Next-generation Sequencing Study of Pathogens in Serum from Patients with Febrile Jaundice in Sierra Leone / 生物医学与环境科学（英文）

OBJECTIVE@#People in Western Africa suffer greatly from febrile jaundice, which is caused by a variety of pathogens. However, yellow fever virus (YFV) is the only pathogen under surveillance in Sierra Leone owing to the undeveloped medical and public health system there. Most of the results of YFV identification are negative. Elucidation of the pathogen spectrum is required to reduce the prevalence of febrile jaundice.@*METHODS@#In the present study, we used Ion Torrent semiconductor sequencing to profile the pathogen spectrum in archived YFV-negative sera from 96 patients in Sierra Leone who presented with unexplained febrile jaundice.@*RESULTS@#The most frequently identified sequencing reads belonged to the following pathogens: cytomegalovirus (89.58%), Epstein-Barr virus (55.21%), hepatitis C virus (34.38%), rhinovirus (28.13%), hepatitis A virus (20.83%), coxsackievirus (10.42%), Ebola virus (8.33%), hepatitis E virus (8.33%), lyssavirus (4.17%), leptospirosis (4.17%), chikungunya virus (2.08%), Crimean-Congo hemorrhagic fever virus (1.04%), and hepatitis B virus (1.04%).@*CONCLUSION@#The distribution of sequencing reads suggests a broader spectrum of pathogens for consideration in clinical diagnostics and epidemiological surveillance in Sierra Leone.

Three-dimensional Culture of Human Airway Epithelium in Matrigel for Evaluation of Human Rhinovirus C and Bocavirus Infections / 生物医学与环境科学（英文）

OBJECTIVE@#Newly identified human rhinovirus C (HRV-C) and human bocavirus (HBoV) cannot propagate in vitro in traditional cell culture models; thus obtaining knowledge about these viruses and developing related vaccines are difficult. Therefore, it is necessary to develop a novel platform for the propagation of these types of viruses.@*METHODS@#A platform for culturing human airway epithelia in a three-dimensional (3D) pattern using Matrigel as scaffold was developed. The features of 3D culture were identified by immunochemical staining and transmission electron microscopy. Nucleic acid levels of HRV-C and HBoV in 3D cells at designated time points were quantitated by real-time polymerase chain reaction (PCR). Levels of cytokines, whose secretion was induced by the viruses, were measured by ELISA.@*RESULTS@#Properties of bronchial-like tissues, such as the expression of biomarkers CK5, ZO-1, and PCK, and the development of cilium-like protuberances indicative of the human respiration tract, were observed in 3D-cultured human airway epithelial (HAE) cultures, but not in monolayer-cultured cells. Nucleic acid levels of HRV-C and HBoV and levels of virus-induced cytokines were also measured using the 3D culture system.@*CONCLUSION@#Our data provide a preliminary indication that the 3D culture model of primary epithelia using a Matrigel scaffold in vitro can be used to propagate HRV-C and HBoV.

Morphogenetic study of human adenovirus type 41 in 293TE cells / 病毒学报

To investigate the morphogenetic process of human adenovirus type 41 (HAdV-41), 293TE cells were infected with purified wild-type HAdV-41, and ultrathin sections of infected cells were prepared and observed under a transmission electron microscope. Results showed that HAdV-41 entered host cells mainly through three ways: non-clathrin-coated pit, clathrin-coated pit, and direct penetration of plasma membrane. In addition, cell microvilli might help HAdV-41 enter cells. After entering into cells, HAdV-41 virus particles could be found in vacuoles or lysosomes or be in a free state in cytoplasm. Only free virus particles could be found near nuclear pores (NP), suggesting that the virus needed to escape from lysosomes for effective infection and viral nucleoprotein entered the nucleus through NP. Progeny viruses were as-sembled in the nucleus. Three types of inclusion bodies, which were termed as fibrillous inclusion body, condense inclusion body, and stripped condense inclusion body, were involved in HAdV-41 morphogenesis. In the late phase of viral replication, the membrane integrity of the infected cells was lost and viral particles were released extracellularly. This study reveals the partial process of HAdV-41 morphogenesis and provides more biological information on HAdV-41.

Characterization of Marburg virus morphology / 病毒学报

Ebola virus (EBOV) and Marburg virus (MARV) belong to the family Filoviridae. Filoviruses cause severe filovirus hemorrhagic fever (FHF) in humans, with high case fatality rates, and represent potential agents for bioterrorism and biological weapons. It is necessary to keep surveillance of filoviruses, even though there is no report of their isolation and patients in China so far. To characterize MARV morphology, the Lake Victoria marburgvirus--Leiden was stained negatively and observed under a transmission electron microscope which is one of important detection methods for filoviruses in emergencies and bioterrorism. MARV showed pleomorphism, with filamentous, rod-shaped, cobra-like, spherical, and branch-shaped particles of uniform diameter but different lengths. Pleomorphism of negatively stained MARV is summarized in this article, so as to provide useful information for possible electron microscopic identification of filoviruses in China.

Characterization of fibrillous inclusion body (FIB) of human adenovirus type 41 (Ad41) using immunoelectron microscopy / 病毒学报

To investigate the components of fibrillous inclusion body (FIB), which was formed in packaging cells during the replication of human adenovirus type 41 (Ad41), Ad41 long fiber knob (LFK) and short fiber knob (SFK) proteins were expressed in prokaryote respectively and then used to immunize BALI mice for preparation of anti-LFK serum and anti-SFK sera. The activity and specificity of anti-LFK and an ti-SFK sera were confirmed with Western blot, indirect immunofluorescence assay (IFA) and immunonegative staining, suggesting these sera could be applied in immuno-colloidal gold labelling electron microscopy (EM). 293TE cells were infected with wild-type Ad41. Ultrathin sections of infected cells were made, and labelled with immuno-colloidal gold technique using anti-Ad41 sera, anti-LFK sera, anti-SFK sera, or anti-fiber monoclonal antibody 4D2, respectively. The labelled sections were observed under EM, and the results demonstrated that both Ad41 long fiber protein and short fiber protein were included FIB.

Establishment of localization ultrathin section for cytopathic cells / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To establish a localization ultrathin section method through which target cytopathic cells could be sectioned in situ.</p><p><b>METHODS</b>Lab-Tek Chamber slide system (177402) was selected as resin embedding mould. Cells infected with Human adenovirus type 5 (Ad5) or A/HN/SWL3/ 2009 (H1N1) influenza virus were embedded in situ as models. Target cytopathic cells were exposed by trimming, sectioned and observed under transmission electron microscope (TEM).</p><p><b>RESULTS</b>Target cells could be sectioned in situ and virus particles could be found easily on sections.</p><p><b>CONCLUSION</b>A localization ultrathin sectioning method was established and this technique could be applied in virus detection in cytopathic cells to improve TEM detection efficiency.</p>

The assemblage, purification and characterization of EV71 VLPs expressed in baculovirus / 病毒学报

To construct a recombinant expression plasmid Bacmid-P1-3CD containing the P1 and 3CD genes of enterovirus 71(EV71), the P1 and 3CD genes were cloned into the same baculovirus shuttle vector (Bacmid). Recombinant AcMNPV-P1-3CD was obtained by transfecting the Bacmid-P1-3CD into the insect cell line of S f9. With the IFA and Western-blot methods for identification of expression products confirmed that the target protein was expressed in interior of infected S f9 cells. Electron microscopy showed that the structural protein capsid P1 was cut by virus-encoded protease 3CD and assembled into EV71 virus like particles (VLPs) about 27nm diameter. Different values of MOI and time points of expression were compared to explore the optimal expression condition, and the results showed that the time point could be a more important factor. Then we used S f9 cells with serum-free medium in CellSTACK-10 Culture Chambers to produce EV71 VLPs in the confirmed condition. After purification of VLPs by density gradient centrifugation, we observed on SDS-PAGE profile the purified sample contained three major proteins whose molecular masses corresponded to those of VP1 (39kD), VP0 (34kD) and VP3 (26kD) as well as the intact structure, which can be greatly used for further study in protein structure and genetic engineering vaccine research.

The genetic stability of recombinant adenovirus expressing human rotavirus VP6 gene which used Ad41 as vector / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To investigate the genetic stability of non-replicating recombinant adenovirus which used Ad41 as vector and could express VP6 gene of group A rotavirus during continous passage, in order to develop the vaccine of rotavirus.</p><p><b>METHODS</b>The recombinant adenovirus rvAd41-VP6 (o) was prepared by our laboratory early, it then was continuously propagated on 293TE7 cells for 14 passages. After that samples of the infected cells were collected at every 2 passages for the detection of the integration of the VP6 gene by PCR, and the expression of the target protein was detected by Western Blot analysis.</p><p><b>RESULTS</b>Analysis by PCR revealed that, there was stable integration of specific VP6 gene in the rvAd41-VP6 (o), Western Blot analysis confirmed that rvAd41-VP6 (o) could stably expressed the group-specific antigen structural protein VP6 (o), and it had preferable genetic stability.</p><p><b>CONCLUSION</b>The recombinant adenovirus rvAd41-VP6 (o) which could stably express the VP6 (o) gene had favorable biological property in vitro, and it has provided a basis for further research of animal immunization.</p>

Optimization of the codon strengthened the human rotavirus VP6 antigen's serum immune responses and protective responses in mice model / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To observe the serum immune responses and protection in mice model of the recombinant adenovirus vector mediated human rotavirus VP6 gene expression through coden optimization (rvAdVP6(o)) in comparison with the wild type (rvAdVP6).</p><p><b>METHODS</b>6-8 week female BALB/c mice were randomly grouped and immunized three times intranasally with 10(8) TCID50 rvAdVP6(o) and rvAdVP6, respectively, then detect the serum IgG level against rotavirus induced by rvAdVP6(o) and rvAdVP6. The amount of sheding viral antigens in feces was detectd after mice rotavirus was taked orally.</p><p><b>RESULTS</b>The serum IgG level against rotavirus induced by rvAdVP6(o) was higher than that of rvAdVP6 after three times of immunization. The immunized mice shed lower amount of viral antigens in feces as compared with the rvAdVP6.</p><p><b>CONCLUSION</b>The recombinant adenovirus which encode optimized human rotavirus VP6 proteins (rvAdVP6(o)) could induce stronger serum immune and protective responses against the challenge of the rotavirus than the wild type (rvAdVP6) at the same immunizing dosage.</p>

Improved expression of human rotavirus VP6 antigen in the recombinant adenoviruse by codon optimization / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To increase the recombinant adenovirus vector mediated human rotavirus VP6 gene expression through coden optimization.</p><p><b>METHODS</b>We have artificially synthesized VP6 gene of group A human rotavirus according to the human biased codon. The modified gene was transfected into 293 cells using adenovirus vector and the gene product, the respective protein was produced. The expression level of optimized gene and wild type gene was detected by Immunofluorescence (IF) and Western Blot.</p><p><b>RESULT</b>A remarkable increase of the expression level of optimized VP6 gene in comparison with the wild-type control.</p><p><b>CONCLUSION</b>The coden optimization indeed help increasing the recombinant adenovirus mediated human rotavirus gene expression, which indicated the potential application of such recombinant adenoviruses in the development of adenoviral-vectored rotavirus vaccines.</p>