Study Of Coxsackievirus A10 Detection Method Based On RT-PCR Technology,synthesis,antimicrobial And Antiviral Activity Of Novel Cryptolepine Analogs

Part 1Hand,foot,and mouth disease(HFMD)is a common disease in children,which is characterized by mild fever along with cutaneous vesicular rashes on the palms,soles,buttocks,and mouth(oropharyngeal ulcers).In rare cases,patients also develop neurological complications,such as encephalomyelitis and aseptic meningitis.Several large epidemics of HFMD have been reported in the Asia-Pacific region,especially in Southeast Asia.Previously,HFMD was predominantly caused by EV71 and CVA16,as well as other enteroviruses such as CVA2,CVA4,CVA5,CVA6,CVA8 and CVA10.Among these,increased incidences of CVA10-associated HFMD epidemics have been reported around the world.Previously,CVA10 had been isolated from in sporadic HFMD cases in New Zealand in 1957 and the first clustering outbreak of HFMD caused by CVA10 was reported in Japan in 1983.CVA10 accounted for 11.8%of all cases in the largest HFMD outbreak in Singapore in 2008 and was one of the main pathogens in the Finland outbreak,which occurred around the same time?A sentinel surveillance study in France in 2010 showed that the predominant serotype was CVA10(39.9%),whereas the infection rate of EV71 and CVA16 was low(23.8%).In mainland China,CVA10 and CVA6 were known to be co-circulating with EV71 and CVA16.Recently,CVA10 has emerged as a key pathogen in the outbreaks of HFMD in mainland China in addition to EV71 and CVA16.Therefore,it is important to investigate the genetic variation and evolution of CVA10 that takes place during HFMD epidemics.And an effective detection method for CVA10 is imperative to strengthen the surveillance of CVA10 infection in HFMD epidemics and control the outbreaks.The main contents of this part are summaried as following:1.A real-time RT-PCR assay for rapid detection of Coxsackievirus A10.Here,we have developed a specific TaqMan real-time RT-PCR assay by analyzing VP1 gene sequences of CVA10 strains from different locations,which is specific,sensitive and quantitative compared to the conventional assay using HEV universal primers.Our results showed high sensitivity of the assay for CVA10 with detection limit as low as 10 copies/?L of CVA10 template.This is the first report on development of a VP1 gene-based Taq Man real-time RT-PCR assay for rapid diagnosis of CVA10 virus.2.Three-step method for the amplification of the coxsackievirus A10 genome.In this study,we present a robust “three-step” protocol performed with A105UF/A820,EVP4/A6141,and A4879/A1005 R for the full-length genome amplification of CVA10.The results revealed that the method is able to accurately and reproducibly amplify three fragments with overlaps of the full-length genome of eight CVA10 strains.Compared with the six-step protocol,this assay is both quick and specific.In addition,the three-step protocol could be capable of amplifying the full-length genomes of CVA10 strains isolated from different countries and regions.Part 2Natural products are important resources for drug development.Cryptolepine is a naturally occurring indoloquinoline alkaloid.It is isolated from the root of the West African shrub Cryptolepis sanguinolenta,and has been used as antimalarial drugs in Central and Western Africa for centuries.Most recent studies further revealed that some structural derivatives of Cryptolepine were associated with a broad spectrum of biological activities including antibacterial,antifungal,tuberculostatic,anthelmintic,antitumor,anti-inflammation,antipyretic,insecticidal and pesticidal properties.In consideration of the therapeutical importance of Cryptolepine,especially as antibacterials and antifungals,we have evaluated the in vitro activity of some new Cryptolepine derivates.We report here the characterisation of eight newly synthesized Cryptolepine derivatives and the evaluation of the in vitro antimicrobial and antiviral activity of the new compounds against Gram-positive,Gram-negative,Candida spp.as well as Coxsackievirus A10.Our results showed that the tested compounds exhibited specific antimicrobial and antiviral activities.Compounds 6d and 6h exhibited the highest activities against the test microorganisms.The MIC evaluation showed that compound 6d has higher activity than Amphotericin B and Ampicillin against all tested fungi and Gram-positive bacteria(Staphylococcus pneumonia)and showed similar activity like Ampicillin against Gram-negative bacteria(Escherichia coli).It is suggested that this kind of compound has the condition to be the lead compound of antibiotics.The compounds showed good antiviral activity against CVA10.Structure-activity relationship analysis showed that the antiviral activity decreased when the cryptolepine analogs with two side chains.And 7,8 and 9 respectively showed a better activity for the substitution of oxygen group,which is the best of the 3c with the oxygen group on the 9 position.But these data do not to fully discuss the structure activity relationship for the small number of compounds.These results have important guiding significance for the structure modification of cryptolepine analogs.