Yiyu Lu

Detection of human novel influenza A (H1N1) viruses using multi-fluorescent real-time RT-PCR

The novel influenza A (H1N1) virus is now rapidly spreading across the world. Early detection is one of the most effective measures to prevent further transmission of the virus. 4 sets of proprietary primers and probes designed for detection of influenza A viruses (InfA), human and swine H1N1 viruses (SH1), the novel H1N1 viruses (NH1) and RNaseP gene (RP) respectively were pooled together in a single tube for a multi-fluorescent real-time RT-PCR assay. The detection limit was found to be one order more sensitive than that employing the WHO recommended protocol. The NH1 probe was negative for all control samples including human seasonal H1N1 virus, other subtypes of human influenza A viruses (H3, H5, H9), human influenza B virus and nasopharyngeal swabs from patients with noninfluenza respiratory diseases, indicating its high specificity, capable of discriminating the novel influenza A virus from the previously identified H1N1 viruses. For confirmation, the PCR amplified fragment of the hemagglutinin gene was sequenced which could provide enough information to identify the novel H1N1 virus as a distinct cluster among all viruses of subtype H1 through average distance clustering analysis. Although these assays should be useful in the current outbreak for rapid detection and discrimination of the novel H1N1 from swine H1N1 and other human seasonal H1N1 viruses, further design improvement is suggested to match possible sequence variations in the detected region along with the course of the epidemic.

Origin and Characteristics of Internal Genes Affect Infectivity of the Novel Avian-Origin Influenza A (H7N9) Virus

Background Human infection with a novel avian-origin influenza A (H7N9) virus occurred continuously in China during the first half of 2013, with high infectivity and pathogenicity to humans. In this study, we investigated the origin of internal genes of the novel H7N9 virus and analyzed the relationship between internal genes and infectivity of the virus. Methodology and Principal findings We tested the environmental specimens using real-time RT-PCR assays and isolated five H9N2 viruses from specimens that were positive for both H7 and H9. Results of recombination and phylogeny analysis, performed based on the entire sequences of 221 influenza viruses, showed that one of the Zhejiang avian H9N2 isolates, A/environment/Zhejiang/16/2013, shared the highest identities on the internal genes with the novel H7N9 virus A/Anhui/1/2013, ranging from 98.98% to 100%. Zhejiang avian H9N2 isolates were all reassortant viruses, by acquiring NS gene from A/chicken/Dawang/1/2011-like viruses and other five internal genes from A/brambling/Beijing/16/2012-like viruses. Compared to A/Anhui/1/2013 (H7N9), the homology on the NS gene was 99.16% with A/chicken/Dawang/1/2011, whereas only 94.27-97.61% with A/bramnling/Beijing/16/2012-like viruses. Analysis on the relationship between internal genes and the infectivity of novel H7N9 viruses were performed by comparing amino acid sequences with the HPAI H5N1 viruses, the H9N2 and the earlier H7N9 avian influenza viruses. There were nine amino acids on the internal genes found to be possibly associated with the infectivity of the novel H7N9 viruses. Conclusions These findings indicate that the internal genes, sharing the highest similarities with A/environment/Zhejiang/16/2013-like (H9N2) viruses, may affect the infectivity of the novel H7N9 viruses.

Inapparent Infection During an Outbreak of Dengue Fever in Southeastern China

Dengue fever (DF) is often asymptomatic in endemic areas. Asymptomatic infection during a DF outbreak in China, where DF is not endemic, has not been reported until now. In this study a total of 365 subjects from 6 villages were recruited from October 4-7, 2009. Overall, 102 subjects (27.95%) were positive for dengue virus (DENV) IgM, and 14 subjects (3.84%) were positive for DENV IgG and IgM. In different age groups, seropositive rates varied from 12.50% to 50.00% for DENV IgM, and from 0% to 11.76% for DENV IgG. Seroprevalence of DENV IgM was significantly higher than that of DENV IgG. Seroprevalence rates of DENV IgM differed among different villages. However, the seroprevalence of DENV IgM was not statistically significantly different among gender and age groups. Asymptomatic DF infection is prevalent in non-endemic areas.

Isolation and characterization of H7N9 avian influenza A virus from humans with respiratory diseases in Zhejiang, China.

In 2013, the novel reassortant avian-origin influenza A (H7N9) virus was reported in China. Through enhanced surveillance, infection by the H7N9 virus in humans was first identified in Zhejiang Province. Real-time reverse-transcriptase-polymerase-chain-reaction (RT-PCR) was used to confirm the infection. Embryonated chicken eggs were used for virus isolation from pharyngeal swabs taken from infected human patients. The H7N9 isolates were first identified by the hemagglutination test and electron microscopy, then used for whole genome sequencing. Bioinformatics software was used to construct the phylogenetic tree and for computing the mean rate of evolution of the HA gene in H7Nx and NA in HxN9. Two novel H7N9 avian influenza A viruses (A/Zhejiang/1/2013 and A/Zhejiang/2/2013) were isolated from the positive infection cases. Substitutions were found in both Zhejiang isolates and were identified as human-type viruses. All phylogenetic results indicated that the novel reassortant in H7N9 originated in viruses that infected birds. The sequencing and phylogenetic analysis of the whole genome revealed the mean rate of evolution of the HA gene in H7NX to be 5.74E-3 (95% Highest posterior density: 3.8218E-3 to 7.7873E-3) while the NA gene showed 2.243E-3 (4.378E-4 to 3.79E-3) substitutions per nucleotide site per year. The novel reassortant H7N9 virus was confirmed by molecular methods to have originated in poultry, with the mutations occurring during the spread of the H7N9 virus infection. Live poultry markets played an important role in whole H7N9 circulation.

Visual Detection of West Nile Virus Using Reverse Transcription Loop-Mediated Isothermal Amplification Combined with a Vertical Flow Visualization Strip.

West Nile virus (WNV) causes a severe zoonosis, which can lead to a large number of casualties and considerable economic losses. A rapid and accurate identification method for WNV for use in field laboratories is urgently needed. Here, a method utilizing reverse transcription loop-mediated isothermal amplification combined with a vertical flow visualization strip (RT-LAMP-VF) was developed to detect the envelope (E) gene of WNV. The RT-LAMP-VF assay could detect 102 copies/μl of an WNV RNA standard using a 40 min amplification reaction followed by a 2 min incubation of the amplification product on the visualization strip, and no cross-reaction with other closely related members of the Flavivirus genus was observed. The assay was further evaluated using cells and mouse brain tissues infected with a recombinant rabies virus expressing the E protein of WNV. The assay produced sensitivities of 101.5 TCID50/ml and 101.33 TCID50/ml for detection of the recombinant virus in the cells and brain tissues, respectively. Overall, the RT-LAMP-VF assay developed in this study is rapid, simple and effective, and it is therefore suitable for clinical application in the field.

Rapid detection of measles virus using reverse transcription loop-mediated isothermal amplification coupled with a disposable lateral flow device.

The measles virus (MeV) causes a highly contagious disease and efforts to reduce its spread are critical. A reverse transcription loop-mediated isothermal amplification assay coupled with a disposable lateral flow device (RT-LAMP-LFD) was developed for the rapid detection of MeV. The assay was performed in 40 min at an optimal temperature of 58 °C, with endpoint results visualized directly. A probe that was complementary to the RT-LAMP amplicon was designed to enhance assay specificity. Detection limit of the assay was 8.8 copies/μL synthetic RNA, which equals the sensitivity of real-time RT-PCR. Clinical specimens were used to validate the RT-LAMP-LFD in provincial Center for Disease Control and Prevention (CDC) (n = 245) and six municipal CDCs (n = 249). The results obtained using RT-LAMP-LFD and real-time RT-PCR were highly concordant. The RT-LAMP-LFD is rapid, stable, and does not require expensive equipment, which can be used for routine MeV monitoring in CDC laboratories.

Establishment of a novel target-based real-time quantitative PCR method for Acinetobacter baumannii detection.

Biofilm formation is a well-known pathogenic mechanism in infections caused by Acinetobacter baumannii. Recently, a biofilm synthesis-associated gene has been found in A. baumannii ATCC19606. Bioinformatic analysis showed 2 transmembrane structures and an hmsS superfamily domain, which was related to biofilm formation. What is more, high homology sequences of the bfs gene were only present in A. baumannii spp., and the similarities of nucleotide sequences of the bfs gene from A. baumannii strains ATCC17978, ACICU, S1, AB307-0294, and AB0057 compared with the reported sequence of bfs (GenBank accession No.: NZ_GG704572) were all above 95%. The distribution and conservation of the bfs gene from clinically derived A. baumannii strains were verified through conventional polymerase chain reaction (PCR). After this, we established a bfs gene-based real-time quantitative PCR assay to detect A. baumannii. Species specificity and sensitivity assays were designed and validated. By using this method, all the A. baumannii strains separated from clinical samples were identified and showed good accordance with the results from biochemical identification. This study is the first report of developing a bfs gene-based quantitative polymerase chain reaction for rapid, stable, and specific detection of A. baumannii. This method can be applied to clinical laboratory diagnosis, and detection of A. baumannii present on medical instruments.

Genotypic heterogeneity of emetic toxin producing Bacillus cereus isolates from China.

&NA; Emetic toxin‐producing Bacillus cereus (emetic B. cereus) is the third member of B. cereus group whose toxins are encoded by megaplasmids, beside anthrax and insecticidal toxins of B. anthracis and B. thuringiensis, respectively. A total of 18 emetic isolates collected from food poisoning events, clinical and non‐random food samples in Zhejiang province of China, were analyzed by plasmid screening, pulse field gel electrophoresis, multilocus sequence typing, and toxic gene identification to investigate their genotypic diversity. In this study, 13 plasmid profile types, 14 pulse types and 6 different STs from emetic isolates were detected, in which ST 1035,1038,1053,1054 and 1065 were first assigned and reported. The toxic gene ces existed on its own, or coexisted with other toxic genes bceT, cytk, entFM and nhe, but never with hbl in emetic isolates. The results demonstrated that the emetic B. cereus strains from China were heterologous at genotypic level.

Redirecting retroviral tropism by insertion of hepatitis B virus PreS1 core peptide into the envelope.

SummaryA potentially powerful approach for in vivo gene delivery is to target retroviral vectors to specific cells through interactions between cell surface receptors and appropriately engineered viral envelope proteins, but this has so far met with little success. We report here an attempt to target ecotropic MLV retroviral vectors to human cells infected by hepatitis B virus (HBV) through the interaction between the HBV PreS1 domain and the receptors on the cell surface. We examined 7 MLV chimeric envelope derivatives that contained either the HBV PreS1 peptide or PreS1 aa 21–47 segment (partial PreS1, pPreS1), which was inserted at various locations within the SU of the MoMLV Env. In addition to infecting host NIH 3T3 cells, some of pseudotyped viruses could transduce human cells. Our results demonstrate that short peptide ligands inserted at appropriate locations in the MLV envelope can selectively target retroviruses to human cells.

Establishment and characterization of an oral gerbil model for a non-mouse-adapted enterovirus 71 strain.

Enterovirus 71 (EV71) is one of the major pathogens causing hand, foot, and mouth disease (HFMD) with neurological and systemic complications worldwide, and it is mostly discovered in infants and young children. It is of great significance to establish suitable animal models of EV71 infection on research of distribution and pathogenesis of the virus. In this study, we established a successful infection of a non-mouse-adapted isolate of EV71 via oral route in 7-day-old Mongolian gerbil (Meriones unguiculatus), all of which were paralyzed and died within 10 days post infection. Analysis of virus loads in twelve tissues showed that virus was first detected in intestine, blood, heart, lung, and muscle one day post-infection, and then in the rest of the tissues/organs within the next few days, among which thymus, spleen, spinal cord and muscles had the highest virus titer at 5 days post infection. Pathological examination showed that severe necrosis was observed in skeletal muscle and spinal cord, and edema was observed in both heart and lung. Comparisons of host gene expression of various tissues from infected and non-infected gerbils revealed a general up-regulation of genes related to anti-viral response and a viral receptor gene (sialic acid-linked glycans), as well as a tissue(gut)-specific up-regulation of genes related to neuronal communication. Collectively, our results showed that EV71 could induce severe neurological complications as well as massive tissue damage all over the body, which indicates that oral infection of 7-day gerbils can be a suitable animal model to study the infection of EV71 in human.