Transmission Efficiency of Cucumber green mottle mosaic virus via Seeds,Soil, Pruning and Irrigation Water

Cucumber green mottle mosaic virus (CGMMV; genus Tobamovirus) infects frequently the grafted watermelon and is widely distributed in China. Investigating the transmission modes and their efficiency is urgently needed to understand the factors contributing to the epidemiology of this viral disease. In the present study, we found that the occurrence of CGMMV in a bottle gourd seed production base reached 100%, while the contamination rate and transmission rate were 100 and 0.92%, respectively. The bottle gourd plants showed obvious mottle symptom on leaves starting 36 days after seed sowing. The long latent period of CGMMV in seedlings implies a potential risk to use contaminated seeds in the production of grafted watermelon. This virus could overwinter in soil with debris of infected plants, and the infection rate of CGMMV from contaminated soils was 10.30%. CGMMV could be transmitted from infected watermelon plants to healthy ones by pruning at least to the ninth plant during the whole growing season. The transmission distance was 1.87 m by drip irrigation and 2.31 m by flow irrigation. This study suggested that contaminated seeds, contaminated soil, pruning and irrigation could transmit CGMMV at different efficiency, and all contribute to the epidemiology of CGMMV.     

Nucleotide sequences of two Korean isolates of Cucumber green mottle mosaic virus

The nucleotide sequences of the genomic RNAs of Cucumber green mottle mosaic virus Korean watermelon isolate (CGMMV-KW) and Korean oriental melon isolate (CGMMV-KOM) were determined and compared to the sequences of other tobamoviruses including CGMMV strains W and SH. Each CGMMV isolate had a genome of 6,424 nucleotides. Each also had 60 and 176 nucleotides of 5' and 3' untranslated regions (UTRs), respectively, and four open reading frames (ORF1-4). ORFs 1 to 4 encode proteins of 129, 186, 29, and 17.4 kDa, respectively. The nucleotide and deduced amino acid sequences of CGMMV-KOM and CGMMV-KW were more than 98.3% identical. When compared to other CGMMV strains in a phylogenetic analysis they were found to form a distinct virus clade, and were more distantly related to other tobamoviruses (23.5-56.7% identity).     

Epidemiological study of Cucumber green mottle mosaic virus in greenhouses enables reduction of disease damage in cucurbit production

Since 2007, the tobamovirus Cucumber green mottle mosaic virus (CGMMV) has become widespread in Israel, causing severe damage to trellised cucumber and melon in greenhouses and watermelon grown in open fields. To reduce disease damage below the economic threshold, this study focused on four objectives: (a) monitoring the patterns of virus distribution within commercial cucumber greenhouses; (b) studying the potential transmission of CGMMV by agrotechnical activities; (c) virus localization in plant tissues; and (d) searching for techniques that might be adapted for mitigating the disease in trellised cucurbit growth. The results of our surveys demonstrated the role of contaminated seeds and soil as primary inoculum sources, and secondary spread caused by agrotechnical activities. The patterns of secondary disease spread were demonstrated in a series of inoculation experiments involving contaminated knives, shears or hands on wet and dry plants, conducted under research‐greenhouse conditions. In parallel experiments using CGMMV‐specific antibody and secondary antibody conjugated to Alexa fluor 488, the viral coat protein was visualized in several plant tissues: phloem, xylem, trichomes and grasping tendrils. In addition, commercial‐greenhouse experiments were aimed at reducing the number of inoculum sources by identifying and removing infected plants from the plots (early monitoring) prior to agrotechnical activities and/or by adding intermediate medium (IM), such as virus‐free compost, to the planting pits at the planting stage. It is suggested that the use of IM combined with early monitoring, awareness of worker mobility (from contaminated structures to young planting areas) and proper sanitation (e.g. disinfection of agrotechnical tools) may reduce the yield losses caused by CGMMV below the economic threshold.     

Differences of reconstitution process between tobacco mosaic virus and cucumber green mottle mosaic virus by synchrotron small angle X-ray scattering using low-temperature quenching

The differences of the reconstitution process of tobacco mosaic virus (TMV) and its mutant, cucumber green mottle mosaic virus (CGMMV) were investigated by the solution X-ray scattering measurements with the synchrotron radiation source using low-temperature quenching. The reconstitution in an aqueous solution is completely stopped below 5°C. The TMV and CGMMV assembly was traced by the small-angle X-ray scattering (SAXS) measurements at 5°C on a series of solutions prepared by low-temperature quenching after incubation at 20°C for an appropriate interval between 0 and 60 min. The SAXS results were analyzed by the Guinier plot, the Kratky plot and the distance distribution function. The incubation of RNA and protein of CGMMV did not reconstitute at the initial reaction stages below 5 min and then began to reconstitute gradually. After 60 min, the radius of gyration for CGMMV reconstitution process reached almost the value for the initial stage of TMV reconstitution process. This is due to the fact the formation of double-layered disk in CGMMV protein is much slower than in TMV protein.     

Comparison of DAS-ELISA and qRT-PCR for the detection of cucurbit viruses in seeds

The importance of seeds as virus vehicles for long-distance dissemination makes essential the availability of adequate methods of analysis to guarantee the quality of seed lots. To improve the repertoire of sensitive methods for seed diagnosis, we have developed quantitative real-time RT-PCR assays (RT-qPCR) based on the TaqMan technology to detect three viruses which are seed transmitted in cucurbits, namely, cucumber green mottle mosaic virus (CGMMV), squash mosaic virus (SqMV) and melon necrotic spot virus (MNSV), and compared these assays with DAS-ELISA, the main method used for virus detection in seeds. The estimated RT-qPCR limits of detection were 96, 97 and 740 RNA target molecules for CGMMV, SqMV and MNSV, respectively. The estimated RT-qPCR analytical sensitivity (highest dilution capable of generating a detectable amplification signal) ranged between 10 and 1 pg/μL for the three viruses. Using RT-qPCR, we could reliably detect a single SqMV- or CGMMV-contaminated seed among 999 uncontaminated seeds in a seed lot, and sensitivities were 1,000 and 10,000 times of those provided by DAS-ELISA for SqMV and CGMMV, respectively. Our RT-qPCR assays have proved to be highly suitable for the analysis of seed lots, and the possibility of their implementation into certification programme should be taken into consideration.     

Virus inactivation in bone tissue transplants (femoral heads) by moist heat with the 'Marburg bone bank system'

Several virus inactivation procedures like heat treatment, gamma irradiation and chemical sterilization are used to increase the safety of bone tissue transplants. In this study we present data on the virus-inactivating effect of heat disinfection on human femoral heads, using the Marburg bone bank system 'Lobator sd-2'. Three enveloped viruses (human immunodeficiency virus type 2 [HIV-2], bovine viral diarrhoea virus as a model for Hepatitis C virus [HCV], and the herpesvirus pseudorabies virus), and three non-enveloped viruses (hepatitis A virus, poliomyelitis virus, and bovine parvovirus) were investigated.In a model system the central part of human femoral heads was contaminated with the respective cell-free virus suspension, establishing a direct contact between virus and native bone tissue. The core temperature in the femoral heads during the sterilization process was determined in additional model experiments. A temperature of 82.5 degrees C, given by the manufacturer as the effective temperature for virus inactivation, was maintained for at least 15 min in decartilaged femoral heads with a diameter of < or = 56 mm. Heat treatment using the Lobator sd-2 inactivated all viruses in human femoral heads below the detection limit (at least by a factor of > or =4 log(10)).By combining a well-focussed anamnesis of the donors and serological testing for relevant infection markers (anti-HIV-1/2, HBsAg, anti-HBcore, anti-HCV, TPHA) with heat treatment of femoral heads in the Lobator sd-2 system, a high safety level is achieved. To further increase virus safety of cadaveric bone transplants, it is recommended that multi-organ donors are tested by nucleic acid testing (i.e. polymerase chain reaction) for HIV, HBV and HCV genome.     

Two related Epstein-Barr virus membrane proteins are encoded by separate genes.

The structures of the 2.3- and 2.0-kilobase Epstein-Barr virus (EBV) mRNAs, partially encoded within the EcoRI J fragment DNA of the viral genome, were determined by analysis of their cDNAs. Both mRNAs are transcribed across the fused terminal repeats of the EBV episome and consist of nine exons. The mRNAs are transcribed from different promoters and have a unique 5' exon from the U5 region of the genome but eight common exons from the U1 region. One principal open reading frame is present in each mRNA and is predicted to encode 54,000- and 40,000-dalton integral membrane proteins. This result was confirmed by in vitro translation of RNAs in the presence of canine pancreatic microsomes. The 2.3-kilobase mRNA is not expressed in Raji cells, owing to the deletion of the 5' regulatory and coding region of this gene, whereas neither mRNA is expressed in Namalwa cells, owing to inactivation as a result of integration of the EBV genome via the terminal repeats. Since these mRNAs are readily detected in largely latently infected cells and do not increase in abundance with EBV replication, these putative latent-infection membrane proteins are tentatively designated LMP-2A and LMP-2B, respectively.     

<Emphasis Type="Italic">Stenotrophomonas maltophilia</Emphasis> HW2 enhanced cucumber resistance against cucumber green mottle mosaic virus

Cucumber green mottle mosaic virus (CGMMV) is a major limiting factor in the production of cucumber plants worldwide. In the present study, we use plant growth-promoting rhizobacteria (PGPR) to control this virus effectively. Stenotrophomonas maltophilia HW2 was isolated from healthy cucumber root, exhibited a good biocontrol efficacy against CGMMV. Here, it is documented that 20 d after virus inoculation, the biocontrol efficacy of HW2 reached 52.61%. HW2 can effectively colonize in cucumber rhizosphere, and also promoted cucumber plants growth. We also examined the effect of HW2 on viral replication and its mechanism. Compared with the control, HW2 pre-treated plants could delay virus replication for more than 3 d and inhibit viral protein genes (CP, MP, Rep) expression in the cucumber leaf. The expression of antioxidant enzyme genes (SOD and CAT) and defense-related genes (PR1 and PR5) were quickly induced by HW2. These results suggest that HW2 induced plant defense responses to CGMMV by increasing the expression of defense response genes. We report for the first time that Stenotrophomonas maltophilia improved cucumber resistance against CGMMV, which highlights the applying of PGPR on controlling of virus diseases.     

Comparative restriction endonuclease maps of proviral DNA of the primate type C simian sarcoma-associated virus and gibbon ape leukemia virus group.

Extrachromosomal DNA was purified from canine thymus cells acutely infected with different strains of infectious primate type C viruses of the woolly monkey (simian) sarcoma helper virus and gibbon ape leukemia virus group. All DNA preparations contained linear proviral molecules of 9.1 to 9.2 kilobases, at least some of which represent complete infectious proviral DNA. Cells infected with a replication-defective fibroblast-transforming sarcoma virus and its helper, a replication-competent nontransforming helper virus, also contained a 6.6- to 6.7-kilobase DNA. These proviral DNA molecules were digested with different restriction endonucleases, and the resultant fragments were oriented to the viral RNA by a combination of partial digestions, codigestion with more than one endonuclease, digestion of integrated proviral DNA, and hybridization with 3'- and 5'-specific viral probes. The 3'- and 5'-specific probes each hybridized to fragments from both ends of proviral DNA, indicating that, in common with those of other retroviruses, these proviruses contain a large terminal redundancy at both ends, each of which consists of sequences derived from both the 3' and 5' regions of the viral RNA. The proviral sequences are organized 3',5'-unique-3',5'. Four restriction enzymes (KpnI, SmaI, PstI, and SstI) recognized sites within the large terminal redundancies, and these sites were conserved within all the isolates tested. This suggests that both the 3' and 5' ends of the genomic RNA of these viruses are extremely closely related. In contrast, the restriction sites within the unique portion of the provirus were not strongly conserved within this group of viruses, even though they were related along most of their genomes. Whereas the 5' 60 to 70% of the RNA of these viruses was more closely related by liquid hybridization experiments than was the 3' 30 to 40%, restriction sites within this region were not preferentially conserved, suggesting that small sequence differences or point mutations or both exist throughout the entire unique portion of the genome among these viruses.     

Interaction between cucumber green mottle mosaic virus MP and CP promotes virus systemic infection

The movement protein (MP) and coat protein (CP) of tobamoviruses play critical roles in viral cell-to-cell and long-distance movement, respectively. Cucumber green mottle mosaic virus (CGMMV) is a member of the genus Tobamovirus. The functions of CGMMV MP and CP during viral infection remain largely unclear. Here, we show that CGMMV MP can interact with CP in vivo, and the amino acids at positions 79–128 in MP are vital for the MP–CP interaction. To confirm this finding, we mutated five conserved residues within the residue 79–128 region and six other conserved residues flanking this region, followed by in vivo interaction assays. The results showed that the conserved threonine residue at the position 107 in MP (MP^T107) is important for the MP–CP interaction. Substitution of T107 with alanine (MP^T107A) delayed CGMMV systemic infection in Nicotiana benthamiana plants, but increased CGMMV local accumulation. Substitutions of another 10 conserved residues, not responsible for the MP–CP interaction, with alanine inhibited or abolished CGMMV systemic infection, suggesting that these 10 conserved residues are possibly required for the MP movement function through a CP-independent manner. Moreover, two movement function-associated point mutants (MP^F17A and MP^D97A) failed to cause systemic infection in plants without impacting on the MP–CP interaction. Furthermore, we have found that co-expression of CGMMV MP and CP increased CP accumulation independent of the interaction. MP and CP interaction inhibits the salicylic acid-associated defence response at an early infection stage. Taken together, we propose that the suppression of host antiviral defence through the MP–CP interaction facilitates virus systemic infection.