Current insights into the role of HIF-PHD axis in renal anemia / 生理学报

Renal anemia, mainly caused by the deficiencies of erythropoietin (EPO) and iron metabolism disorder, is one of the most common complications of chronic kidney disease. Hypoxia-inducible factor (HIF) is a class of transcription factors responsible for maintaining homeostasis during oxygen deprivation. In normoxia, HIF is degraded by prolyl hydroxylase (PHD). While under hypoxic conditions, the hydroxylation activity of PHD is inhibited, and the cellular concentration of HIF is elevated, resulting in an increase in endogenous EPO production and iron absorption. Therefore, this regulating pathway, also termed as the HIF-PHD axis, has become a promising therapeutic target of treating renal anemia. Several innovative drugs acting as selective HIF-PHD inhibitors have been successfully developed in the past years, and some of them are undergoing clinical trials. In this review, we will introduce the definition and regulatory mechanism of HIF-PHD axis, as well as current insights into its physiologic and therapeutic role in renal anemia.

Establishment and application of TaqMan real-time RT-PCR for the detection of hepatitis A virus / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To establish a specific TaqMan-based Real-time PCR assay for the detection of hepatitis A virus in serum samples.</p><p><b>METHODS</b>According to the references, primers-probe sets which were located in 5'-NCR, the most conservative part of HAV genome were designed and therefore we established a TaqMan real-time RT-PCR assay with great performance of specificity, sensitivity and reproducibility. And then it was used in the detection of HAV RNA in serum from HAV patients.</p><p><b>RESULTS</b>The HAV Real-time RT-PCR assay established in this study were able to detect HAV RNA and its detection limit ranged from 0.1CCID50/reaction to 0.01CCID50/reaction. When the detection of a same sample was repeated for three times, coefficients of varistion (CV) of intra- and inter-assay were calculated and they were all less than 2.0% and 2.6% respectively. Our data suggested that there were 5.18 x 10(2) - 4.93 x 10(7) RNA copies in 1 ml of the serum from acute HAV patients.</p><p><b>CONCLUSION</b>The TaqMan-based Real-time PCR assay established in this study was specific and precise for the rapid detection of HAV RNA. It was applied successfully in the pathogen detection of clinical samples.</p>

Hepatitis A virus mimotope mapping by phage display peptide library / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>A 12 mer phage display peptide library was used to identify hepatitis A virus mimotopes of antigenic determinants, to provide the feasibility of virus epitope mapping by using this approach.</p><p><b>METHODS</b>Using purified anti-hepatitis A virus monoclonal antibody as affinity selective molecule, phage display peptide library was biopanned and positive clones were selected by ELISA, competition assay and DNA sequencing.</p><p><b>RESULTS</b>10 ELISA positive clones were chosen for DNA sequencing, and the displayed peptide sequences were deduced. 9 of them showed identical nucleotide sequence, and similarity in their amino acid sequence with VP1 of HAV HM175 was found, but no sequence homology was found between the other phage clone and the capsid proteins of HAV. Those peptides may behave as mimotopes of HAV.</p><p><b>CONCLUSION</b>The mimotope of HAV was selected by using phage display peptide library screening. The results provide the potential of this method to search for the mimotopes of the virus.</p>

Characterization of capsid protein VP3-VP1 genes of hepatitis A virus prevalent strains circulated in China / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To analyse the genetic characteristics of the capsid protein VP3-VP1 region of hepatitis A virus strains circulated in China.</p><p><b>METHODS</b>The nucleotide sequences of VP3-VP1-2A region of 42 HAV IgM positive serum samples were sequenced and analysed for nucleotide and amino acid identities and genetic characteristics of the VP3-VP1 region.</p><p><b>RESULTS</b>The nucleotide and amino acid identities in the VP1-2A junction region among the 42 strains were 89.1% - 100% and 97.3% - 100%; while in the complete VP3-VP1 region, the identities were 87.6%-100% and 98.8%-100%. Strains with identical nucleotide sequences in the VP1-2A junction region had 98.4%-100% nucleotide identity in the complete VP3-VP1 region and 0-2 amino acid differences in this region. There were no amino acid changes at neutralizing antigenetic sites of VP3-VP1 region within the 42 HAV strains.</p><p><b>CONCLUSION</b>All the 42 HAV strains belonged to genotype I, with 40 strains clustering to sub-genotype IA and 2 to sub-genotype IB. Different HAV strains analysed in this paper differed in the nucleotide sequences of the VP3-VP1 region, but the amino acid sequences were highly conserved with no changes at neutralizing antigenetic sites. Both the nucleotide and amino acid sequences of the strains with the same VP1-2A junction region were identical or closely related when compared in the complete VP3-VP1 region.</p>

The characterization analysis of HAV recombinant antigen was expressed by vaccinia virus vector / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To find a suitable cell line for hepatitis A antigen expressed by vaccinia virus vector and to find a way of inactivation and preservation of the HAV recombinant antigen. Methods Series of cell lines such as K4,143, HEL, Hep-2 and Vero were inoculated with vaccinia virus that can express HAV recombinant antigen. ELISA was used to determine the contents of expression antigen. The characterization of the HAV antigen expressed by vaccinia virus was then analyzed after it was treated with different methods.</p><p><b>RESULTS</b>The expression of HAV recombinant antigen in K4,143 and HEL cell lines was a little more than expression in Hep-2 and Vero cell lines. The antigenicity is obviously higher when HAV recombinant antigen was inactivated by beta-propiolactone other than it was inactivated by formalin. It was best to preserve the prepared HAV recombinant antigen under -40 degrees C condition.</p><p><b>CONCLUSIONS</b>The application of vaccinia virus vector in hepatitis A antigen preparation was very useful and promising.</p>

Genetic analysis of HAV strains isolated from patients with acute hepatitis in Hebei Shijiazhuang of China / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To characterize the hepatitis A virus (HAV) wild type strains circulating in Hebei Shijiazhuang of China during 2005-2007, to provide the bases for further investigation of the sources of HAV infection.</p><p><b>METHODS</b>The VP1/P2A junction regions were detected by RT-PCR from HAV IgM positives serum samples during 2005 and 2007, the 34 RT-PCR positive samples were sequenced and subjected to phylogenetic analysis by Neighbor Joining (NJ) method.</p><p><b>RESULTS</b>All the detected HAV strains were identified as sub-genotype I A, the homology of nucleotide sequence in the VP1-2A imation region ranged from 95%-100%, the amino acid sequences of HAV strains almost had no difference.</p><p><b>CONCLUSION</b>There are different HAV strains existing in Hebei Shijiazhuang of China, same HAV strain may exist in different areas; or in one area, identical or different HAV strains may be detected. This work provides the bases for further investigation of the sources of HAV infection and also for effectively control measures to prevent the spread of the disease.</p>

Genotyping of hepatitis A virus prevalent strains in Xinjiang Hetian of China in 2006 / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To analysis the genotypes of wild type hepatitis A virus circulated in Xinjiang Hetian of China in 2006.</p><p><b>METHODS</b>The Vp1-2A region of HAV genome was amplified and sequenced from serum samples collected in Xinjiang Hetian of China in 2006, and subjected to phylogenetic analysis by Neighbor Joining (NJ) method.</p><p><b>RESULTS</b>The nucleotide sequence differences in the VP1-2A region among Xinjiang Hetian HAV strains ranged from 0%-3.9%, all belonged to sub-genotype 1A. Genetically similar strains were identified among Xinjiang Hetian 2006 and Xinjiang Yili 2005 of China isolates. Only 0-2 amino acid differences were found among the Xinjiang Hetian HAV isolates in the VP1-2A region.</p><p><b>CONCLUSION</b>There were different HAV strains existing in the investigated areas, these strains may have different transmission pathways for the spread of the disease. The results indicate the usefulness of molecular epidemiological methods in studying changes in the circulating HAV strains and in tracing transmission routes, and also for effectively control measures to prevent the spread of the disease.</p>

Development of an extraction and concentration method for the detection of hepatitis A virus in different samples / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To develop an extraction and concentration method for the detection of hepatitis A virus (HAV) in shellfish, water, serum and saliva samples by nested RT-PCR.</p><p><b>METHODS</b>HAV were artificially inoculated into the above samples, calm sample was extracted using glycine buffer pH9.5, PEG precipitation; water sample was PEG precipitated directly; then all the samples including serum and saliva samples were extracted using Trizol regent, followed by nested RT-PCR detection using primers from HAV VP1-2A region.</p><p><b>RESULTS</b>The detection limit for HAV in cultured cell lysis was 0.1TCID50; in water, serum or salva sample was 1TCID50 respectively, in calm sample was 1-10 TCID50. HAV RNA was detected in water and sera samples collected from the HAV outbreak region, sequenced and analysis.</p><p><b>CONCLUSION</b>The method developed here is convenient, specific and capable of detecting low levels of HAV in different samples, would be useful for diagnostic laboratories in order to perform HAV analysis in cases of foodborne infections or for molecular epidemiology investigation of HAV outbreaks.</p>

Baculovirus expression of two human recombinant neutralizing IgG monoclonal antibodies to hepatitis A virus / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To develop human recombinant neutralizing IgG monoclonal antibodies to hepatitis A virus (HAV) by baculovirus expression system.</p><p><b>METHODS</b>The heavy and light chain genes of two human-derived neutralizing Fab antibodies to HAV were cloned into baculovirus expression vector Pac-kappa-Fc and Pac-L-Fc, and further expressed in insect cells as IgG antibodies. The IgG products were purified and well characterized.</p><p><b>RESULTS</b>The baculovirus expressed McAb HAFc16 fully retained the specificity of binding to hepatitis A virus and the competition with mouse anti-hepatitis A virus McAb using ELISA. The viral neutralization assay in vitro demonstrated the retention of antibody function after expression of the human antibody in insect cells. The other expressed antibody HAFc78 also has the neutralizing activity but it is directed against different epitopes of HAV when compared with HAFc16.</p><p><b>CONCLUSION</b>The recombinant baculovirus/insect cells expressed human neutralizing IgG antibodies to hepatitis A virus retained all biological functions specific for hepatitis A virus. The results provided the possibility of using these antibodies to rapidly protect high risk or early exposure populations from hepatitis A virus infection.</p>