CMV Strains Research Articles

Grafting to manage infections of top stunting and necrogenic strains of cucumber mosaic virus in tomato

AbstractCucumber mosaic virus (CMV) lists among the most important etiological agents of tomato diseases. Some isolates of CMV function as helper virus for replication, encapsidation and transmission of satellite RNAs (satRNA), which may exacerbate symptoms induced by CMV in certain hosts. Outbreaks of CMV strains supporting hypervirulent variants of satRNAs are recurrent in tomato with devastating effects on crop production and efficient control measures are still unavailable. In this study, we examined the dynamics of infection of the CMV strains tomato top stunting (TTS) and 77 supporting replication of satRNA variants that codetermine top stunting (TTS‐satRNA) and necrotic (77‐satRNA) phenotypes in two tomato cultivars denoted Solanum lycopersicum Manduria (Sl‐Ma) and S. lycopersicum UC82 (Sl‐UC). Sl‐Ma but not Sl‐UC recovered from disease symptoms induced by CMV‐TTS while both the cultivars succumbed to the infection of CMV‐77 and its necrogenic satRNA. Ability to recover of the Sl‐Ma plants was transmitted by grafting to the susceptible genotype Sl‐UC. More interestingly, recovery was observed also against the challenge inoculation of CMV plus 77‐satRNA in plants grafted on Sl‐Ma and in self‐grafted plants of both the Sl‐Ma and Sl‐UC cultivars. Analysis of small RNAs and genes of the defence plant response based on RNA interference (RNAi) suggested that RNAi is involved in the recovery of Sl‐Ma against CMV with hypervirulent satRNAs and in scions grafted on this rootstock. The response of Sl‐Ma to the inoculation of CMV‐77 plus 77‐satRNA was compared with that of the transgenic tomato line S. lycopersicum transgenic line UCTC5.9.2 that expresses constitutively the benign variant of the satRNA denoted Tfn‐satRNA. Comparative analysis suggested that the response may operate via similar mechanisms, which involve RNAi, the graft and the presence of the satRNA.

Identification and comparison of RCMV ALL 03 open reading frame (ORF) among several different strains of cytomegalovirus worldwide

BackgroundRat cytomegalovirus ALL-03 (Malaysian strain) which was isolated from a placenta and uterus of a house rat, Rattus rattus diardii has the ability to cross the placenta and infecting the fetus. To further elucidate the pathogenesis of the Malaysian strain of Rat Cytomegalovirus ALL-03 (RCMV ALL-03), detailed analysis on the viral genome sequence is crucial. MethodsGenome sequencing of RCMV ALL-03 was carried out in order to identify the open reading frame (ORF), homology comparison of ORF with other strains of CMV, phylogenetic analysis, classifying ORF with its corresponding conserved genes, and determination of functional proteins and grouping of gene families in order to obtain fundamental knowledge of the genome. ResultsThe present study revealed a total of 123 Coding DNA sequences (CDS) from RCMV ALL-03 with 37 conserved ORF domains as with all herpesvirus genomes. All the CDS possess similar function with RCMV-England followed by RCMV-Berlin, RCMV-Maastricht, and Human CMV. The phylogenetic analysis of RCMV ALL-03 based on conserving genes of herpes virus showed that the Malaysian RCMV isolate is closest to RCMV-English and RCMV-Berlin strains, with 99% and 97% homology, respectively. Similarly, it also demonstrated an evolutionary relationship between RCMV ALL-03 and other strains of herpesviruses from all the three subfamilies. Interestingly, betaherpesvirus subfamily, which has been shown to be more closely related with gammaherpesviruses as compared to alphaherpesviruses, shares some of the functional ORFs. In addition, the arrangement of gene blocks for RCMV ALL-03, which was conserved among herpesvirus family members was also observed in the RCMV ALL-03 genome. ConclusionGenomic analysis of RCMV ALL-03 provided an overall picture of the whole genome organization and it served as a good platform for further understanding on the divergence in the family of Herpesviridae.

The Microtubule Inhibitor Podofilox Inhibits an Early Entry Step of Human Cytomegalovirus.

Human cytomegalovirus is a ubiquitous β-herpesvirus that infects many different cell types through an initial binding to cell surface receptors followed by a fusion event at the cell membrane or endocytic vesicle. A recent high-throughput screen to identify compounds that block a step prior to viral gene expression identified podofilox as a potent and nontoxic inhibitor. Time-of-addition studies in combination with quantitative-PCR analysis demonstrated that podofilox limits an early step of virus entry at the cell surface. Podofilox was also able to drastically reduce infection by herpes simplex 1, an α-herpesvirus with a very similar entry process to CMV. Podofilox caused a reduced maximal plateau inhibition of infection by viruses with single step binding processes prior to fusion-like Newcastle disease virus, Sendai virus, and influenza A virus or viruses that enter via endocytosis like vesicular stomatitis virus and a clinical-like strain of CMV. These results indicate that microtubules appear to be participating in the post-binding step of virus entry including the pre- and post-penetration events. Modulation of the plasma membrane is required to promote virus entry for herpesviruses, and that podofilox, unlike colchicine or nocodazole, is able to preferentially target microtubule networks at the plasma membrane.

A novel strain of cynomolgus macaque cytomegalovirus: implications for host-virus co-evolution.

BackgroundCytomegaloviruses belong to a large, ancient, genus of DNA viruses comprised of a wide array of species-specific strains that occur in diverse array of hosts.MethodsIn this study we sequenced the ~217 Kb genome of a cytomegalovirus isolated from a Mauritius cynomolgus macaque, CyCMV Mauritius, and compared it to previously sequenced cytomegaloviruses from a cynomolgus macaque of Filipino origin (CyCMV Ottawa) and two from Indian rhesus macaques (RhCMV 180.92 and RhCMV 68–1).ResultsThough more closely related to CyCMV Ottawa, CyCMV Mauritius is less genetically distant from both RhCMV strains than is CyCMV Ottawa. Several individual genes, including homologues of CMV genes RL11B, UL123, UL83b, UL84 and a homologue of mammalian COX-2, show a closer relationship between homologues of CyCMV Mauritius and the RhCMVs than between homologues of CyCMV Mauritius and CyCMV Ottawa. A broader phylogenetic analysis of 12 CMV strains from eight species recovers evolutionary relationships among viral strains that mirror those amongst the host species, further demonstrating co-evolution of host and virus.ConclusionsPhylogenetic analyses of rhesus and cynomolgus macaque CMV genome sequences demonstrate co-speciation of the virus and host.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2588-3) contains supplementary material, which is available to authorized users.

Management of Cytomegalovirus Retinitis in HIV and Non-HIV Patients.

As CMVR continues to affect HIV-positive and non-HIV immunosuppressed patients, ophthalmologists must continue to tailor diagnostics and therapeutics to individual cases. In HIV-related disease, ocular fluid sampling and intravitreal drug delivery are considerations, but systemic antiviral therapy is paramount in the initial management from both ophthalmic and systemic morbidity standpoints. Non-HIV-related disease should be approached with a multidisciplinary team, including an ophthalmologist/vitreoretinal/uveitis specialist for consideration of intravitreal antiviral therapy with qualitative and quantitative aqueous PCR monitoring, and consideration of PCR genome sequencing for CMV strains that may become resistant to antiviral therapies from long-term antiviral prophylactic exposure. Hematologists or oncologists may help with patients who remain bone marrow-suppressed following transplantation or systemic chemotherapy. Because of related toxicities of the anti-CMV medications and immunosuppressive medications (eg, bone marrow suppression and cytopenias), infectious disease consultation can help in the treatment and monitoring of side effects.

ФИЛОГЕНЕТИЧЕСКИЕ ВЗАИМОСВЯЗИ ИЗОЛЯТОВ ВИРУСА ОГУРЕЧНОЙ МОЗАИКИ (CUCUMBER MOSAIC VIRUS) ИЗ ОГУРЦА (CUCUMIS SATIVUS), ПЕРЦА (CAPSICUM ANNUUM) И АГЕРАТУМА (AGERATUM HOUSTONIANUM)

The nucleotide sequences of two strains of CMV isolated from cucumber and ageratum in the Primorsk region and one strain from pepper isolated in the North Korea have been studied. The phylogenetic linkages among isolated strains and strains of CMV from Genbank were revealed. The intragroup position of the isolated strains of CMV widespread in the Far East was specified.

First report of Cucumber mosaic virus in African Impatiens (Impatiens walleriana) in Korea

Virus-like symptoms including stunt, severe mosaic with malformation of leaves, fern-like leaves and abnormal petals were observed from an African impatiens (Impatiens walleriana) grown in a plant nursery in Icheon, Korea. Serological analysis using immuno-strip kits for viruses reported in African impatiens indicated that Cucumber mosaic virus (named CMV-Im) was a causal agent for the symptomatic African impatiens. Biological properties of CMV-Im were analyzed using responses of host plant species, suggesting that CMV-Im is a typical strain that belongs to CMV subgroup I. RT-PCR analysis verified CMV-Im infection from naturally infected African impatiens or mechanically inoculated some host species. Analysis of multiple alignments of CMV capsid protein (CP) sequences showed that CMV-Im shared high CP amino acids identities with other CMV strains. Phylogenetic tree analysis for the CP sequences of CMV-Im and representative CMV strains confirmed that CMV is a typical member of CMV subgroup I. To our knowledge, it is the first report of CMV in African impa tiens in Korea.

Genetic Diversity of Cucumber Mosaic Virus Strain Soybean from Several Areas

Cucumber mosaic virus strain soybean (CMV-S) is one of economically important virus infecting soybean in Indonesia. However, it is very few information related with the CMV-S Indonesia isolates. Thus, the aim of present work is to detect and identify CMV-S isolates from different origin. Leaf samples collected from six different soybean fields in Java and North Sumatera. Molecular detection was carried out by reverse transcription polymerase chain reaction (RT-PCR) using specific primer of the coat protein (CP) gene and DNA sequencing. RT-PCR were successfully amplified the entire CP gene size ~657 bp from all samples and encoded 218 amino acids. Analysis of nucleotide and amino acid sequences showed high homology among isolates ranging from 99.5% and 100%. However isolate from Sindang Laut either nucleotides or amino acids showed lower homology to that of five isolates ranging from 89.4%-89.6% and 91.7%, respectively. The differences between CMV-S with other non legume strain found in six amino acid positions in the CP gene, suggesting the differences might related with host specificity. Phylogenetic analysis of amino acid of six isolates to other CMV strain showed that the five CMV-S isolates were in the same cluster, while Sindang Laut isolate was in different cluster together with the non-legume CMV from India. It is indicating the present of genetic variability among CMV-S isolates in Java.

1357Viral Diversity and Neutralizing Antibody Responses in Infants with Symptomatic Congenital CMV Disease receiving Antiviral Therapy

Background: Congenital cytomegalovirus (cCMV) is the most common congenital infection and an important cause of childhood sensorineural hearing loss (SNHL). Extensive genetic diversity among CMV strains, infection with multiple CMV strains, and strain-specific neutralizing antibody responses appear to be common. However, the association between virus diversity and strain-specific antibody responses is not known. Methods: Residual samples were available from 22 infants with symptomatic cCMV enrolled in two NIAID Collaborative Antiviral Study Group studies, both evaluating antiviral treatment of cCMV. All subjects received 6 weeks of antiviral therapy from enrollment and then subjects in one study were randomized to receive either active drug (valganciclovir) or placebo for a full 6 months. Urine samples obtained at enrollment (less than 3 weeks of age) and at age 6 months were analyzed for genotypes of glycoproteins gB, gH and gN. Neutralization response was measured in sera at enrollment using gN recombinant viruses at enrollment and at 6 months. A 3-fold difference in neutralizing titers against at least two gN recombinant viruses was considered evidence of strain specific response. Results: All 22 subjects had >1 CMV genotype detected at enrollment, with a range of 2-5 per subject. Six subjects (30%) had an increase in number of genotypes over time, while 9 (45%) had a decrease. There did not appear to be a significant effect of antiviral treatment on genotypic diversity. Neutralization titers were decreased from initial to follow up at 6 months (mean 2400 vs 970, respectively, p = 0.006). Eighteen subjects (82%) showed strain-specific neutralizing responses. If a particular genotype was found to be absent at enrollment, neutralization titers were found to be increased (2600 vs 3600 for gN1, 1200 vs 3800 for gN2; 1200 vs 2200 for gN3). Conclusion: All study infants were infected with multiple CMV strains and most had strain-specific neutralizing antibody responses. Overall the viral diversity decreased over time; however, this did not appear to be accounted for only by antiviral treatment. Virus neutralization at birth was significantly better than neutralization at 6 months of life, suggesting maternal antibodies account for much of the neutralization within the first 6 months of life. There was a detectible trend towards higher neutralization titers (implying improved neutralization) if the cooresponding viral strain is absent, suggesting humoral immune response plays a role in genotypic diversity of congenitally infected infants. Further studies are needed to better classify the role humoral immune response plays in congenital CMV.

Altered microRNA expression after infection with human cytomegalovirus leads to TIMP3 downregulation and increased shedding of metalloprotease substrates, including MICA.

Proteolytic shedding of ligands for the NK group 2D (NKG2D) receptor is a strategy used by tumors to modulate immune recognition by NK cells and cytotoxic T cells. A number of metalloproteases, especially those of the A disintegrin and metalloprotease (ADAM) family, can mediate NKG2D ligand cleavage and this process can be modulated by expression of the thiol isomerase ERp5. In this article, we describe that an increased shedding of the NKG2D ligand MICA is observed postinfection with several strains of human CMV due to an enhanced activity of ADAM17 (TNF-α converting enzyme) and matrix metalloprotease 14 caused by a reduction in the expression of the endogenous inhibitor of metalloproteases tissue inhibitors of metalloproteinase 3 (TIMP3). This decrease in TIMP3 expression correlates with increased expression of a cellular miRNA known to target TIMP3, and we also identify a human CMV-encoded microRNA able to modulate TIMP3 expression. These observations characterize a novel viral strategy to influence the shedding of cell-surface molecules involved in immune response modulation. They also provide an explanation for previous reports of increased levels of various ADAM17 substrates in the serum from patients with CMV disease. Consistent with this hypothesis, we detected soluble MICA in serum of transplant recipients with CMV disease. Finally, these data suggest that it might be worthwhile to prospectively study ADAM17 activity in a larger group of patients to assay whether this might be a useful biomarker to identify patients at risk for development of CMV disease.

Pre-Transplant T-Cell Subtype Assessment as a Predictive Tool for CMV Viremia After Transplant.

Background: Some studies have suggested that pre-transplant T-cell responses may be useful for predicting the risk of CMV reactivation following transplantation. We tested this hypothesis in a large cohort of CMV seropositive transplant recipients. Methods: We assessed CMV specific T-cell subsets pre-transplant using PBMCs from CMV seropositive (R+) transplant patients enrolled in 5 centers. No patient received antiviral prophylaxis and all had regular virologic monitoring as part of a pre-emptive strategy. PBMCs were exposed for 6 hours to CMV strain Towne at MOI 0.03, followed by antibody staining for flow cytometry immunophenotyping of T-cell subsets including CD4+, CD8+, Treg populations (CD25+, FOXP3+), interferon-γ (IFN-γ). The primary outcome was viremia requiring antiviral therapy within the first 3-months post-transplant. Results: A total of 272 CMV R+ transplant patients were analyzed. Transplant types included kidney (65.4%), liver (23.5%), and other (12%). Outcomes included any CMV viremia (n=123/272; 45.2%), CMV viremia requiring antiviral therapy (n=83/272; 30.7%) and CMV disease (n=10/272; 3.7%). A positive pre-transplant CD4+ and CD8+ IFN-γ CMV-specific T-cell response was seen in 14.1% and 42.6% respectively. In terms of the primary outcome (clinically significant CMV viremia), a positive CD4+, IFN-γ response was seen in 12.3% (no viremia) vs. 18.1% (viremia) of patients (p=0.28). A CD8+ IFN-γ response was seen in 41.2% (no viremia) vs. 45.8% (viremia) of patients (p=0.57). Median CD4+ and CD8+ IFN-γ + T-cell frequencies were also similar in the viremic vs. non-viremic groups (p=NS). Also neither CD4+ or CD8+ frequencies predicted symptomatic CMV disease [p=NS for all comparisons]. Analysis of the D+ and D- subgroups did not yield different results. Mean T-reg frequency following viral stimulation was 1.43% ± 1.37%. Compared to controls, viral stimulation resulted in a significant decline in T-reg expression of FOXP3 by 6-hours (median 2.2 fold decline; p<0.001). Higher baseline T-reg% correlated with increased risk of CMV reactivation post-transplant (p=0.035). Conclusions: This study suggests that pre-transplant testing for CMV specific CD4+ and CD8+ T-cell responses are unlikely to be of significant clinical utility for predicting CMV viremia or disease post-transplant. The predictive value of Treg measurements is a novel finding. DISCLOSURE:Kumar, D.: Grant/Research Support, Roche, Astellas, Other, Oxford Immunotec, Advisory Board Member. Humar, A.: Grant/Research Support, Roche, Other, Astellas, Advisory board member, Chimerix, Advisory board member.

Microarray analysis for the influence of CMV infection on tomato genomes (617.1)

Functional genomics is the study of gene function through parallel expression measurements of a genome. The most common tools used to carry out these measurements include complementary DNA microarrays, oligonucleotide microarrays or serial analysis of gene expression (SAGE)3. A custom‐designed Affymetrix tomato GeneChip array was used to study the gene expression changes in response to viral inoculation with a mild and severe CMV strains. The basic technique involves extraction of RNA from tomato leaf samples and then copied, while incorporating either fluorescent nucleotides or a tag that is later stained with fluorescence. Labeled RNA is then hybridized to a microarray for a period of time, then excess is washed off and the microarray is scanned under laser light. Different expression responses were resulted due to the type of viral infection.Total number of expressed genes were 3213, Out of this 1660 genes were down‐regulated and 1553 were up‐regulated. The response to CMV‐16 infection led to 1365 gene expression, 642 were down‐regulated and 723 genes were up‐regulated Specific seven different genes in tomato genome (Hsp70,ChMDH, Wif1, rccr, AAP2, PIIF &amp; GAPDH endogenous gene used as a control) were analyzed and resulted in down regulation of Hsp70 and upper regulation of both ChMDH and PIIF. Gene expression results were confirmed with relative quantitative RT‐PCR technology.

Nuclear-Cytoplasmic Partitioning of Cucumber Mosaic Virus Protein 2b Determines the Balance between Its Roles as a Virulence Determinant and an RNA-Silencing Suppressor

ABSTRACTThe Cucumber Mosaic Virus (CMV) 2b protein is an RNA-silencing suppressor that plays roles in CMV accumulation and virulence. The 2b proteins of subgroup IA CMV strains partition between the nucleus and cytoplasm, but the biological significance of this is uncertain. We fused an additional nuclear localization signal (NLS) to the 2b protein of subgroup IA strain Fny-CMV to create 2b-NLS and tested its effects on subcellular distribution, silencing, and virulence. The additional NLS enhanced 2b protein nuclear and nucleolar accumulation, but nuclear and nucleolar enrichment correlated with markedly diminished silencing suppressor activity in patch assays and abolished 2b protein-mediated disruption of microRNA activity in transgenic Arabidopsis. Nucleus/nucleolus-localized 2b protein possesses at least some ability to inhibit antiviral silencing, but this was not sufficient to prevent recovery from disease in younger, developing leaves in Arabidopsis. However, enhanced nuclear and nucleolar accumulation of 2b increased virulence and accelerated symptom appearance in older leaves. Experiments with Arabidopsis lines carrying mutant Dicer-like alleles demonstrated that compromised suppressor activity explained the diminished ability of 2b-NLS to enhance virus accumulation. Remarkably, the increased virulence that 2b-NLS engendered was unrelated to effects on microRNA- or short interfering RNA-regulated host functions. Thus, although nucleus- and nucleolus-localized 2b protein is less efficient at silencing suppression than cytoplasm-localized 2b, it enhances CMV virulence. We propose that partitioning of the 2b protein between the cytoplasmic and nuclear/nucleolar compartments allows CMV to regulate the balance between virus accumulation and damage to the host, presumably to maximize the benefit for the virus.IMPORTANCE In this work, the main finding is that nucleus/nucleolus-localized 2b protein is strongly associated with CMV virulence, which is independent of its effect on small RNA pathways. Moreover, this work supports the contention that the silencing suppressor activity of CMV 2b protein is predominantly exerted by that portion of the 2b protein residing in the cytoplasm. Thus, we propose that partitioning of the 2b protein between the cytoplasmic and nuclear/nucleolar compartments allows CMV to regulate the balance between virus accumulation and damage to the host, presumably to maximize the benefit for the virus.

Genotypic Diversity and Mixed Infection in Newborn Disease and Hearing Loss in Congenital Cytomegalovirus Infection

Congenital cytomegalovirus (cCMV) is a common congenital infection and a leading nongenetic cause of sensorineural hearing loss (SNHL). CMV exhibits extensive genetic variability, and infection with multiple CMV strains (mixed infection) was shown to be common in congenital CMV. The role of mixed infections in disease and outcome remains to be defined. Genotyping of envelope glycoproteins, UL55 (gB), UL73 (gN) and UL75 (gH), was performed on saliva specimens of 79 infants from the ongoing CMV and Hearing Multicenter Screening (CHIMES) Study and on blood and urine specimens of 52 infants who participated in natural history studies at the University of Alabama at Birmingham. Genotyping of UL144 and US28 was also performed in the CHIMES cohort. The association of individual genotypes and mixed infection with clinical findings at birth and SNHL was examined. Thirty-seven of 131 infants (28%) were symptomatic at birth and 26 (20%) had SNHL at birth. All known genotypes of UL55, UL75, UL73 and US28 were represented, and no particular genotype was associated with symptomatic infection or SNHL. UL144 subtype C was more common in symptomatic infants but not associated with SNHL. Mixed infection was observed in 59 infants (45%) and not associated with symptoms (P = 0.43) or SNHL at birth (P = 0.82). In the cohort of 52 infants with long-term hearing outcome, mixed infection at birth was not predictive of SNHL. Mixed infection is common in infants with congenital CMV but is neither associated with symptomatic infection nor associated with SNHL.

Is IL-17 an Accomplice Contributing to Salivary Gland Damage During CMV Infection?

Aim: This study aimed to explore the potential role of IL-17 in the pathological damage to the salivary gland during CMV infection. Materials &amp; methods: For all studies, 4-week-old female BALB/c mice were used. Mouse embryo fibroblast cells were isolated from embryos of pregnant mice. The Smith murine CMV (MCMV) strain was propagated and obtained from homogenates of the salivary glands. BALB/c mice were randomly divided into two groups. Mice in one group were infected with Smith MCMV to establish disseminated infection; mice in the other group were controls. Four mice of each group were randomly chosen to be harvested on days 3, 7, 14 and 28. Viral titers in salivary gland tissues were determined using a standard plaque assay; IL-17+ T cells in the spleen were analyzed by flow cytometry; the expressions of IL-17 and IL-17 receptor (IL-17R) mRNA was measured by reverse transcription PCR; and the pathology of the salivary glands in infected mice were assessed by hematoxylin and eosin staining. Results: The viral titers in salivary gland tissues, which were much higher than those of other organs, elevated on day 7 and peaked on day 14, then gradually declined on day 28. The percentages of CD4+IL-17+ T cells were increased in MCMV-infected mice compared with controls, with the main increase of CD4+IL-17+ T cells being Th17 cells (p &lt; 0.05). In salivary gland tissues, the expression of IL-17 and IL-17R mRNA increased significantly and achieved the highest level on day 14, which was significantly higher than that of the controls (p &lt; 0.05). Compared with controls, serious inflammatory cellular infiltrates in the salivary glands were present on day 7 after MCMV infection, and on day 14, many of the cellular infiltrate became larger and coalesced with neighboring foci. We found that IL-17 and IL-17R were enriched in the salivary glands when the tissues were mainly injured. Conclusion: The results illustrated that the inflammatory factors, IL-17 and IL-17R, possibly contributed to the process of viral infection and played a part in inducing pathological damage of the salivary gland.

Glycoprotein B genotyping in congenital/perinatal cytomegalovirus infection in symptomatic infants

Molecular epidemiological studies on circulating strains of CMV in cogenital/perinatal infections have not been done earlier in this region. To study the glycoprotein B genotypes in babies with symptomatic congenital/perinatal CMV infection and to assess the possible influence of genotype on the outcome of the infection. Clinical samples (blood and urine) of symptomatic babies are sent to the Virology Department of NCDC, Delhi for the diagnosis of congenital infections. 375 clinical samples of infants (newborn - 6 months old) were included for the study. Serum samples were subjected to ELISA for detection of IgM antibodies against CMV. DNA isolation and amplification of CMV genomic DNA targeting gB gene fragment by nested PCR, was carried out in the samples. The amplified fragment including the cleavage site was subjected to RFLP using restriction enzymes Rsal and Hinf1. They were also verified by sequencing using Big Dye Terminator chemistry. 75 samples out of 375 tested were confirmed positive for CMV infection by serology and PCR. Both RFLP and sequencing of gB gene fragment showed that gB 1, 2 and 3 genotypes were in circulation. gB 3 was the most prevalent genotype in symptomatic infants. Hepatosplenomegaly was the most common feature in gB-3 genotype of CMV. gB2 congenital CMV infection was more commonly associated with long term sequelae.

Antibody inhibition of human cytomegalovirus spread in epithelial cell cultures

Anti-cytomegalovirus (CMV) antibodies reduce the incidence of CMV transmission and ameliorate the severity of CMV-associated disease. Neutralizing activity, measured as the ability of antibodies to prevent entry of cell-free virus, is an important component of natural immunity. However, in vivo CMV amplification may occur mainly via spread between adjacent cells within tissues. Thus, inhibition of cell-to-cell spread may be important when evaluating therapeutic antibodies or humoral responses to infection or immunization. In vitro CMV cell-to-cell spread is largely resistant to antibodies in fibroblast cultures but sensitive in endothelial cell cultures. In the present study antibodies in CMV hyperimmuneglobulin or seropositive human sera inhibited CMV cell-to-cell spread in epithelial cell cultures. Spread inhibition activity was quantitated with a GFP reporter assay employing GFP-tagged epithelialtropic variants of CMV strains Towne or AD169. Measurement of spread inhibition provides an additional parameter for the evaluation of candidate vaccines or immunotherapeutics and to further characterize the role of antibodies in controlling CMV transmission and disease.

Natural Killer Cell Dependent Within-Host Competition Arises during Multiple MCMV Infection: Consequences for Viral Transmission and Evolution

It is becoming increasingly clear that many diseases are the result of infection from multiple genetically distinct strains of a pathogen. Such multi-strain infections have the capacity to alter both disease and pathogen dynamics. Infection with multiple strains of human cytomegalovirus (HCMV) is common and has been linked to enhanced disease. Suggestions that disease enhancement in multi-strain infected patients is due to complementation have been supported by trans-complementation studies in mice during co-infection of wild type and gene knockout strains of murine CMV (MCMV). Complementation between naturally circulating strains of CMV has, however, not been assessed. In addition, many models of multi-strain infection predict that co-infecting strains will compete with each other and that this competition may contribute to selective transmission of more virulent pathogen strains. To assess the outcome of multi-strain infection, C57BL/6 mice were infected with up to four naturally circulating strains of MCMV. In this study, profound within-host competition was observed between co-infecting strains of MCMV. This competition was MCMV strain specific and resulted in the complete exclusion of certain strains of MCMV from the salivary glands of multi-strain infected mice. Competition was dependent on Ly49H+ natural killer (NK) cells as well as the expression of the ligand for Ly49H, the MCMV encoded product, m157. Strains of MCMV which expressed an m157 gene product capable of ligating Ly49H were outcompeted by strains of MCMV expressing variant m157 genes. Importantly, within-host competition prevented the shedding of the less virulent strains of MCMV, those recognized by Ly49H, into the saliva of multi-strain infected mice. These data demonstrate that NK cells have the strain specific recognition capacity required to meditate within-host competition between strains of MCMV. Furthermore, this within-host competition has the capacity to shape the dynamics of viral shedding and potentially select for the transmission of more virulent virus strains.

CLONING AND CHARACTERIZATION OF CUCUMBER MOSAIC VIRUS COAT PROTEIN GENE FROM INFECTED BANANA PLANTS IN EGYPT

Cucumber mosaic virus was detected in banana plants showing typical symptoms of CMV infection using DAS-ELISA. Coat protein cDNA was ampli- fied, by RT-PCR using specific primers, and cloned into pGEM®-T Easy Vector, then transformed into E. coli Top 10 cells. The complete sequence of coat protein gene from CMV-Egy strain has been de- termined and compared to other known CMV-CP sequences. The alignment of the amino acid sequences revealed high ho- mology (98-100%) between CMV-Egy and different CMV strains. Phylogenetic analysis also revealed that CMV-Egy is closely related with CMV-Fny isolated from USA and Sny isolated from Israel.

Application of a Reassortant Cucumber mosaic virus Vector for Gene Silencing in Tomato and Chili Pepper Plants

We developed a reassortant RNA virus vector derived from <TEX>$Cucumber$</TEX> <TEX>$mosaic$</TEX> <TEX>$virus$</TEX> (CMV), which has advantages of very wide host range and can efficiently induce gene silencing in a few model plants. Certain CMV isolates, however, show limited host ranges presumably because they naturally co-evolved with their own hosts. We used a reassortant comprised of two strains of CMV, Y-CMV and Gn-CMV, to broaden the host range and to develop a virus vector for virus-induced gene silencing (VIGS). Gn-CMV could infect chili pepper and tomato more efficiently than Y-CMV. Gn-CMV RNA1, 3 and Y-CMV RNA2-A1 vector were newly reconstructed, and the transcript mixture of RNA1 and 3 genomes of Gn-CMV and RNA2 genome of Y-CMV RNA2 containing portions of the endogenous phytoene desaturase (PDS) gene (CMV2A1::PDSs) was inoculated onto chili pepper (cv. Chung-yang), tomato (cvs. Bloody butcher, Tigerella, Silvery fir tree, and Czech bush) and <TEX>$Nicotiana$</TEX> <TEX>$benthamiana$</TEX>. All the tested plants infected by the reassortant CMV vector showed typical photo-bleaching phenotypes and reduced expression levels of <TEX>$PDS$</TEX> mRNA. These results suggest that the reassortant CMV vector would be a useful tool for the rapid induction of the RNA silencing of endogenous genes in chili pepper and tomato plants.