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.     

Pepper Mild Mottle Virus as an Indicator of Fecal Pollution

Accurate indicators of fecal pollution are needed in order to minimize public health risks associated with wastewater contamination in recreational waters. However, the bacterial indicators currently used for monitoring water quality do not correlate with the presence of pathogens. Here we demonstrate that the plant pathogen Pepper mild mottle virus (PMMoV) is widespread and abundant in wastewater from the United States, suggesting the utility of this virus as an indicator of human fecal pollution. Quantitative PCR was used to determine the abundance of PMMoV in raw sewage, treated wastewater, seawater exposed to wastewater, and fecal samples and/or intestinal homogenates from a wide variety of animals. PMMoV was present in all wastewater samples at concentrations greater than 1 million copies per milliliter of raw sewage. Despite the ubiquity of PMMoV in human feces, this virus was not detected in the majority of animal fecal samples tested, with the exception of chicken and seagull samples. PMMoV was detected in four out of six seawater samples collected near point sources of secondary treated wastewater off southeastern Florida, where it co-occurred with several other pathogens and indicators of fecal pollution. Since PMMoV was not found in nonpolluted seawater samples and could be detected in surface seawater for approximately 1 week after its initial introduction, the presence of PMMoV in the marine environment reflects a recent contamination event. Together, these data demonstrate that PMMoV is a promising new indicator of fecal pollution in coastal environments.Existing wastewater treatment practices are not always effective at removing the large number of pathogens (bacteria, protists, and viruses) present in human feces (17, 42, 47-49, 51). Therefore, wastewater discharges into the environment can have a negative impact on human health. Recreational waters throughout the United States are monitored for the presence of fecal pollution as a means of limiting public exposure to pathogens in areas impacted by wastewater discharges (44). The presence of pathogenic viruses in aquatic environments is an important parameter to consider in the evaluation of water quality. However, the bacterial indicators currently used to detect fecal contamination, such as fecal coliforms and enterococci, often do not correlate with the presence of feces-associated viruses and other pathogens (5, 10, 26, 33, 37, 51). In response, several researchers have proposed the use of viral indicators as a more effective method for monitoring wastewater contamination and the associated risks to public health (11, 14, 31).To date, the majority of the proposed viral indicators of fecal pollution are enteric viruses transmitted via the fecal-oral route (4). Enteric viruses present in raw sewage (including members of the families Adenoviridae, Caliciviridae, Picornaviridae, and Reoviridae and of the genus Anellovirus) have been used in several previous studies to identify fecal pollution in the environment (7, 8, 11, 12, 13, 18, 19, 27, 28, 32-36, 38, 50, 51). Of the enteric viruses that have been used as indicators, only the adenoviruses were ubiquitously found in raw sewage samples collected throughout the United States (41). Picobirnaviruses and Torque teno virus are abundant in raw sewage from some regions and thus have also been proposed as indicator viruses (15, 41). However, one potential problem with the use of human viruses as indicators is that their abundance in wastewater depends on the degree of infection and shedding in the human population at any given time.In addition to viruses infecting humans, other viruses shed in feces may be useful for indicating wastewater pollution. The plant pathogen Pepper mild mottle virus (PMMoV) was the most abundant virus found in a metagenomic survey of RNA viruses from human feces (52). PMMoV is a positive-sense, single-stranded RNA virus that belongs to the Tobamovirus genus and infects hot, bell, and ornamental peppers (Capsicum spp.) (9). The nonenveloped, rod-shaped PMMoV virions are extremely stable (9) and have been demonstrated to retain their infectivity for plants after passage through the human gut (52). PMMoV originates from processed pepper products (e.g., hot sauce and curry) and is excreted in human feces at concentrations of 1 million to 1 billion viruses per g (dry weight) (52). Since the presence of PMMoV in human feces is dietary in origin, this plant pathogen may be more abundant in the healthy human population than viruses that cause human disease.This study analyzed the presence of PMMoV in raw sewage and treated wastewater samples collected from wastewater treatment facilities throughout the coastal United States. To determine if PMMoV is a human-specific indicator useful for tracking the source of fecal pollution, fecal samples from numerous animals were tested for this virus. Finally, the presence of PMMoV in marine environments exposed to wastewater was determined and compared to that of other microbial indicators. The results of this work demonstrate that PMMoV is a promising indicator of fecal pollution.     

Biological and Molecular Characterization of a Crucifer Tobamovirus Infecting Oilseed Rape

In China, the tobamovirus that infects oilseed rape has been misdiagnosed as Tobacco mosaic virus (TMV) based on its morphological similarity and serological relatedness. Recently, a tobamovirus has been isolated from oilseed rape in China, which we named Youcai mosaic virus Br (YoMV-Br), according to its biological and molecular characteristics. It had strong infectivity to Cruciferae but less to Solanaceae, Leguminosae, and Cucurbitaceae, and its virion morphology was consistent with that of the tobamoviruses. At high concentrations, it serologically cross reacted with TMV antiserum. The 3′ terminal sequence (2,283 nucleotides) of YoMV-Br was determined, including the 3′ noncoding region, the CP and MP genes, and the C-terminal part of the replicase gene. Between the MP and CP genes, 77 nucleotides overlapped. Compared with homologous regions of 21 recognized species of Tobamovirus, YoMV-Br had a much higher identity to crucifer species than to other tobamoviruses. Phylogenetic analysis demonstrated that YoMV-Br was closely related to the YoMV cluster of tobamoviruses and distantly to TMV, so that they likely belong to different strains of the same species.     

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.     

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%.     

广西黄瓜绿斑驳花叶病毒（CGMMV）黄瓜分离物的RT—PCR检测及CP基因序列分析

利用黄瓜绿斑驳花叶病毒（Cucumber Green Mottle Mosaic Virus,CGMMV）的特异性引物对来自广西一温室栽培的黄瓜病样进行RT—PCR检测,结果扩增得到了与预期大小相符的目的片段（650bp）。序列分析表明,该目的片段包含有CGMMV完整的CP基因序列、部分运动蛋白基因（MP）及3’端非编码区（3＇-UTR）序列,其中CP基因全长486bp,与已报道的CP基因序列同源性为91．2％～99．4％。经系统发育分析,明确该GX-CS分离物与日本、法国、印度等分离物属于CGMMV同一类群,并推测该分离物与广西的葫芦（GX—BG）分离物具有相同的起源关系。     

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.     

侵染辣椒的黄瓜花叶病毒CP基因的序列分析、原核表达及抗血清制备

从吉林长春感病辣椒上获得一黄瓜花叶病毒(Cucumber mosaic virus,CMV)分离物(CMV-CC),根据GenBank中已登录的CMV外壳蛋白(coat protein,CP)基因核苷酸序列设计简并引物,通过RT-PCR的方法克隆到了长度为657 bp的目的片段。序列分析表明,CMV-CC与CMVI组各分离物核苷酸同源性为93.2%-97.9%。根据完整CP基因核苷酸序列构建的系统进化树显示:38个CMV分离物可分为3个组,CMV-CC属于CMV的IB亚组。将CMV-CC CP基因与原核表达载体pET-22b(+)连接,在大肠杆菌BL21(DE3)诱导表达出分子量约27 kD的融合蛋白。表达的融合蛋白经树脂纯化后免疫家兔制备了抗血清。用间接ELISA测定抗血清效价为1/4 096。Western blotting分析表明制备的抗血清对CP有高度特异性,为准确、快速地检测CMV奠定了基础。     

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.     

Response of Resistant and Susceptible Bell Pepper (Capsicum annuum) to a Southern California Meloidogyne incognita Population from a Commercial Bell Pepper Field

To determine the presence and level of root-knot nematode (Meloidogyne spp.) infestation in Southern California bell pepper (Capsicum annuum) fields, soil and root samples were collected in April and May 2012 and analyzed for the presence of root-knot nematodes. The earlier samples were virtually free of root-knot nematodes, but the later samples all contained, sometimes very high numbers, of root-knot nematodes. Nematodes were all identified as M. incognita. A nematode population from one of these fields was multiplied in a greenhouse and used as inoculum for two repeated pot experiments with three susceptible and two resistant bell pepper varieties. Fruit yields of the resistant peppers were not affected by the nematodes, whereas yields of two of the three susceptible pepper cultivars decreased as a result of nematode inoculation. Nematode-induced root galling and nematode multiplication was low but different between the two resistant cultivars. Root galling and nematode reproduction was much higher on the three susceptible cultivars. One of these susceptible cultivars exhibited tolerance, as yields were not affected by the nematodes, but nematode multiplication was high. It is concluded that M. incognita is common in Southern California bell pepper production, and that resistant cultivars may provide a useful tool in a nonchemical management strategy.     

A Tobamovirus Causing Heavy Losses in Protected Pepper Crops in Spain

During a four-year (1982–1985) survey of plant viruses infecting pepper cultivars grown under plastic in the Southeastern region of Spain, a tobamovirus was found to be the major disease agent of this crop. The virus produces slight or no symptoms on the leaves, but causes chlorotic mottling, malformation and reduction in size with occasional necrosis on the fruits and was able to infect all commercial pepper cultivars tested, including those resistant to other tobamoviruses, causing a catastrophic disease. The biological and serological characterization of the virus showed that it is very similar to pepper mild mottle virus (PMMV) (Wetter et al. 1984) and therefore we have termed it as Spanish strain of PMMV (PMMV-S). The need of grouping all the so-called “pepper strains” of tobacco mosaic virus (TMV) as a new distinct member of the tobamovirus group with the name of PMMV is emphasized.     

Molecular Characterization of a Distinct Begomovirus Infecting Euphorbia pulcherrima in China

Virus isolate G35 was obtained from Euphorbia pulcherrima showing leaf curl and vein thickening symptoms in Tianyang, Guangxi Province, China. The virus was transmitted by whiteflies to Nicotiana tabacum, Lycopersicon esculentum, Datura stramonium and E. pulcherrima. DNA‐A contains 2746 nucleotides, with two open reading frames (ORFs) in the virion‐sense DNA and four ORFs in the complementary‐sense DNA. When compared with the DNA‐A sequence of other begomoviruses, the total DNA‐A of isolate G35 was most closely related to that of Ageratum enation virus (79.9% sequence identity). However, the deduced coat protein of G35 is most like that of Pepper leaf curl virus from Bangladesh (94.9% amino acid sequence identity), and the AC1 of G35 is most like that of Cotton leaf curl Multan virus‐Okra (87.2% amino acid sequence identity). The molecular data showed that G35 is a distinct Begomovirus species, for which the name Euphorbia leaf curl virus (ELCV) is proposed.