A zinc finger protein Tsip1 controls Cucumber mosaic virus infection by interacting with the replication complex on vacuolar membranes of the tobacco plant

? In Cucumber mosaic virus (CMV) RNA replication, replicase-associated protein CMV 1a and RNA-dependent RNA polymerase protein CMV 2a are essential for formation of an active virus replicase complex on vacuolar membranes. ? To identify plant host factors involved in CMV replication, a yeast two-hybrid system was used with CMV 1a protein as bait. One of the candidate genes encoded Tsi1-interacting protein 1 (Tsip1), a zinc (Zn) finger protein. Tsip1 strongly interacted with CMV 2a protein, too. ? Formation of a Tsip1 complex involving CMV 1a or CMV 2a was confirmed in vitro and in planta. When 35S::Tsip1 tobacco (Nicotiana tabacum) plants were inoculated with CMV-Kor, disease symptom development was delayed and the accumulation of CMV RNAs and coat protein was decreased in both the infected local leaves and the uninfected upper leaves, compared with the wild type, whereas Tsip1-RNAi plants showed modestly but consistently increased CMV susceptibility. In a CMV replication assay, CMV RNA concentrations were reduced in the 35S::Tsip1 transgenic protoplasts compared with wild-type (WT) protoplasts. ? These results indicate that Tsip1 might directly control CMV multiplication in tobacco plants by formation of a complex with CMV 1a and CMV 2a.     

T and B cell responses to cytomegalovirus antigens in healthy blood donors and bone marrow transplant recipients

Abstract We measured the production of interferon-gamma (IFN-γ) from single T cells and the T cell proliferative response to different cytomegalovirus (CMV) antigens in healthy blood donors and bone marrow transplant recipients. The antigens consisted of a CMV nuclear antigen (CMV na) containing the pp65-kDa matrix protein and the immediate early antigens but lacking CMV glycoproteins, and an antigen comprising native CMV glycoproteins (CMV gp). We also measured the IgG antibodies to CMV na and CMV gp. The T cells reacted to CMV na in CMV seropositive blood donors both with the production of IFN-γ and with proliferation, while bone marrow transplant recipients had a deficient T cell response. After stimulation with CMV gp, no T cell response could be observed in CMV seropositive subjects. IgG antibodies to CMV na coexisted in plasma with similar levels of antibodies to CMV gp.     

First Report of Cucumber mosaic virus Infecting Chinese Mallow in China

The virus in naturally infected, stunted Chinese mallow plants and mosaic leaves was identified as Cucumber mosaic virus (CMV). Six symptomatic plants and one symptomless plant were collected in Chongqing, China. DAS‐ELISA suggested CMV was likely associated with the diseased Chinese mallow. Double‐stranded RNA was extracted from the samples, analysed by RT‐PCR, and the coding sequences of their coat proteins (CPs) were sequenced. The results further confirmed CMV was the pathogen causing Chinese mallow stunted, mosaic disease. The isolate was named CMV‐DXC. The full sequence of CMV‐DXC CP was determined, and it had the highest nucleotide identity (99.4%) of those of CMV‐lily, CMV‐WSJ and CMV‐Hnt, respectively. Phylogenetic analysis shows that CMV‐DXC belongs to CMV subgroup II. To our knowledge, this is the first report of CMV infecting Chinese mallow in China.     

Evaluation of the indirect and IgM‐capture anti‐human cytomegalovirus IgM ELISA methods as confirmed by cytomegalovirus IgG avidity

Primary cytomegalovirus (CMV) infection during pregnancy often results in congenital CMV infection with severe clinical complications. IgM antibodies are one of the indices of primary infection. The IgG avidity index (AI) is also known to remain low for 3 months after primary infection. Here, we evaluated and compared the performance of CMV IgM and IgG avidity assays. Because sensitivity and specificity reportedly differ between CMV IgM kits, CMV IgM detection was compared between the two commercially available ELISA kits that are most commonly used in Japan. Sera for CMV IgM were first screened using a traditional indirect ELISA kit. Selected samples were then tested for CMV IgM and CMV AI using a CMV IgM‐capture ELISA kit and a CMV IgG avidity assay, respectively. The rate of concordance between the IgM kits was 89% (42/47), indicating the absence of any significant difference. Most of the CMV IgM‐positive plasma samples showed high CMV IgG AI; however, 18 commercially available plasma samples with low CMV IgG AI were all CMV IgM‐positive. One plausible explanation for this discrepancy is that the duration of low IgG AI is shorter than that of IgM positivity. Alternatively, CMV IgM tests may generate pseudo‐positive readouts in cases of congenital infection. Nevertheless, our study confirms that CMV IgG AI can be a reliable indicator of CMV primary infection.     

Hypersensitive response in cucumber mosaic virus-inoculated Arabidopsis thaliana

The responses of 12 Arabidopsis ecotypes to cucumber mosaic virus strain Y (CMV(Y)) or strain O (CMV(O)) were characterized. Except for ecotype C24, all ecotypes were susceptible to both strains of CMV. In C24, CMV(O) multiplied systemically, but CMV(Y) did not spread systemically and induced only local necrotic spots in virus-inoculated leaves 21–27 h after inoculation. In CMV(Y)-inoculated C24 leaves, virus was confined to the inoculated leaves, and the amount of the pathogenesis-related-1 protein increased during the progress of local necrotic spot formation. These results indicate that C24 mounts a hypersensitive response (HR) to CMV(Y). By genetic analysis of crosses between C24 and ecotype Columbia or Landsberg (erecta) which are susceptible to CMV(Y) infection, the HR to CMV(Y) in C24 was found to be determined by a single major dominant gene whose function was influenced by a modifier gene from the Landsberg ecotype. Comparison of the responses between C24 leaves inoculated with pseudorecombinants of both strains of CMV suggested that the HR was controlled by CMV RNA3. The molecular interaction between the single major gene and CMV(Y) RNA3 is likely to induce the HR in CMV(Y)-inoculated C24.     

RCY1-mediated resistance to Cucumber mosaic virus is regulated by LRR domain-mediated interaction with CMV(Y) following degradation of RCY1

RCY1, which encodes a coiled coil nucleotide-binding site leucine-rich repeat (LRR) class R protein, confers the hypersensitive response (HR) to a yellow strain of Cucumber mosaic virus (CMV[Y]) in Arabidopsis thaliana. Nicotiana benthamiana transformed with hemagglutinin (HA) epitope-tagged RCY1 (RCY1-HA) also exhibited a defense response accompanied by HR cell death and induction of defense-related gene expression in response to CMV(Y). Following transient expression of RCY1-HA by agroinfiltration, the defense reaction was induced in N. benthamiana leaves infected with CMV(Y) but not in virulent CMV(B2)-infected N. benthamiana leaves transiently expressing RCY1-HA or CMV(Y)-infected N. benthamiana leaves transiently expressing HA-tagged RPP8 (RPP8-HA), which is allelic to RCY1. This result suggests that Arabidopsis RCY1-conferred resistance to CMV(Y) could be reproduced in N. benthamiana leaves in a gene-for-gene manner. Expression of a series of chimeric constructs between RCY1-HA and RPP8-HA in CMV(Y)-infected N. benthamiana indicated that induction of defense responses to CMV(Y) is regulated by the LRR domain of RCY1. Interestingly, in CMV(Y)-infected N. benthamiana manifesting the defense response, the levels of both RCY1 and chimeric proteins harboring the RCY1 LRR domain were significantly reduced. Taken together, these data indicate that the RCY1-conferred resistance response to CMV(Y) is regulated by an LRR domain-mediated interaction with CMV(Y) and seems to be tightly associated with the degradation of RCY1 in response to CMV(Y).     

Direct relationship between suppression of virus-specific immunity and emergence of cytomegalovirus disease in simian AIDS

Although opportunistic infections like cytomegalovirus (CMV) are common sequelae of end-stage AIDS, the immune events leading to CMV reactivation in human immunodeficiency virus (HIV)-infected individuals are not well defined. The role of cellular and humoral CMV-specific immune responses in immune control of latent CMV infection was evaluated prospectively in a cohort of 11 simian immunodeficiency virus (SIV)-infected CMV-seropositive rhesus macaques, 6 of whom had histologic evidence of CMV disease at death. Macaques with CMV disease differed from macaques without CMV disease in having significantly higher levels of plasma SIV RNA and CMV DNA and significantly lower titers of anti-CMV binding antibodies (Abs) at the time of death. A significant decline in anti-CMV Abs and CMV-specific CD4(+) and CD8(+) T lymphocytes over time was observed in the macaques with CMV disease, but not in the macaques without CMV disease. Reduction in CMV-specific CD8(+) T lymphocytes and anti-CMV neutralizing Abs was significantly correlated with a decline in CMV-specific CD4(+) T lymphocytes. Although declines in CMV-specific T lymphocytes alone were sufficient for reactivation of low-level CMV viremia, high-level viremia (>1,000 copies of CMV DNA per ml of plasma) was observed when anti-CMV neutralizing and binding Abs had also declined. Thus, the occurrence of CMV reactivation-associated disease in AIDS is associated with suppression of both cellular and humoral CMV-specific immune responses. The underlying mechanism may be a dysfunction of memory B and CD8(+) T lymphocytes associated with SIV-induced impairment of CMV-specific CD4(+) T-cell help.     

Time to initiation of pre-emptive therapy for cytomegalovirus impacts overall survival in pediatric hematopoietic stem cell transplant recipients

Background aimsCytomegalovirus (CMV) reactivation is a significant complication following allogeneic hematopoietic stem cell transplant (HSCT) and affects upwards of 40% of pediatric HSCT patients. Pre-emptive therapy remains the only effective treatment strategy available for pediatric patients following CMV reactivation. Little is known about how the timing of induction treatment following CMV reactivation impacts outcomes in pediatric patients, especially following ex vivo T-cell-depleted (TCD) HSCT.MethodsThe authors evaluated how the timing of induction treatment after CMV reactivation impacts overall survival (OS) and CMV disease in pediatric patients undergoing TCD HSCT at a single institution. The authors retrospectively analyzed patients treated on the pediatric service who received an initial ex vivo TCD HSCT at Memorial Sloan Kettering Cancer Center (MSKCC) from January 2010 to June 2018. CMV reactivation was defined as ≥1 CMV polymerase chain reaction >500 copies/mL in whole blood or >137 IU/mL in plasma within the first 180 days after allogeneic HSCT. To analyze the impact of the timing of induction treatment, the authors’ primary study outcome was OS and secondary outcome was CMV disease.ResultsA total of 169 patients who underwent an initial allogeneic TCD HSCT on the pediatric service at MSKCC from January 2010 to June 2018 were included in the analysis. Thirty-seven (22%) patients reactivated CMV during the first 180 days following HSCT. Of those patients who reactivated CMV, CMV donor/recipient (D/R) serostatus was as follows: D+/R+ n = 28 (76%) and D–/R+ n = 9 (24%). There was no CMV reactivation observed among recipients who were CMV-seronegative irrespective of donor serostatus. In those patients who reactivated CMV, the median time from HSCT to CMV reactivation was 24 days (interquartile range, 20–31). Eleven patients ultimately developed CMV disease in addition to CMV viremia, whereas the remaining patients had only CMV viremia. The cumulative incidence of CMV reactivation at 60 days was 45.2% (95% confidence interval [CI], 32.8–57.5) in the D+/R+ subgroup and 31% (95% CI, 14.2–47.9) in the D–/R+ subgroup. For those patients who reactivated CMV, 30 (81%) received induction treatment with ganciclovir or foscarnet. To analyze the impact of the timing of induction treatment on clinical outcomes, the authors restricted the analysis to those patients who reactivated CMV and received induction treatment (n = 30). The timing of induction treatment was significantly associated with OS, with optimal timing of initiation within a week of CMV reactivation (P = 0.02). There was no significant impact on the timing of induction treatment and risk of CMV disease (P = 0.30).ConclusionsIn ex vivo TCD HSCT in pediatric patients, early initiation of induction treatment after CMV reactivation is associated with improved OS.     

Human cytomegalovirus induced cyclooxygenase-2 in human retinal pigment epithelial cells augments viral replication through a prostaglandin pathway

Cytomegalovirus (CMV) retinitis is characterized by alterations in retinal cell function and host responses to virus replication. The goal of this study was to evaluate the induction of cyclooxygenase-2 (COX-2) and prostaglandin (PGE) in CMV infected human retinal pigment epithelial (RPE) cells and to determine their effect on virus replication. CMV immediate early (IE) protein and COX-2 proteins were identified in RPE cells in retinal tissue sections from patients with CMV retinitis. COX-2 mRNA and protein were induced after CMV infection of human RPE cell cultures. CMV infection of RPE cells induced translocation of NF-kappaB from the cytoplasm to the nucleus. PGE1 and PGE2 were significantly (p<0.001) increased in human RPE cell cultures infected with CMV. Inhibition of CMV IE gene by antisense oligonucleotides abrogated induction of mRNA for COX-2 and protein synthesis of COX-2 and PGE2. PGE enhanced CMV plaque formation and real time PCR analysis revealed that PGE treatment significantly increased CMV DNA copy numbers. These studies demonstrate that when CMV replicates within human RPE cells, COX-2 induction augments virus replication via the PGE pathway. The induction of COX-2 and PGE during retinal CMV infection may augment virus replication and alter a variety of retinal physiological responses.     

Cytomegalovirus (CMV) DNA Load Is an Independent Predictor of CMV Disease and Survival in Advanced AIDS

The impact of cytomegalovirus (CMV) on human immunodeficiency virus type 1 (HIV-1) disease progression has been controversial. In this study, we sought to determine if CMV viral load is independent of HIV-1 viral load in predicting CMV disease and survival. Our findings indicate that in patients with advanced AIDS, CMV DNA load is an independent marker of CMV disease and survival and is more predictive than HIV-1 RNA load. Moreover, patients who respond to preemptive therapy with oral ganciclovir, with resulting undetectable levels of CMV DNA, in their plasma, have a significantly lower risk of developing CMV disease and higher rates of survival, despite stable or increasing HIV-1 RNA loads. These data provide support for CMV as an independent risk factor for mortality in persons with advanced AIDS and further suggest that effective preemptive therapy for CMV can improve patient survival rates.     

Transgenic Melon and Squash Expressing Coat Protein Genes of Aphid-borne Viruses do not Assist the Spread of an Aphid Non- transmissible Strain of Cucumber Mosaic Virus in the Field

Transgenic melon and squash containing the coat protein (CP) gene of the aphid transmissible strain WL of cucumber mosaic cucumovirus (CMV) were grown under field conditions to determine if they would assist the spread of the aphid non-transmissible strain C of CMV, possibly through heterologous encapsidation and recombination. Transgenic melon were susceptible to CMV strain C whereas transgenic squash were resistant although the latter occasionally developed chlorotic blotches on lower leaves. Transgenic squash line ZW-20, one of the parents of commercialized cultivar Freedom II, which expresses the CP genes of the aphid transmissible strains FL of zucchini yellow mosaic (ZYMV) and watermelon mosaic virus 2 (WMV 2) potyviruses was also tested. Line ZW-20 is resistant to ZYMV and WMV 2 but is susceptible to CMV. Field experiments conducted over two consecutive years showed that aphid-vectored spread of CMV strain C did not occur from any of the CMV strain C-challenge inoculated transgenic plants to any of the uninoculated CMV-susceptible non- transgenic plants. Although CMV was detected in 3% (22/764) of the uninoculated plants, several assays including ELISA, RT- PCR-RFLP, identification of CP amino acid at position 168, and aphid transmission tests demonstrated that these CMV isolates were distinct from strain C. Instead, they were non-targeted CMV isolates that came from outside the field plots. This is the first report on field experiments designed to determine the potential of transgenic plants expressing CP genes for triggering changes in virus-vector specificity. Our results indicate that transgenic plants expressing CP genes of aphid transmissible strains of CMV, ZYMV, and WMV 2 are unlikely to mediate the spread of aphid non-transmissible strains of CMV. This finding is of practical relevance because transgenic crops expressing the three CP genes are targeted for commercial release, and because CMV is economically important, has a wide host range, and is widespread worldwide.     

A Novel Satellite RNA of Cucumber Mosaic Virus Induces Unique Line-pattern Mosaic Symptoms in Tobacco

The CMV(YW) isolate of cucumber mosaic virus (CMV) induced unique line‐pattern mosaic symptoms in systemically infected leaves of tobacco (Nicotiana tabacum cv. Ky57). By northern hybridization analysis using cDNA to CMV(Y) satellite RNA as a probe, it was confirmed that CMV(YW) contained a satellite RNA. which was designated sat‐YW RNA; this was 388 nucleotides in length and did not have either a conserved domain that induces necrosis in tomato or chlorosis in tobacco. CMV(YW) free of sat‐YW RNA. which was isolated by the single lesion isolation method using Chenopodium amaranticolor, did not induce the unique line‐pattern mosaic symptom. Furthermore, the sat‐YW RNA‐mediated line‐pattern mosaic symptom was also induced by in vitro transcribed infectious sat‐YW RNA in tobaccos infected with either CMV(YW) or CMV(Y) genomic RNA. These results clearly demonstrated that sat‐YW RNA induces the unique line‐pattern mosaic symptom on CMV‐infected tobaccos.     

ACE inhibition facilitates sodium and water excretion during PEEP in conscious volume-expanded dogs.

Increased activity of the renin-angiotensin system may be involved in sodium and water retention during controlled mechanical ventilation (CMV) with positive end-expiratory pressure (PEEP). We therefore evaluated renal, hemodynamic, and hormonal effects of an acute angiotensin-converting enzyme inhibition (ACEI) during PEEP and extracellular volume expansion in five trained chronically tracheotomized dogs. Three protocols were performed: control, 4 h spontaneous breathing with continuous positive mean airway pressure (Paw) of 4 cmH2O (CPAP 4); CMV 20, CPAP for 1st h, CMV with 20 cmH2O Paw for 2 h (2nd and 3rd h), and 1 h of CPAP (4th h); and CMV20-ACEI, ACEI (Ramipril, 2 mg/kg body wt) followed by the same protocol as in CMV 20. During control, sodium excretion (UNaV) and urine volume (V) increased continuously to 56.2 +/- 2.7 (SE) mumol.min-1.kg body wt-1 and 482 +/- 23 microliters.min-1.kg body wt-1, respectively. UNaV and V increased less during PEEP in CMV 20 and CMV 20-ACEI. However, significantly more sodium and water were retained in CMV 20 than in CMV 20-ACEI (2.3 +/- 0.3 vs. 1.0 +/- 0.3 mmol/kg body wt, and 20 +/- 3 vs. 11 +/- 2 ml/kg body wt) because of a decrease of glomerular filtration rate and fractional UNaV in CMV 20. Heart rate did not change in control, CMV 20, or CMV 20-ACEI. Mean arterial pressure increased during control by 13 mmHg, did not change during CMV 20, and was decreased by 7 mmHg in CMV 20-ACEI.(ABSTRACT TRUNCATED AT 250 WORDS)     

Amiloride inhibition of human cytomegalovirus replication.

Amiloride, an inhibitor of Na+/H+ exchange, interfered with cytomegalovirus (CMV) DNA synthesis, blocked the formation of nuclear inclusions, and reduced CMV infectious yields. The reduction of CMV infectious yields was concentration dependent with an ED90 of 46 microM. Amiloride at a concentration of 150 microM reduced CMV yields by about 100-fold. Reduction of infectious yields appeared to be related to interference with the formation of nuclear inclusions and to inhibition of CMV DNA synthesis. Nuclear inclusions were much reduced in size and demonstrated poorly defined cellulae in the amiloride-treated cells. CMV DNA synthesis was inhibited by approximately 70% when cells were treated with 150 microM amiloride. The reduction in CMV yields could not be related to the reported inhibitory effect of amiloride on protein synthesis. In amiloride (150 microM)-treated, CMV-infected cells, late, yet not immediate-early or early, protein synthesis was markedly decreased relative to untreated, CMV-infected cells. Accordingly, CMV DNA synthesis and the replication of CMV may be related to Na+ entry through an amiloride-sensitive pathway.     

Comparative analysis of expressed sequence tags in resistant and susceptible ecotypes of Arabidopsis thaliana infected with cucumber mosaic virus

Arabidopsis thaliana ecotype Columbia (Col-0) is susceptible to the yellow strain of cucumber mosaic virus [CMV(Y)], whereas ecotype C24 is resistant to CMV(Y). Comprehensive analyses of approximately 9,000 expressed sequence tags in ecotypes Col-0 and C24 infected with CMV(Y) suggested that the gene expression patterns in the two ecotypes differed. At 6, 12, 24 and 48 h after CMV(Y) inoculation, the expression of 6, 30, 85 and 788 genes, respectively, had changed in C24, as opposed to 20, 80, 53 and 150 genes in CMV(Y)-infected Col-0. At 12, 24 and 48 h after CMV(Y) inoculation, the abundance of 3, 10 and 55 mRNAs was altered in both ecotypes. However, at 6 h after CMV(Y) inoculation, no genes were co-induced or co-suppressed in both ecotypes. This differential pattern of gene expression between the two ecotypes at an early stage of CMV(Y) infection indicated that the cellular response for resistance may differ from that resulting in susceptibility at the level detectable by the macroarray. According to the expression pattern at various stages of infection, the expression of many genes could be grouped into clusters using cluster analysis. About 100 genes that encode proteins involved in chloroplast function were categorized into clusters 1 and 4, which had a differentially lower expression in CMV(Y)-inoculated C24. The expression of various genes encoding proteins in the endomembrane system belonged to clusters 2 and 4, which were induced in CMV(Y)-inoculated C24 and Col-0 leaves. Characterization of CMV(Y)-altered gene expression in the two ecotypes will contribute to a better understanding of the molecular basis of compatible and incompatible interactions between virus and host plants.     

Co-electroporation of rice protoplasts with RNAs of cucumber mosaic and tobacco mosaic viruses

Cucumber mosaic virus (CMV) RNA was used to study electroporation conditions suitable for protoplasts from rice suspension cultures. Rice protoplasts required a stronger and shorter electric pulse than tobacco protoplasts for introduction of viral RNA. Under optimized conditions, CMV infection was established in 65 % of electroporated protoplasts. In contrast, electroporation with tobacco mosaic virus (TMV) RNA did not result in infection of rice protoplasts. However, when TMV RNA was electroporated into rice protoplasts together with CMV RNA, TMV production was demonstrated in 15 % of protoplasts. Differential staining with fluorescent antibodies against the two viruses showed that the protoplasts producing TMV were without exception also infected by CMV. The results show that CMV replicates in rice protoplasts by itself, whereas TMV does so only with the aid of CMV.Abbreviations CMV cucumber mosaiv virus - PBS phosphate buffered saline - TMV tobacco mosaic virus.     

Occurrence and distribution of lettuce mosaic disease in Tehran province from Iran

Lettuce mosaic virus (LMV) Cucumber mosaic virus (CMV) and Tomato spotted wilt virus (TSWV) were identified in fields of Tehran province. In this study 452 leaf samples were collected from the fields throughout the Tehran province during 2002 and 2003. Distribution of Lettuce mosaic virus (LMV), Cucumber mosaic virus (CMV), Tomato spotted wilt virus (TSWV)and Arabis mosaic virus (ArMV) was determined with DAS-ELISA. Percentage of single Infection to LMV. CMV or TSWV was 20.58, 15.93 and 9.96% respectively. Also 15.28% of samples were co- infected with LMV+CMV, 8.19% with LMV+TSMV and 7.74% with CMV+TSWV. 4.65% of samples were Infected to all of these three viruses. LMV was found in 48.69%, CMV in 43.59% and TSWV in 30.54% of samples totally. Therefore LMV is major dominant agent of lettuce mosaic disease in Tehran province. This is the first report of occurrence of TSWV on lettuce in Iran and first report of CMV and LMV in Tehran province.     

Biological and Molecular Characterization of Taiwanese Isolates of Cucumber mosaic virus Associated with Banana Mosaic Disease

Banana mosaic disease (BMD) caused by Cucumber mosaic virus (CMV) has become an important threat to the banana industry. We collected and characterized 10 CMV isolates associated with BMD in Taiwan and compared their biological characteristics and coat protein sequences. The isolates fell into four pathotypes on the basis of the symptoms they induce on banana, Nicotiana glutinosa and Vigna unguiculata (cowpea). Double-stranded RNA analysis revealed that the different pathotypes are not related to the presence of CMV satellite RNA. Phylogenetic analysis of worldwide CMV coat protein sequences revealed that among the currently known CMV subgroups IA, IB and II, subgroup IB is phylogenetically unresolved. Our CMV isolates form a new subgroup, IT, within subgroup I. In addition, we resolved another new CMV subgroup, IS, within subgroup I. The analysis also revealed that isolates within different subgroups can infect the banana.