Detection Of Poliovirus SabinⅠin SARS Patients And Rescue Of SabinⅠ

Although the causative agent of SARS has been identified as a novel coronavirus,called SARS-associated coronavirus (SARS-CoV), other pathogens, includingadenoviruses, respiratory syncytial virus, hendra virus, nipah virus, influenza virus andhuman metapneumovirus have also been detected in some patients with SARS.However, the detection of poliovirus in SARS patients has not been reported so far.In 2003, when we tried to isolate SARS-CoV in the throat swab specimen of aSARS patient, poliovirus was detected by electron microscope detection andreverse-transcriptase polymerase chain reaction (RT-PCR) assay. To confirm this result,poliovirus was isolated and purified by using human rhabdomyosarcoma (RD) cell line,which was recommended by World Health Organization as suitable manipulation forpoliovirus. The isolation was further characterized by neutralization test and indirectimmunofluorescence staining assay, as well the whole genome sequence by RT-PCRand 5'RACE amplification. All of the results indicated that our isolation was OPV-likepoliovirus type 1. In addition, to investigate whether this poliovirus type 1 wasprevalent in other SARS patients, we collected the specimens from SARS patientsincluding urine, throat swab, sputum, saliva and stool specimen. Viral RNA wasextracted and amplified by primers targeted the 5′-UTR, VP3–VP1 and 2C-3A regionsof poliovirus SabinⅠ. It indicated that 38.5%(5/13) of saliva specimens, 37.5%(3/8) ofsputum specimens, 71.4%(5/7) of urine specimens, 100%(5/5)of throat swabs and100%(6/6) of stool specimens were positive for OPV-like poliovirus 1, which showedthat 15 of 21 (71.4%) SARS patients were positive for OPV-like poliovirus 1, with thereverse mutation of 480G→A and 2795A→G. By contrast, only 1 of 50 (2%) stoolspecimens from 25 healthy individuals and 25 patients with hepatitis A or hepatitis Binfection, was positive for OPV-like poliovirus 1 (p﹤0.01). On the other side, wedemonstrated the distribution of OPV-like poliovirus 1 among the different organs inSARS patient, with that kidney and small intestine were positive and brain, lung, liver,spleen or pancreas were negative.Although the impossibility of vaccine-associated paralytic poliomyelitis in SARSpatients, the OPV-like poliovirus 1 existing in adult patients of SARS suggested that the virological testing and antibody testing should be strengthened in adults especially inpopulations with severe respiratory infection or impaired immunity, besides childrenyounger than 15-years old. It also indicated that adults with poor immunity againstpoliovirus should be inoculated with poliovirus vaccine to strengthen their immunityagainst this virus. However, the inadequate data of SARS specimens collection limitedthe analysis of the exact relationship between OPV-like poliovirus 1 andSARS/SARS-CoV. The role of OPV-like poliovirus 1 on SARS was unknown andneeds further research.The existence of OPV-like poliovirus 1 in SARS patients indicated that we shouldgive more attention to this virus. For the extensive use of OPV worldwide currently it isurgent to find the way to decrease the reverse mutation rate of SabinⅠgenome so as toreduce the vaccine derived poliovirus and thus vaccine-associated paralyticpoliomyelitis. If the reverse mutation rate could be lowered by modifying theSabinⅠgenome, with the same immunological effect and safety, it will facilitate thereduction of morbidity of vaccine-associated paralytic poliomyelitis. Construction ofinfectious cDNA clone to rescue virus was the important way to study the function ofvirus genome. Thus, the fragments of 1nt-2497nt, 2412nt-5616nt and 5588nt-7441ntwere amplified and cloned into T vector, followed by digestion with restriction enzymeand ligation step by step, using the NheⅠsite in 2470nt and BglⅡsite in 5601nt. Thefull-length SabinⅠinfectious cDNA clone was then constructed successfully. Instead ofexpensive and difficult extracellular transcription, we used nonviral eukaryoticexpression vector to express T7 RNA polymerase intracellularly to transcript SabinⅠRNA and then resue the virus. This successful poliovirus SabinⅠrescuing systemprovided important implications for further research on function of viral gene.Nuclear localization signals can help to transport proteins from cell plasma tonuclei. It has not been related to poliovirus rescuing in previous reports. In present studyeukaryotic vector expressing T7 RNA polymerase with/without SV40 large T antigennuclear localization signal PKKKRKV was constructed. After coinfection withSabinⅠinfectious cDNA clone on vero cells, the vector expressing T7 RNA polymerasewith nuclear localization signal showed better result of rescuing virus than that withoutnuclear localization signal. It indicated that the advantageous position of RNAtranscription with SabinⅠinfectious cDNA clone was probably existed in cell nucleus,which may due to more amount of transcription associated molecules in nucleus thanthose in cytoplasm. This is the first report on research of enterovirus rescuing mechanism.Moreover, to find the way of decreasing the reverse mutation rate, rescued viruswas acquired with Gly→Ser in 64 position in 3D polymerase region, and the primaryresults showed that the mutation contributed to decreased rescuing efficiency. It lays thefoundation for further research on characteristics of mutated rescued poliovirus.