A Rapid and Sensitive One Step-SYBR Green Based Semi Quantitative Real Time RT-PCR for
the Detection of peste des petits ruminants Virus in the Clinical Samples
摘要： A sensitive and rapid single step real time (rt) RT-PCR was standardized
using one-step Brilliant SYBR Green kit? for detection and semi-quantitation of
peste des petitis ruminants virus (PPRV) using the virus RNA and matrix (M) protein
gene-specific primers and compared with established conventional RT-PCR and
TaqMan RT-PCR. The assay amplifies a 124 bp fragment of the PPRV M gene with
Tm of 78.28 to 78.50. The assay was linear within a range of 50 ng to 0.5 fg total
virus RNA with a detection limit (sensitivity) of 0.5 fg. Based on the serial dilution
of the live-attenuated PPR vaccine virus, the detection limit was ~0.0001 cell culture
infectious dose 50% units (TCID50). Additionally, swab materials spiked with
known titre of vaccine virus were equally well detected in the assay. The
standardized rt RT-PCR was easily employed for the detection of PPRV nucleic acid
directly in the field and experimental clinical samples. The assay detected the PPRV
nucleic acid as early as 3 day post infection (dpi) and up to 20 dpi in swab materials
from the experimental samples. The assay was rapid and more sensitive than
TaqMan and conventional RT-PCR in the detection of PPRV nucleic acid from the
PPR suspected clinical samples of sheep and goats. Therefore, the established,
simplified SYBR green rt RT-PCR is an alternative test to the already existing
various diagnostic assays and could be useful for rapid clinical diagnosis with
advantage in reducing risk of contamination.
关键词： PPR M gene SYBR green RT-PCR
Early diagnosis
Clinical samples
Genetic Analysis of the P1 Region of Human Enterovirus 71 Strains and Expression of the 55 F
StrainVP1 Protein
Abstract: Enterovirus 71 (EV71) is a member of the Entero-virus genus of the
Picornaviridae family and is the major cause of Hand, foot, and mouth disease
(HFMD) in children. Different strains from Gansu were cloned and the P1 protein
was sequenced and analysed. Results indicate that there are three kinds of EV71
infections prevalent in Gansu. The VP1 protein from one of these strains, 55F, was
expressed. The recombinant protein was expressed with high level and reacted
specifically with the EV71 patient antibody, the recombinant protein was also
applied to raise antiserum in rabbits and after the fourth injection a high titer of
antiserum was detected by ELISA assay. These data are useful for further
clarification of prevalent EV71 strains in the north of China at the molecular level
and provide a basis for EV71 diagnosis.
Keywords: EV71 Genetic analysis
P1 region Expression
VP1 protein
Inhibitory Effects of Ginsenoside Rb1 on Apoptosis Caused by HSV-1 in Human Glioma Cells
摘要： To investigate the inhibitory effects of Ginsenoside Rb1 (GRb1) on apoptosis
caused by Herpes Simplex Virus-1(HSV-1) in Human Glioma Cells (U251), U251
cells were infected by HSV-1 at a multiplicity of infection of 5 and GRb1,
GRb1+HSV-1, HSV-1 and control groups. MTT and cell apoptosis assays were used
to detect the inhibitory effects of GRb1 on the apoptosis of U251 cells that caused by
HSV-1 infection for various concentrations of drug and virus treatments by MTT
assay. We found that in the 400 μg/mL GRb1 and 400 μg/mL GRb1+HSV-1 groups,
MTT values were higher than control group at all times (P<0. 05). Moreover, the
apoptosis rate in the 400 μg/mL GRb1+HSV-1 group was lower than the HSV-1 group
(P<0. 05). These results confirmed that, at appropriate concentrations, GRb1 could
inhibit nerve cell apoptosis in HSV-1 infections.
Molecular and in vitro Characterization of Field Isolates of Bovine Herpesvirus-1
摘要： Bovine Herpesvirus-1 (BoHV-1) is distributed worldwide and is a major
pathogen in cattle, being the causal agent of a variety of clinical syndromes. The aim of
this study was to isolate and to characterize (molecular and biological characterization)
BoHV-1 from 29 immunosuppressed animals. It was possible to obtain 18 isolates,
each from a different animal, such as from the respiratory and reproductive tracts. In
some cases the cytopathic effect was visible 12 hours post-inoculation, and became
characteristic after 36-48 hours. Biological characteristics were evaluated and compared
with Iowa and Colorado-1 reference strains, and differences were found in plaque size,
virus titer measured by TCID50 and PFU/mL, and one step virus curves. These results
showed that some isolates had a highly virulent-like behavior in vitro, compared to the
reference strains, with shorter eclipse periods, faster release of virus into the
supernatants, and higher burst size and viral titer. There were no differences in
glycoprotein expression of BoHV-1 isolates, measured by Western blot on monolayers.
Moreover, using restriction endonucleases analysis, most of the viruses were confirmed
as BoHV-1.1 and just one of them was confirmed as BoHV-1.2a subtype. These
findings suggest that some wild-type BoHV-1 isolates could be useful as seeds to
develop new monovalent vaccines.
关键词：Bovine herpesvirus 1 Genital infection Respiratory infection Latent infection
HCV NS5A and NS5B Enhance Expression of Human Ceramide Glucosyltransferase
Gene
摘要：Host genes involved in lipid metabolism are differentially affected during the
early stages of hepatitis C virus (HCV) infection. Here we demonstrate that artificial
up-regulation of fatty acid biosynthesis has a positive effect on the replication of the
HCV full-length replicon when cells were treated with nystatin. Conversely, the HCV
RNA replication was decreased when fatty acid biosynthesis was inhibited with
25-hydroxycholesterol
and
PDMP(D-threo-1-phenyl-2-decanoylamino-3-
morpholino-1-propanol). In agreement with these results, the expression level of
GlcT-1(ceramide glucosyltransferase), a host glucosyltransferase in the first step of
GSL (glycosphingolipid) biosynthesis, was found to be closely associated with the
expression and replication of HCV RNA. On the other hand, the viral RNA can also
activate GlcT-1 in the early stage of viral RNA transfection in vitro. To identify viral
factors that are responsible for GlcT-1 activation, we constructed ten stable Vero cell
lines that express individual HCV proteins. Based on the analyses of these cell lines and
transient transfection assay of the GlcT-1 promoter regions, we conclude that HCV
proteins, especially NS5A and NS5B, have positive effects on the expression of GlcT-1.
It is possible that NS5A and NS5B stimulate transcription factor(s) to activate the
expression of GlcT-1 by increasing its transcription level.
关键词：
Hepatitis C virus
Fatty acid biosynthesis
Ceramide glucosyltransferase
Stable cell
lines
Development of Multiple ELISAs for the Detection of Antibodies against Classical Swine Fever
Virus in Pig Sera
摘要： The major immunogenic proteins (Erns, E2 and NS3) of classical swine fever
virus (CSFV) (Shimen strain) were expressed in E. coli and purified by affinity
chromatography. The recombinant antigens were applied to develop multiple
enzyme-linked immunosorbent assays (ELISAs) for the detection of specific
antibodies in pig sera. Optimum cut-off values were determined by receiver operating
characteristic (ROC) analysis after testing 201 sera of vaccinated pigs and 64 negative
sera of unvaccinated piglets. The multiple ELISAs were validated with 265 pig sera
yielding high sensitivity and specificity in comparison with the virus neutralization
results. The results demonstrated that multiple ELISAs can be a valuable tool for the
detection of CSFV infection and serological surveys in CSFV-free countries or for the
evaluation of the antibody responses in pigs induced by a live attenuated C-strain
vaccination.
The Protamine-like DNA-binding Protein P6.9 Epigenetically Up-regulates
Autographa alifornica Multiple Nucleopolyhedrovirus Gene Transcription in the
Late Infection Phase
摘 要 ： Protamines are a group of highly basic proteins first discovered in
spermatozoon that allow for denser packaging of DNA than histones and will result in
down-regulation of gene transcription[1]. It is well recognized that the Autographa
californica
multicapsid
nucleopolyhedrovirus
(AcMNPV)
encodes
P6.9,
a
protamine-like protein that forms the viral subnucleosome through binding to the viral
genome[29]. Previous research demonstrates that P6.9 is essential for viral
nucleocapsid assembly, while it has no influence on viral genome replication[31]. In
the present study, the role of P6.9 in viral gene transcription regulation is
characterized. In contrast to protamines or other protamine-like proteins that usually
down-regulate gene transcription, P6.9 appears to up-regulate viral gene transcription
at 12-24 hours post infection (hpi), whereas it is non-essential for the basal level of
viral gene transcription. Fluorescence microscopy reveals the P6.9’s co-localization
with DNA is temporally and spatially synchronized with P6.9’s impact on viral gene
transcription, indicating the P6.9-DNA association contributes to transcription
regulation. Chromatin fractionation assay further reveals an unexpected co-existence
of P6.9 and host RNA polymerase II in the same transcriptionally active chromatin
fraction at 24 hpi, which may probably contribute to viral gene transcription
up-regulation in the late infection phase
Ribosome Inactivating Proteins from Plants Inhibiting Viruses
摘要：Many plants contain ribosome inactivating proteins (RIPs) with N-glycosidase
activity, which depurinate large ribosomal RNA and arrest protein synthesis. RIPs so
far tested inhibit replication of mRNA as well as DNA viruses and these proteins,
isolated from plants, are found to be effective against a broad range of viruses such
as human immunodeficiency virus (HIV), hepatitis B virus (HBV) and herpes
simplex virus (HSV). Most of the research work related to RIPs has been focused on
antiviral activity against HIV; however, the exact mechanism of antiviral activity is
still not clear. The mechanism of antiviral activity was thought to follow inactivation
of the host cell ribosome, leading to inhibition of viral protein translation and host
cell death. Enzymatic activity of RIPs is not limited to depurination of the large
rRNA, in addition they can depurinate viral DNA as well as RNA. Recently, Phase
I/II clinical trials have demonstrated the potential use of RIPs for treating patients
with HIV disease. The aim of this review is to focus on various RIPs from plants
associated with anti-HIV activity.
关键词： Ribosome inactivating protein Human immunodeficiency virus Hepatitis B
virus Herpes simplex virus
Caspase Work Model During Pathogen Infection
Caspases
are
an
evolutionarily
conserved
family
of
aspartate-specific
cystein-dependent proteases with essential functions in apoptosis and normally exist
in cells as inactive proenzymes. In addition to the inflammatory caspases, the
initiator and effector caspases have been shown to have an important role in
regulating the immune response, but are involved in different ways. We give a brief
introduction on the benefit of apoptosis on the clearance of invasive pathogens, and
the caspase functions involved in the immune response. Then we construct a
working model of caspases during pathogen invasion. A detailed description of the
three modes is given in the discussion. These three modes are regulated by different
inhibitors, and there may be a novel way to treat intracellular pathogen and
autoimmune diseases based on the specific inhibitors.
关键词： Caspases Immune Response Pathogen Infection
Phylogenetic Analysis of the Non-structural (NS) Gene of Influenza A Viruses
Isolated in Kazakhstan in 2002-2009*
摘要： Although the important role of the non-structural (NS1 and NEP) gene of
influenza A in virulence of the virus is well established, our knowledge about the
extent of variation in the NS gene pool of influenza A viruses in their natural
reservoirs in Kazakhstan is incomplete. 17 influenza A viruses of different subtypes
were studied in this paper. Seven types of haemagglutinin and five different
neuraminidase subtypes in eight combinations were found among the isolated viruses.
A comparison of nucleotide sequences of isolated viruses revealed a substantial
number of silent mutations, which results in high degree of homology in amino acid
sequences. By phylogenetic analysis it was shown that two distinct gene pools,
corresponding to both NS allele A with 5 Clades and B, were present at the same time
in Kazakhstan. The degree of variation within the alleles was very low. In our study
allele A viruses had a maximum of 5% amino acid divergence in Clade while allele B
viruses had only 4% amino acid divergence
Emerging Trends of Drug-Resistant HIV-1 among Drug-Treated Patients in
Former Blood Donors in Hubei, China: a Three-Year Surveillance from 2004 to
2006*
摘要： This study aimed to evaluate emerging trends of drug resistance to
nucleoside reverse transcriptase inhibitors (NRTIs) and nonnucleoside reverse
transcriptase inhibitors (NNRTIs) among 290 former blood donor HIV-1 infected
patients in Hubei, China, from 2004 to 2006, all of whom had received anti-HIV-1
therapy. The presence of NRTI- and NNRTI-associated mutations were established
by sequencing; genotypic and predicted phenotypic drug resistance were evaluated
using
HIVdb
Program
version
5.0.1
(http://hivdb.stanford.edu/
pages/algs/HIVdb.html). Genotypic drug resistance analysis showed significant
increases in percentages of patients carrying HIV-1 strains with M41L, T215Y/F,
D67N, K103N, G190A/S, Y181C/F or L210W mutations. Of the variants’ predicted
phenotypic drug resistance, highly significant increases were detected in
percentages of patients carrying HIV-1 with high resistance to zidovudine (AZT) or
stavudine (D4T) in NRTIs, and to delavirdine (DLV), efavirenz (EFV) or
nevirapine (NVP) in NNRTIs; intermediate resistance to abacavir (ABC), AZT,
D4T, didanosine (DDI) or tenofovir disoproxil fumarate (TDF) in NRTIs, and to
etravirine (ETR) in NNRTIs; and low and potential low resistance to lamivudine
(3TC), ABC, emtricitabine (FTC) or TDF in NRTIs, and to ETR in NNRTIs.
关键词： HIV-1 Drug-Resistant mutation Former blood donors Drug-treated
Molecular Characterization of Banana streak virus Isolate from Musa
Acuminata in China*
摘要：Banana streak virus (BSV), a member of genus Badnavirus, is a causal agent
of banana streak disease throughout the world. The genetic diversity of BSVs from
different regions of banana plantations has previously been investigated, but there
are relatively few reports of the genetic characteristic of episomal (non-integrated)
BSV genomes isolated from China. Here, the complete genome, a total of 7722bp
(GenBank accession number DQ092436), of an isolate of Banana streak virus (BSV)
on cultivar Cavendish (BSAcYNV) in Yunnan, China was determined. The genome
organises in the typical manner of badnaviruses. The intergenic region of genomic
DNA contains a large stem-loop, which may contribute to the ribosome shift into the
following open reading frames (ORFs). The coding region of BSAcYNV consists of
three overlapping ORFs, ORF1 with a non-AUG start codon and ORF2 encoding
two small proteins are individually involved in viral movement and ORF3 encodes a
polyprotein. Besides the complete genome, a defective genome lacking the whole
RNA leader region and a majority of ORF1 and which encompasses 6525bp was
also isolated and sequenced from this BSV DNA reservoir in infected banana plants.
Sequence analyses showed that BSAcYNV has closest similarity in terms of genome
organization and the coding assignments with an BSV isolate from Vietnam
(BSAcVNV). The corresponding coding regions shared identities of 88% and ~95%
at nucleotide and amino acid levels, respectively. Phylogenetic analysis also
indicated BSAcYNV shared the closest geographical evolutionary relationship to
BSAcVNV among sequenced banana streak badnaviruses.
关键词： BSAcYNV Complete genome Defective genome Identity Phylogenetic
analysis
Bovine Herpesvirus 1 Protein bICP0 Represses the Transcription of bISG15 in
Fetal Bovine Lung Cells
摘要： The ubiquitin-like modifier bISG15 is an antiviral protein found in fetal
bovine lung (FBL) cells. Bovine Herpesvirus 1(BHV-1), which is a viral pathogen of
cattle, can infect FBL cells and induce cytopathic effects. Real-time PCR assays
showed that BHV-1’s infection could repress the basal or inducible transcription of
bISG15 in FBL cells. It demonstrates that this repression effect depends on BHV-1
viral infection and new protein synthesis. Our previous work showed that bIRF-3 was
the key factor in the stimulation of bISG15 in FBL cells, so the effect of BHV-1 viral
protein on bIRF-3 activating the promoter of bISG15 was confirmed. The luciferase
assay showed the BHV-1 viral protein bICP0 inhibited the activation of bISG15
promoter stimulated by bIRF-3. Taken together, our work suggested that BHV-1 had
some molecular mechanism to resist the cellular bISG15’s antiviral functions.
Surface Display of Domain III of Japanese Encephalitis Virus E Protein on
Salmonella Typhimurium by Using an Ice Nucleation Protein
摘要： A bacterial cell surface display technique based on an ice nucleation protein
has been employed for the development of live vaccine against viral infection. Due to
its ubiquitous ability to invade host cells, Salmonella typhimurium might be a good
candidate for displaying viral antigens. We demonstrated the surface display of
domain III of Japanese encephalitis virus E protein and the enhanced green
fluorescent protein on S. typhimurium BRD509 using the ice nucleation protein. The
effects of the motif in the ice nucleation protein on the effective display of integral
protein were also investigated. The results showed that display motifs in the protein
can target integral foreign protein on the surface of S. typhimurium BRD509.
Moreover, recombinant strains with surface displayed viral proteins retained their
invasiveness, suggesting that the recombinant S. typhimurium can be used as live
vaccine vector for eliciting complete immunogenicity. The data may yield better
understanding of the mechanism by which ice nucleation protein displays foreign
proteins in the Salmonella strain.
Genetic Characteristics of 2009 Pandemic H1N1 Influenza A Viruses Isolated
from Mainland China*
摘要： A total of 100 H1N1 flu real-time-PCR positive throat swabs collected from
fever patients in Zhejiang, Hubei and Guangdong between June and November 2009,
were provided by local CDC laboratories. After MDCK cell culture, 57 Influenza A
Pandemic (H1N1) viruses were isolated and submitted for whole genome sequencing.
A total of 39 HA sequences, 52 NA sequences, 36 PB2 sequences, 31 PB1 sequences,
40 PA sequences, 48 NP sequences, 51 MP sequences and 36 NS sequences were
obtained, including 20 whole genome sequences. Sequence comparison revealed they
shared a high degree of homology (96% ～99%) with known epidemic strains
(A/California/04/2009(H1N1). Phylogenetic analysis showed that although the
sequences were highly conserved, they clustered into a small number of groups with
only a few distinct strains. Site analysis revealed three substitutions at loop 220
(221–228) of the HA receptor binding site in the 39 HA sequences: A/Hubei/86/2009
PKVRDQEG→PKVRDQEA,
A/Zhejiang/08/2009
PKVRDQEG→PKVRDQER,
A/Hubei/75/2009 PKVRDQEG→PKVRDQGG, the A/Hubei/75/2009 was isolated
from an acute case, while the other two were from patients with mild symptoms.
Other key sites such as 119, 274, 292 and 294 amino acids of NA protein,627 of PB2
protein were conserved. Meanwhile, all the M2 protein sequences possessed the
Ser32Asn mutation, suggesting that these viruses were resistant to adamantanes.
Comparison of these sequences with other H1N1 viruses collected from the NCBI
database provides insight into H1N1 transmission and circulation patterns
Similar Neutralizing Activity in the HIV-1 Infected Long Term Non-progressors(LTNPs) and
Typical Progressors(TPs)*
Abstract: Neutralizing antibodies are considered to be an important protective
parameter used in HIV-1 vaccine evaluation. However, the exact role that neutralizing
antibodies plays in controlling the disease progression of HIV-1 infected peoples is
still undetermined. In this paper, we compared the protective function of the
neutralizing antibody response in the plasma from LTNP and TP against clade B and
clade C pseudoviruses. No difference in the neutralizing activities between the plasma
from LTNP and TP was found, which was consistent with the most recent reports. In
addition, no correlations between the titer or breadth and CD4+ or viral load in HIV-1
infected individuals were found. The protective roles played by neutralizingantibodies
in controlling disease progression of HIV-1 infected people need to be considered in a
new viewpoint.
抗狂犬病病毒单克隆抗体的制备及鉴定
Preparation and Identification of Anti-rabies Virus Monoclonal Antibodies*
Abstract:
To provide a foundation for the development of rapid and specific methods for the
diagnosis of rabies virus infection, anti-rabies virus monoclonal antibodies were prepared and
rabies virus nucleoprotein and human rabies virus vaccine strain (PV strain) were used as
immunogens to immunize 6-8 week old female BALB/c mice. Spleen cells and SP2/0 myeloma
cells were fused according to conventional methods: the monoclonal cell strains obtained were
selected using the indirect immunofluorescence test; this was followed by preparation of
monoclonal antibody ascitic fluid; and finally, systematic identification of subclass, specificity and
sensitivity was carried out. Two high potency and specific monoclonal antibodies against rabies
virus were obtained and named 3B12 and 4A12, with ascitic fluid titers of 1:8000 and 1:10000,
respectively. Both belonged to the IgG2a subclass. These strains secrete potent, stable and specific
anti-rabies virus monoclonal antibodies, which makes them well suited for the development of
rabies diagnosis reagents.
摘要： 制备抗狂犬病病毒单克隆抗体，为研发快速特异的狂犬病病毒诊断方法奠定基础。以狂犬病病毒核
蛋白、狂犬病病毒人用疫苗株（PV 株）为免疫原免疫 6-8 周龄雌性 BALB/c 小鼠，取其脾细胞与 SP2/0 骨
髓瘤细胞按常规方法进行融合；利用间接免疫荧光法筛选所获单抗细胞株；将获得特异稳定分泌抗狂犬病
病毒单克隆抗体的杂交瘤细胞株经小鼠腹腔注射制备单抗腹水；对所得单抗进行了亚类及特异性和敏感性
的系统鉴定。获得 2 株（3B12 和 4A12）特异稳定分泌抗狂犬病病毒单抗细胞株，其腹水效价分别为 1：8000
和 1：10000，分泌单抗为抗狂犬病病毒 IgG2a 亚类。所得两株单抗细胞株能高效价稳定特异分泌抗狂犬病
病毒单抗，且具有良好的稳定性和特异性，适宜应用于下一步狂犬病检测试剂的研发。