Detection and characterization of hepatitis A virus and Norovirus in estuarine water samples using ultrafiltration – RT-PCR integrated methods

Aims:  Waterborne outbreaks of hepatitis A and Norovirus disease have been reported and associated with contaminated water supply in various countries. However, in Mexico, there are no studies that report HAV and NV presence in water. This study reports the application of ultrafiltration and RT-nested PCR methods to concentrate and identify these viruses.
Methods and Results:  Forty estuarine water samples were collected from the Huizache Caimanero Lagunary Complex. Samples were concentrated by ultrafiltration system (UFS) and RT-nested PCR was performed for HAV and NV identification. These viruses were found in 80% and 70% of the samples collected respectively and both were present in 57·5%. The DNA sequences analysis showed that 21 estuarine water samples were associated with HAV and 13 with NV. Faecal coliforms were isolated in 48·57% of the samples, while Escherichia coli were found in 34·28%.
Conclusions:  DNA sequencing showed that the genotype IB for HAV and GII for NV were predominant in México. No significant relationships were detected between indicators and viruses ( P  < 0·05).
Significance and Impact of the Study:  This study shows that the UFS is adequate for viral concentration. This is the first study analysing the genetic sequence of HAV and NV isolated from Mexican estuarine water.     

鸭甲型病毒性肝炎的研究进展

以下综述了鸭甲型肝炎病毒的命名与溯源、分子遗传与进化,分析了鸭甲型病毒性肝炎的流行病学、临床特征及监测手段,总结了国内外鸭甲型病毒性肝炎的研究现状,进一步阐述了研究该病的必要性和紧迫性,旨在为从事该领域的科研人员提供参考依据。     

Detection of hepatitis B virus in blood samples negative for surface antigen,by DNA probe hybridization assay

A DNA hybridization assay was developed using a cloned hepatitis B viral genome to detect the presence of infectious virions in human serum. The merit of this assay was to put in evidence virus particles in 7 out of 133 sera that were negative for surface antigen (HBsAg) using routine serological methods. The usefulness of this assay was confirmed by actual visualization of the virus under electron microscope. Some serum samples although positive for surface antigen, did not give a hybridization signal by dot blot assay and might indicate cases of acute hepatitis     

Expression and characterization of hepatitis C virus core protein fused to hepatitis B virus core antigen

Recombinant plasmids were constructed by fusing the gene fragments encoding the full-length (1-191aa) and the truncated (1-40aa and 1-69aa) HCV core proteins (HCc) respectively to the core gene of HBV at the position of amino acid 144 and expressed in E. coli. The products were analyzed by ELISA, Western blotting as well as the immunization of the mice. The results showed that those fusion proteins (B144C191, B144C69, B144C40) possessed the dual antigenicity and immunogenicity of both hepatitis B virus core antigen (HBcAg) and hepatitis C virus core protein (HCc). Analysis by electron microscopy and CsCl density gradient ultra-centrifugation revealed that similar to the HBcAg itself, all fusion proteins were able to form particles. Comparison of the antigenicity and immunogenicity of those fusion proteins showed that the length of HCc gene fused to HBeAg had no much effect on the antigenicity and immunogenicity of HBcAg, however, B144C69 and B144C40 induced higher titres antibodies against HCc than B14d     

Detection of Legionella pneumophila in water samples by species-specific real-time and nested PCR assays

AIMS: Legionella pneumophila is a contaminant of man-made water systems, including potable water, cooling towers, water systems of large buildings, etc. It is the most common causative agent of legionellosis, a respiratory infection, which may give rise to restricted outbreaks. To survey environmental water samples from hospitals and private habitations in Bologna, we developed a species-specific nested and a TaqMan real-time PCR for the detection of L. pneumophila. We compared the two assays and both to cultural isolation. METHODS AND RESULTS: The targeted gene was macrophage infectivity potentiator (mip), conserved in L. pneumophila, and divergent in other legionellae. One assay was based on a nested PCR and the other on a TaqMan real-time PCR protocol. Their sensitivities were 14 % or 5% higher than that of cultural isolation respectively. The detection limits were 1-2 genome equivalents per 50-microl reaction. Specificity was assessed using DNA from nine target and 20 nontarget organisms. CONCLUSIONS: When applied to water samples, both assays detected L. pneumophila at 80% or higher frequency. SIGNIFICANCE AND IMPACT OF THE STUDY: The species-specific molecular diagnosis of L. pneumophila by means of nested PCR does not require a specific instrumentation, exhibits a high sensitivity, and is advantageous over the cultural isolation and real-time PCR detection. It allows to quickly monitor water samples for the risk assessment of environmental contaminations.     

Incidence and molecular characterization of hepatitis A viruses in Korean surface water between 2007 and 2010

Several bodies of surface water in Korea were surveyed for the presence of hepatitis A virus (HAV) between 2007 and 2010. Of 265 surface water samples, 9 (3.4%) were HAV‐positive. HAVs were mainly detected in the summer (3/62, 4.8%) and spring (4/96, 4.2%) seasons. Comparing different water sources, the highest prevalence (6.6%) of positive samples was seen in lake water, four HAV‐positive samples being from lakes. Comparing prevalence rates across the four representative Korean basin systems, no HAVs were found in the Han or Nakdong river basins. The highest HAV prevalence was found in samples from the Yeongsan river and other basins (6.3%); the Geum/Seom river was also found to have a high HAV prevalence (5.7%). HAVs from the nine positive samples were then sequenced and analyzed phylogenetically. Two of the HAVs belong to genotype IA and fall within the same cluster as HAVs 6‐3(ASAN4) (EU049548), KANSAN‐PS1 (EU049554), and ASAN‐KM (EU049563), which were collected from the stools of patients with gastroenteritis in Korea. The seven other HAV nucleotide sequences belong to the genotype IB cluster. This is the first nationwide surveillance of HAV in major Korean water sources.     

Interference of hepatitis A virus replication by small interfering RNAs

The rate of acute liver failure due to hepatitis A virus (HAV) has not decreased, and therapy of severe infections is still of major interest. Using a DNA-based HAV replicon cell culture system, we demonstrate that small interfering RNAs (siRNAs) targeted against viral sequences or a reporter gene contained in the viral genome specifically inhibit HAV RNA replication in HuhT7 cells. Combinations of siRNAs were more effective suppressors of HAV RNA replication. Also, siRNAs targeted against HAV 2C and 3D inhibited the expression of the respective protein. Expressions of endogenous beta-actin and double-stranded-specific RNA-activated serin/threonine kinase (PKR) were unaltered, demonstrating that the siRNA inhibitory effect was not connected to interferon inhibition, but rather was specifically targeted against HAV RNA. These results suggest that RNA interference might ultimately be useful in treatment of severe HAV infection with or without chronic liver diseases.     

鸭肝炎病毒新血清型基因组序列分析

[目的]为了揭示鸭肝炎病毒新血清型(DHV-N)G株的演化及其与DHV-1的基因序列差异.[方法]运用RT-PCR结合5'-/3'-RACE策略对DHV-N G株基因组进行扩增,克隆测序后进行序列分析.[结果]DHV-NG株全基因组序列除12 nt poly(A)尾外全长共7774 nt,含有一个大的开放阅读框,编码2251 aa的蛋白前体,其基因组结构:5'UTR-L-VP0-VP3-VP1-2A1-2A2-2B-2C-3A-3B-3C-3D-3'UTR;3'UTR长为366 nt,比1型鸭肝炎病毒C80株多52 nt;VP1蛋白与DHV-1相比发生较大的变异,特别在其第140～221位;其基因组与DHV-1、韩国DHV-N和台湾DHV-N的同源率分别为72.8%～73.4%、96.3%～96.5%和78.3%.[结论]DHV-N G株基因组结构与DHV-1存在明显差异,在DHV-N成员DHV-N G株与韩国DHV-N关系更为密切.     

西北农林科技大学动物医学院，兽医免疫学研究所，杨凌712100

禽戊型肝炎病毒(Hepatitis E virus,HEV)与人、猪HEV同属于肝炎病毒属,它们在遗传性和抗原性上有一定的相关性。自禽HEV被分离鉴定以来,许多国家从血清学或分子流行病学方面证实了该病毒的存在和流行。目前,GenBank上共有5个禽HEV的全基因组或接近全基因组的序列,分为3个基因型,并且其全基因组包含3个ORFs,其中ORF2基因编码病毒的衣壳蛋白,包含病毒主要的抗原表位,是血清学检测和疫苗设计的主要靶蛋白。禽HEV由于其对家禽养殖业的危害以及人畜共患的可能性,正被引起越来越多的关注。本文结合国内禽HEV的分离鉴定从禽HEV病原学、致病性以及衣壳蛋白抗原性等方面进行了总结概述。     

Mechanistic and kinetic characterization of hepatitis C virus NS3 protein interactions with NS4A and protease inhibitors

The mechanism and kinetics of the interactions between ligands and immobilized full‐length hepatitis C virus (HCV) genotype 1a NS3 have been characterized by SPR biosensor technology. The NS3 interactions for a series of NS3 protease inhibitors as well as for the NS4A cofactor, represented by a peptide corresponding to the sequence interacting with the enzyme, were found to be heterogeneous. It may represent interactions with two stable conformations of the protein. The NS3–NS4A interaction consisted of a high‐affinity (K_D = 50 nM) and a low‐affinity (K_D = 2 µM) interaction, contributing equally to the overall binding. By immobilizing NS3 alone or together with NS4A it was shown that all inhibitors had a higher affinity for NS3 in the presence of NS4A. NS4A thus has a direct effect on the binding of inhibitors to NS3 and not only on catalysis. As predicted, the mechanism‐based inhibitor VX 950 exhibited a time‐dependent interaction with a slow formation of a stable complex. BILN 2061 or ITMN‐191 showed no signs of time‐dependent interactions, but ITMN‐191 had the highest affinity of the tested compounds, with both the slowest dissociation (k_off) and fastest association rate, closely followed by BILN 2061. The k_off for the inhibitors correlated strongly with their NS3 protease inhibitory effect as well as with their effect on replication of viral proteins in replicon cell cultures, confirming the relevance of the kinetic data. This approach for obtaining kinetic and mechanistic data for NS3 protease inhibitor and cofactor interactions is expected to be of importance for understanding the characteristics of HCV NS3 functionality as well as for anti‐HCV lead discovery and optimization. Copyright © 2010 John Wiley & Sons, Ltd.     

Detection of replication-defective hepatitis A virus based on the correlation between real-time polymerase chain reaction and ELISA in situ results

ELISA in situ can be used to titrate hepatitis A virus (HAV) particles and real-time polymerase chain reaction (RT-PCR) has been shown to be a fast method to quantify the HAV genome. Precise quantification of viral concentration is necessary to distinguish between infectious and non-infectious particles. The purpose of this study was to compare cell culture and RT-PCR quantification results and determine whether HAV genome quantification can be correlated with infectivity. For this purpose, three stocks of undiluted, five-fold diluted and 10-fold diluted HAV were prepared to inoculate cells in a 96-well plate. Monolayers were then incubated for seven, 10 and 14 days and the correlation between the ELISA in situ and RT-PCR results was evaluated. At 10 days post-incubation, the highest viral load was observed in all stocks of HAV via RT-PCR (10⁵ copies/mL) (p = 0.0002), while ELISA revealed the highest quantity of particles after 14 days (optical density = 0.24, p < 0.001). At seven days post-infection, there was a significant statistical correlation between the results of the two methods, indicating equivalents titres of particles and HAV genome during this period of infection. The results reported here indicate that the duration of growth of HAV in cell culture must be taken into account to correlate genome quantification with infectivity.     

Detection of hepatitis E virus antibodies among Cercopithecidae and Hominidae monkeys in Cameroon

We screened hepatitis E from 15 species of non‐human primates. Anti‐HEV IgG was detected in 11.1% (1/9) Mandrillus sphinx, 14.3% (2/14) Gorilla gorilla, 5.9% (4/67) pan troglodytes and 8.7% (2/23) Mandrillus leucophaeus, whereas anti‐HEV IgM was detected in 1.5% (1/18) papio Anubis, 28.6% (2/7) Cercocebus agilis and 1.5% (1/67) pan troglodyte.     

乙型肝炎病毒B2和C2亚型中SSRs的比较分析

乙型肝炎病毒(hepatitis B virus,HBV)属于嗜肝DNA病毒科(hepadnaviridae),它分布在世界各地并严重危害人类的健康。本文从NCBI的GenBank中下载了106条B2亚型和130条C2亚型的基因组全长序列,以下载的数据为材料,分析简单重复序列(simple sequence repeats,SSRs)在B2和C2亚型基因组序列中的分布情况。结果显示,简单重复序列的重复次数比较少;二型简单重复序列在研究的五种简单重复序列类型中占绝对优势;这可能与B2和C2亚型基因组序列有较高的突变率有关。同时还发现最普遍的SSRs、序列间SSRs的平均相对丰度和平均相对密度在B2和C2亚型中分布情况相似。     

Using immunoglobulin Y as an alternative antibody for the detection of hepatitis A virus in frozen liver sections

An increasing amount of research has been conducted on immunoglobulin Y (IgY) because the use of IgY offers several advantages with respect to diagnostic testing, including its easy accessibility, low cost and translatability to large-scale production, in addition to the fact that it can be ethically produced. In a previous work, immunoglobulin was produced and purified from egg yolks (IgY) reactive to hepatitis A virus (HAV) antigens. In the present work, this anti-HAV-specific IgY was used in an indirect immunofluorescence assay to detect viral antigens in liver biopsies that were obtained from experimentally infected cynomolgus monkeys. Fields that were positive for HAV antigen were detected in liver sections using confocal microscopy. In conclusion, egg yolks from immunised hens may be a reliable source for antibody production, which can be employed for immunological studies.     

Detection of hepatitis C virus RNA in the hearts of patients with hepatogenic cardiomyopathy

We examined the Hepatitis C virus (HCV) genome in the myocardium and liver obtained at autopsy from seven patients with HCV-positive liver cirrhosis and hepatocellular carcinoma (HCC) by in situ hybridization and histopathological studies. The HCV virus genome was detected in the myocardium of one patient as well as in the liver in three out of seven patients. However, Epstein-Barr (EB) virus genome could not be detected in liver or myocardium. In the patient who showed positive reaction to HCV in myocardium, both serum HCV and Hepatitis B virus (HBV) antibodies were positive. It is unknown whether this was related to an immunological abnormality of the host or to an interaction between RNA and DNA viruses. In conclusion, we could identify the HCV genome in the myocardium of a patient with hepatogenic myocardosis.     

Construction and characterization of the chimeric antibody 8C11 to the hepatitis E virus

Homodimers of the truncated hepatitis E virus (HEV) capsid proteins, E2 and p239, were conformed to model the dominant antigenic determinants of HEV. Using E2 as an immunogen, two neutralizing monoclonal antibodies (mAbs), namely 8C11 and 8H3, were produced. We constructed a mouse-human chimeric antibody derived from 8C11 and its expression in Chinese hamster ovary (CHO) cells. cDNAs encoding variable regions of heavy and light chains were isolated from hybridoma cells and inserted into mammalian expression vectors containing cDNA of human gamma-1 and kappa constant regions, respectively. The vectors were then cotransfected into CHO cells, and a stable cell line was established. Results from indirect enzyme-linked immunosorbent assay (ELISA) and Western blot analysis showed that the chimeric antibody was assembled correctly to the native IgG molecule and could be secreted from the cells. Similar to the original mAb, the expressed chimeric antibody displayed HEV antigen-binding activity and an enhancement effect on 8H3 binding to HEV antigen. The chimeric antibody could specifically inhibit the binding of p239 to HepG2 cells and compete with HEV IgG in positive serum by antibody-competitive ELISA. The chimeric antibody is expected to be less immunogenic in human and more suitable for antibody therapy of hepatitis E.