Molecular Detection Of Pepper Mild Mottle Virus And Location In Tissues

Pepper mild mottle virus?PMMoV?is a kind of positive-strand RNA virus which belongs to the Tobamovirus.In the world,PMMoV is one of the main viruses that threatened pepper's production,it can infect Capsicum and causes serious damage to the growth of pepper.In the study of environmental monitoring,PMMoV also can be used as an indicator to detect the water which is polluted by animal waste.In this paper,we use molecular biology,immunology and viral metagenomics methods to study distribution of PMMoV in plants,water and Cyprinus carpio.The main contents are as follows:1.Cyprinus carpio was used as experimental material,total viral nucleic acid extracted from intestinal tissue was amplified randomly.At last,viral metagenomics was analyzed by high-throughput sequencing.The results showed that it contained a lot of viral specific sequence,such as Cercopithecine herpesvirus 5,Pepper mild mottle virus,Alphapapillomavirus 9,Cauliflower mosaic virus,Choristoneura occidentalis granulovirus,Enterobacteria phage p1,Enterobacteria phage lambda,Escherichia phage TL₂011b,Hepatitis B virus,Human endogenous retrovirus,Human endogenous retrovirus H,Human endogenous retrovirus K,Human herpesvirus 5 and Semliki forest virus.It is the first report that nucleotide sequences of PMMoV were found in fish intestinal tissue.In other tissues of Cyprinus carpio,PMMoV was also tested with reverse transcription PCR,and the results showed that PMMoV was widely present in various tissues of fish.2.Total RNA of pepper leaf was extracted and amplified by revers transcription PCR,then PCR products were sequenced.The results showed that they were PMMoV specific fragment.Frozen-sections of pepper leaves,anther,fruits,seeds and other tissues were prepared,we found PMMoV was located in different tissues by in situ hybridization,Immunohistochemistry and Immunofluorescence.The results showed that PMMoV distributed widely in the pepper.PMMoV can locate in the reproductive tissues,such as pollen and seeds,it may be the main way for the spread of PMMoV.The results of whole-mount in situ hybridization of leaves,stems,roots and flowers showed that PMMoV were more obvious distribution in active areas of mesophyll cells and stem,also found it in the root and petals.In situ hybridization can reflect the distributed of PMMoV in pepper.Compared with in situ hybridization,whole-mount in situ hybridization can clearly reflect the macroscopic characteristics of tissues and the overall structure of the mesophyll cells,stem,root cells and flowers.The fragmentation of tissue and cell was an important shortcoming of in situ hybridization,and it can be made up by whole-mount in situ hybridization.Combination the two methods,we can effectively analyze the different characteristics of different pepper tissues infected by PMMoV.3.Four samples of water from the channels of Majiagou,Hejiagou,Songhua River and tap water in Harbin were used experimental material,PMMoV was detected by reverse transcription PCR.The result showed that the former three samples were positive,and tap water was negative.In order to avoid false-positive,dot-blot hybridization was also used to detect PCR products for further confirming the RT-PCR results,the result of dot-blot hybridization of was the same to that of RT-PCR.The 3 positives results indicated that they have been contaminated by feces and PMMoV may be widely distributed in natural water.In addition,PMMoV in the polluted water may further spread around with water,which contributed to the long-distance spread of the virus.