Yiyue Ge

Serum MicroRNA Expression Profile Distinguishes Enterovirus 71 and Coxsackievirus 16 Infections in Patients with Hand-Foot-and-Mouth Disease

Altered circulating microRNA (miRNA) profiles have been noted in patients with microbial infections. We compared host serum miRNA levels in patients with hand-foot-and-mouth disease (HFMD) caused by enterovirus 71 (EV71) and coxsackievirus 16 (CVA16) as well as in other microbial infections and in healthy individuals. Among 664 different miRNAs analyzed using a miRNA array, 102 were up-regulated and 26 were down-regulated in sera of patients with enteroviral infections. Expression levels of ten candidate miRNAs were further evaluated by quantitative real-time PCR assays. A receiver operating characteristic (ROC) curve analysis revealed that six miRNAs (miR-148a, miR-143, miR-324-3p, miR-628-3p, miR-140-5p, and miR-362-3p) were able to discriminate patients with enterovirus infections from healthy controls with area under curve (AUC) values ranged from 0.828 to 0.934. The combined six miRNA using multiple logistic regression analysis provided not only a sensitivity of 97.1% and a specificity of 92.7% but also a unique profile that differentiated enterovirial infections from other microbial infections. Expression levels of five miRNAs (miR-148a, miR-143, miR-324-3p, miR-545, and miR-140-5p) were significantly increased in patients with CVA16 versus those with EV71 (p<0.05). Combination of miR-545, miR-324-3p, and miR-143 possessed a moderate ability to discrimination between CVA16 and EV71 with an AUC value of 0.761. These data indicate that sera from patients with different subtypes of enteroviral infection express unique miRNA profiles. Serum miRNA expression profiles may provide supplemental biomarkers for diagnosing and subtyping enteroviral HFMD infections.

Cytokine and Chemokine Levels in Patients Infected with the Novel Avian Influenza A (H7N9) Virus in China

H7N9 avian influenza is an emerging viral disease in China caused by avian influenza A (H7N9) virus. We investigated host cytokine and chemokine profiles in serum samples of H7N9 patients by multiplex-microbead immunoassays. Statistical analysis showed that IP-10, IL-6, IL-17, and IL-2 were increased in H7N9 infected patients. Furthermore, IL-6 and the chemokine IP-10 were significantly higher in severe H7N9 patients compared to nonsevere H7N9 cases. We suggest that proinflammatory cytokine responses, characterized by a combined Th1/Th17 cytokine induction, are partially responsible for the disease progression of patients with H7N9 infection.

Whole-Genome Sequence of a Reassortant H5N6 Avian Influenza Virus Isolated from a Live Poultry Market in China, 2013.

ABSTRACT An avian influenza virus, A/environment/Zhenjiang/C13/2013(H5N6), was isolated from a live poultry market in eastern China. Phylogenetic analysis showed that the isolate was a novel reassortant virus with a neuraminidase (NA) gene from H6N6 viruses and the other seven genes from H5N1 viruses, which may pose a potential threat to human and animal health.

Detection of severe fever with thrombocytopenia syndrome virus by reverse transcription-cross-priming amplification coupled with vertical flow visualization.

ABSTRACT A virus known as severe fever with thrombocytopenia syndrome virus (SFTSV) was recently identified as the etiological agent of severe fever with thrombocytopenia syndrome (SFTS) in China. Reliable laboratory detection and identification of this virus are likely to become clinically and epidemiologically desirable. We developed a nearly instrument-free, simple molecular method which incorporates reverse transcription–cross-priming amplification (RT-CPA) coupled with a vertical flow (VF) visualization strip for rapid detection of SFTSV. The RT-CPA-VF assay targets a conserved region of the M segment of the SFTSV genome and has a limit of detection of 100 copies per reaction, with no cross-reaction with other vector-borne bunyaviruses and bacterial pathogens. The performance of the RT-CPA-VF assay was determined with 175 human plasma specimens collected from 89 clinically suspected SFTS patients and 86 healthy donors. The sensitivity and specificity of the assay were 94.1% and 100.0%, respectively, compared with a combination of virus culture and real-time RT-PCR. The entire procedure, from specimen processing to result reporting, can be completed within 2 h. The simplicity and nearly instrument-free platform of the RT-CPA-VF assay make it practical for point-of-care testing.

Rapid and Sensitive Detection of Novel Avian-Origin Influenza A (H7N9) Virus by Reverse Transcription Loop-Mediated Isothermal Amplification Combined with a Lateral-Flow Device

A severe disease in humans caused by a novel avian-origin influenza A (H7N9) virus emerged in China recently, which has caused at least 128 cases and 26 deaths. Rapid detection of the novel H7N9 virus is urgently needed to differentiate the disease from other infections, and to facilitate infection control as well as epidemiologic investigations. In this study, a reverse transcription loop-mediated isothermal amplification combined with a lateral flow device (RT-LAMP-LFD) assay to rapidly detect H7N9 virus was developed and evaluated. The RT-LAMP primers were designed to target the haemagglutinin (HA) and neuraminidase (NA) genes of H7N9 virus. Results of 10-fold dilution series assays showed that analysis of RT-LAMP products by the LFD method was as sensitive as real-time turbidity detection, and that the analytic sensitivities of the HA and NA RT-LAMP assays were both 10 copies of synthetic RNA. Furthermore, both the assays showed 100% clinical specificity for identification of H7N9 virus. The performance characteristics of the RT-LAMP-LFD assay were evaluated with 80 clinical specimens collected from suspected H7N9 patients. The NA RT-LAMP-LFD assay was more sensitive than real time RT-PCR assay. Compared with a combination of virus culture and real-time RT-PCR, the sensitivity, specificity, positive predictive value, and negative predictive value of the RT-LAMP-LFD assay were all 100%. Overall, The RT-LAMP-LFD assay established in this study can be used as a reliable method for early diagnosis of the avian-origin influenza A (H7N9) virus infection.

Detection of novel swine origin influenza A virus (H1N1) by real-time nucleic acid sequence-based amplification

Rapid detection of novel swine origin influenza A virus (S-OIV) (H1N1) is crucial for timely implementation of infection control measures. In this study, a haemagglutinin (HA) gene-based real-time nucleic acid sequence-based amplification (NASBA) assay was developed for the specific detection of S-OIV (H1N1). The assay was evaluated and validated by comparing it with existing detection methods for S-OIV (H1N1). Results obtained in a 10-fold dilution series assay demonstrated the analytic sensitivity of the present assay was comparable to that of a commercial S-OIV (H1N1) real-time RT-PCR kit and higher than that of the Centers for Disease Control and Prevention (CDC) TaqMan assay. The actual detection limit of the real-time NASBA assay was approximately 50 copies per reaction. Compared with reference methods (viral culture, conventional RT-PCR, and real-time RT-PCR), the sensitivity, specificity, positive predictive value, and negative predictive value of the present assay were all 100%. Overall, the results showed that the real-time NASBA assay could be used for sensitive and specific detection of S-OIV (H1N1).

Dysregulated microRNA expression in serum of non-vaccinated children with varicella.

Circulating microRNAs (miRNAs) may play an important role in pathogen-host interactions and can serve as molecular markers for the detection of infectious diseases. To date, the relationship between circulating miRNAs and varicella-zoster virus (VZV) caused varicella has not been reported. Using TaqMan Low-Density Array (TLDA) analysis, expression levels of miRNAs in serum samples from 29 patients with varicella and 60 patients with Bordetella pertussis (BP), measles virus (MEV) and enterovirus (EV) were analyzed. The array results showed that 247 miRNAs were differentially expressed in sera of the varicella patients compared with healthy controls (215 up-regulated and 32 down-regulated). Through the following qRT-PCR confirmation and receiver operational characteristic (ROC) curve analysis, five miRNAs (miR-197, miR-629, miR-363, miR-132 and miR-122) were shown to distinguish varicella patients from healthy controls and other microbial infections with moderate sensitivity and specificity. A number of significantly enriched pathways regulated by these circulating miRNAs were predicted, and some of them were involved in inflammatory response, nervous system and respiratory system development. Our results, for the first time, revealed that a number of miRNAs were differentially expressed during VZV infection, and these five serum miRNAs have great potential to serve as biomarkers for the diagnosis of VZV infection in varicella patients.

A novel pyrosequencing assay for the detection of neuraminidase inhibitor resistance-conferring mutations among clinical isolates of avian H7N9 influenza virus

Abstract A novel reassortant avian influenza A virus (H7N9) emerged in humans in Eastern China in late February 2013. All virus strains were resistant to adamantanes (amantadine and rimantadine), but susceptible to neuraminidase inhibitors (NAIs) (oseltamivir and zanamivir). One strain (A/shanghai/1/2013) contained the R294K substitution in the neuraminidase (NA) gene, indicating resistance to oseltamivir. Pyrosequencing has proven to be a useful tool in the surveillance of drug resistance in influenza A viruses. Here, we describe a reverse transcription (RT)-PCR assay coupled with pyrosequencing to identify the NA residues E120, H276, and R294 (N9 numbering) of H7N9 viruses. A total of 43 specimens (26 clinical samples and 17 isolates) were tested. Only one isolate containing the E120V heterogenic mutation was detected by pyrosequencing and confirmed by Sanger sequencing. However, this mutation was not detected in the original clinical specimen. Since virus isolation might lead to the selection of variants that might not fully represent the virus population in the clinical specimens, we suggest that using pyrosequencing to detect NAI resistance in H7N9 viruses directly from clinical specimens rather than from cultured isolates. No cross-reactions with other types of influenza virus and respiratory tract viruses were found, and this assay has a sensitivity of 100 copies of synthetic RNA for all three codons. The high sensitivity and specificity of the assay should be sufficient for the detection of positive clinical specimens. In this study, we provide a rapid and reliable method for the characterization of NAI resistance in H7N9 viruses.

Simultaneous detection of hemagglutinin and neuraminidase genes of novel influenza A (H7N9) by duplex real-time reverse transcription polymerase chain reaction

Abstract A novel reassortant influenza A (H7N9) virus emerged recently in China. In this study, a duplex real-time reverse transcription polymerase chain reaction (rRT-PCR) assay was developed for the simultaneous detection of hemagglutinin (HA) and neuraminidase (NA) genes of H7N9 influenza viruses. The sensitivity of the assay was determined to be 10 RNA copies per reaction for both HA and NA genes. No cross-reactivity was observed with other influenza virus subtypes or respiratory tract viruses. One hundred and forty-six clinical and environmental specimens were tested and compared with reference methods and were found to be consistent. The assay is suitable for large-scale screening due to short turnaround times and high specificity, sensitivity, and reproducibility.

Identification of GRP75 as a novel PreS1 binding protein using a proteomics strategy

The PreS1 region of the L protein is important in cell attachment and consequently in hepatitis B virus (HBV) infectivity. To identify novel PreS1 interacting protein, we performed Glutathione-S-transferase (GST) pull-down, two-dimensional gel electrophoresis (2-DE) and mass spectrometry assays. Glucose-regulated proteins (GRP) 78 and 75 were found to bind PreS1. The interactions between PreS1 and GRP75 were confirmed by a co-immunoprecipitation experiment. GRP78 and GRP75 may play important roles in mediating HBV virion entering into hepatocyte and regulating proper folding of the L protein due to their critical functions in protein folding and trafficking. The finding of novel PreS1 binding protein enriches our knowledge about molecular mechanism of HBV infection.