Development Of Two-panel Reactions Of Real-time PCR For Detection Of 18 Types/Subtypes Of Respiratory Viruses And Its Application For Epidemiological Investigation

Respiratory tract infection, caused mostly (70%-80%) by virus, is very common in clinics. Many kinds of respiratory viruses cause the infections with similar clinical features, which makes the syndrome-based etiological diagnosis difficult. Therefore, establishing an accurate, low-cost assay for rapid identification of the pothgens is necessary for appropriate interventions and avoiding antibiotics misuse. In addition, new emerging respiratory viruses like SARS-CoV (Severe acute respiratory syndrome coronavirus), caused severe infectious disease, and even threatened public health. It is so difficult to prevent and control infectious diseases spread by respiratory tracts because of the widely and fast spreading. Therefore, building a surveillance platform to monitor the pathogenic spectrum of febrile respiratory syndrome (FRS) in real time, is of great significance in immediate prevention and pre-alarming of major and emerging infectious diseases. At present, the FRS monitoring platform is one of the major monitoring platforms established in our country, and this platform covers many respiratory viruses, such as influenza virus (IFV), parainfluenza virus (PIV), human Rhinovirus (HRV),adenovirus (ADV), Respiratory syncytial virus (RSV), human coronavirus (HCoV), human Metapneumovirus (hMPV) and human bocavirus (HBoV) etc.Therefore, a fast and simple assay to detect multiple respiratory pathogens is required for rapid diagnosis of common respiratory viruses and construction of a FRS monitoring platform. At present, however, the commonly used PCR with a single reaction condition is labor-intensive and time-consuming, and a new technique (Respiratory Virus Panel Fast Array, RVP Fast) based on bead-based suspension arrays is too expensive to be widely used in clinical practice in China. To address problems above, we successfully developed two-panel reactions of real-time PCR for detection of 18 types/subtypes of respiratory viruses according to primers and probes reported by references. The two panels of real-time PCR not only can assist clinicians of rapid identification of different types/subtypes common respiratory viruses, but also provide favorable technical support for establishment of a febrile respiratory syndrome monitoring platform and the application of epidemiological investigation.Among the viruses mentioned above, we also pay a particular attention on human coronavirus (HCoV), one of the pathogens in the FRS monitoring platform, although this virus is underemphasized that it usually causes common cold. In 1965 and 1967, human coronavirus HCoV-229E and HCoV-OC43 were isolated respectively, which only caused human common cold. Thereafter, HCoV-NL63 and HCoV-HKU1, mostly causing severe lower respiratory tract infections and even death in elderly patients or immunocompromised patients, were identified. To investigate the epidemiology of HCoV-229E,-HCoV-OC43, HCoV-NL63, HCoV-HKU1, we analyzed the epidemiology and clinical features of the four HCoVs among FRS patients from November 2011 to December 2014 in Guangzhou City, detecting the four HCoVs by real-time PCR established.As well as the four HCoVs above, we further focus on two novel coronaviruses, SARS-CoV and MERS-CoV (Middle east respiratory syndrome coronavirus). In 2002-2003, SARS-CoV continued to roam the planet and caused SARS, fatality up to 10%. In 2012, a new HCoV, MERS-CoV emerged, with clinical symptoms similar to those of SARS, fatality up to more than 50%. The emergence of SARS-CoV and MERS-CoV broken the traditional concept completely that coronaviruses only caused mild respiratory infections, which made them attract considerable attention. Study showed that SARS-CoV was closely related to bats coronavirus HKU3, and MERS-CoV closely related to bats coronavirus HKU4 and HKU5. Both have potential to cross the species barrier from bats to human. Novel coronaviruses mutated from those in animals can easily cause emergent infectious diseases in a pandemic, having attracted more attention of scientists. On one hand, not only because of fast variation of coronavirus and various subtypes, but also novel coronavirus mutated by animal coronavirus can transmit to human, while virus-specific PCR fail to cover a broad spectrum of coronaviruses, a pan-coronavirus RT-PCR screening a broad spectrum of coronaviruses is the chief choice to monitor the distribution of novel coronavirus in the population at present.On the other hand, due to unknown etiology, atypical pneumonia caused by SARS-CoV affected people’s prophylaxis and treatment, and resulted in mass epidemic. This public health hazard kept warning us that we need pay attention to unexplained pneumonia (UP). It is more significant and cost-effective for surveillance of novel coronavirus in UP cases than that widely screening in the population, assisting clinicians for early diagnosis and treatment. For these reasons, and based on the highly conserved RNA-dependent RNA polymerase gene (RdRp), we investigated the distribution of novel coronavirus in UP cases in 2013-2014 by a pan-coronavirus RT-PCR previously established according to primers of the RdRp gene friendly provided by Wuhan Institute of Virologyfor screening a broad spectrum of coronaviruses, in response to preventing and alerting major infectious diseases and emerging infectious diseases.Considering that SARS-CoV haven’t reemerged up to now, the study aimed to SARS-CoV gradually decreased, and for now there is a lack of studies about the change of SARS-CoV specific antibody levels in patients. Moreover, SARS-CoV nucleocapsid protein(SARS-NP)as an ideal target in immunodiagnosis, having been proved by our previous study, an indirect ELISA for serological study based on recombinant SARS-NP of prokaryotic expression. To optimize the indirect ELISA based on SARS-NP and make it an alternative for the serological epidemiology investigation and diagnosis of the potential SARS outbreak, we developed an indirect ELISA based on SARS-NP, expressed by baculovirus-insect cells. Using the developed indirect ELISA assay, the serum antibodies against SARS-NP in SARS patients before and after 10 years.There are three parts in this study as follows.Part I. Development of two-panel reactions of real-time PCR for detection of 18 types/subtypes of respiratory virusesThere are many types of common respiratory viruses resulting infections, in which clinical features are too similar to make an etiology diagnosis. Two panels of real-time PCR for detection of 18 types/subtypes of respiratory viruses were successfully developed according to primers and probes reported by references. The results of verifying the sensitivity, specificity and accuracy of the assays showed that the lowest limit of detection was 10 copies/μL, coefficient of variation was 1.27%-3.44%, and the assays showed a fine specificity. Among 408 specimens, the detection rates of nasal swabs and bronchoalveolar lavage fluid were 63.2% (225/356) and 25%(13/52) respectively. This suggests that real-time PCR assays we developed is simple, specific, sensitive and stable.Part Ⅱ. Epidemiological investigation of four common coronavirus in 2011-2014 in Guangzhou and the distribution of novel coronavirus in unexplained pneumonia patients in 2013-2014Epidemiology investigation of HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1 among FRS patients in 2011-2014 in Guangzhou was conducted detecting these four coronavirus by real-time PCR mentioned above. The total detection rate of coronaviruses was 4.42%(104/2355),while HCoV/HRV coinfection (17.3%,18/104) and HCoV/RSV coinfection (14.4%,15/104) were the most common. No significance of the detection rates of four coronaviruses among age groups(P=0.188>0.05)and different types of specimens(P=1.000>0.05) were observed.In addition, the prevalence of HCoVs during the long term observation (during November 2011 to December 2014) as follows:HCoVs were observed in all seasons; HCoV-OC43 and HCoV-NL63 were both predominant in 2012 and 2014, displaying a typical characteristic of biennial peaks; HCoV-OC43 was observed in all seasons,while HCoV-NL63 was predominant peaking in summer and autumn (from June to October); HCoV-229E and HCoV-HKUl were detected without distinct characteristic. The most common clinical symptom of HCoV infections is fever. Furthermore, most (66.3%) of HCoV infections suffered from low respiratory tract infection, and severe pneumonia (6.73%) and even death (1.92%). It was suggested that HCoV infection manifested not only mild respiratory symptoms, but also more serious events.We further analyzed the potential existed novel coronavirus in UP cases in 2013-2014 using a pan-coronavirus nest-PCR and gene sequencing. No novel coronavirus was found in 272 respiratory specimens (204 nasal swabs,68 bronchoalveolar lavage specimens) from UP patients. However, severe pneumonia and death accounted for 6.73% and 1.92% of UP cases respectively, suggesting that UP resulted in serious outcome.Part III. Serological study of SARS-NP antibody titer changes in SARS patients before and after 10 yearsUsing the recombinant SARS-NP expressed by baculovirus-insect cell expression systems in previous study as antigen, we further established and optimized an indirect ELISA to detect and compare SARS-NP specific IgG in SARS patients’convalescence sera before and after 10 years. No cross reaction against SARS-NP detected by the indirect ELISA was observed in the convalescence sera of other pathogens. Among 532 specimens from healthy adults,6(1.13%) was detected as positive by indirect ELISA, but only 4 (0.75%) was confirmed as positive by western blot. However, a total of 57 to 60 convalescence sera specimens before and after 10 years from 13 SARS patients were detected positive with the sensitivity of 95%. Among them,54 of 55 convalescence sera specimens (98.2%), collected in 2003-2004, just half to one year after the onset of disease, were detected as positive, while 3 of 5 convalescence sera specimens from 5 of 13 SARS patients collected in 2015, were detected as positive. The false positive rate of the method was 0.38%, with specificity of 99.62%. Furthermore, the highest titers of anti-SARS-NP IgG were from 1:160 to 1:5120 in sera of 5 SARS patients before 10 years, and those in sera of 5 SARS patients after 10 years were from 1:10 to 1:160. A significant decline of anti-SARS-NP IgG was observed during 10 years, however, a high titer of anti-SARS-NP IgG was still observed in some patients, suggesting that anti-SARS-NP IgG was lasting in vivo for a long period of time.SummaryThe necessity and significances of this work are as follows.1. A real-time PCR using two-panel reactions to detect all 18 types/subtypes respiratory viruses, was successfully established and evaluated by clinical specimens. Our real-time PCR assays are convenient, cover various viruses to detect, low-cost and of high clinical values. Besides, it provides a powerful technical support for establishment of FRS monitoring platform and application of epidemiological investigation.2. The epidemiological and clinical features of HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1 among FRS patients were observed and analyzed. The potentially existing novel coronavirus in UP cases was further detected among FRS patients, as well as these UP patients’clinical features were analyzed. Surveillance of respiratory pathogenic spectrum and the prevalence, is of great importance to prevent and warn the occurrence and development of major and emerging infectious diseases.3. An indirect ELISA based on the recombinant SARS-NP by baculovirus expression to detect SARS specific antibody was developed, and the levels of anti-SARS-NP IgG both in healthy adults and SARS patients were investigated. Besides of using in diagnosis by indirect ELISA, the serological studies based on observation of antibody level is also helpful for a better understanding of the body’s humoral defense mechanism against SARS.