MATERIALS AND METHODS

Source of virus isolate: Samples showing distinctive viral symptoms of Beta vulgaris L. cv. Gazella plants naturally infected were collected randomly from Kafr El-Sheikh Governorate.

The CMV was detected serologically by DAS-ELISA according to Clark and Adams (1977), using polyclonal antibody specific against CMV, kindly provided from Prof. Dr. Stephan Winter, Head of Plant Virus Department Leibniz-Institut Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), German collection of microorganisms and cell cultures, Inhoffenstrasse 7B, 38124 Braunschweig, Germany.

The collected infected samples which gave positive serological reaction using IgG-CMV by DAS-ELISA, were used for virus isolation and propagation. The virus was isolated biologically by single local lesion method on C. amaranticolor (Noordam, 1973; Megahed et al., 2012). For biological purification, one single local lesion was separated and grinding in 1 mL of 1:2 (w/v) of 0.1 M phosphate buffer, pH 7.2, containing 1% Na₂SO₃, 0.02 M EDTA, 0.1% β-mercaptoethanol and 1.5% Triton X-100. The extracted sap was mechanically inoculated on middle leaves of indicator plants. The extraction of resulted local lesions was inoculated on healthy Beta vulgaris cv. Gazella plants as a CMV propagative host. The inoculated plants were kept in an insect proof under greenhouse conditions (25-28°C) for 21 days until symptoms appeared.

Dot and tissue printing immunoassay (DBIA and TPIA) were used for confirmation of CMV-Beet-EG isolate using specific CMV-polyclonal antibodies as described by Lin et al. (1990).

Molecular identification: Total RNA was extracted from infected sugar beet leaves by using a method described by Gibbs and Mackenzie (1997), the extracted RNA was evaluated by electrophoresis and spectrophotometer and then, keept at -80°C.

Amplification of CMV-Beet-EG/cp gene: The cDNA was synthesis using (200 U μL^-1) reverse transcriptase (Promega). The amplification was carried out by the UNOII thermocycler system (Biometra, Germany) and using 0.2 mL micro amplification PCR tubes using each complementary reverse primer 5'-AAC ACG GAA TCA GAC TGG GAG-3' and forward primer 5'-TTG AGT CGA GTC ATG GAC AAA TC-3' (Lin et al., 2004; Sofy and Soliman, 2011). Hard denaturation of the DNA was performed at 94°C for 3 min, followed by 35 cycles of amplification with denaturation at 94°C for 1 min annealing at 45°C for 2 min and extension 72°C for 1.5 min. A single trailing cycle of long extension at 72°C for 7 min ended the run. The gel was visualized with UV light after staining for 10 min with 10 mg mL^-1 of ethidium bromide using Gel Documentation System (Gel Doc 2000 BIO RAD 1000/115 V~50/60 Hz-150 VA).

DNA sequencing of CMV-Beet-EG coat protein gene: DNA fragments were purified from agarose gel using the gel slicing and melting method described by Wieslander (1979). The nucleotide sequence was determined using an automated DNA sequencer (ABI Prism 3730XL DNA analyzer) and analyzed by FinchTV^TM version 1.4.0 software.

Sequence analysis: The sequence data of multiple nucleotides, deduced amino acids and multiple alignment of the cp gene for CMV-Beet-EG were translated, analyzed and has been submitted to GenBank Database (accession No. JX826591) by using DNAMAN program (DNAMAN V 5.2.9 package, Madison, Wisconsin, USA). Phytogenetic relationship and antigenic index of with the available sequences were carried out using ClustalW (Ver.1.74) program (Thompson et al., 1994).

RESULTS

Virus isolation: The CMV-Beet-EG isolate was detected in the collected naturally infected sugar beet plants showing vein clearing, mottling, mosaic and blister, where gave positive results by DAS-ELISA using specific CMV-IgG. The virus isolate was biologically isolated by single local lesion from C. amaranticolor produce necrotic L.L. surrounded with little halo-edge without brown color. It was propagated in healthy sugar beet plants and exhibited vein clearing, narrow leaf and deformation (Fig. 1). This result was confirmed by DAS-ELISA.

Fig. 1(a-b):	(a) Infected sugar beet and (b) C. amaranticolor leaves inoculated with CMV-Beet-EG, showing external systematic and local symptoms

Fig. 2:	Dot and tissue printing immunoassay for CMV-Beet-EG precipitation against specific IgG-CMV polyclonal, -ve: Negative samples +ve: Positive samples

Fig. 3(a-b):	The 1% agarose gel electrophoresis showing, (a) Total RNA and (b) Amplified PCR product of CMV-Beet-EG/cp gene of the correct size 820 bp, M: Molecular weight of DNA Marker, Lane 1: amplified CMV-Beet-EG/cp gene isolate

CMV confirmation: The sap extract of inoculated sugar beet was serologically precipitated reaction against specific polyclonal IgG-CMV by immunoblotting and printing and revealed a purplish blue color developed, whereas other extract from healthy plants remain green in the negative reactions (Fig. 2).

Molecular identification: Total RNA was extracted from CMV-Beet-EG infected sugar beet isolate. The integrity and quantity of the purified RNA were confirmed by gel electrophoresis (Fig. 3a) and UV spectrophotometer. The concentration of RNA was 95 μg/0.2 g of infected tissues and the purity was measured by A_260/280 absorbance ratio which it was 1.5.

The total RNAs prepared from infected sugar beet were reverse transcribed by MMLV reverse transcriptase. The reverse transcription was primed for RNA using Oligo (dt) as minus sense primer (5'-AAC ACG GAA TCA GAC TGG GAG-3') for CMV. The resulting complementary DNA (cDNA) of cp gene/CMV-Beet-EG was amplified by PCR after adding one set of primer CMV-Beet-EG, for cp-gene. The efficiency of DNA amplification from CMV-Beet-EG infected leaf tissue was detected by analysis of PCR product using 1.0% agarose gel electrophoresis. The size of amplified CMV-DNA fragments was as expected 820 bp (Lane 1) (Fig. 3b).

Partial nucleotide sequence of CMV-Beet-EG/cp gene: The partial nucleotide sequence (619 nucleotides) of the CMV-Beet-EG/cp gene was aligned with other coat protein sequences of CMV isolates published in GeneBank as shown in Fig. 4 using DNAMAN program (DNAMAN V 5.2.9 package, Madison, Wisconsin, USA).

Fig. 4:	Partial nucleotide sequence alignment of the cp-gene for CMV-Beet-EG using DNAMAN program

It was noticed the variation in nucleotide sequence of CMV-Beet-EG/cp gene sequence and other sequences due to replacement of either pyrimidines with purines or opposite revealed there are 31 position of replacement.

The changes of nucleotide at position 18 and 19 of CMV-Beet-EG and Gera-EG replaced T and G by G and C, respectively. At position 42 G replaced by A and at 51 T replaced by C.

The Gd-Austria isolate C replaced by T at 66, the ZM-South Korea at position 90 A replaced by G, the CMV-Beet-EG at positions 93 and 94 (C and T) replaced by G while, Brazil isolate the G replaced by A at 105.

Fig. 5(a-b):	(a) Similarity index and (b) Consensus phylogenic tree of nucleic acid sequence constructed from the multiple alignment of the CP/CMV-Beet-EG (under study) and nine CMV isolates

CMV-Beet-EG at positions 152, 154, 157 and 168; the A, C, C and A replaced by T in all previous position. J-Poland isolate G replaced by A at 185 position while CMV-Beet-EG A replaced by T at 211 and Brazil isolate C replaced by T at position 249. At position 262, T replaced by C for both CMV-Beet-EG and Gera-EG isolates, where at 265 C replaced by T for each ZM-South Korea and J-Poland. CMV-Beet-EG at 347 position C replaced by T while in Gd-Austria isolate sequence C replaced by T at 402, on the other position 420, C replaced by T of Brazil and J-Poland. At position 487, C replaced by T of ZM-South Korea, Brazil and J-Poland, where at 498 T replaced by C of Gd-Austria. The Brazil replaced G and C by A and T at 268 and 286, respectively while T replaced by G at 594 of Gd-Austria isolate and finally the CMV-Beet-EG at position 600, the nucleotide was deleted.

The similarity index of CMV-Beet-EG presented in Fig. 5a, revealed the nucleotide sequence of CMV-Beet-EG under study which has 97.3% identified to Gera-EG (Accession No. JQ013954) isolate and 96.4% to Fny-USA (Accession No. MCVCP3A1), Ny-Australia (Accession No. CMU22821), ToCMV5-3-Syria (Accession No. AB448693) and 702-07-Serbia (Accession No. GQ340670) CMV isolates.

Table 1:	Bases composition of partial CP sequence CMV-Beet-EG and different three CMV isolates published in GeneBank

The results in Fig. 5b, represented the phylogenetic tree of CMV-Beet-EG and other nine CMV isolates revealed that, CMV-Beet-EG has identified 96% with sub-group consist of Gera-EG (99%); Fny-USA, Ny-Australia, ToCMV5-3-Syria and 702-07-Serbia (99%) and Gd-Austria, ZM-South Korea, J-Poland and Brazil (99%).

The nitrogen base composition variation of cp gene for CMV-Beet-EG Table 1 revealed that, the highest content for Thymine (T) was 164 (26.5%) followed by Cytosine (C) 163 (26.3%), then Adenine (A) 148 (23.9%) and Guanine (G) 144 (23.3%). Comparison between nitrogen base composition of partial cp gene sequence for CMV-Beet-EG (Accession No. JX826591) and three CMV isolates published in GeneBank, Gera-EG (Accession No. JQ013954), Fny-USA (Accession No. MCVCP3A1) and Brazil (Accession No. AY380812) was done to determine the ratio of A/T and G/C and percentage of A+T and G+C Table 1. The ratio of A/T, G/C were 0.902 and 0.883 of CMV-Beet-EG (Accession No. JX826591); 0.896 and 0.873 of Gera-EG (Accession No. JQ013954); 0.890 and 0.878 of Fny-USA (Accession No. MCVCP3A1) and 0.880 and 0.888 of Brazil (Accession No. AY380812). The percentage of A+T and G+C in Table 1 were found to be 50.4 and 49.6%; 49.8 and 50.2%; 50.0 and 50.0% and 51.0 and 49.0% for CMV-Beet-EG (Accession No. JX826591), Gera-EG (Accession No. JQ013954), Fny-USA (Accession No. MCVCP3A1) and Brazil (Accession No. AY380812), respectively.

Deduced amino acids sequence of CMV-Beet-EG/cp gene: The predicted numbers of amino acids produced from translation of partial cp gene nucleotide sequence of CMV-Beet-EG were 206 amino acids starting with Serine (S) and ended with Alanine (A) (Fig. 6).

The deduced amino acids sequence of CMV-Beet-EG/cp gene, 206 amino acids was aligned with other deduced amino acids sequence of CP/CMV isolates published in GeneBank as shown in Fig. 7, CMV-Beet-EG has 6 different amino acids present at position from 200-206 which absent in all other aligned isolates. The presented data in Fig. 7 revealed that, there are 17 differences relative to replacement position of amino acid as following.

The CMV-Beet-EG at positions 5, 7, 27, 32, 43, 45, 51, 52, 53, 71 and 116, the amino acids Serine, Glycine, Alanine, Leucine, Leucine, Alanine, Asparagine, Histidine, Proline, Threonine and Serine replaced with Arginine, Arginine, Aspartic acid, Valine, Phenylalanine, Glutamic, Isoleucine, Tyrosine, Serine, Serine and Phenylalanine, respectively. AAQ89596-Brazil at 190 Alanine replaced by Threonine, where at position 2 Glutamic replaced by Glycine while in isolate Lucknow-India Aspartic acid replaced by Threonine at 194. At positions 134, Alanine replaced by Threonine for Mf-South Korea, where at position 16 and 190, the amino acids Arginine and Alanine replaced by Cysteine and Threonine, respectively for Pf-USA isolate.

Fig 6:	Predicted amino acid of the cp-gene of the isolated CMV-Beet-EG

Fig. 7:	Deduced amino acids sequence alignment of the CP for CMV-Beet-EG using DNAMAN program

Fig. 8(a-b):	(a) Similarity index and (b) Consensus phylogenic tree of deduced amino acids sequence constructed from the multiple alignment of the CP/CMV-Beet-EG (under study) and nine CP /CMV isolates

The similarity index of deduced amino acids sequence constructed from the multiple alignment of the CP/CMV-Beet-EG (under study) and nine CP/CMV isolates (Fig. 8a) revealed the deduced amino acids sequence of CMV-Beet-EG has 95.5% identified to Gera-EG (Accession No. AEZ03836) isolate; 94.0% to AAQ89571-Brazil (Accession No. AAQ89571) and Fny-USA (Accession No. NP-040777) and 93.5% to Woj-Poland (Accession No. ACM89097), AAQ89596-Brazil (Accession No. AAQ89596), RT88-Spain (Accession No. CAH17692), Lucknow-India (Accession No. ABC18318), Mf-South Korea (Accession No. CAB77390) and Pf-USA (Accession No. CAB41491) CP/CMV isolates.

Table 2:	The predicted amino acids of partial cp gene sequence for CMV-Beet-EG isolate and different three CP/CMV isolates published in GeneBank

A phylogenetic tree in Fig. 8b, revealed that, CMV-Beet-EG has identified 94% and with 5 sub-group for other nine CMV isolates, sub group 1 has 100% consist of 6 minor groups for Gera-EG, sub-group, (AAQ89571-Brazil, Fny-USA and Woj-Poland), RT88-Spain, Lucknow-India, Mf-South Korea and (AAQ89596-Brazil and Pf-USA). Sub-group 2 has 99% and sub-group 3 has 98%. Comparison between the predicted amino acids of partial cp gene sequence of CMV-Beet-EG (Accession No. JX826591) and three CP/CMV isolates published in GeneBank, Gera-EG (Accession No. AEZ03836), Fny-USA (Accession No. NP-040777) and AAQ89596-Brazil (Accession No. AAQ89596) was done in Table 2.

The amino acid Serine (S) has the highest content No. of all amino acids 25 (12.1%) for CMV-Beet-EG (Accession No. JX826591), 22 (11.0%) for Gera-EG (Accession No. AEZ03836), 23 (11.5%) for each Fny-USA (Accession No. NP-040777) and AAQ89596-Brazil (Accession No. AAQ89596). The deduced amino acids sequence of cp gene for CMV-Beet-EG isolated from sugar beet, Table 2 have a M.wt 22.983 kDa, isolelectric point 9.95 of CP and the type of amino acids consists of 20 amino acids; the lowest content of amino acid was 1 (0.5%) to the each two amino acid Histidine (H) and Trpyptophan (W). The M.Wt and isolelectric point of deduced CP/CMV-Gera-EG isolated from Geranium (Accession No. AEZ03836) were 22.238 kDa and 10.19, respectively and Trpyptophan (W) has the lowest content of amino acid 1 (0.5%) of 200 total no of amino acid (Table 2). The CP of deduced amino acids consist of 200 amino acid specific to cp gene of Fny-USA (Accession No. NP-040777) isolated from cucumber and AAQ89596-Brazil (Accession No. AAQ89596) isolated from Amazon Lily have a M.wt 22.123 and 22.153 kDa, respectively and the same isolelectric point 10.18. Also the two amino acid Cysteine (C) and Trpyptophan (W) have the same lowest amino acid content 1 (0.5%) of two each CP/CMV isolates (Table 2).

DISCUSSION

In the present study, the CMV was detected in naturally infected B. vulgaris L. cv. Gazella which showed distinctive viral symptoms in the form of vein clearing, mottling, mosaic and blister. The detected samples gave ⁺ve reaction against specific antiserum by using DAS-ELISA technique, as consistent with Abdelkader et al. (2006). CMV-Beet-EG was biologically purified by single local lesion assay on C. amaranticolor and sugar beet was used as a propagative host which reacted as vein clearing, narrow leaf and deformation symptoms as reported by many investigators (El-Afifi et al., 2007; Sofy and Soliman, 2011; Megahed et al., 2012).

The results of DBIA and TPIA were found to be sensitive techniques for CMV immunogene detection in all infected sugar beet plants, when the antigenic determinants of CMV-Beet-EG isolate were identified by using polyclonal antibodies specific CMV which give serologically precipitation reacted in DAS-ELISA and in DBIA and TPIA where the purple color appeared clearly, the obtained results in accordance with many authors (Hu and Chang, 2006; Megahed et al., 2012).

Total RNA of CMV-coat protein gene beet Egyption has 95 μg/0.2 g of infected tissues and the purity was measured by A_260/280 absorbance ratio 1.5 indicated haigh yield and purity of the extracted RNA. CMV-coat protein gene beet Egyption isolate was successfully amplified using RT-PCR from infected sugar beet in fragment of about 820 bp using specific synthesized primers according to Lin et al. (2004). Other expected size were obtained by authors; Abdelkader et al. (2006) detected the size of amplified cDNA fragment of CMV-EG isolated from sugar beet as 650 bp; Yu et al. (2005) the size of DNA fragment was 600 bp for CMV/cp of cucumber isolate while the result of the CMV-cp amplified from Capsicum annuum showed the band of the fragment with 740 bp (Wu et al., 2007). As well as, Sofy and Soliman (2011) found the amplified fragment of CMV/cp Gera-EG was 657 bp.

The nucleotide sequence of coat protein gene of CMV-Beet-EG isolate under study revealed 619 nucleotides of cp-sequence has similarity CMV isolates belonging to subgroup IA in accordance with Abdelkader et al. (2006) and Sofy and Soliman (2011), in a different percentages; 97.3% identified similarity to Gera-EG-Egypt (Accession No. JQ013954) isolate and 96.4% to Fny-USA (Accession No. MCVCP3A1), Ny-Australia (Accession No. CMU22821), ToCMV5-3-Syria (Accession No. AB448693) and 702-07-Serbia (Accession No. GQ340670) CMV isolates.

The nucleotide sequence of coat protein gene of CMV-Beet-EG showed the highest content for Thymine (T) 164 (26.5%) followed by Cytosine (C) 163 (26.3%), then Adenine (A) 148 (23.9%) and Guanine (G) 144 (23.3%). The similarity index of deduced amino acids sequence constructed from the multiple alignment of the CP/CMV-Beet-EG and nine CP/CMV isolates revealed the deduced amino acids sequence of CMV-Beet-EG is 95.5% identity to Gera-EG-Egypt (Accession No. AEZ03836), 94.0% identity to AAQ89571-Brazil (Accession No. AAQ89571) and Fny-USA (Accession No. NP-040777) and 93.5% identity to other isolates. The deduced amino acids sequence of cp gene for CMV-Beet-EG isolated from sugar beet have a M.Wt 22.983 kDa, isolelectric point 9.95 of 206 amino acids consists of 20 amino acids.

CONCLUSION

It was concluded that, based on the results obtained by the RT-PCR, sequence and bioinformatics analyses, the CMV-Beet-EG isolated from sugar beet cultivated areas in Egypt is a new CMV isolate belongs to subgroup IA and recorded in GeneBank with Accession No. JX826591.

ACKNOWLEDGMENT

This study was sponsored financially by National Research Centre (NRC), Dokki, Cairo, Egypt. The authors thanks Prof. Dr. Stephan Winter, Head of Plant Virus Dept., Leibniz-Institut Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), German collection of microorganisms and cell cultures, Inhoffenstrasse 7B, 38124 Braunschweig, Germany for his kindly providing polyclonal antibody specific against CMV.