VSITA, an Improved Approach of Target Amplification in the Identification of Viral Pathogens.

OBJECTIVE: Unbiased next generation sequencing (NGS) is susceptible to interference from host or environmental sequences. Consequently, background depletion and virome enrichment techniques are usually needed for clinical samples where viral load is much lower than background sequences. METHODS: A viral Sequence Independent Targeted Amplification (VSITA) approach using a set of non-ribosomal and virus-enriched octamers (V8) was developed and compared with traditionally used random hexamers (N6). Forty-five archived clinical samples of different types were used in parallel to compare the V8 and N6 enrichment performance of viral sequences and removal performance of ribosomal sequences in the step of reverse transcription followed by quantitative PCR (qPCR). Ten sera samples from patients with fever of unknown origin and 10 feces samples from patients with diarrhea of unknown origin were used in comparison of V8 and N6 enrichment performance following NGS analysis. RESULTS: A minimum 30 hexamers matching to viral reference sequences (sense and antisense) were selected from a dataset of random 4,096 (46) hexamers (N6). Two random nucleotides were added to the 5' end of the selected hexamers, and 480 (30 × 42) octamers (V8) were obtained. In general, VSITA approach showed higher enrichment of virus-targeted cDNA and enhanced ability to remove unwanted ribosomal sequences in the majorities of 45 predefined clinical samples. Moreover, VSITA combined with NGS enabled to detect not only more viruses but also achieve more viral reads hit and higher viral genome coverage in 20 clinical samples with diarrhea or fever of unknown origin. CONCLUSION: The VSITA approach designed in this study is demonstrated to possess higher sensitivity and broader genome coverage than traditionally used random hexamers in the NGS-based identification of viral pathogens directly from clinical samples.

Identification and control of a poliomyelitis outbreak in Xinjiang, China.

BACKGROUND: The last case of infection with wild-type poliovirus indigenous to China was reported in 1994, and China was certified as a poliomyelitis-free region in 2000. In 2011, an outbreak of infection with imported wild-type poliovirus occurred in the province of Xinjiang. METHODS: We conducted an investigation to guide the response to the outbreak, performed sequence analysis of the poliovirus type 1 capsid protein VP1 to determine the source, and carried out serologic and coverage surveys to assess the risk of viral propagation. Surveillance for acute flaccid paralysis was intensified to enhance case ascertainment. RESULTS: Between July 3 and October 9, 2011, investigators identified 21 cases of infection with wild-type poliovirus and 23 clinically compatible cases in southern Xinjiang. Wild-type poliovirus type 1 was isolated from 14 of 673 contacts of patients with acute flaccid paralysis (2.1%) and from 13 of 491 healthy persons who were not in contact with affected persons (2.6%). Sequence analysis implicated an imported wild-type poliovirus that originated in Pakistan as the cause of the outbreak. A public health emergency was declared in Xinjiang after the outbreak was confirmed. Surveillance for acute flaccid paralysis was enhanced, with daily reporting from all public and private hospitals. Five rounds of vaccination with live, attenuated oral poliovirus vaccine (OPV) were conducted among children and adults, and 43 million doses of OPV were administered. Trivalent OPV was used in three rounds, and monovalent OPV type 1 was used in two rounds. The outbreak was stopped 1.5 months after laboratory confirmation of the index case. CONCLUSIONS: The 2011 outbreak in China showed that poliomyelitis-free countries remain at risk for outbreaks while the poliovirus circulates anywhere in the world. Global eradication of poliomyelitis will benefit all countries, even those that are currently free of poliomyelitis.

Fever with thrombocytopenia associated with a novel bunyavirus in China.

BACKGROUND: Heightened surveillance of acute febrile illness in China since 2009 has led to the identification of a severe fever with thrombocytopenia syndrome (SFTS) with an unknown cause. Infection with Anaplasma phagocytophilum has been suggested as a cause, but the pathogen has not been detected in most patients on laboratory testing. METHODS: We obtained blood samples from patients with the case definition of SFTS in six provinces in China. The blood samples were used to isolate the causal pathogen by inoculation of cell culture and for detection of viral RNA on polymerase-chain-reaction assay. The pathogen was characterized on electron microscopy and nucleic acid sequencing. We used enzyme-linked immunosorbent assay, indirect immunofluorescence assay, and neutralization testing to analyze the level of virus-specific antibody in patients' serum samples. RESULTS: We isolated a novel virus, designated SFTS bunyavirus, from patients who presented with fever, thrombocytopenia, leukocytopenia, and multiorgan dysfunction. RNA sequence analysis revealed that the virus was a newly identified member of the genus phlebovirus in the Bunyaviridae family. Electron-microscopical examination revealed virions with the morphologic characteristics of a bunyavirus. The presence of the virus was confirmed in 171 patients with SFTS from six provinces by detection of viral RNA, specific antibodies to the virus in blood, or both. Serologic assays showed a virus-specific immune response in all 35 pairs of serum samples collected from patients during the acute and convalescent phases of the illness. CONCLUSIONS: A novel phlebovirus was identified in patients with a life-threatening illness associated with fever and thrombocytopenia in China. (Funded by the China Mega-Project for Infectious Diseases and others.).

Enhanced surveillance of acute flaccid paralysis following importation of wild poliovirus in Xinjiang Uygur Autonomous Region, China.

BACKGROUND: After being polio free for more than 10 years, an outbreak occurred in China in 2011 in Xinjiang Uygur Autonomous Region (Xinjiang) following the importation of wild poliovirus (WPV) originating from neighboring Pakistan. METHODS: To strengthen acute flaccid paralysis (AFP) surveillance in Xinjiang, "zero case daily reporting" and retrospective searching of AFP cases were initiated after the confirmation of the WPV outbreak. To pinpoint all the polio cases in time, AFP surveillance system was expanded to include persons of all ages in the entire population in Xinjiang. RESULTS: Totally, 578 AFP cases were reported in 2011 in Xinjiang, including 21 WPV cases, 23 clinical compatible polio cases and 534 non-polio AFP cases. Of the 44 polio cases, 27 (61.4%) cases were reported among adults aged 15-53 years. Strengthening AFP surveillance resulted in an increase in the number of non-polio AFP cases in 2011 (148 children < 15 years) compared with 76 cases < 15 years in 2010. The AFP surveillance system in Xinjiang was sensitive enough to detect polio cases, with the AFP incidence of 3.28/100,000 among children < 15 years of age. CONCLUSIONS: Incorporating adult cases into the AFP surveillance system is of potential value to understand the overall characteristics of the epidemic and to guide emergency responses, especially in countries facing WPV outbreak following long-term polio free status. The AFP surveillance system in Xinjiang was satisfactory despite limitations in biological sample collection.

Clinical progress and risk factors for death in severe fever with thrombocytopenia syndrome patients.

BACKGROUND: Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease caused by the SFTS virus (SFTSV) with an average fatality rate of 12%. The clinical factors for death in SFTS patients remain unclear. METHODS: Clinical features and laboratory parameters were dynamically collected for 11 fatal and 48 non-fatal SFTS cases. Univariate logistic regression was used to evaluate the risk factors associated with death. RESULTS: Dynamic tracking of laboratory parameters revealed that during the initial fever stage, the viral load was comparable for the patients who survived as well as the ones that died. Then in the second stage when multi-organ dysfunction occurred, from 7-13 days after disease onset, the viral load decreased in survivors but it remained high in the patients that died. The key risk factors that contributed to patient death were elevated serum aspartate aminotransferase, lactate dehydrogenase, creatine kinase, and creatine kinase fraction, as well as the appearance of CNS (central nervous system) symptoms, hemorrhagic manifestation, disseminated intravascular coagulation, and multi-organ failure. All clinical markers reverted to normal in the convalescent stage for SFTS patients who survived. CONCLUSIONS: We identified a period of 7-13 days after the onset of illness as the critical stage in SFTS progression. A sustained serum viral load may indicate that disease conditions will worsen and lead to death.

An Analysis of Predictive Factors for Severe Neonatal Infection and the Construction of a Prediction Model.

Objective: To investigate the primary predictive factors for the occurrence of severe neonatal infection, construct a prediction model and assess its effectiveness. Methods: A total of 160 neonates hospitalised in the Department of Neonatology at Suixi County Hospital from January 2019 to June 2022 were retrospectively analysed. Clinical data was analyzed to determine the primary predictive factors for the occurrence of severe neonatal infection. Predictive efficacy was evaluated using a receiver operating characteristic curve, and a nomogram model was constructed according to the predictors. A bootstrap technique was used to verify the accuracy of the model. Results: The neonates were divided, based on the degree of infection, into a mild infection group (n = 80) and a severe infection group (n = 80) according to a 1:1 ratio. Multivariate logistic regression analysis showed that compared with the recovery stage, white blood cell count (WBC) and platelet count (PLT) in the two groups were significantly decreased in the early stage of infection, and the ratio of mean platelet volume to PLT, as well as C-reactive protein (CRP) and procalcitonin levels, was elevated (P < 0.05). The area under the curves (AUCs) of decreased WBC, decreased PLT and elevated CRP levels, and the combination of these three indicators, were 0.881, 0.798, 0.523 and 0.914, respectively. According to the filtered indicators, two models (a dichotomous variable equation model and a nomogram model) of continuous numerical variables were constructed, and their AUCs were 0.958 and 0.914, respectively. The calibration curve of the nomogram model was validated with a consistency index of 0.908 (95% confidence interval [0.862, 0.954]). Conclusion: Decreased WBC and PLT levels and an elevated CRP level were the primary independent predictors of severe neonatal infection.

[Survival analysis of patients with spontaneous rupture of hepatocellular carcinoma].

OBJECTIVE: To explore the prognostic factors influencing overall survival (OS) in patients with spontaneous rupture of hepatocellular carcinoma (HCC-SR). METHODS: The medical records of 44 patients with HCC-SR treated in our department from January 1, 2005 to April 1 2011 were retrospectively reviewed. The clinical and prognostic data of 19 HCC-SR patients who received curative hepatectomy were compared with data of 137 HCC patients with no SR who were managed by curative hepatectomy during the same period. Type of HCC-SR was defined according to previously established criteria. The clinicopathological data were evaluated for possible associations with OS of HCC-SR by univariate analysis with the Kaplan-Meier method followed by multivariate analysis with the Cox proportional hazard model. RESULTS: While some clinical features differed between the HCC-SR patients and non-HCC-SR patients, the postoperative prognosis was comparable between the two groups: (1) The 1-, 2-, 3- and 5-year postoperative cumulative recurrence rates were 78.9% (15/19), 89.5% (17/19), 94.7% (18/19) and 94.7% (18/19) in the HCC-SR group but 43.1% (59/137), 54.0% (74/137), 59.1% (81/137) and 66.4% (91/137) in the non-HCC-SR group respectively, and the differences reached statistical significance (P = 0.006, 0.003, 0.002, and 0.014); (2) The 1-, 2-, 3- and 5-year postoperative disease-free survival rates were 10.5% (2/19), 5.3% (1/19), 5.3% (1/19) and 5.3% (1/19) in the HCC-SR group but 40.1% (55/137), 21.2% (29/137), 12.4% (17/137) and 4.4% (6/137) in the non-HCC-SR group respectively, and only the 1-year disease-free survival rate was significantly different (P = 0.032); (3) The 1-, 2-, 3- and 5-year postoperative OS rates were 42.1% (8/19), 10.5% (2/19), 5.3% (1/19) and 5.3% (1/19) in the HCC-SR group but 59.1% (81/137), 32.8% (45/137), 19.0% (26/137) and 6.6% (9/137) in the non-HCC-SR group, and none of the differences reached statistical significance (P = 1.972, 0.061, 0.200, 1.000). Multivariate analysis identified that severity of concomitant liver cirrhosis, levels of alpha fetoprotein (AFP), choice of treatment modality, and type of HCC-SR acted as factors influencing OS. CONCLUSIONS: Patients with HCC-SR receiving curative hepatectomy have higher postoperative recurrence rates than their non-HCC-SR counterparts, but the two groups have similar postoperative OS rates. OS is influenced by severity of concomitant liver cirrhosis, level of AFP, choice of treatment modality, and type of HCC-SR.

SARS patients-derived human recombinant antibodies to S and M proteins efficiently neutralize SARS-coronavirus infectivity.

OBJECTIVE: To develop a specific SARS virus-targeted antibody preparation for emergent prophylaxis and treatment of SARS virus infection. METHODS: By using phage display technology, we constructed a naive antibody library from convalescent SARS patient lymphocytes. To obtain the neutralizing antibody to SARS virus surface proteins, the library panning procedure was performed on purified SARS virions and the specific Fab antibody clones were enriched by four rounds of repeated panning procedure and screened by highthroughput selection. The selected Fab antibodies expressed in the periplasma of E. coli were soluble and further purified and tested for their binding properties and antiviral function to SARS virus. The functional Fab antibodies were converted to full human IgG antibodies with recombinant baculovirus/insect cell systems and their neutralizing activities were further determined. RESULTS: After four rounds of the panning, a number of SARS-CoV virus-targeted human recombinant Fab antibodies were isolated from the SARS patient antibody library. Most of these were identified to recognize both natural and recombinant SARS spike (S) proteins, two Fab antibodies were specific for the virus membrane (M) protein, only one bound to SARS-CoV nucleocapsid protein. The SARS-CoV S and M protein-targeted Fab or IgG antibodies showed significant neutralizing activities in cytopathic effect (CPE) inhibition neutralization test, these antibodies were able to completely neutralize the SARS virus and protect the Vero cells from CPE after virus infection. However, the N protein-targeted Fab or IgG antibodies failed to neutralize the virus. In addition, the SARS N protein-targeted human Fab antibody reacted with the denatured N proteins, whereas none of the S and M protein specific neutralizing antibodies did. These results suggested that the S and M protein-specific neutralizing antibodies could recognize conformational epitopes which might be involved in the binding of virions to cellular receptors and the fusion activity of the virus. CONCLUSION: The SARS-CoV spike protein and membrane proteins are able to elicite efficient neutralizing antibodies in SARS patients. The neutralizing antibodies we generated in this study may be more promising candidates for prophylaxis and treatment of SARS infection.

Efficacy of mild hypothermia for the treatment of patients with cardiac arrest.

BACKGROUND: Therapeutic hypothermia has been recommended for the treatment of cardiac arrest patients who remain comatose after the return of spontaneous circulation. The aim of this study was to evaluate the effectiveness and safety of mild hypothermia on patients with cardiac arrest by conducting a meta-analysis. METHODS: The relevant trials were searched in Cochrane Library, PubMed, Web of Science, Embase, CNKI and Wan Fang Data from the date of their establishment to October 2014. Thereafter, the studies retrieved were screened based on predefined inclusion and exclusion criteria. Data were extracted, and the quality of the included studies was evaluated. A meta-analysis was conducted using the Cochrane Collaboration Review Manager 5.2 software. RESULTS: Six randomized controlled trials involving 531 cases were included, among which 273 cases were assigned to the treatment group and the other 258 cases to the control group. The meta-analysis indicated that mild hypothermia therapy after cardiac arrest produced significant differences in survival rate (relative risk [RR] =1.23, 95% confidence interval [CI]: 1.02-1.48, P = 0.03) and neurological function (RR = 1.33, 95% CI: 1.08-1.65, P = 0.007) after 6 months compared with normothermia therapy. However, no significant differences were observed in the survival to the hospital discharge (RR = 1.35, 95% CI: 0.87-2.10, P = 0.18), favorable neurological outcome at hospital discharge (RR = 1.53, 95% CI: 0.95-2.45, P = 0.08) and adverse events. CONCLUSIONS: The meta-analysis demonstrated that mild hypothermia can improve the survival rate and neurological function of patients with cardiac arrest after 6 months. On the other hand, regarding the survival to hospital discharge, favorable neurological outcome at hospital discharge, and adverse events, our meta-analysis produced nonsignificant results.

[Chlamydia-like and coronavirus-like agents found in dead cases of atypical pneumonia by electron microscopy].

OBJECTIVE: To explore the causative agents of the atypical pneumonia (also SARS) occurred recently in some regions of our country. METHOD: Organ samples of 7 dead cases of SARS were collected from Guangdong, Shanxi, Sichuan Provinces and Beijing for electron microscopic examination. 293 cell line was inoculated with the materials derived from the lungs to isolate causative agent(s). The agents in the organs and cell cultures were revealed by immunoassay. RESULTS: Both Chlamydia-like and coronavirus-like particles were found in EM. Inclusion bodies containing elementary bodies, reticulate antibodies and intermediate bodies of Chlamydia-like agent were visualized in multiple organs from the 7 dead cases, including lungs (7 cases), spleens (2 cases), livers (2 cases), kidneys (3 cases) and lymph nodes (1 cases), by ultrathin section electron microscopy (EM). In some few sections, coronavirus-like particles were concurrently seen. A coronavirus RNA- polymerase segment (440 bp) was amplified from the lung tissues of two cases of the SARS. After inoculated with materials from the lung samples, the similar Chlamydia-like particles were also found in the inoculated 293 cells. Since the Chlamydia-like agents visualized in both organs and cell cultures could not react with the genus specific antibodies against Chlamydia and monoclonal antibodies against C. pneumoniae and C. psittaci, the results might well be suggestive of a novel Chlamydia-like agent. CONCLUSION: Since the novel Chlamydia-like agent was found co-existing with a coronavirus-like agent in the dead cases of SARS, it looks most likely that both the agents play some roles in the disease. At the present time, however, one can hardly determining how did these agents interact each other synergetically, or one follows another, need further study.

[Study on serological cross-reactivity of six pathogenic phleboviruses].

This article aimed to study the antigenicity of nucleocapsid proteins (NPs) in six pathogenic phleboviruses and to provide theoretical evidence for the development of serological diagnostic reagents. NPs of six pathogenic phleboviruses were expressed and purified using a prokaryotic expression system and rabbits were immunized with individual recombinant NPs. Cross-reactions among NPs and rabbit sera were determined by both indirect ELISA and Western blotting analyses, and the sera titer was determined by indirect ELISA. Furthermore, sera from SFTS patients were also detected by each recombinant NP as a coating antigen using indirect ELISA. The cross-reactions and the sera titer were subsequently determined. Both the concentration and purity of recombinant NPs of six pathogenic phleboviruses met the standards for immunization and detection. The results of indirect ELISA and Western blotting showed that each anti-phlebovirus NP rabbit immune serum had potential serological cross-reactivity with the other five virus NP antigens. Furthermore, the sera from SFTS patients also had cross-reactivity with the other five NP antigens to a certain extent. Our preliminary study evaluated the antigenicity and immune reactivity of six pathogenic phleboviruses NPs and laid the foundation for the development of diagnostic reagents.

[Study on adjuvant effect of oral recombinant subunit vaccine formulated with chitosan against human enterovirus 71].

To evaluate the adjuvant effect of recombinant enterovirus 71 (EV71) subunit vaccine formulated with chitosan, rabbits were orally immunized with recombinant VP1 (rVP1) or rVP1 mixed with chitosan adjuvant. Levels of virus-specific IgG and IgA antibodies in sera, mucosal wash buffer (intestine, nasal cavity, and lung), and feces were determined by indirect enzyme-linked immunosorbent assay (ELISA). The titers of neutralizing antibodies against EV71 were determined using cytopathic effect-based neutralizing assay, and levels of cytokines (IFN-gamma and IL-4) secreted from in vitro-cultured rabbit splenic lymphocytes under antigen stimulation were also determined by ELISA. Results showed that immunization with rVP1 alone could only induce low levels of serum IgG and mucosal IgA, while rVP1 combined with chitosan adjuvant were able to induce significantly higher levels of antibodies, rVP1 can only induce neutralizing antibodies when used in combination with chitosan. Levels of IFN-gamma and IL-4 in the group immunized with rVP1 plus chitosan were significantly higher than those in the group immunized with rVP1 only or those in the control groups. Our study lays the foundation for development of oral VP1 vaccine against EV71 infection.

[Severe fever with thrombocytopenia syndrome virus nucleoprotein specifically binds to 60kD SSA/Ro protein in host cells].

This study aims to investigate whether the nucleoprotein (NP) of severe fever with thrombocytopenia syndrome virus (SFTSV) can impact the cellular immunity of host cells. Gene segments that encode the NP and non-structural protein (NSs) of SFTSV were inserted into eukaryotic expression vector VR1012. Host proteins that interact with NP and affect immunity were identified with co-immunoprecipitation (IP), SDS-PAGE, mass spectrometry (MS), and Western blot. Co-localization of NP and the identified host proteins was confirmed by confocal microscopy. A 60kD SSA/Ro, a protein related to immunity, interacted with NP, as found by IP and MS. Confocal microscopy showed that NP and SSA/Ro were co-localized in cytoplasm. These results indicated that SFTSV NP may specifically bind to 60kD SSA/Ro and cause a series of immune responses and clinical symptoms.

[[Virus-like particle-based immunoglobulin M capture enzyme-linked immunosorbent assay for the detection of IgM antibodies against Chikungunya virus].

To establish a MacELISA method for the detection of IgM antibodies against Chikungunya virus (CHIKV), we prepared virus like particle (VLP) antigens of CHIKV using the whole structural protein C-E3-E2-6K-E1 encoding gene with a baculovirus expression system in Sf9 insect cells. The VLPs were purified and used to immunize Kunming mice. Then, polyclonal antibodies were purified from the samples of ascites with a protein G HiTrap SP column and labeled with horseradish peroxidase. A MacELISA method for the detection of IgM antibodies against CHIKV was assembled with goat anti-human IgM antibody, VLP antigens and an enzyme-labeled polyclonal antibody. The results were evaluated with a serum panel containing serum samples from laboratory-confirmed CHIK, HFRS patients, healthy donors, and commercially available CHIKV IgM as a quality control. It was shown that the MacELISA had a specificity of 99% (99/100), the coefficients of variation (CoV) within a plate were <10%, and the CoV of different ELISA plates in terms of the plate variation coefficient was <15%. A comparative analysis was performed to compare the current method against a commercial CHIKV IgM antibody detection kit for IIFA-IgM. The detection limit of MacELISA was significantly lower than that of the IIFA-IgM commercial kit (P< 0.0001). Here, we demonstrate that the VLP-based MacELISA is a promising tool for the early diagnosis and epidemiological investigation of CHIKV infection, with a high level of sensitivity and specificity for the detection of IgM antibodies against CHIKV.

[Laboratory diagnosis of viral hemorrhagic fevers].

Viral hemorrhagic fevers (VHFs) refer to a group of acute infections with high case fatality rates that are caused by four distinct families of RNA viruses belonging to the families Bunyaviridae, Flaviviridae, Filoviridae and Arenaviridae, the main clinical symptoms of these diseases are accompanied by fever and bleeding. For the reason that these infections have similar primary clinical symptoms, it is difficult to diagnose and distinguish them; rapid and reliable laboratory diagnostic tests are required in suspected cases for epidemiological investigation and controlling the spread of VHFs. This review addresses the laboratory diagnostics of VHFs, covering etiological classification and different diagnostic techniques, such as virus isolation, nucleic acid detection, as well as antigen and antibody assays. Prospects for novel diagnostic tools are also discussed.

[Epidemic situation and surveillance on SFTS in China, 2011-2012].

OBJECTIVE: To analyze the data of surveillance on severe fever with thrombocytopenia syndrome (SFTS), from 2011 to 2012 in China. METHODS: Descriptive methods were conducted to analyze the surveillance data from 2011 to 2012 which were collected from the internet-based National Notifiable Disease Reporting System. RESULTS: From 2011 to 2012, a total of 1229 SFTS cases and 107 deaths were reported in China with the average annual incidence rate of 0. 046/100 000 and case fatality rate of 8.7%. Compared to 2011, morbidity of 2012 has increased by 23.5% and mortality has decreased by 32%. 16 provinces reported SFTS cases. More cases occurred in spring and summer seasons,with the peak in May to July, during this period, 69% of the total cases were reported. The ages of the patients ranged from 1 to 85 years, 44.2% of total case was 55 to 70 years old, there were no differences in sex. Of all the cases 86. 8% was farmer. CONCLUSION: Severe fever with thrombocytopenia syndrome in widely distributed in China, especially in the central and eastern regions, the incidence has obvious seasonal. Surveillance and immigration quarantine should be strengthened.

[Investigation of RNA viral genome amplification by multiple displacement amplification technique].

In order to facilitate the detection of newly emerging or rare viral infectious diseases, a negative-strand RNA virus-severe fever with thrombocytopenia syndrome bunyavirus, and a positive-strand RNA virus-dengue virus, were used to investigate RNA viral genome unspecific amplification by multiple displacement amplification technique from clinical samples. Series of 10-fold diluted purified viral RNA were utilized as analog samples with different pathogen loads, after a series of reactions were sequentially processed, single-strand cDNA, double-strand cDNA, double-strand cDNA treated with ligation without or with supplemental RNA were generated, then a Phi29 DNA polymerase depended isothermal amplification was employed, and finally the target gene copies were detected by real time PCR assays to evaluate the amplification efficiencies of various methods. The results showed that multiple displacement amplification effects of single-strand or double-strand cDNA templates were limited, while the fold increases of double-strand cDNA templates treated with ligation could be up to 6 X 10(3), even 2 X 10(5) when supplemental RNA existed, and better results were obtained when viral RNA loads were lower. A RNA viral genome amplification system using multiple displacement amplification technique was established in this study and effective amplification of RNA viral genome with low load was achieved, which could provide a tool to synthesize adequate viral genome for multiplex pathogens detection.

[A double antibody sandwich ELISA based assay for titration of severe fever with thrombocytopenia syndrome virus].

OBJECTIVE: To develop an assay for titration of severe fever with thrombocytopenia syndrome virus (SFTSV) based on double antibody sandwich ELISA. METHODS: A double antibody sandwich ELISA was developed for detection of SFTSV based on SFTSV nucleocapsid (N) protein specific poly- and monoclonal antibodies, procedures were optimized and evaluated. This ELISA based titration assay was compared with fluorescence assasy and plaque assay based titration method. RESULTS: The results suggested that the titers obtained by ELISA based method are consistent with those obtained by IFA based method (R = 0.999) and the plaque assay titration method (R = 0.949). CONCLUSION: The novel ELISA based titration method with high sensitivity and specificity is easy to manage and perform, and can overcome the subjectivity associated with result determination of the fluorescence assay and plaque assay based methods. The novel ELISA based titration method can also be applied to high throughput detection.

[Hepatitis A virus mimotope mapping by phage display peptide library].

OBJECTIVE: A 12 mer phage display peptide library was used to identify hepatitis A virus mimotopes of antigenic determinants, to provide the feasibility of virus epitope mapping by using this approach. METHODS: Using purified anti-hepatitis A virus monoclonal antibody as affinity selective molecule, phage display peptide library was biopanned and positive clones were selected by ELISA, competition assay and DNA sequencing. RESULTS: 10 ELISA positive clones were chosen for DNA sequencing, and the displayed peptide sequences were deduced. 9 of them showed identical nucleotide sequence, and similarity in their amino acid sequence with VP1 of HAV HM175 was found, but no sequence homology was found between the other phage clone and the capsid proteins of HAV. Those peptides may behave as mimotopes of HAV. CONCLUSION: The mimotope of HAV was selected by using phage display peptide library screening. The results provide the potential of this method to search for the mimotopes of the virus.