Isolation and Antigenic and Molecular Characterization of G10 of Group A Rotavirus in Camel
Eman Abo Hatab,
We are here reporting the successful isolation and characterization of G10 serotype of group A rotaviruses from fecal samples collected from camel farms suffering from diarrhea in Alexandria and Esmalia Governorates. After preparation of fecal samples and inoculation on MA 104 cell line for five passages, 8 isolates were successfully isolated with a clear and reproducible CPE on the inoculated cells. The isolates were identified antigenically using VP6Monoclonal Antibodies (MAbs) based antigens capture ELISA that able to detect any group A rotavirus. The viral RNA was extracted from the tissue culture harvest of the propagated viruses and RT-PCR using primers specific for VP6 and VP7 of group A rotaviruses was employed and confirmed the molecular characterization of the isolates viruses with the correct and expected bands. The RT-PCR specific band of VP7 gene of two selected isolates was eluted from the agarose gel and sequenced using VP7 specific primers sequence. The obtained sequence was analyzed using computer software (BLAST) which revealed that both isolates had maximum identity to the G10 serotype of group A bovine rotaviruses ranging from 90-93%. This is the first report on the circulation of G10 serotype of group A rotaviruses in camel.
Rotavirus (RV) is recognized as the single most important cause of sever acute
dehydrating diarrhea in the young human and many animals species including calves
(Kapikian and Chanock, 1996). High mortality and morbidity
among young calves due to rotavirus disease is a serious cause of economic loss
to animal farms and diary industry (Deleeuw et al.,
1980; House, 1978; Saif and
Fernandez, 1996; Woode and Bridger, 1975). The calf
rotavirus infection has a worldwide distribution and associated with 40-48%
of the neonatal calves (Morin et al., 1976; Snodgrass
and Wells, 1976). The morbidity and mortality rates can reach up to 30 and
90%, respectively due to camel calf diarrhea (Schwartz and
Dioil, 1992). Only few reports on viral causes of camel calf diarrhea were
published (Mahin Schwers, et al., 1983). In general
there is a lack of details study on the role of rotavirus in camel calf diarrhea.
Rotavirus is composed of triple-layered protein capsid which encloses a genome
of eleven segments of double-stranded ds RNA (Estes, 1996).
The genome primarily encodes six structure and six non structure protein (Estes
and Cohen, 1989). VP4 and VP7, encoded respectively by gene segments
4 and 7, 8 or 9 depend on the strain. Specify two distinct serotype specificities
termed P (protease sensitive) and G (glycoprotein) serotypes (Estes
and Cohen, 1989). The intermediate capsid protein VP6 possesses the
group and subgroup specific epitopes. Rotaviruses are classified into 7 groups
(A-G) based on the antigenic properties of VP6 protein. Group A rotaviruses
constitute the major pathogens in human and animals (Kapikian
and Chanock, 1996). On the bases of the VP7 and VP4 proteins and
their coding nucleic acid, group A rotaviruses are classified into different
serotypes and genotypes. 15 G serotypes and 16 G genotypes have been identified
in diarrheic calves (Estes and Kapikian, 2007; Gulati
et al., 2007) and there is 14 P serotypes and 27 P genotypes (Khamrin
et al., 2007). The most predominant G serotypes in diarrheic calves
in Egypt are G6 and G10 (Hussein et al., 1993,
1999). There is no available data on the circulating
camel rotaviruses in Egypt. In this study, trial for isolation, antigenic and
genetic characterization of rotaviruses in fecal samples of diarrheic camel
calves was achieved.
MATERIALS AND METHODS
Eighty five fecal samples from 2 weeks till 4 months day old camel calves
were collected from four different governorates in Egypt (Fayoum, Alexandria,
Ismailia and Giza) during period 2004-2005. Fecal samples were tested for group
A rotavirus by Mabs-based ELISA (Hussein et al.,
1995). Positive fecal samples for RV were used for isolation trial.
Positive fecal samples in ELISA were examined with electron microscopy (Alain
et al., 1987) for shown the characteristic feature of rotavirus particles.
Eight positive fecal samples and tissue culture supernatant in Mabs based-ELISA
were propagated after treatment with trypsin on rhesus monkey kidney (MA104)
cells in the presence of 0.5 μg of trypsin per mL as described by Saif
et al. (1988).
Extraction of Rotavirus ds RNA
The dsRNA was extracted from fecal samples using RNA extraction kit [GIBCO]
according to recommended procedures that involved dissociating cells by Trizol
then chloroform, isopropanol and ethanol 75% with different centrifugation then
suspended the extracted RNA in nuclease free water (Chomczynski
and Sacchi, 1987) the RNA suspension was kept at -85°C till used for
reverse transcriptase RT-PCR.
Primers was designed according to publish database (El-Sabagh,
2006) to be used for amplification of full length VP6 gene, the sequence
of forward primer is (5-GGCTTTTAAACGAAGTCTT CAACATGG-3) and the
VP6 reverse primer (5-GGTCACATCCTCTCACTACGC-3) were used
to amplify of 1356 bp fragment of rotavirus VP6 gene. The VP7
forward primer is (5-GCGGTTAG CTCCTTTTAATGTATGG-3) and the reverse
primer is (5-GGTCACATCATATACAACCTC TAATCTAACAT-3) were used to amplify
1030 bp fragment of VP7 gene.
The RT-PCR was performed by modification according to Chang
et al. (1996) the 30 μL of RNA were mixed with 5 μL of
dimethylsulfoxide and the mixture incubated at 95°C for 5 min followed by
rapid cooling on ice the denaturated ds RNA of VP6 and VP7 genes
were amplified using one step RT-PCR kit. The first-strand cDNA synthesis was
accomplish by incubating the mixture for 30 min at 47°C then at 94°C
for 2 min and then 35 amplification cycles of 95°C for 45 sec (denaturation),
55°C for 45 sec (annealing), 72°C for 1.5 min (extension) and conducted
followed by a (final extension cycle) of 5 min at 72°C. The PCR products
10 μL were loaded on to agarose gel was prepared by dissolving of 1.25
g agarose gel in 100 mL (1X) TAE buffer with 0.5 μg mL-1 ethidium
bromide. Electrophoresis was conducted for 1 h at 120 V and the gels were photographed
under UV light according to Sambrook et al. (1989)
the bands of expected correct size were cut from gel and gel slices containing
DNA bands were placed in montage DNA gel extraction device. Then the eluted
DNA was sent to Agricultural Genetic Engineering Research Institute (AGERI)
with forward primer to be sequenced. The sequencing was analyzed by Blast computer
utility of the National Center for Biotechnology and Information (NCBI) web
RESULTS AND DISCUSSION
In a total of 85 fecal samples obtained from the diarrheic calves, eight were positive for Mabs- based ELISA as shown in Table 1, then the eight positive samples were concentrated by ultracentrifugation and examined under electron microscopy as in Fig. 1.
The RT-PCR amplification of RNA extracted from fecal samples revealed a specific bands of VP6 gene at the predicted size of 1356 bp in only one sample (No. 19) as shown in Fig. 2.
Trail for isolation of CRV from positive fecal samples of diarrheic camel calves in MA104 cell culture till 5th passage.
The propagated eight samples were identified using Mabs- based ELISA in harvested tissue culture supernatants shown in Table 2. The cytopathic behavior of the inoculated samples on MA104 cell line after five passages are shown in Table 3.
After 3rd and 5th passages, the RT-PCR using VP6 primers was carried
out on the extracted RNA from tissue culture supernatant 7 out of 8 samples
produced the specific band 1356 bp as in Fig. 3 and 4.
The RT-PCR using VP7 specific primers was carried out on the extracted
RNA from tissue culture supernatant of the 5th passage. Figure
5 showing 6 out of 8 propagated samples produce the specific band of VP7
at 1030 bp.
The RT-PCR product for VP7 gene of two selected isolates (10, 25) was
eluted from gel and sent for sequencing using VP7 specific primers as
shown in Fig. 6.
||The characteristic feature of rotavirus particles under electron
||Different absorbances of field fecal samples of diarrheic
calves in ELISA reader
||Ethidium bromide stained agarose gel electrophoresis of the
RT-PCR products of VP6 gene of CRV in fecal samples along with 1
kbp DNA ladder. M: 1 kbp DNA ladder. Lane 4: The amplified product of CRV
in sample No. 19. Lanes 1, 2, 3, 5, 6, 7 and 8: The negative samples for
||RT-PCR products of VP6 (1356 bp) of CRV isolates after
3rd passage in ethidium bromide stained agarose gel electrophoresis, along
with 1 kbp DNA ladder (M) that contains 13 size bands ranged between 250
and 10.000 bp. M: Bands of molecular sizes of (10,000, 3000, 2000, 1500,
1000, 750, 500, 250 bp): Lane 1, 3, 4, 5, 6 and 7: Amplified product of
correctly VP7 gene size 1356 bp of isolates No. 3, 10, 19, 25, 33
and 34. Lane 2, 8: The negative for amplification isolates No. 6 and 67
||The optical densities of the harvested tissue culture supernatant
of the eight propagated isolates in comparison with normal control
||The cytopathic behavior of the inoculated samples on MA104
cell line after five passages
||RT- PCR products of VP6 (1356 bp) of CRV propagated
in tissue cell culture MA104 of 5th passage in ethiduim bromide stained
agarose gel electrophoresis, along with 1 kbp DNA ladder (M). M: 1 kbp DNA
ladder. Lanes 1, 3, 4, 5, 6, 7 and 8: The amplified product of VP6
genes For the positive propagated isolates No. 3, 10, 19, 25, 33, 34 and
67. Lane 2: The negative isolates No. 6
||RT-PCR products of VP7 (1030 bp)of 5th passage of propagated
isolates in MA104 cells of CRV in ethidium bromide stained agarose gel electrophoresis,
along with 1 kbp DNA ladder M: 1 kbp DNA ladder. Lanes 1, 3, 4, 5, 6, 7
and 8: The amplified product of VP7 genes for the seven propagated
isolates No. 3, 10, 19, 25, 33, 34 and 67. Lane 2: The negative isolate
||Ethidium bromide stained agarose gel before and after cutting
of the amplified VP7 gene of CRV isolates coded 10 and 25 to be eluted
and send for sequencing. M: 10,000 bp; Lane 1 and 2: The amplified bands
of correct size (1030) of VP7 gene of CRV
Analysis of the Nucleotide Sequence of VP7 Gene Fragments of CRV
The purified DNA representing the outer capsid VP7 gene of CRV was
1030bp in size only sequence of 580 bp as obtained. The obtained nucleotide
sequence shown in the following box:
The obtained sequence was analyzed using computer software (BLAST) via., the
which revealed that the nucleotide sequence was highly related to strains of
G10 bovine rotavirus in percentage range between 88 to 92% so, the CRV were
related to G10 BRV lineage as shown in Table 4.
In this study, we have detected of group A rotavirus in fecal samples of diarrheic
camel calves using Mabs-based ELISA and electron microscopy (House,
1978; Yolken et al., 1978; Herrman
et al., 1985; Hussein et al., 1995).
The availability of local strain of CRV was limited so, important to continue
in trail to isolate CRV using MA104 cell culture (Babuik
et al., 1977). The cytopathic effect Fig. 7 (CPE)
on the inoculated cells were varied between the samples among the applied five
passages (Saif and Theil, 1985).
||Homology percentage of the two highly positive samples sequenced
fragment to the published group A rotavirus sequences in the gene bank (NCBI)
The application of Mabs- based ELISA to identify rotavirus in cell culture
was of great help (Hussein et al., 1995).
The molecular characterization of CRV is most important in current study. The
PCR has proved its efficacy in detecting rotavirus in the samples of both fecal
and tissue culture (Estes and Kapikian, 2007; Gentsch
et al., 1993; Isegawa et al., 1993;
Gouvea et al., 1993, 1994)
and the developed RT-PCR genotyping assay based detecting of G6 and G10 serotypes
of group A bovine (Hussein et al., 1996) the
RT-PCR based assay for genotyping of CRV was used in present study to genomic
characterization of local 7 isolated stains based on extraction of ds RNA and
amplification of CRV (VP6) gene 1356 bp and VP7 gene 1030 bp of
the RT product using specific primers complementary to full length of each VP6
and VP7 genes (Hussein et al., 1995).
The obtained bands from RT-PCR were detected in the accurate size 1356 and 1030
bp for both VP6 and VP7 gene. Also, the original samples give
one band which was detected in predicted size for VP6 1356 bp in Fig.
2. After 3rd passages of the inoculated samples in cell culture, RT-PCR
products revealed six positive isolates bands at size 1356 bp for VP6
gene at (Fig. 3). After the 5th passage of isolates in tissue
cell culture, RT-PCR amplified products were seven positive isolates with bands
at predict size 1356 bp for VP6 gene and six positive isolates with 1030
bp of the VP7 gene (Fig. 4, 5). Two
highly positive isolates coded No. (10, 25). Extracted dsRNA which was amplified
by RT-PCR for VP7 gene. The eluted (purified) DNA after electrophoresis
of the products was sent to be sequenced with the forward primer to be sequenced.
The obtained sequence results of only 580 bp fragment of the amplified 1030
bp PCR product revealed the specificity of the product to rotaviruses. Nucleotide
sequence analysis has indicated the high identity of the sequenced fragment
of both isolates (Estes and Kapikian, 2007; Morin
et al., 1976) to the G10 serotype of group A rotavirus.
||Showing the sequential appearance of the characteristic CPE
of rotavirus in comparison with control cells. (a) Non-inoculated cell control
with high power, (b) Non-inoculated cell with low power, (c) Rounding and
clumping of inoculated cells high power, (d) Cell rounding and clumping
cells with low power, (e) Detachment of inoculated cells sheet with high
power and (f) detachment of inoculated cells with low power
This is the first report on the existence of G10 serotype of group A rotavirus
in camel. Characterization of such serotype in camel population will add more
interest on gathering the diversity information of rotaviruses.
Alain, R., F. Nadon, C. Seguin, P. Payment and Trudel, 1987. Rapid virus subunit visualization by direct sedimentation of samples on electron microscope gride. J. Virogical Meth., 16: 209-216.
Babuik, L.A., K. Mohammed, M. Spence, M. Fauvel and R. Petro, 1977. Rotavirus isolation and cultivation in the presence of trypsin. J. Clin. Microbiol., 6: 610-617.
Chang, K.O., A.V. Parwani and L.J. Saif, 1996. The characterization of VP7 (G type) and VP4 (p type) genes of bovine group A rotaviruses from field samples using RT-PCR and RFLP analysis. Arch. Virol., 141: 1727-1739.
Chomczynski, P. and N. Sacchi, 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem., 162: 156-159.
CrossRef | PubMed | Direct Link |
Deleeuw, P.W., D.J. Ellens and P.J. Staver, 1980. Rotavirus infections in calves in dairy herds. Res. Vet. Sci., 29: 135-141.
El-Sabagh, 2006. Coloning and expression of bovine rotavirus genes. Ph.D. Thesis.
Estes, M.K. and A.Z. Kapikian, 2007. Rotaviurses. In: Fields Virology, Fields, B.N., D.M. Knipe and P.M. Howley (Eds.). Wolters Kuwer Health/Lippincortt, Wiliams and Wilkins, Philadelphia, PA., pp: 1917-1973.
Estes, M.K. and J. Cohen, 1989. Rotavirus gene structure and function. Microbiol. Rev., 53: 410-449.
Direct Link |
Estes, M.K., 1996. Rotavirus and their Replication. In: Fields Virology, Fields, B.N., D.M. Knipe, P.M. Howely, R.N. Chanock, J.K. Melnick, T.P. Monath, B. Roizman and S.E. Straus (Eds.). Lippincott-Raven, Philadelphia, pp: 1625-1655.
Gentsch, J.R., B.K. Das and B. Jiang, 1993. Similarity of the VP4 protein of human rotavirus strain 116E to that of the bovine B223 strain. Virology, 194: 424-430.
Gouvea, V., C. Ramirez and B. Li, 1993. Restriction endonuclease analysis of the VP7 genes of human and animal rotaviruses. J. Clin. Microbiol., 31: 917-923.
Gouvea, V., N. Santos and M. do Carom Timenetsky, 1994. VP4 typing of bovine and porcine group A rotaviruses by PCR. J. Clin. Microbiol., 32: 1333-1337.
Gulati, B.R., R. Deepa, K. Singh and C.D. Rao, 2007. Diversity in Indian equine rotaviruses: Identification of G10, P6  and G1 type and a new VP7 genotype (G16) strains in diarrheic foals in India. J. Clin. Microbiol., 45: 972-978.
Direct Link |
Herrman, J.E., N.R. Blacklow, D.M. Perron, G. Cukor, P.J. Krause, J.S. Hyams, H.H. Barrett and P.L. Ogra, 1985. Enzyme immuno assay with monoclonal antibodies for the detection of rotavirus in stool specimens. J. Infect. Dis., 152: 830-832.
House, J.A., 1978. Economic impact of rotavirus and other neonatal disease agents of animals. J. Am. Vet. Med. Assoc., 173: 573-576.
Hussein, H.A., A.A. EL-Sanousi, M.A. Shalaby, M.S. Saber and I.M. Reda, 1993. Stereotypical differentiation of group A rotaviruses from field cases on the basis of G-types monoclonal antibody based ELISA in Egypt. Proceedings of the TAHRP 2nd Scientific Workshop, June 9-14, 1993, Cairo and Alexandria, Egypt -.
Hussein, H.A., A.M. Samy, M.A. Shalaby, M.S. Saber and I.M. Reda, 1999. Bovine rotavirus in Egypt: Antigenic characterization of G6 and G10 serotypes within field strains of group A bovine rotavirus in dairy calves using monoclonal antibodies. Proceedings of the 5th Science Congress Egyptian Society for Cattle Diseases, November 28-30, 1999, Assiut, Egypt -.
Hussein, H.A., E. Cornaglia, M.S. Saber and Y. El-Azhary, 1995. Prevalence of serotypes G6 and G10 group A rotaviruses in dairy calves in Quebec. Can. J. Vet. Res., 59: 235-237.
Hussein, H.A., E. Frost, B. Talbot, M. Shalaby, E. Cornaglia and Y. El-Azhary, 1996. Comparison of polymerase chain reaction monoclonal antibodies for G-typing of group A bovine rotavirus directly from fecal material vet. Microbiology, 51: 11-17.
Isegawa, Y., O. Nakagomi, T. Nakagomi, S. Ishida, S. Uesugi and S. Ueda, 1993. Determination of bovine rotavirus G and P serotypes by polymerase chain reaction. Mol. Cell. Probes, 7: 277-284.
Kapikian, A.Z. and R.M. Chanock, 1996. Rotaviruses Field's Virology. Microbiological observation made on spontaneous cases of acute neonatal calf diarrhea. Can. J. Comp. Med., 40: 228-240.
Khamrin, P., N. Maneekarn, S. Peerakoma, W. Chan-It, F. Yagyu, S. Okitsu and H. Ushijima, 2007. Novel Porcine rotavirus of genotype P shares new phylogenic lineage with G2 Porcine rotavirus strain. Virology, 361: 243-252.
Mahin Schwers, L., A. Chadli, M. Maenhoudt and P.P. Pastoret, 1983. Receptivite du dromadaire (Camelus dromedaries) al'infection par rotavirus. Rev. Elev. Med. Vet. Pays. Trop., 36: 251-252.
Morin, M., S. Larviere and R. Lallier, 1976. Pathological and microbiological observation made on spontaneous cases of acute neonatal calf diarrhea. Can. J. Comp. Med., 40: 288-290.
Saif, L.F. and F.M. Fernandez, 1996. Group a rotavirus veterinary vaccines. J. Infect. Dis., 174: S98-S106.
Saif, L.J. and K.W. Theil, 1985. Antigentically distinct rotaviruses of human and animal origin. Proceedings of the Infect Diarrhea Young, (IDY'85), Elsevier, Amsterdam, the Netherlands, pp: 208-214.
Saif, L.J., B. Rosen, S. Kang and K. Miller, 1988. Cell culture propagation of rotaviruses. J. Tissue Cult. Meth., 11: 147-151.
Sambrook, J., E.F. Fritsch and T.A. Maniatis, 1989. Molecular Cloning: A Laboratory Manual. 2nd Edn., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA., ISBN-13: 9780879695774, Pages: 397.
Schwartz, H.J. and M. Dioil, 1992. Diseases of the Gastro-Intestinal System. In: The One Humped Camel in Eastern Africa, Schwartz, H.J., M. Dioli Verlag (Eds.). Josef Margraf-Scientific Books, Wekersheim, Germany, pp: 195-198.
Snodgrass, D.R. and P.W. Wells, 1976. Rotavirus infection in lambs: Studies on passive protection. Arch. Virol., 52: 201-205.
Woode, G.N. and J.C. Bridger, 1975. Viral enteritis of calves. The Vet. Record, 96: 85-88.
Direct Link |
Yolken, R.H., B. Babbour, R.G. Wyatt, A.R. Kalica, A.Z. Kapikian and R.M. Chanock, 1978. Enzyme linked immunosorbent assay for identification of rotaviruses from different animal species. Science, 201: 259-262.