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Isolation and Antigenic and Molecular Characterization of G10 of Group A Rotavirus in Camel



Eman Abo Hatab, H.A. Hussein, I.M. El-Sabagh and M.S. Saber
 
ABSTRACT

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.

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Eman Abo Hatab, H.A. Hussein, I.M. El-Sabagh and M.S. Saber, 2009. Isolation and Antigenic and Molecular Characterization of G10 of Group A Rotavirus in Camel. International Journal of Virology, 5: 18-27.

DOI: 10.3923/ijv.2009.18.27

URL: https://scialert.net/abstract/?doi=ijv.2009.18.27

INTRODUCTION

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

Fecal Samples
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.

Electron Microscopy
Positive fecal samples in ELISA were examined with electron microscopy (Alain et al., 1987) for shown the characteristic feature of rotavirus particles.

Tissue Culture
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.

Oligonucleotide Primers
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.

RT-PCR

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 of internet.

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.

Fig. 1: The characteristic feature of rotavirus particles under electron microscopy

Table 1: Different absorbances of field fecal samples of diarrheic calves in ELISA reader

Fig. 2: 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 amplification

Fig. 3: 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

Table 2: The optical densities of the harvested tissue culture supernatant of the eight propagated isolates in comparison with normal control

Table 3: The cytopathic behavior of the inoculated samples on MA104 cell line after five passages

Fig. 4: 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

Fig. 5: 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 No. 6

Fig. 6: 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 Internet (http://www.ncbi.nlh.gov.) 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).

Table 4: 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.

Fig. 7: 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.

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