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Distribution of G (VP7) and P (VP4) genotypes of group A bovine rotaviruses from Turkish calves with diarrhea, 1997–2008
Veterinary Microbiology 141 (2010) 231–237
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Distribution of G (VP7) and P (VP4) genotypes of group A bovine rotaviruses from Turkish calves with diarrhea, 1997–2008 Feray Alkan a,*, Aykut Ozkul a, Tuba C. Oguzoglu a, Mehmet O. Timurkan b, Elvin Caliskan c, Vito Martella d, Ibrahim Burgu a a
Ankara University, Faculty of Veterinary Medicine, Irfan Bastug Cad, Diskapi, 06110 Ankara, Turkey Atatu¨rk University, Faculty of Veterinary Medicine, Erzurum, Turkey Veterinary Control and Research Institute, Etlik, Ankara, Turkey d Department of Veterinary Public Health, University of Bari, Italy b c
A R T I C L E I N F O
A B S T R A C T
Article history: Received 15 April 2009 Received in revised form 3 September 2009 Accepted 22 September 2009
Group A rotaviruses are major enteric pathogens of calves. In order to investigate the genetic diversity of bovine rotaviruses (BRVs), a collection of 53 BRVs, detected from diarrheic calves from several Turkish geographical areas, between 1997 and 2008 was analyzed by RT-PCR for specificities of the outer capsid proteins VP7 (G type) and VP4 (P type), for the first time. Overall, G6 was the predominant G type, detected in 40/53 samples (75.4%), while P[11] was the predominant P type, detected in 52/53 samples (98.1%). The most common VP7/VP4 combinations were G6P[11] (60.3%) and G10P[11] (24.5%). Mixed infections were identified in 7/53 samples (13.2%). In the VP7 region the G6P[11] viruses were similar to other ones detected worldwide, forming an independent G6 lineage, distantly related to the G6 gene of the vaccine G6P[1] strain NCDV (90.1% amino acid identity), and suggesting that G6P[11] viruses represent a genetically stable BRV strain. The study of G and P type diversity is pivotal to understand the efficacy of the existing rotavirus vaccines and to provide the basis of future prophylaxis tools against rotaviral diarrhea of calves. ß 2009 Elsevier B.V. All rights reserved.
Keywords: Rotavirus RT-PCR Genotyping Bovine
1. Introduction Group A rotaviruses are major causative agents of severe diarrhea in humans and animals. The rotavirus genome consists of 11 segments of double-stranded RNA (dsRNA), encoding 6 structural (VP1–VP4, VP6 and VP7) and 6 non-structural proteins (NSP1–NSP6). The outer capsid proteins, VP7 and VP4, elicit the production of neutralizing antibodies (Hoshino et al., 1985; Snodgrass et al., 1991; Xu et al., 1993) and define the antigenic specificities, referred to as G type (glycoprotein) and P type (protease-sensitive protein), respectively (Estes, 2001).
* Corresponding author. Tel.: +90 312 3170315x363; fax: +90 312 3164472. E-mail address: [email protected] (F. Alkan). 0378-1135/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.vetmic.2009.09.016
Antigenic characterization of the VP7 and VP4 substantially overlaps the genetic characterization. Thus far 14 G serotypes (G1 to G14) and 22 G genotypes are recognized, while 14 P serotypes (P1A, P1B, and P2 to P13) and 32 P genotypes have been characterized (Martella et al., 2005, 2006, 2007; Matthijnssens et al., in press; Mc Neal et al., 2005; Rahman et al., 2005; Rao et al., 2000; Redmond et al., 1992; Steyer et al., 2007). The main G types of BRVs are G6, G8 and G10 (Martella et al., 1999; Falcone et al., 1999; Fukai et al., 1999; Parwani et al., 1993), although other G types (G1, G2, G3, G7, G11 and G15) have also been detected in calves (Adah et al., 2001; Alfieri et al., 2004; Hussein et al., 1993). The most common P types of BRVs are P[1], P[5] and P[11] (Falcone et al., 1999; Gulati et al., 1999; Reidy et al., 2006). The role of type-specific (homotypic) versus crossprotective (heterotypic) immunity for protection against
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rotavirus infection and disease is still debated. Several reports suggest that protection against rotavirus infection is mainly serotype-specific and human vaccines containing rotavirus strains matching the VP7/VP4 antigenic specificities of the more epidemiologically relevant strains have been developed and licensed (Hussein et al., 1993; Heaton and Ciarlet, 2007). Accordingly, gathering data on the genetic/antigenic diversity of human and animal rotaviruses is pivotal to assess the efficacy of currently available vaccines and to provide the basis for the development of future vaccines (Fukai et al., 1998; Garaicoechea et al., 2006; Lu et al., 1994; Reidy et al., 2006). Although BRV infection have been reported in Turkey on previous occasions (Alkan et al., 2004; Ok et al., 2009; Ozkul et al., 2002; Sahna-Can and Alkan, 2003), there is no information on the VP4 and VP7 diversity of the BRVs circulating locally. In this study we analyzed, for the first time, a collection of bovine rotaviruses (BRV) isolated from diarrheic calves from several Turkish geographical areas between 1997 and 2008. 2. Materials and methods 2.1. Reference virus strains The BRV strains B223 (G10P[11]), UK (G6P[5]) and NCDV-Lincoln (G6P[1]) were used as reference viruses. The viruses were kindly provided by Dr. Peter Nettleton (Moredun Research Institute, Moredun, England). 2.2. Fecal samples and RNA extraction Fecal samples were collected from diarrheic calves less than 1 month of age and screened by either a commercial ELISA test (BioK 067, Bio-X Diagnostic, Belgium) or by PAGE for the presence of group A rotaviruses. A total of 53 samples were found to be BRV-positive, 26 collected between 1997 and 2003, and 27 collected between 2005 and 2008. The extraction of rotaviral dsRNA was done by the procedure of Chomcyznski and Sacchi (1987). The resulting dsRNA pellet was dissolved in 20 ml of RNase-free nanopure water and used for cDNA synthesis. 2.3. RT-PCR for the detection and genotyping of BRV All samples were tested for BRV by RT-PCR, using rotavirus generic primers targeting the VP4 and VP7 protein coding genes. Subsequently, G- and P-genotyping of rotaviruses was carried out by RT-PCR using oligonucleotides specific for the major BRV G and P genotypes. Synthesis of cDNA was achieved following denaturation of RNA at 70 8C for 5 min. The cDNA was synthesized using Moloney Murine Leukemia Virus (MMLV) reverse transcriptase (RT) (Fermentas, Lithuania) and random hexamers (Fermentas, Lithuania), by incubating at 25 8C for 10 min, and thereafter at 37 8C for 1 h. MMLV-RT was therefore inactivated at 70 8C for 10 min (Iturizza-Gomara et al., 1999). Detection and characterization of BRV was performed as described elsewhere (Gouvea et al., 1990, 1994; Isegawa et al., 1993) with minor modifications using oligonucleotide primers designed by same researchers.
Briefly, PCR amplification was carried out in 30 ml of total reaction mixture by adding 3 ml of cDNA into a reaction cocktail containing 75 mM Tris–HCl (pH 8.8), 20 mM NH4(SO4)2, 2.4 mM MgCl2, 10 pmol of each primer, 0.2 mM dNTP, and 5 U of Taq DNA polymerase (MBI, Fermentas, Lithuania). The steps of thermal cycling were set up as follows; initial denaturation for 6 min at 94 8C, followed by 40 cycles of at 94 8C for 1 min, 48–52 8C for 1 min, 72 8C for 2 min, with a final extension at 50 8C for 1 min and 72 8C for 10 min. The resulting amplicons were analyzed on 1.5% agarose gel after electrophoresis at 80 V for 30 min and visualized under ultraviolet light. 2.4. Sequence analysis Since RT-PCR genotyping of the VP7 and VP4 genes unexpectedly characterized the vast majority of the samples as G6P[11] and G10P[11], a finding which is rather unusual in comparison with the epidemiological data available for other countries, the VP7 and VP4 amplicons of a G10P[11] and of a G6P[11] strain were subjected to sequencing. The PCR amplicons were excised from agarose gel, purified using commercial PCR/Gel purification kit (GeneMark Technology Co., Ltd., Tainan, Taiwan) and cloned into pTZ57R TA plasmid (Fermentas, Lithuania). Plasmid DNA was than subjected directly to sequencing in CEQ 8000 Genetic Analyzer (Beckmann Coulter, USA) using the Dye Termination Cycle Sequencing Kit (DTCS, Beckmann Coulter, USA). Sequence editing and multiple alignments were performed with Bioedit software package vers. 2.1 (Hall, 1999). Phylogenetic analysis (neighbor-joining) with bootstrap analysis (1000 replicates) and Kimura 2-parameter correction was conducted by using the MEGA software package v3.0 (Kumar et al., 2004). 3. Results Amplicons of the expected sizes were obtained in the generic RT-PCRs targeting the VP4 (856 bp) and VP7 (1013 bp) from the reference viruses (Fig. 1). Correct characterization was achieved by RT-PCR VP7 genotyping of the reference G6 viruses NCDV and UK (288 bp), and G10 virus B223 (715 bp). By VP4 RT-PCR genotyping, amplicons of 463 bp (P[1]-specific), 662 bp (P[5]-specific) and 335 bp (P[11]-specific) were generated with the viruses NCDV, UK and B223, respectively (Fig. 2). By RT-PCR genotyping of the Turkish BRV strains, one strain was characterized as G6P[5], 32 as G6P[11], 13 as G10P[11], 5 as G6P[5] + [11], 1 as G6 + 10P[11] and 1 as G6 + 10P[5] + P[11] (Table 1). Table 1 shows the distribution of the various G and P types and of the G/P combinations identified in the 53 field BRV strains. Analysis of fecal samples from two dairy herds (herds IV and X) gave the opportunity to monitor the circulation of rotavirus during an extended time span, since BRV infection was identified in those herds on more occasions. In the herd IV the same BRV strain, G6P[11], was detected in 2002 and in 2007, while in herd X, a G6P[11] BRV was identified in 2005, 2006 and 2008 (Table 2). Sequence analysis of the VP7 and VP4 genes of field G6P[11] and
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Fig. 1. PCR results based on VP4 and VP7 coding regions of BRV reference strains and BRV field strains. Lines 1 and 12, 3000 bp DNA ladder (Fermentas, Lithuania); lines 2 and 7 BRV NCDV strain (G6P[1]), lines 3 and 8 BRV UK strain (G6P[5]); lines 4 and 9 BRV B223 strain (G10P[11]); lines 5 and 6, and 10 and 11 BRV field strains.
Fig. 2. The results of RT-PCR products of reference viruses and of rotavirus field strains in fecal samples. (A) G genotypes: lines 1 and 5, 100 bp DNA ladder (Fermentas, Lithuania); line 2, BRV B223 strain (G10P[11]); line 6, BRV NCDV strain (G6P[1]); lines 3 and 4, and 7 and 8 BRV field strains; (B) P genotypes: lines 1 and 11, 100 bp DNA ladder (Fermentas, Lithuania); lines 2, BRV NCDV strain (G6P[1]); line 7, BRV UK strain (G6P[5]); line 8, BRV B223 strain (G10P[11]); lines 3–6, and 9 and 10, BRV field strains.
G10P[11] BRV strains (Turkey421-05/Ko/Bo and Turkey324060/Ko/Bo, respectively) confirmed the results obtained by PCR genotyping. In the VP7, the G6P[11] Turkish strain displayed 96.8% nt and 98% aa identity to the G10P[11] reference virus B233, while in the VP8* portion of the VP4, the identity to strain B233 was 95.8% nt and 97.8% aa. A G6P[11] strain, the predominant combination found in this study, was also analyzed. In the VP7 region, the highest identity (97.8–96.7% nt and 98.4–96.5 aa) was found to G6 strains VMRI-29, C8336, MC27 and RN-4, all which are P[11] and form a G6 VP7 lineage distantly related to the G6 strain included in the vaccines (NCDV). Several amino acids were found to differ between G6P[11] viruses and NCDV-like G6 strains in the antigenic regions A (87-Val ! Ile, 99-Asp ! Asn), B (148-Gln ! Leu), C (221Table 1 The G and P genotypes of bovine rotavirus strains in Turkey during 1997– 2008. Genotype
G6
G10
G6 + G10
Total
P[5] P[11] P[5] + P[11]
1 32 5
– 13 –
– 1 1
1 46 6
Total
38
13
2
53
Thr/Met ! Ala/Gln) and F (238-Asn ! Asp, 242-Ala ! Thr). Identity to strain NCDV was 90.1% aa and only 72% nt. In the VP8* portion of the VP4, the virus resembled P[11] strains (95.6% nt and 98.2% aa identity to strain B233). Phylogenetic analysis based on VP7 protein of G6 RVs revealed that Turkish BRV isolate (Turkey421-05/Ko/Bo; GeneBank Access. # FJ878799) were clustered together [P11] viruses in the tree (Fig. 3). 3.1. Nucleotide sequence accession numbers GenBank accession numbers FJ878797 (VP4 of G6P[11] genotype), FJ878798 (VP4 of G10P[11] genotype), FJ878799 (VP7 of G6P[11] genotype) and FJ878800 (VP7 of G10P[11] genotype) were assigned to rotavirus field strains isolated in this study 4. Discussion BRV is the most common etiological agent of severe diarrhea in calves. Preventing the spread of BRV infections in herds requires good hygiene and sanitation measures, along with vaccine prophylaxis, based on immunization of pregnant cows with either inactivated or attenuated vaccines in order to confer adequate passive immunity
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234 Table 2 Distribution of BRV field strains in herds. Herd Code
Sample Number
Genotype Location
Year
Combination
Number
Vaccine strain(s)
I
9
Ankara
1997
G6P[11]
9
–
II
4
Konya
1997
G6P[11] G6 + G10P[5] + [11]
3 1
– –
III
2
Bursa
2000
G6P[11] G6 + G10 P[11]
1 1
G6P[1] G6P[5]
IV
10
Ankara
2002 2007
G6P[11] G6P[11]
9 1
G6P[1] G6P[1] G6P[5]
V
1
Kirklareli
2005
G6P[11]
1
–
11
Samsun
2006
G10P[11] G6P[5] + [11]
8 3
G6P[1]
VII VIII IX
3 1 1
Samsun Denizli Igdir
2006 1997 2003
G10P[11] G6P[5] G6P[5] + P[11]
3 1 1
– – –
X
5
Aksaray
2005 2006 2008
G6P[5] + P[11] G6P[11] G6P[11]
1 2 2
G6P[1] G6P[1] –
XI
2
Eskisehir
2008
G6P[11]
2
–
XII
2
Sivas
2008
G10P[11]
2
G6P[1] G6P[5]
XIII XIV
1 1
Ankara Balikesir
2007 2008
G6P[11] G6P[11]
1 1
– G6P[5]
VI
to calves (Alkan et al., 2004; Barreiros et al., 2004; Kohara et al., 1997). Vaccine failures or breakthroughs may be accounted for by several reasons, chiefly vaccination management. It is unclear whether antigenic mismatches between the vaccine antigens and the antigenic make up of field viruses may also affect, to some extent, the efficacy of the vaccines and therefore the study of the antigenic/genetic diversity of field BRV strains is paramount to the investigation of this issue. Surveillance of the human RV types involved in gastroenteritis episodes throughout the world has been used to define the appropriate vaccine formulations to prevent human RV infections in pediatric populations. The current human RV vaccines have been targeted to the most epidemiologically relevant VP7 and VP4 antigens, i.e. G1, G2, G3, G4 and P[8] (Heaton and Ciarlet, 2007). Whether a similar approach may be used to implement the current BRV vaccines must be considered. In a study in Brazil, vaccinated herds displayed unusual combinations of G and P types, while strains matching the vaccine strain, G6[P1] were much rarer than in unvaccinated animals (Barreiros et al., 2004). As evidenced by several epidemiological studies worldwide, the majority of BRV strains are G6 and G10 (Alfieri et al., 2004; Garaicoechea et al., 2006; Reidy et al., 2006) while G8 BRVs appear to be less common (Falcone et al., 1999; Parwani et al., 1993), although in a study in Japan, G8 was the predominant serotype (Fukai et al., 1999). In this study, the majority (40/53) of the BRVs detected in Turkey between 1997 and 2008 were characterized as G6, followed by G10 (15/53), while G8 viruses were not
detected. Interestingly, the most common VP4 type in this study was P[11], and it was identified in 52 out of 53 samples, followed by P[5] (7/53). When considering the frequency of detection in the various herds, the P[11] and P[5] types were detected in 92.8% (13/14) and 35.7% (5/14) of the herds, respectively, a finding that mirrors data reported elsewhere (Falcone et al., 1999). The most common binary combinations in group A bovine rotaviruses are G6P[1], G6P[5], G10P[11] and G8P[1], which are exhibited by the reference strains NCDV, UK, B223 and A5, respectively (Elleman et al., 1983; Taniguchi et al., 1991; Woode et al., 1983). The most common VP7/VP4 combination in BRVs identified from Turkish calves were G6P[11] (60.4%, 32/53) and G10P[11] (24.5%, 13/53) (Table 1). Similar high detection rates of G6P[11] and G10P[11] BRVs have been observed in Brazil (Alfieri et al., 2004), Italy (Falcone et al., 1999) and India (Gulati et al., 1999) but not in other countries. In USA and UK, G6P[1] and G10P[1] BRVs strains were found to be more common (Parwani et al., 1993; Redmond et al., 1992), while the most prevalent combination in a study in Japan was G6P[5], followed by G10P[11], G6P[11] and G10P[5] (Ishizaki et al., 1996). Similarly, G6P[5] has been also reported as the most prevalent BRV genotype in Italy (Falcone et al., 1999) and Ireland (Reidy et al., 2006). Analysis of the VP7 and VP4 of the predominant Turkish strains, G6P[11] and G10P[11], confirmed the results of RTPCR genotyping. The Turkish G10P[11] strain was similar to the prototype G10P[11] virus B223, isolated in USA in the early 1980s in both in the VP7 and VP4 proteins (Woode et al., 1983). Despite the fact that sequenced
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Fig. 3. Phylogenetic tree based on the VP7 of G6 human and animal RV strains. The tree was inferred using the nucleotide alignment by the neighbor-joining method, with the Kimura’s two parameters distance correction. Statistical support was obtained by bootstrapping analysis over 1000 replicates. The Turkish G6P[11] strain is in boldface and underlined.
Turkish G10P[11] strain was identified about 25 years later form the North American strain, both viruses were conserved in both outer capsid proteins (96.8% nt and 98% aa in the VP7; 95.8% nt and 97.8% aa in the VP8* portion of the VP4). In a similar fashion, analysis of the predominant G6P[11] virus showed marked conservation with respect to other G6P[11] viruses detected from various geographical areas (VMRI-29, C8336, MC27 and KN-4), not only in the VP7 but also in the VP4. Indeed, the G6P[11] viruses tended to form a unique G6 lineage (Fig. 3), distantly related (only 90.1 aa identity) to the G6 strain NCDV, included in the vaccines. Such intra-serotype genetic/antigenic heterogeneity, coupled with the different VP4 antigen, could help G6P[11] strains to escape the immune pressure induced by the G6P[1] and G6P[5] vaccines. This observation would be consistent with the high prevalence of G6P[11] viruses observed in herds vaccinated with the G6P[1] NCDV-based vaccine (Barreiros et al., 2004).
In this study, rotavirus vaccines were used in six out of 14 herds, and the vaccines contained G6P[1] and G6P[5] strains (Table 2). Most of the strains detected in the vaccinated herds were characterized as either G6P[11] or G10P[11]. Co-segregation between G6 lineages and P types might be indicative of multiple reassortment events occurring in the context of a constant pattern of linear evolution (Martella et al., 2003). Cross-neutralization with immune sera obtained form naturally infected and vaccinated animals could help understand the real impact of the genetic heterogeneity oberved in BRVs. In conclusion, the data presented here emphasize the need for monitoring the distribution of rotavirus genotypes in bovine herds. The epidemiological information gathered on BRV genetic/antigenic heterogeneity will be useful in understanding the patterns of replacement over time and to improve vaccination strategies. A 4-years study in Japan has demonstrated that BRV strains may
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show marked temporal fluctuations in a restricted geographical area (Fukai et al., 2002) and similar findings have been observed during a 25-year long surveillance for human RVs in Palermo, Italy (Arista et al., 2006), suggesting that temporal replacement of the various RV strains and variants is a mechanism able to decrease rotavirus-specific immunity in local populations. In addition, epidemiological studies are necessary to monitor the onset of unusual animal strains and to gather baseline information for the understating of rotavirus ecology in various animal species. Acknowledgements The authors thank Dr. Peter Nettleton, Edinburgh, United Kingdom for providing the laboratory strains used in the present study. This project was supported by a grant of Turkish Scientific and Technical Research Council (Project No: TOVAG-2068). References Adah, M.I., Wade, A., Taniguchi, K., 2001. Molecular epidemiology of rotavirus in Nigeria: detection of unusual strains with G2P[6] and G8P[1] specificities. J. Clin. Microbiol. 39, 3969–3975. Alfieri, A.F., Alfieri, A.A., Barreiros, M.A., Leite, J.P., Richtzenhain, L.J., 2004. G and P genotypes of group A rotavirus strains circulating in calves in Brazil, 1996–1999. Vet. Microbiol. 99, 167–173. Alkan, F., Burgu, I., Can-S¸ahna, K., C¸okc¸alıs¸kan, C., 2004. Yeni dog˘an buzag˘ı ishallerine kars¸ı ticari as¸ı ile as¸ılanan sıg˘ırlardan dog˘an ¨ niv. Vet. Fak. Derg. yavrularda pasif bag˘ıs¸ıklık du¨zeyi. Ankara U 51, 47–53. Arista, S., Giammanco, G.M., De Grazia, S., Ramirez, S., Lo Biundo, C., Colomba, C., Cascio, A., Martella, V., 2006. Heterogeneity and temporal dynamics of evolution of G1 human rotaviruses in a settled population. J. Virol. 80, 10724–10733. Barreiros, M.A.B., Alfieri, A.F., Medici, K.C., Leite, J.P.G., Alfieri, A.A., 2004. G and P genotypes of Group A rotavirus from diarrhoeic calves born to cows vaccinated against the NCDV (P[1], G6) rotavirus strain. J. Vet. Med. B 51, 104–109. Chomcyznski, P., Sacchi, N., 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction. Anal. Biochem. 162, 156–159. Elleman, T.C., Hoyne, P.A., Dyall-Smith, M.L., Holmes, I.H., Azad, A.A., 1983. Nucleotide sequence of the gene encoding the serotype-specific glycoprotein of UK bovine rotavirus. Nucleic Acids Res. 11, 4689– 4701. Estes, M.K., 2001. Rotavirus and their replication. In: Knipe, D.M., Howley, P.M. (Eds.), Fields Virology. 4th ed. Lippincott, Williams and Wilkins, Philadelphia, PA, pp. 1747–1785. Falcone, E., Tarantino, M., Di Trani, L., Cordioli, P., Lavazza, A., Tollis, M., 1999. Determination of bovine rotavirus G and P serotypes in Italy by PCR. J. Clin. Microbiol. 37, 3879–3882. Fukai, K., Sakai, T., Kamata, H., 1998. Distribution of G serotypes and P genotypes of bovine group A rotavirus isolated in Japan. Aust. Vet. J. 76, 418–422. Fukai, K., Sakai, T., Hirose, M., Itou, T., 1999. Prevalence of calf diarrhea caused by bovine group A rotavirus carrying G serotype 8 specificity. Vet. Microbiol. 66, 301–311. Fukai, K., Maeda, Y., Fujimoto, K., Itou, T., Sakai, T., 2002. Changes in the prevalence of rotavirus G and P types in diarrheic calves from the Kagoshima prefecture in Japan. Vet. Microbiol. 86, 343–349. Garaicoechea, L., Bok, K., Jones, L.R., Combessies, G., Odeon, A., Fernandez, F., Parreno, V., 2006. Molecular characterization of bovine rotavirus circulating in beef and dairy herds in Argentina during a 10-year period (1994–2003). Vet. Microbiol. 118, 1–11. Gouvea, V., Glass, R.I., Woods, P., Taniguchi, K., Clark, H.F., Forrester, B., Fang, Z.Y., 1990. Polymerase chain reaction amplification and typing of rotavirus nucleic acids from stool specimens. J. Clin. Microbiol. 28, 276–282. Gouvea, V., Santos, N., Timenetsky, Mdo, C., 1994. Identification of bovine and porcine rotavirus G types by PCR. J. Clin. Microbiol. 32, 1338– 1340.
Gulati, R.B., Nakagomi, O., Koshimura, Y., Nakagomi, T., Pandey, R., 1999. Relative frequencies of G and P types among rotavirus from Indian diarrheic cow and buffalo calves. J. Clin. Microbiol. 37, 2074–2076. Hall, T.A., 1999. BioEdit: a user-friendly biological sequence alignment and analysis program for Windows 95/98/NT. Nucleic Acids Symp. 41, 95–98. Heaton, P.M., Ciarlet, M., 2007. The pentavalent rotavirus vaccine: discovery to licensure and beyond. Infect. Clin. Dis. 45, 1618– 1624. Hoshino, Y., Sereno, M.M., Midthun, K., Flores, J., Kapikian, A.Z., Channock, R.M., 1985. Independed segregation of two antigen specifities (VP3 and VP7) involved in neutralization of rotavirus infectivity. Proc. Natl. Acad. Sci. U.S.A. 82, 8701–8704. Hussein, H.A., Parwani, A.V., Rosen, B.I., Lucchelli, A., Saif, L.J., 1993. Detection of rotavirus serotypes G1, G2, G3 and G11 in feces of diarrheic calves by using polymerase chain reaction derived cDNA probes. J. Clin. Microbiol. 31, 2491–2496. Isegawa, Y., Nakagomi, O., Nakagomi, T., Ishida, S., Uesugi, S., Ueda, S., 1993. Determination of bovine rotavirus G and P serotypes by polymerase chain reaction. Mol. Cell. Probes 7, 277–284. Ishizaki, H., Sakai, T., Shirahata, T., Taniguchi, K., Urasawa, T., Urasawa, S., Goto, H., 1996. The distribution of G and P types within isolates of bovine rotavirus in Japan. Vet. Microbiol. 48, 367–372. Iturizza-Gomara, M., Green, J., Brown, D.W., Desselberger, U., Gray, J.J., 1999. Comparison of specific and random priming in the reverse transcription polymerase chain reaction for genotyping group A rotaviruses. J. Virol. Methods 78, 93–103. Kohara, J., Hirai, T., Mori, K., Ishizaki, H., Tsunemitsu, H., 1997. Enhancement of passive immunity with maternal vaccine against newborn calf diarrhea. J. Vet. Med. Sci. 62, 219–221. Kumar, S., Tamura, K., Nei, M., 2004. MEGA3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform. 5, 150–163. Lu, W., Duhamel, E.D., Benfield, A.B., Grotelueschen, D.M., 1994. Serological and genotypic characterization of group A rotavirus reassortants from diarrheic calves born to dams vaccinated against rotavirus. Vet. Microbiol. 42, 159–170. Martella, V., Ciarlet, M., Banyai, K., Loursso, E., Arista, S., Lavazza, A., Pezzotti, G., Decaro, N., Cavalli, A., Lecente, M.S., Corrente, M., Elia, G., Camero, M., Tempesta, M., Buonavoglia, C., 2007. Identification of group A porcine rotavirus strains bearing a novel VP4 (P) genotype in Italian swine herds. J. Clin. Microbiol. 45, 577–580. Martella, V., Ciarlet, M., Banyai, K., Loursso, E., Cavalli, A., Corrente, M., Elia, G., Arista, S., Camero, M., Decaro, N., Lavazza, A., Buonavoglia, C., 2006. Identification of a novel VP4 genotype carried by a serotype G5 porcine rotavirus strain. Virology 346, 301–311. Martella, V., Ciarlet, M., Baselga, R., Arista, S., Elia, G., Loursso, E., Banyai, K., Terio, V., Madio, A., Ruggeri, F.M., Falcone, E., Camero, M., Decaro, N., Buonavoglia, C., 2005. Sequence analysis of the VP7 and VP4 genes identifies a novel VP7 gene allele of porcine rotavirus, sharing a common evolutionary origin with human G2 rotavirus. Virology 337, 111–123. Martella, V., Ciarlet, M., Pratelli, A., Arista, S., Terio, V., Elia, G., Cavalli, A., Gentile, M., Decaro, N., Greco, G., Cafiero, M.A., Tempesta, M., Buonavoglia, C., 2003. Molecular analysis of the VP7, VP4, VP6, NSP4 and NSP5/6 genes of a buffalo rotavirus strain: identification of the rare P[3] rhesus rotavirus-like VP4 gene allele. J. Clin. Microbiol. 41, 5665– 5675. Martella, V., Pratelli, A., Pinto, O., Ferrara, G., Tempesta, M., Buonavoglia, C., 1999. Typing by polymerase chain reaction of buffalo rotaviruses isolated in Italy. Zentralbl. Veterinarmed. B 46, 499–502. Matthijnssens, J., Bilcke, J., Ciarlet, M., Martella, V., Ba´nyai, K., Rahman, M., Zeller, M., Beutels, P., Van Damme, P., Van Ranst, M., in press. Rotaviruses burden of disease. Future Microbiol. Rev. Mc Neal, M.M., Sestak, K., Choi, A.H., Basu, M., Cole, M.J., Aye, P.P., Bohm, R.P., Ward, R.L., 2005. Development of a rotavirus-shedding model in rhesus macaques, using a homologous wild-type rotavirus of a new P genotype. J. Virol. 79, 944–954. Ok, M., Gu¨ler, L., Turgut, K., Ok, U., Sen, I., Gu¨ndu¨z, I.K., Birdane, M.F., Gu¨zelbektes, H., 2009. The studies on the aetiology of diarrhoea in neonatal calves and determination of virulence gene markers of Escherichia coli strains by multiplex PCR. Zoonoses Public Health 56, 94–101. Ozkul, A., Yes¸ilbag˘, K., Karaog˘lu, T., Burgu, I., 2002. Elecrophoretypes of bovine rotavirus detected in Turkey. Turk. J. Vet. Anim. Sci. 26, 359– 362. Parwani, A.V., Hussein, H.A., Rosen, B.I., Luchelli, A., Navarro, L.J., 1993. Characterisation of field strains of group A bovine rotaviruses by using polymerase chain reaction-generated G and P type specific cDNA probes. J. Clin. Microbiol. 31, 2010–2015.
F. Alkan et al. / Veterinary Microbiology 141 (2010) 231–237 Rahman, M., Matthijnssens, J., Nahar, S., Podder, G., Sack, D.A., Azim, T., Van Ranst, M., 2005. Characterization of a novel P[25], G11 human group a rotavirus. J. Clin. Microbiol. 43, 3208–3212. Rao, C.D., Gowda, K., Reddy, B.S.Y., 2000. Sequence analysis of VP4 and VP7 genes of nontypeable strains identifies a new pair of outer capsid proteins representing novel P and G genotypes in bovine rotaviruses. Virology 276, 104–113. Redmond, D.L., Inglis, N.F., Fitzgerald, T.A., Snodgrass, D.R., Herring, A.J., 1992. A liquid-hybridization method for typing the VP4 and VP7 genes of bovine rotaviruses. J. Virol. Methods 39, 165–177. Reidy, N., Lennon, G., Fanning, S., Power, E., O’Shea, H., 2006. Molecular characterization and analysis of bovine rotavirus strains circulating in Ireland 2002–2004. Vet. Microbiol. 117, 242–247. Sahna-Can, K., Alkan, F., 2003. Sıg˘ırlarda Rotavirus Enfeksiyonunun Epi¨ niversitesi Sag˘lık Bilimleri demiyolojisinde Gebelig˘in Rolu¨. Fırat U Dergisi 17, 203–209. Snodgrass, D.R., Fitzgerald, T.A., Campbell, I., Browning, G.F., Scott, F.M.M., Hoshino, Y., Davies, R.C., 1991. Homotypic and heterotypic serological
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Distribution of G (VP7) and P (VP4) genotypes of group A bovine rotaviruses from Turkish calves with diarrhea, 1997–2008 Feray Alkan a,*, Aykut Ozkul a, Tuba C. Oguzoglu a, Mehmet O. Timurkan b, Elvin Caliskan c, Vito Martella d, Ibrahim Burgu a a
Ankara University, Faculty of Veterinary Medicine, Irfan Bastug Cad, Diskapi, 06110 Ankara, Turkey Atatu¨rk University, Faculty of Veterinary Medicine, Erzurum, Turkey Veterinary Control and Research Institute, Etlik, Ankara, Turkey d Department of Veterinary Public Health, University of Bari, Italy b c
A R T I C L E I N F O
A B S T R A C T
Article history: Received 15 April 2009 Received in revised form 3 September 2009 Accepted 22 September 2009
Group A rotaviruses are major enteric pathogens of calves. In order to investigate the genetic diversity of bovine rotaviruses (BRVs), a collection of 53 BRVs, detected from diarrheic calves from several Turkish geographical areas, between 1997 and 2008 was analyzed by RT-PCR for specificities of the outer capsid proteins VP7 (G type) and VP4 (P type), for the first time. Overall, G6 was the predominant G type, detected in 40/53 samples (75.4%), while P[11] was the predominant P type, detected in 52/53 samples (98.1%). The most common VP7/VP4 combinations were G6P[11] (60.3%) and G10P[11] (24.5%). Mixed infections were identified in 7/53 samples (13.2%). In the VP7 region the G6P[11] viruses were similar to other ones detected worldwide, forming an independent G6 lineage, distantly related to the G6 gene of the vaccine G6P[1] strain NCDV (90.1% amino acid identity), and suggesting that G6P[11] viruses represent a genetically stable BRV strain. The study of G and P type diversity is pivotal to understand the efficacy of the existing rotavirus vaccines and to provide the basis of future prophylaxis tools against rotaviral diarrhea of calves. ß 2009 Elsevier B.V. All rights reserved.
Keywords: Rotavirus RT-PCR Genotyping Bovine
1. Introduction Group A rotaviruses are major causative agents of severe diarrhea in humans and animals. The rotavirus genome consists of 11 segments of double-stranded RNA (dsRNA), encoding 6 structural (VP1–VP4, VP6 and VP7) and 6 non-structural proteins (NSP1–NSP6). The outer capsid proteins, VP7 and VP4, elicit the production of neutralizing antibodies (Hoshino et al., 1985; Snodgrass et al., 1991; Xu et al., 1993) and define the antigenic specificities, referred to as G type (glycoprotein) and P type (protease-sensitive protein), respectively (Estes, 2001).
* Corresponding author. Tel.: +90 312 3170315x363; fax: +90 312 3164472. E-mail address: [email protected] (F. Alkan). 0378-1135/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.vetmic.2009.09.016
Antigenic characterization of the VP7 and VP4 substantially overlaps the genetic characterization. Thus far 14 G serotypes (G1 to G14) and 22 G genotypes are recognized, while 14 P serotypes (P1A, P1B, and P2 to P13) and 32 P genotypes have been characterized (Martella et al., 2005, 2006, 2007; Matthijnssens et al., in press; Mc Neal et al., 2005; Rahman et al., 2005; Rao et al., 2000; Redmond et al., 1992; Steyer et al., 2007). The main G types of BRVs are G6, G8 and G10 (Martella et al., 1999; Falcone et al., 1999; Fukai et al., 1999; Parwani et al., 1993), although other G types (G1, G2, G3, G7, G11 and G15) have also been detected in calves (Adah et al., 2001; Alfieri et al., 2004; Hussein et al., 1993). The most common P types of BRVs are P[1], P[5] and P[11] (Falcone et al., 1999; Gulati et al., 1999; Reidy et al., 2006). The role of type-specific (homotypic) versus crossprotective (heterotypic) immunity for protection against
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rotavirus infection and disease is still debated. Several reports suggest that protection against rotavirus infection is mainly serotype-specific and human vaccines containing rotavirus strains matching the VP7/VP4 antigenic specificities of the more epidemiologically relevant strains have been developed and licensed (Hussein et al., 1993; Heaton and Ciarlet, 2007). Accordingly, gathering data on the genetic/antigenic diversity of human and animal rotaviruses is pivotal to assess the efficacy of currently available vaccines and to provide the basis for the development of future vaccines (Fukai et al., 1998; Garaicoechea et al., 2006; Lu et al., 1994; Reidy et al., 2006). Although BRV infection have been reported in Turkey on previous occasions (Alkan et al., 2004; Ok et al., 2009; Ozkul et al., 2002; Sahna-Can and Alkan, 2003), there is no information on the VP4 and VP7 diversity of the BRVs circulating locally. In this study we analyzed, for the first time, a collection of bovine rotaviruses (BRV) isolated from diarrheic calves from several Turkish geographical areas between 1997 and 2008. 2. Materials and methods 2.1. Reference virus strains The BRV strains B223 (G10P[11]), UK (G6P[5]) and NCDV-Lincoln (G6P[1]) were used as reference viruses. The viruses were kindly provided by Dr. Peter Nettleton (Moredun Research Institute, Moredun, England). 2.2. Fecal samples and RNA extraction Fecal samples were collected from diarrheic calves less than 1 month of age and screened by either a commercial ELISA test (BioK 067, Bio-X Diagnostic, Belgium) or by PAGE for the presence of group A rotaviruses. A total of 53 samples were found to be BRV-positive, 26 collected between 1997 and 2003, and 27 collected between 2005 and 2008. The extraction of rotaviral dsRNA was done by the procedure of Chomcyznski and Sacchi (1987). The resulting dsRNA pellet was dissolved in 20 ml of RNase-free nanopure water and used for cDNA synthesis. 2.3. RT-PCR for the detection and genotyping of BRV All samples were tested for BRV by RT-PCR, using rotavirus generic primers targeting the VP4 and VP7 protein coding genes. Subsequently, G- and P-genotyping of rotaviruses was carried out by RT-PCR using oligonucleotides specific for the major BRV G and P genotypes. Synthesis of cDNA was achieved following denaturation of RNA at 70 8C for 5 min. The cDNA was synthesized using Moloney Murine Leukemia Virus (MMLV) reverse transcriptase (RT) (Fermentas, Lithuania) and random hexamers (Fermentas, Lithuania), by incubating at 25 8C for 10 min, and thereafter at 37 8C for 1 h. MMLV-RT was therefore inactivated at 70 8C for 10 min (Iturizza-Gomara et al., 1999). Detection and characterization of BRV was performed as described elsewhere (Gouvea et al., 1990, 1994; Isegawa et al., 1993) with minor modifications using oligonucleotide primers designed by same researchers.
Briefly, PCR amplification was carried out in 30 ml of total reaction mixture by adding 3 ml of cDNA into a reaction cocktail containing 75 mM Tris–HCl (pH 8.8), 20 mM NH4(SO4)2, 2.4 mM MgCl2, 10 pmol of each primer, 0.2 mM dNTP, and 5 U of Taq DNA polymerase (MBI, Fermentas, Lithuania). The steps of thermal cycling were set up as follows; initial denaturation for 6 min at 94 8C, followed by 40 cycles of at 94 8C for 1 min, 48–52 8C for 1 min, 72 8C for 2 min, with a final extension at 50 8C for 1 min and 72 8C for 10 min. The resulting amplicons were analyzed on 1.5% agarose gel after electrophoresis at 80 V for 30 min and visualized under ultraviolet light. 2.4. Sequence analysis Since RT-PCR genotyping of the VP7 and VP4 genes unexpectedly characterized the vast majority of the samples as G6P[11] and G10P[11], a finding which is rather unusual in comparison with the epidemiological data available for other countries, the VP7 and VP4 amplicons of a G10P[11] and of a G6P[11] strain were subjected to sequencing. The PCR amplicons were excised from agarose gel, purified using commercial PCR/Gel purification kit (GeneMark Technology Co., Ltd., Tainan, Taiwan) and cloned into pTZ57R TA plasmid (Fermentas, Lithuania). Plasmid DNA was than subjected directly to sequencing in CEQ 8000 Genetic Analyzer (Beckmann Coulter, USA) using the Dye Termination Cycle Sequencing Kit (DTCS, Beckmann Coulter, USA). Sequence editing and multiple alignments were performed with Bioedit software package vers. 2.1 (Hall, 1999). Phylogenetic analysis (neighbor-joining) with bootstrap analysis (1000 replicates) and Kimura 2-parameter correction was conducted by using the MEGA software package v3.0 (Kumar et al., 2004). 3. Results Amplicons of the expected sizes were obtained in the generic RT-PCRs targeting the VP4 (856 bp) and VP7 (1013 bp) from the reference viruses (Fig. 1). Correct characterization was achieved by RT-PCR VP7 genotyping of the reference G6 viruses NCDV and UK (288 bp), and G10 virus B223 (715 bp). By VP4 RT-PCR genotyping, amplicons of 463 bp (P[1]-specific), 662 bp (P[5]-specific) and 335 bp (P[11]-specific) were generated with the viruses NCDV, UK and B223, respectively (Fig. 2). By RT-PCR genotyping of the Turkish BRV strains, one strain was characterized as G6P[5], 32 as G6P[11], 13 as G10P[11], 5 as G6P[5] + [11], 1 as G6 + 10P[11] and 1 as G6 + 10P[5] + P[11] (Table 1). Table 1 shows the distribution of the various G and P types and of the G/P combinations identified in the 53 field BRV strains. Analysis of fecal samples from two dairy herds (herds IV and X) gave the opportunity to monitor the circulation of rotavirus during an extended time span, since BRV infection was identified in those herds on more occasions. In the herd IV the same BRV strain, G6P[11], was detected in 2002 and in 2007, while in herd X, a G6P[11] BRV was identified in 2005, 2006 and 2008 (Table 2). Sequence analysis of the VP7 and VP4 genes of field G6P[11] and
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233
Fig. 1. PCR results based on VP4 and VP7 coding regions of BRV reference strains and BRV field strains. Lines 1 and 12, 3000 bp DNA ladder (Fermentas, Lithuania); lines 2 and 7 BRV NCDV strain (G6P[1]), lines 3 and 8 BRV UK strain (G6P[5]); lines 4 and 9 BRV B223 strain (G10P[11]); lines 5 and 6, and 10 and 11 BRV field strains.
Fig. 2. The results of RT-PCR products of reference viruses and of rotavirus field strains in fecal samples. (A) G genotypes: lines 1 and 5, 100 bp DNA ladder (Fermentas, Lithuania); line 2, BRV B223 strain (G10P[11]); line 6, BRV NCDV strain (G6P[1]); lines 3 and 4, and 7 and 8 BRV field strains; (B) P genotypes: lines 1 and 11, 100 bp DNA ladder (Fermentas, Lithuania); lines 2, BRV NCDV strain (G6P[1]); line 7, BRV UK strain (G6P[5]); line 8, BRV B223 strain (G10P[11]); lines 3–6, and 9 and 10, BRV field strains.
G10P[11] BRV strains (Turkey421-05/Ko/Bo and Turkey324060/Ko/Bo, respectively) confirmed the results obtained by PCR genotyping. In the VP7, the G6P[11] Turkish strain displayed 96.8% nt and 98% aa identity to the G10P[11] reference virus B233, while in the VP8* portion of the VP4, the identity to strain B233 was 95.8% nt and 97.8% aa. A G6P[11] strain, the predominant combination found in this study, was also analyzed. In the VP7 region, the highest identity (97.8–96.7% nt and 98.4–96.5 aa) was found to G6 strains VMRI-29, C8336, MC27 and RN-4, all which are P[11] and form a G6 VP7 lineage distantly related to the G6 strain included in the vaccines (NCDV). Several amino acids were found to differ between G6P[11] viruses and NCDV-like G6 strains in the antigenic regions A (87-Val ! Ile, 99-Asp ! Asn), B (148-Gln ! Leu), C (221Table 1 The G and P genotypes of bovine rotavirus strains in Turkey during 1997– 2008. Genotype
G6
G10
G6 + G10
Total
P[5] P[11] P[5] + P[11]
1 32 5
– 13 –
– 1 1
1 46 6
Total
38
13
2
53
Thr/Met ! Ala/Gln) and F (238-Asn ! Asp, 242-Ala ! Thr). Identity to strain NCDV was 90.1% aa and only 72% nt. In the VP8* portion of the VP4, the virus resembled P[11] strains (95.6% nt and 98.2% aa identity to strain B233). Phylogenetic analysis based on VP7 protein of G6 RVs revealed that Turkish BRV isolate (Turkey421-05/Ko/Bo; GeneBank Access. # FJ878799) were clustered together [P11] viruses in the tree (Fig. 3). 3.1. Nucleotide sequence accession numbers GenBank accession numbers FJ878797 (VP4 of G6P[11] genotype), FJ878798 (VP4 of G10P[11] genotype), FJ878799 (VP7 of G6P[11] genotype) and FJ878800 (VP7 of G10P[11] genotype) were assigned to rotavirus field strains isolated in this study 4. Discussion BRV is the most common etiological agent of severe diarrhea in calves. Preventing the spread of BRV infections in herds requires good hygiene and sanitation measures, along with vaccine prophylaxis, based on immunization of pregnant cows with either inactivated or attenuated vaccines in order to confer adequate passive immunity
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234 Table 2 Distribution of BRV field strains in herds. Herd Code
Sample Number
Genotype Location
Year
Combination
Number
Vaccine strain(s)
I
9
Ankara
1997
G6P[11]
9
–
II
4
Konya
1997
G6P[11] G6 + G10P[5] + [11]
3 1
– –
III
2
Bursa
2000
G6P[11] G6 + G10 P[11]
1 1
G6P[1] G6P[5]
IV
10
Ankara
2002 2007
G6P[11] G6P[11]
9 1
G6P[1] G6P[1] G6P[5]
V
1
Kirklareli
2005
G6P[11]
1
–
11
Samsun
2006
G10P[11] G6P[5] + [11]
8 3
G6P[1]
VII VIII IX
3 1 1
Samsun Denizli Igdir
2006 1997 2003
G10P[11] G6P[5] G6P[5] + P[11]
3 1 1
– – –
X
5
Aksaray
2005 2006 2008
G6P[5] + P[11] G6P[11] G6P[11]
1 2 2
G6P[1] G6P[1] –
XI
2
Eskisehir
2008
G6P[11]
2
–
XII
2
Sivas
2008
G10P[11]
2
G6P[1] G6P[5]
XIII XIV
1 1
Ankara Balikesir
2007 2008
G6P[11] G6P[11]
1 1
– G6P[5]
VI
to calves (Alkan et al., 2004; Barreiros et al., 2004; Kohara et al., 1997). Vaccine failures or breakthroughs may be accounted for by several reasons, chiefly vaccination management. It is unclear whether antigenic mismatches between the vaccine antigens and the antigenic make up of field viruses may also affect, to some extent, the efficacy of the vaccines and therefore the study of the antigenic/genetic diversity of field BRV strains is paramount to the investigation of this issue. Surveillance of the human RV types involved in gastroenteritis episodes throughout the world has been used to define the appropriate vaccine formulations to prevent human RV infections in pediatric populations. The current human RV vaccines have been targeted to the most epidemiologically relevant VP7 and VP4 antigens, i.e. G1, G2, G3, G4 and P[8] (Heaton and Ciarlet, 2007). Whether a similar approach may be used to implement the current BRV vaccines must be considered. In a study in Brazil, vaccinated herds displayed unusual combinations of G and P types, while strains matching the vaccine strain, G6[P1] were much rarer than in unvaccinated animals (Barreiros et al., 2004). As evidenced by several epidemiological studies worldwide, the majority of BRV strains are G6 and G10 (Alfieri et al., 2004; Garaicoechea et al., 2006; Reidy et al., 2006) while G8 BRVs appear to be less common (Falcone et al., 1999; Parwani et al., 1993), although in a study in Japan, G8 was the predominant serotype (Fukai et al., 1999). In this study, the majority (40/53) of the BRVs detected in Turkey between 1997 and 2008 were characterized as G6, followed by G10 (15/53), while G8 viruses were not
detected. Interestingly, the most common VP4 type in this study was P[11], and it was identified in 52 out of 53 samples, followed by P[5] (7/53). When considering the frequency of detection in the various herds, the P[11] and P[5] types were detected in 92.8% (13/14) and 35.7% (5/14) of the herds, respectively, a finding that mirrors data reported elsewhere (Falcone et al., 1999). The most common binary combinations in group A bovine rotaviruses are G6P[1], G6P[5], G10P[11] and G8P[1], which are exhibited by the reference strains NCDV, UK, B223 and A5, respectively (Elleman et al., 1983; Taniguchi et al., 1991; Woode et al., 1983). The most common VP7/VP4 combination in BRVs identified from Turkish calves were G6P[11] (60.4%, 32/53) and G10P[11] (24.5%, 13/53) (Table 1). Similar high detection rates of G6P[11] and G10P[11] BRVs have been observed in Brazil (Alfieri et al., 2004), Italy (Falcone et al., 1999) and India (Gulati et al., 1999) but not in other countries. In USA and UK, G6P[1] and G10P[1] BRVs strains were found to be more common (Parwani et al., 1993; Redmond et al., 1992), while the most prevalent combination in a study in Japan was G6P[5], followed by G10P[11], G6P[11] and G10P[5] (Ishizaki et al., 1996). Similarly, G6P[5] has been also reported as the most prevalent BRV genotype in Italy (Falcone et al., 1999) and Ireland (Reidy et al., 2006). Analysis of the VP7 and VP4 of the predominant Turkish strains, G6P[11] and G10P[11], confirmed the results of RTPCR genotyping. The Turkish G10P[11] strain was similar to the prototype G10P[11] virus B223, isolated in USA in the early 1980s in both in the VP7 and VP4 proteins (Woode et al., 1983). Despite the fact that sequenced
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Fig. 3. Phylogenetic tree based on the VP7 of G6 human and animal RV strains. The tree was inferred using the nucleotide alignment by the neighbor-joining method, with the Kimura’s two parameters distance correction. Statistical support was obtained by bootstrapping analysis over 1000 replicates. The Turkish G6P[11] strain is in boldface and underlined.
Turkish G10P[11] strain was identified about 25 years later form the North American strain, both viruses were conserved in both outer capsid proteins (96.8% nt and 98% aa in the VP7; 95.8% nt and 97.8% aa in the VP8* portion of the VP4). In a similar fashion, analysis of the predominant G6P[11] virus showed marked conservation with respect to other G6P[11] viruses detected from various geographical areas (VMRI-29, C8336, MC27 and KN-4), not only in the VP7 but also in the VP4. Indeed, the G6P[11] viruses tended to form a unique G6 lineage (Fig. 3), distantly related (only 90.1 aa identity) to the G6 strain NCDV, included in the vaccines. Such intra-serotype genetic/antigenic heterogeneity, coupled with the different VP4 antigen, could help G6P[11] strains to escape the immune pressure induced by the G6P[1] and G6P[5] vaccines. This observation would be consistent with the high prevalence of G6P[11] viruses observed in herds vaccinated with the G6P[1] NCDV-based vaccine (Barreiros et al., 2004).
In this study, rotavirus vaccines were used in six out of 14 herds, and the vaccines contained G6P[1] and G6P[5] strains (Table 2). Most of the strains detected in the vaccinated herds were characterized as either G6P[11] or G10P[11]. Co-segregation between G6 lineages and P types might be indicative of multiple reassortment events occurring in the context of a constant pattern of linear evolution (Martella et al., 2003). Cross-neutralization with immune sera obtained form naturally infected and vaccinated animals could help understand the real impact of the genetic heterogeneity oberved in BRVs. In conclusion, the data presented here emphasize the need for monitoring the distribution of rotavirus genotypes in bovine herds. The epidemiological information gathered on BRV genetic/antigenic heterogeneity will be useful in understanding the patterns of replacement over time and to improve vaccination strategies. A 4-years study in Japan has demonstrated that BRV strains may
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