辣椒温和斑点病毒(PMMV)外壳蛋白基因的克隆和序列测定

从本院分离的辣椒温和斑点病毒株系PMMV-QD中提取RNA.根据已有报道的外壳蛋白(CP)自行设计引物P1、P2,用RT-PCR扩增PMMV-QD的CP基因的cDNA,插入pMD 18-T载体,转化感受态受体菌XL1-B1ue,以蓝白菌落筛选转化株,用PCR检测白色菌落,确认获得了含cDNA的阳性重组子.序列测定和比较的结果表明该片段确含有PMMV的CP基因编码区,此种cDNA与国外报道的PMMV病毒株系核苷酸序列的同源性达到97.7%.     

Methanobrevibacter ruminantium as an Indicator of Domesticated-Ruminant Fecal Pollution in Surface Waters

A PCR-based assay (Mrnif) targeting the nifH gene of Methanobrevibacter ruminantium was developed to detect fecal pollution from domesticated ruminants in environmental water samples. The assay produced the expected amplification product only when the reaction mixture contained DNA extracted from M. ruminantium culture, bovine (80%), sheep (100%), and goat (75%) feces, and water samples from a bovine waste lagoon (100%) and a creek contaminated with bovine lagoon waste (100%). The assay appears to be specific and sensitive and can distinguish between domesticated- and nondomesticated-ruminant fecal pollution in environmental samples.     

Bell Pepper Mottle Virus, a Distinct Tobamovirus Infecting Pepper

Bell pepper mottle virus (BPeMV) can be distinguished by symptomatology and host range from other tobamoviruses but a reliable identification needs serological tests. The relationships of BPeMV to tobacco mosaic virus (TMV), Odontoglossum ringspot virus (ORSV), tobacco mild green mosaic virus (TMGMV), and pepper mild mottle virus (PMMV) were investigated using precipitin drop tests on slides, immunodiffusion gel tests, double antibody sandwich enzyme-linked immunosorbent assay (ELISA), and indirectELISA using enzyme-linked goat anti-rabbit globulins for the determination of antiserum titers and serological differentiation indices (SDI). Comparisons of SDIs and amino acid composition data demonstrated that BPeMV is a new species of the tobamovirusgroup. BPeMV, ORSV, PMMV, and TMGMV form a cluster within the genus (group) and could be considered as a subgenus of tobamoviruses.     

Use of Bifidobacterium dentium as an Indicator of the Origin of Fecal Water Pollution

A new, simple, and specific protocol to discriminate between human and animal fecal pollution is described. The procedure is based on the detection of certain Bifidobacterium species in the samples. Two 16S rRNA gene-targeted probes are described. One of these probes (BDE) has as its target a region of the 16S rRNA gene of Bifidobacterium dentium, a Bifidobacterium species of exclusively human origin. The other probe (BAN) is based on the sequence of a region of 16S rRNA gene for several Bifidobacterium species related with animal origins. The specificity of both probes was evaluated by using 24 Bifidobacterium species, and their threshold detection limit was established by DNA-DNA hybridization. DNA-DNA hybridization with the BDE probe showed it to be specific for B. dentium, whereas that with the BAN probe showed it to be specific for B. animalis, B. asteroides, B. coryneforme, B. cuniculi, B. globosum, B. magnum, B. minimum, and B. subtile. A simple and specific protocol was also developed for the detection of their target species in environmental samples (sewage and feces). DNA-DNA hybridization with the BAN probe was only positive for samples from cattle and goats. Thus, this probe is not suitable for the identification of any animal fecal pollution. Whereas all samples with human fecal pollution showed a positive DNA-DNA hybridization result with the BDE probe, none of those with animal fecal pollution did. Therefore, this finding supports the potential use of this probe in detecting fecal pollution of human origin.     

Further evidence for the recognition of Ullucus Mild Mottle Virus as a Distinct Tobamovirus

The affinities of Ullucus mild mottle virus (UMMV). purified by a modified procedure. were examined by immunoblotting and probing with antisera to five distinct tobamoviruses. RNA. isolated from purified virus, was used for in vitro protein translation in a wheat germ system and the products examined by denaturing polyacrylamide gel electrophoresis. The results of these investigations, together with a study of the double-stranded RNAs associated with infection. confirm that UMMV is a distinct, tobamovirus which has close affinities with tobacco mosaic, tomato mosaic and cucumber green mottle tobamoviruses and more distant relationships with ribgrass mosaic and odontoglossum ringspot tobamoviruses.     

Inhibitory Effects of Pepper Mild Mottle Virus Infection by Supernatants of Five Bacterial Cultures in Capsicum annuum L.

Pepper mild mottle virus (PMMoV), one of the most prevalent viruses in chili pepper (Capsicum annuum L.) is a non-enveloped, rod-shaped, single-stranded positive-sense RNA virus classified in the genus Tobamovirus. The supernatants of five bacterial cultures (Pseudomonas putida [PP], Bacillus licheniformis [BLI], P. fluorescens [PF], Serratia marcescens [SER], and B. amyloliquifaciens [BA]) were analyzed to find novel antiviral agents to PMMoV in chili pepper. Foliar spraying with supernatants (1:1, v/v) obtained from Luria-Bertani broth cultures of PP, BLI, PF, SER, and BA inhibited PMMoV infection of chili pepper if applied before the PMMoV inoculation. Double-antibody sandwich enzyme-linked immunosorbent assay showed that treatments of five supernatants resulted in 51–66% reductions in PMMoV accumulation in the treated chili pepper. To identify key compounds in supernatants of PP, BLI, PF, SER, and BA, the supernatants were subjected to gas chromatography-mass spectrometry. The 24 different types of compounds were identified from the supernatants of PP, BLI, PF, SER, and BA. The compounds vary from supernatants of one bacterial culture to another which includes simple compounds—alkanes, ketones, alcohols, and an aromatic ring containing compounds. The compounds triggered the inhibitory effect on PMMoV propagation in chili pepper plants. In conclusion, the cultures could be used to further conduct tissue culture and field trial experiments as potential bio-control agents.     

Ultrastructure of Soybean Leaf Cells Infected with Cowpea Mild Mottle Virus

Cowpea mild mottle virus (CMMV), a whitefly-transmitted, rod-shaped virus isolated in Thailand, induced feather-like structures in the cytoplasm of infected soybean cells. These structures were the results of a complex arrangement of virus particles and occurred in all types of cells observed. An organized arrangement of virus particles in the form of layers was also observed in the cytoplasm of the infected cells. In ultrathin sections, the particles measured about 10 nm wide and more than 600 nm long, which corresponded to the size reported for the purified preparations of CMMV. No feather-like structures or virus particles were observed in the comparable healthy tissues.     

Pepper Mild Mottle Virus,a Plant Virus Associated with Specific Immune Responses,Fever, Abdominal Pains,and Pruritus in Humans

Background

Recently, metagenomic studies have identified viable Pepper mild mottle virus (PMMoV), a plant virus, in the stool of healthy subjects. However, its source and role as pathogen have not been determined.

Methods and Findings

21 commercialized food products containing peppers, 357 stool samples from 304 adults and 208 stool samples from 137 children were tested for PMMoV using real-time PCR, sequencing, and electron microscopy. Anti-PMMoV IgM antibody testing was concurrently performed. A case-control study tested the association of biological and clinical symptoms with the presence of PMMoV in the stool. Twelve (57%) food products were positive for PMMoV RNA sequencing. Stool samples from twenty-two (7.2%) adults and one child (0.7%) were positive for PMMoV by real-time PCR. Positive cases were significantly more likely to have been sampled in Dermatology Units (p<10⁻⁶), to be seropositive for anti-PMMoV IgM antibodies (p = 0.026) and to be patients who exhibited fever, abdominal pains, and pruritus (p = 0.045, 0.038 and 0.046, respectively).

Conclusions

Our study identified a local source of PMMoV and linked the presence of PMMoV RNA in stool with a specific immune response and clinical symptoms. Although clinical symptoms may be imputable to another cofactor, including spicy food, our data suggest the possibility of a direct or indirect pathogenic role of plant viruses in humans.     

A Serious Disease of Groundnut Caused by Cowpea Mild Mottle Virus in the Sudan

A serious disease of groundnut (Arachis hypogaea L.) characterized by stunting of plants, downward rolling, mottling, general chlorosis and reduced size of the leaflets was observed in the Sudan. Surveys conducted from 1992 to 1994 showed that this disease was restricted to irrigated groundnut crops grown between the two Niles. The virus had slightly flexuous filamentous particles (626 nm long) and was transmitted by whiteflies. It was identified serologically as cowpea mild mottle virus (CPMMV). This appears to be the first record of natural occurrence of CPMMV on groundnut in the Sudan and the first evidence that it causes a disease of major economic importance.     

Serodiagnosis of Pepper Veinal Mottle Virus in West Africa using Specific Monoclonal Antibodies in DAS-ELISA

In order to develop a reliable diagnostic test for pepper veinal mottle virus (PVMV), a panel of monoclonal antibodies (mAbs), raised to a Nigerian isolate (PVMW-Ni), was evaluated by double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) on infected pepper samples from seven West African countries. PVMV from different geographical locations was serologically very homogeneous and no significant epitope differences were detected even with a very sensitive double-mAb sandwich ELISA. This ELISA format was optimized with mAb 6C12 and its application in a diagnostic test resulted in the identification of PVMV in Burkina Faso, Togo and Sénégal where it had not been previously reported.     

The Natural Occurrence, Transmission, Properties and Possible Affinities of Cowpea Mild Mottle Virus

Cowpea mild mottle virus (CMMV), although thought to be of only local importance when first found in 1973 infecting cowpeas (Vigna unguiculata) in Ghana, has since been shown to have a very extensive geographical distribution and a wide natural host range. It occurs in Africa, Asia, South America and Oceania in tomato (Lycopersicon esculenturri) and a range of leguminous crops including cowpeas, groundnuts (Arachis hypogaea, soybean (Glycine max) and French beans (Phaseolns vulgaris). The virus has physico-chemical properties resembling those of aphid-borne carlaviruses; it has filamentous particles ca. 650 nm long which contain a single polypeptide of 31–33 KDa and single-stranded RNA of 2. 5 × 10⁶. Unlike carlaviruses, however, CMMV is transmitted in a non-persistent manner by whiteflies (Bemisia tabaci) and the particles occur in vivo in characteristic brush-like inclusions. It is also seed-borne in some, but not all, cultivars of cowpea and soybean, but seed-transmission is probably dependent upon the interaction of genotype, virus strain, time of infection and environmental factors. CMMV is serologically closely related to, and thus probably synonymous with, viruses previously designated groundnut crinkle, Psophocarpus necrotic mosaic, Voandzeia mosaic and tomato pale chlorosis. It is, however, serologically unrelated to 18 recognized carlaviruses; because it is also transmitted by whiteflies and induces the formation of unusual brush-like inclusions, it is probably best left unclassified or tentatively placed in a sub-group of the carlavirus group until the taxonomic significance of these features has been fully evaluated.     

Percentage of Fecal Moss in Arctic Ungulates as an Indicator of Wintering Area Quality

Abstract: I investigated local and regional differences in percent moss in the feces of muskoxen (Ovibos moschatus) and reindeer or caribou (Rangifer tarandus) in northwestern Alaska, USA, and related fecal moss to forage availability, snow conditions, animal density, and terrain ruggedness on wintering areas. Reindeer are a partially domesticated form of Rangifer tarandus originating from Europe and Asia and differ physiologically and ecologically from caribou. Percent moss in feces of muskoxen differed locally among individual wintering areas. Because of the large local variation in moss content of muskox feces, regional differences between the 2 study areas were difficult to resolve. Percent of moss in the feces of reindeer–caribou did not differ between wintering areas within the same study area but did differ between study areas. On muskox wintering areas, fecal moss correlated negatively with graminoid cover and snow hardness and positively with moss cover and muskox density, but fecal moss did not correlate with snow depth or terrain ruggedness. On reindeer–caribou wintering areas, fecal moss correlated positively with moss availability but not with lichen cover or snow depth or hardness. Because muskox groups in Alaska are isolated from each other in winter, even groups wintering on neighboring hills may face different foraging availability and might, therefore, exhibit differences in growth or productivity. Reindeer–caribou are more mobile than muskoxen in winter, and fecal samples may not be representative of vegetative and snow conditions at the wintering area where I collected them. I conclude that managers can use fecal moss as an indicator of overgrazed ranges, severe snow conditions, or crowded conditions on muskox wintering areas, but that the association between fecal moss and range conditions has to be drawn with caution for the more mobile reindeer–caribou.     

The Role of Non-Vectored Soil Transmission as a Primary Source of Infection by Pepper Mild Mottle and Cucumber Mosaic Viruses in Glasshouse-Grown Capsicum in Australia

Polyclonal rabbit antibodies, prepared against partially purified mycoplasmalike-organisms (MLO) from Grapevine Flavescence Dorée-infected plant extracts, were used to detect MLO in situ in excised salivary glands of the experimental vector Euscelidius variegates Kbm by an indirect immunofluorescent staining technique. Glands were examined from the fourth to the tenth week after the start of MLO acquisition. MLO detection was compared using this method, the transmission test and the ELISA technique applied to insects corpses after removal of salivary glands.     

Characterization of Two Strains of Cowpea Mild Mottle Virus Naturally Infecting Groundnut (Arachis hypogaea L.) in India

Two viruses were isolated from commercial groundnut plants showing mild mottle (MM) and severe mottle (SM) symptoms in the Rayalaseema area of Andhra Pradesh State, India. The host range of both the isolates was restricted to Fabaceae. The physical properties of both isolates were: DEP 10^-6 to 10^-7. TIP 70–85 C and LIV 5–8 days. Partially purified virus preparations contained slightly flexuous filamentous particles. In ELISA and gel diffusion tests both isolates reacted similarly with anusera to the carlaviruses cowpea mild mottle, cassia mild mosaic and potato M. Particle morphology. sedimentation coefficient, and estimated sizes of the coat proteins and RNAs gave additional evidence that the viruses were carlaviruses. Bemisia labaci transmitted only the MM isolate and the two isolates also differed in electrophoretic mobility of intact particles and amino acid composition of the coat proteins. The isolates are identified as distinct strains of cowpea mild mottle virus.