAAATT 459–479 772F-RVG CAGATATGGCGAGRGGTGAT 772–791 RGVP6.R1 AATTCTATTACTATATCACC 786–805 RGVP6.R2 AGCTTAGCTGTATCATATGC 447–466 1229R-RVG AAACTCTCCTCCACAGCCGA 1229–1248 RGNSP2.F1 GAGTGCGTCGTGAGAAGGGAG NSP2 20–40 RGNSP2.F3 GTTTTTGARGATGTATTTGAA 380–400 RGNSP2.R1 CAGCGCTCAATGAATGGATTT 978–998 RGNSP2.R3 GTACTGTTCTAACATGTCCGT 750–770 RGNSP4.F3 GATKCATTTAAGATTCTTT NSP4 105–123 RGNSP4.F4 CARAATTCGAAGGAAATGGT 384–403 RGNPS4.R3 AACCCATCGGCTCTGCACC 740–758 RGNSP4.R4 TCCATYGATTCTTCAATTCC 534–553 RGNSP5.F1 GGAATATTAAAGTGTCGCTTGGTG NSP5 1–24 RGNSP5.F2 GGTGGCTGGAAACACTGAGTGG 21–42 RGNSP5.R1 CCCAGAAATAATCAGTGGAGTC 657–678 RGNSP5.R2 TCAACTTCATCHGCCCAGTT 315–334 RGNSP5.R3 CTAATTTTCGAWATTTCAT 445–463
Table A2 RVF and RVG reference strains GenBank access code.
Viral Strain Origin Gene VP1 VP2 VP3 VP4 VP6 VP7 NSP1 NSP2 NSP3 NSP4 NSP5 RVF Ch-03V0568 Germany JN596591 JQ919995 JQ919997 HQ403603 JQ919998 JQ919999 JQ920000 JQ920002 JQ920003 Ch-361bR-k141_94516 China MZ218346 MZ218350 MZ218351 Ch-D62 South Korea KM254212 KM254213 KM254215 KM254216 KM254217 KM254218 KM254219 KM254221 KM254222 Ch-D11 South Korea KM254224 Ch-PB56-SII36 Switzerland OM469208 Ch-ITA-956-1 Italy KT073228 Ch-BR-15-4S-8 Brazil MG846380 Ch-RS-BR-15-4S-7 Brazil MG846381 Ch-RS-BR-15-5R Brazil MG846379 Ch-AVRVFBR01 Brazil KF926653 Ch-AVRVFBR02 Brazil KF926654 Ch-AVRVFBR03 Brazil KF926655 Ch-AVRVFBR04 Brazil KF926656 Ch-AVRVFBR05 Brazil KF926657 Ch-BRA27 Brazil KP824796 Ch-BRA54 Brazil KP824800 Ch-BRA57 Brazil KP824801 Ch-BRA71 Brazil KP824803 Ch-BRA81 Brazil KP824808 RVG Ch-03V0567 Germany HQ403604 JQ920012 Ch-MRC-DPRU1679 South Africa KJ752088 KJ752083 Ty-Minnesota-1 USA KY689681 Ty-Minnesota-2 USA MF120219 Pg-HK18 Hong Kong KC876014 Hg-H01-10385 Netherlands KP057507 Pr-956-2 Italy KT073229 Ch-BRA41 Brazil KP824797 Ch-BRA43 Brazil KP824798 Ch-BRA49 Brazil KP824799 Ch-BRA68 Brazil KP824802 Ch-BRA75 Brazil KP824804 Ch-BRA76 Brazil KP824805 Ch-BRA77 Brazil KP824806 Ch-BRA78 Brazil KP824807
Table A3 Length of fragments analyzed for each RVF and RVG gene.
Gene Segment Virus Strains RVF RVG VP1 Complete ORF (3258 bp): BJ12 - VP2 Partial ORF (1200 bp): BJ1, BJ7, BJ12 - VP3 - - VP4 Complete ORF (2204 bp): BJ1, BJ7 - VP6 Complete ORF (1188 bp): BJ1, BJ7, BJ12 Complete ORF: MF48 (1173 bp) VP7 Partial ORF (398 bp): BJ1, BJ7, BJ12 - NSP1 Complete ORF (1641 bp): BJ12 - NSP2 Complete ORF (954 bp): BJ1 Partial ORF (953 bp): MF48 NSP3 - - NSP4 Complete ORF (507 bp): BJ1, BJ7, BJ12, BJ37, MF104, MF110 - NSP5 Complete ORF (654 bp): BJ1, BJ7, BJ10, BJ11, BJ12 Complete ORF (543 bp): MF40, MF41, MF48
By Mariana S. Pinheiro; Juliana B. L. Dias; Melissa P. Petrucci; Carlos E. P. F. Travassos; Gabriella S. Mendes and Norma Santos
Reported by Author; Author; Author; Author; Author; Author