Laboratory confirmation of the first influenza A (H1N1) imported case in Mainland China / 病毒学报

The clinical throat swab specimen of an imported suspected case of influenza A (H1N1) was detec ted with real-time PCR, RT-PCR and subsequently confirmed by gene sequencing. The presence of influ enza A (H1N1) virus confirmed the first case with A (H1N1) infection in Mainland China.

Histologic and ultrastructural studies of the patient died of highly pathogenic H5N1 avian influenza virus infection in China / 中华病理学杂志

<p><b>OBJECTIVE</b>To explore histopathologic and ultrastructural characteristics of human avian influenza (AI) infection and related etiological pathogenesis.</p><p><b>METHODS</b>Postmortem lung and heart samples were collected from the patient who died of avian influenza virus infection on November 29, 2003 in China. Light and electron microscopy, immunohistochemistry and histochemistry were used to investigate the pathological changes.</p><p><b>RESULTS</b>The main pathological findings included extensive pulmonary consolidation, hemorrhage, pulmonary edema and local hemorrhagic infarct. The lamina of alveoli and bronchioles were abundantly filled with protein-rich fluid, erythrocytes, fibrin and cell debris admixed with many neutrophilis, macrophages, lymphocytes and a few of monokaryon and multinuclear giant cells. Hyaline membranes were formed. Local pulmonary tissues were heavily damaged by hemorrhage and necrosis. Alveolar septum was disintegrated. Mesenchymal edema with a few of macrophages infiltration of heart was found. Electron microscopy showed the avian influenza A virus-like particles (type C and type A) of 80 - 120 nm diameter and envelopes in the cytoplasm of pneumocytes and endothelial cells.</p><p><b>CONCLUSIONS</b>Fatal pneumonia associated with highly pathogenic avian influenza A virus (H5N1) infection leads to extensive pulmonary consolidation, edema and marked hemorrhagic necrosis and inflammation. Electron microscopy can identify avian influenza A virus-like particles. The findings may offer an important theoretical basis for clinical diagnosis and treatment.</p>

Transfection with a novel cationic gene carrier: PEI-PBLG / 生物工程学报

This study mainly deals with cell transfection and cytotoxicity for PEI(10kD)-PBLG, a novel cationic copolymer, to observe its potential as a gene carrier. Size measurement and SEM were used to show the modality of the PEI-PBLG/pDNA complexes. Cytotoxicity of PEI (10kD)-PBLG was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay and compared with PEI(25kD)-PBLG, PEI(10kD), and PEI(25kD). Furthermore, pEGFP that can express the enhanced green fluorescent protein was chosen as a reporter to observe the transfection efficiency directly. Then, PEI (10kD)-PBLG/pEGFP complexes were transfected into several cell lines, such as Hela, COS-7, Vero-E6, and ECV-304, and effects of the transfection conditions were evaluated. The efficiencies were measured by FACS. Size measurement of complex particles indicated that PEI-PBLG/pDNA tended to form smaller nanoparticles compared with PEI/pDNA. The representative size of the PEI(10kD)-PBLG/pDNA complex was approximately 100 - 200 nm. SEM images showed that the particles were condense and compact. This can be suitable for their entry into cells. Cytotoxicity studies suggested that PEI (10kD)-PBLG had considerably lower toxicity than the other three materials. In the transfection tests, PEI (10kD)-PBLG/pDNA complexes could be transfected into all the cell lines that were tested. These provided the highest level of EGFP expression (45.02%) in Hela cells, which was considerably higher than that of PEI(10kD)/pEGFP (29.16%). Being less affected by the serum during transfection, PEI-PBLG/pDNA complexes offered greater biocompatibility than PEI. PEI-PBLG copolymer reduces the cytotoxicity of PEI, improves the transfection efficiency, and offers greater biocompatibility than PEI. It shows considerable potential as an efficient nonviral carrier for gene delivery.

Expression of tick-borne encephalitis virus prM-E protein in insect cells and studies on its antigenicity / 中华实验和临床病毒学杂志

<p><b>BACKGROUND</b>To express the prM-E protein in Sf9 cells, and lay a basis for further study on the function of the viral proteins and development of specific diagnostic reagents.</p><p><b>METHODS</b>The recombinant prM-E protein of tick-borne encephalitis virus was expressed in insect cell Sf9 by RT-PCR amplification of prM-E gene, construction of donor plasmid of Bac-to-Bac baculovirus expression system, homologous recombination of donor plasmid with bacmid DNA at the site of Tn7 and transfection of insect cell Sf9.</p><p><b>RESULTS</b>Recombinant subviral particles, about 30 nm in diameter, consisting of prM-E were observed by electron microscope in the supernatant of infected cells, which indicated that infected cells released virus-like particles (VLPs) into the culture medium. The results of Western-blot and the indirect immunofluorescence assay (IFA) showed that the recombinant proteins retained antigenic and conformational structures similar to those of native virus proteins. Using the recombinant prM-E as antigens to detect samples of patient sera by ELISA and IFA, all of 16 sera from patients with tick-borne encephalitis were positive and all of 6 sera from other patients were negative.</p><p><b>CONCLUSION</b>The prM-E protein expressed in insect cells retains good antigenicity.</p>

The screening and identification of an unknown virus by DNA microarray / 中华检验医学杂志

Objective In our previous study,we established DNA microarray technology for identification of medical viruses on genus levels and arboviruses on species levels.In this study,we employed these microarrays to determine the pathogen of newly isolated unknown virus in July,2006 from pig brain in Shanxi province.Methods The pathogen isolated from pig brains were inoculated in BHK21 cells.After CPE were observed,the supernatants were collected and RNA was extracted.After reverse transcription and random PCR amplification,the labeled nucleic acids were hybridized with DNA microarrays.Results The hybridization results with medical viruses DNA microarray indicated that the unknown virus belonged to Flavivirus.Combined with epidemiological investigation,we presumed that it might be a kind of arbovirus. Then the labeled specimen were further hybridized with arbovirus DNA microarray and the results confirmed that it was Japanese encephalitis virus(JBV).This coincided with PCR and sequencing analysis.Conclusions The DNA microarray we established previously could be employed to identify unknown viruses.This method provides a new method for determining new viral pathogens.

The application of indirect immuno-fluorescence assay in the diagnosis of severe acute respiratory syndrome / 中华流行病学杂志

<p><b>OBJECTIVE</b>To explore the temporal profile of serum antibody against coronavirus in patients with severe acute respiratory syndrome (SARS), and to evaluate the reliability of indirect immuno-fluorescence assay (IFA) in the diagnosis of SARS.</p><p><b>METHODS</b>Clinically confirmed SARS patients, suspected SARS patients, and controls were included in the study. IFA was used to detect the serum antibody against SARS coronavirus. General information about the subjects was collected using a standard questionnaire.</p><p><b>RESULTS</b>The positive rates of specific IgG and IgM against SARS virus within 10 days after onset of the disease were 55.1% and 16.3% respectively and then increased up to 89.8% for IgG and 65.3% for IgM. After 25 days of the onset of the disease, 90.9% patients became positive for both IgG and IgM. Results from chi-square for trend test revealed that the positive rates of both IgG and IgM increased with time (chi(2) for trend = 16.376, P = 0.00005 for IgG; chi(2) for trend = 28.736, P = 0.00000 for IgM). Sensitivity, specificity and agreement value of IFA regarding the diagnosis of SARS were all higher than 90%.</p><p><b>CONCLUSION</b>IFA can be used to assist diagnosis of SARS after 10 days of the onset of disease.</p>

Ultrastructural characteristics of SARS associated virus in infected cells / 中华病理学杂志

<p><b>OBJECTIVE</b>Electron microscopical study of infected cells to identify the pathogenic agent of SARS.</p><p><b>METHODS</b>Vero E6 cells infected with lung autopsy samples or nasopharyngeal swabs from SARS patients of Beijing and Guangzhou were inoculated. The supernatant and cultured cells exhibiting identifiable cytopathic effect (CPE) were prepared for electron microscopic study.</p><p><b>RESULTS</b>Examination of CPE cells on thin-section revealed characteristic coronavirus particles within the cisternae of endoplasmic reticulum, Golgi apparatus, vesicles and extracellular space. They were mainly spherical or oval in shape, annular or dense, about 80 nm in diameter. Negative-stain electron microscopy identified coronavirus particles in culture supernatant, 80 - 120 nm in diameter, with club-shaped surface projections. Elongated, rod-, kidney- or other irregular shaped virons with the size of 100 - 200 nm by 60 - 90 nm were also found in the cultured cells infected with the lung samples from the Guangdong patients. Infectious virons entered cells by endocytosis or membrane fusion and released through a budding process.</p><p><b>CONCLUSION</b>These data indicate a novel coronavirus as the causative agent of SARS. Most viral particles showed typical characteristics of coronavirus. The potential role of special shape viruses is expected to be further investigated.</p>

Advance in Research into the Infection of Avian Influenza Virus in Humans Crossing the Species Barrier / 微生物学通报

avian influenza virus (AIV) can not only infect avian and cause pandemics,but also result infection and initiate pandemics in humans and other mammal animals,crossing the species barrier.There have been some advance in research into the nonspecific species barrier of human respiratory tract against AIV infection and the mechanism of the infection of AIV in humans in recent years.