Acute transverse myelitis and hepatitis C virus

In the last few years, substantial evidence has been provided on peripheral nervous involvement in infection by hepatitis C virus (HCV), whilst central nervous involvement is rare. Here, we report a case of acute transverse myelitis in a woman developing 4 years after documented HCV seropositivity, associated with intrathecal anti-HCV protein IgG. Isoelectrofocusing of all CSF samples before and after immunoabsorption with recombinant structural HCV proteins revealed disappearance or marked decrease of some oligoclonal IgG bands suggesting binding to HCV proteins. To our knowledge, this is the first report of acute myelitis associated with intrathecal immune response against HCV proteins. This finding suggests that in acute myelitis of unknown aetiology, testing CSF for HCV RNA and related antibodies is warranted.     

含恩替卡韦耐药基因的HBV全基因组逆转录病毒载体的构建及包装

目的：构建含恩替卡韦耐药基因的HBV全基因组逆转录病毒载体,同时进行逆转录病毒载体的包装,为进一步产生含恩替卡位耐药基因的假病毒,进一步感染肝源性细胞,进行耐药机制的研究做准备。方法: 采用基因扩增、重组和克隆的方法,从构建好的含恩替卡位耐药基因的重组质粒中,扩增出含恩替卡位耐药基因的HBV全基因组,然后插到pLEGFP-N1载体上,再进行重组质粒的转化、提取、纯化和测序,然后将测序成功的重组质粒扩大培养,并转染PT67细胞,最后通过荧光倒置显微镜观察和提取细胞培养上清进行病毒DNA提取。结果: 凝胶电泳分析血清HBV DNA扩增可见3.2 kb条带,HBV DNA和pLEGFP-N1载体连接后可见10.1 kb目的条带,该重组质粒双酶切后可见3.2 kb和6.9 kb目的条带。突变前质粒测序显示含有HBV基因型为C型血清型adr的全基因组,突变后测序证实存在HBV逆转录酶169、184、202、250位点的预期碱基突变。荧光倒置显微镜显示,转染后的PT67细胞有明显的荧光表达,培养上清DNA提取后,经过核酸分析仪检测,最后换算结果显示培养上清假病毒颗粒可达104-105/cfu。结论: 成功构建了含恩替卡韦耐药基因的HBV全基因重组质粒,并进行了包装,产生了含恩替卡位耐药基因的假病毒颗粒。     

Analysis of the full-length genome of genotype 4 hepatitis E virus isolates from patients with fulminant or acute self-limited hepatitis E

It was suggested that hepatitis E virus (HEV) genotype 4 is associated more closely with the severity of hepatitis E than genotype 3, although the virological basis remains unknown. The aim of this study was to examine whether genomic differences among genotype 4 HEVs are responsible for the development of fulminant hepatitis. Full-length sequences of genotype 4 HEVs from three patients with fulminant hepatitis and six patients with acute self-limited hepatitis were determined. The sequences were analyzed with those of 13 genotype 4 HEV isolates whose entire nucleotide sequence is known. Analysis of 22 full-length sequences (fulminant hepatitis, 5; acute hepatitis, 17) revealed that C at nt 1816 and U at nt 3148 (U3148), both of which do not change the amino acid sequences, were significantly associated with fulminant hepatitis (P = 0.0489, respectively). When partial nucleotide sequences containing nt 1816 or nt 3148 were determined in 16 additional HEV isolates of genotype 4, a closer association between U3148 and fulminant hepatitis (P = 0.0018) was observed. The comparison of 86 HEV isolates of all four genotypes showed that U3148 had a stronger association with fulminant hepatitis than other nucleotides at nt 3148 (P = 0.0006). Patients infected with HEV with U3148 had a significantly lower value of the lowest prothrombin activity (P = 0.0293). Nt 3148 is located within the RNA helicase domain, and 22-nt sequence including nt 3148 was well conserved among all genotypes. A silent substitution of U3148 in HEV may be associated with the development of fulminant hepatitis. Further studies are needed to clarify the underlying mechanism.     

Sustained elimination of hepatitis B virus from serum induced in a patient with chronic hepatitis B and advanced human immunodeficiency virus infection

A 48-year-old male patient was admitted with acquired immunodeficiency syndrome (stage III, Centers for Disease Control 1993) and viremic hepatitis B. Blood CD4 count was 15/l. Discontinuation of prednisolone, previously prescribed by the patient's family practitioner because of elevated liver enzymes, resulted in severe hepatitis (alanine aminotransferase > 300U/1). Administration of interferon-, (9 × 10⁶U s.c. 3 × weekly) was initiated. Serum markers of viral replication disappeared, and aminotransferase levels returned to normal within a few weeks. The patient's serum was found negative for HBsAg after 3 months. Immunohistochemical analysis of liver biopsies before and during interferon therapy showed disappearance of all hepatitis B virus antigens and a marked reduction in inflammatory activity. Hepatitis B virus seroconversion remained stable until the patient died from the syndrome 2 years later. This case shows that in spite of severe HIV-associated immune deficiency with CD4 counts constantly below 100/l, interferon- can lead to sustained serological and histological improvement of viremic hepatitis B. Previous administration and discontinuation of cortisone may have helped to reach this effect.Abbreviations HBV hepatitis B virus - HIV human immunodeficiency virus - IFN interferon Correspondence to: G. Gerken     

High replicative full-length lamivudine-resistant hepatitis B virus isolated during acute exacerbations

During chronic HBV infections, exacerbations of disease usually occur without clearly understood mechanisms. In this study, full-length HBV genomes were analyzed from four chronic hepatitis B patients who developed resistance to lamivudine [-2'-deoxy-3'-thiacytidine, LMV] accompanied by acute exacerbation of disease. Paired full-length HBV isolates were cloned from the sera of patients prior to LMV treatment and after drug resistant breakthrough isolates emerged with exacerbation. Compared to the isolates before treatment, isolates from all four patients during exacerbation showed marked increase in replicative competence by cell transfection study. Viral genome amplification and direct sequencing was used further to study the sequence differences between the dominant species and the clones used for functional analysis. Apart from mutations at the YMDD motif, no shared mutations were shown between all isolates. The isolates from the one patient who recovered from the exacerbation showed a lower number of mutations, and in particular, lacked basal core promoter (BCP) mutations at 1762/1764. In contrast, BCP mutations were found in isolates from the other three patients. Thus, in patients with acute exacerbation, high replicative strains might be selected from the total HBV quasispecies during treatment, and amongst these strains, those with core promoter mutations were most likely to be associated with severe clinical exacerbations.     

Development of a drug assay system with hepatitis C virus genome derived from a patient with acute hepatitis C

We developed a new cell culture drug assay system (AH1R), in which genome-length hepatitis C virus (HCV) RNA (AH1 strain of genotype 1b derived from a patient with acute hepatitis C) efficiently replicates. By comparing the AH1R system with the OR6 assay system that we developed previously (O strain of genotype 1b derived from an HCV-positive blood donor), we demonstrated that the anti-HCV profiles of reagents including interferon-γ and cyclosporine A significantly differed between these assay systems. Furthermore, we found unexpectedly that rolipram, an anti-inflammatory drug, showed anti-HCV activity in the AH1R assay but not in the OR6 assay, suggesting that the anti-HCV activity of rolipram differs depending on the HCV strain. Taken together, these results suggest that the AH1R assay system is useful for the objective evaluation of anti-HCV reagents and for the discovery of different classes of anti-HCV reagents.     

流行性乙型脑炎病毒脑脊液分离株"47株"全基因组特征分析

目的 对1950年分离自我国黑龙江省患者脑脊液的乙脑病毒"47株"进行全基因组序列的测定和分析,全面了解其全基因组特征.方法 复苏毒种提取病毒RNA,使用自行设计的乙脑病毒全基因组扩增测序引物,完成对病毒全基因组序列的测定.采用DNAStar、Modeltest、Phylip等生物软件完成全基因组核苷酸、氨基酸序列差异分析和乙脑病毒全基因组的系统进化分析.结果 乙脑病毒"47株"全长10 977个核苷酸.96至10 391位为开放读码框ORF,共10 296个核苷酸,编码3432个氨基酸."47株"与5株疫苗株在全基因组水平的核苷酸差异在2.4%～4.4%之间,氨基酸差异在0.3%～1.1%之间.乙脑病毒全基因组最适进化模型为GTR+I+G.全基因组进化分析显示"47株"属于基因Ⅲ型乙脑病毒.结论 "47株"全基因组核苷酸和氨基酸高度保守,属于基因Ⅲ型乙脑病毒.     

丙型肝炎病毒全长cDNA模板的构建及鉴定

目的 构建具有功能的丙型肝炎病毒(HCV)全长cDNA克隆。方法 应用长模板RTPCR法扩增1份上海地区HCV感染者血清的RNA(基因型为1b)，分段扩增、融合拼接成9．2kb的基因片段，克隆人含有HCV基因两端非编码区序列的载体作为模板。为检测此过程是否发生对特定变异株的选择，分析4个独立克隆的HVRl序列。对原核细胞中表达的HCV核心蛋白、NS3蛋白酶及解旋酶，以蛋白印迹实验确证其免疫反应性。并构建NS3／4A-SEAP表达系统检验NS3的蛋白酶活性。结果 获得丙型肝炎病毒全长cDNA模板。不同克隆间HVR1序列存在较大差异，提示长模板RT-PCR所制备HCVcDNA具有HCV基因准种(quasispecies)的特性。该模板编码基因在原核细胞内得到高效表达，并具有良好的免疫反应性。在NS3／4A-SEAP表达系统内，NS3可切割、释放其下游的SEAP，具有蛋白酶活性。结论 本工作为构建全长功能性HCV cDNA模板及感染性克隆奠定了基础。     

Analysis of hepatitis B virus quasispecies distribution in a Korean chronic patient based on the full genome sequences

Although Korea is a hepatitis B virus (HBV) endemic area, relatively few full-length genome sequences are available. In particular, no comparative analysis has been performed on the full-genome sequences of different HBV quasispecies from a single Korean patient. This report describes the full-length sequences of five HBV clones (two clones with shorter PCR amplicons and three clones with longer amplicons). Large deletions, that is, 685-bp, 487-bp, and 144-bp, that might interfere with the production of normal proteins were observed in four of five clones. Double mutations in the basal core promoter (BCP) region (T1762/A1764) were detected in two clones but no precore mutations (A1896) were detected in any of the five clones. These data support previous results that genotype C, in particular Korean clones of this genotype, is prone to mutations. Two independent mechanisms, namely, the deletions of long lengths and amino acid substitutions followed by BCP double mutations might contribute to the diversity of HBV quasispecies. Considering the importance of HBV quasispecies as HBV variant sources, the distribution of HBV quasispecies in mutation prone genotype C prevalent areas like Korea, should be monitored to improve the management of chronic HBV infections and to control HBV variants that arise due to the administration of vaccine or antiviral therapy.     

Analysis of the full-length genome of hepatitis E virus isolates obtained from farm pigs in Mongolia

Although no outbreaks of hepatitis E have been reported in Mongolia, a significant proportion of the general population had antibodies to hepatitis E virus (HEV). To investigate whether pigs are possible reservoirs of HEV in Mongolia, serum samples obtained from 243 2- or 3-month-old pigs on four swine farms surrounding Ulaanbaatar, the capital city of Mongolia, were tested for the presence of anti-HEV antibodies and HEV RNA. Overall, 223 pigs (91.8%) tested positive for anti-HEV, while 89 pigs (36.6%) had detectable HEV RNA. The 89 HEV isolates obtained from the viremic pigs were 78.7-100% identical to each other, and 80.9-85.9% similar to the prototype genotype 3 HEV isolate (US1) in the 412-nucleotide (nt) sequence within open reading frame 2. They were classified into two novel phylogenetic groups within genotype 3, differing by 16.4-21.3%. The swMN06-A1288 and swMN06-C1056 isolates, representing each of the two clusters within genotype 3, had a genomic length of nucleotides (nt) 7,222 nt and 7,223 nt, respectively, excluding the poly(A) tail, and shared only 81.6% over the entire genome. Upon comparison with the 25-reported genotype 3 HEV isolates over the entire genome, swMN06-A1288 had identities of merely up to 84.9%, while swMN06-C1056 of only up to 85.9%. Phylogenetic analysis confirmed the remote relatedness of the Mongolian swine isolates to the genotype 3 HEV isolates reported thus far. These results indicate that farm pigs in Mongolia are frequently infected with presumably indigenous HEV strains of genotype 3 and could be a source of HEV infections in humans in Mongolia.     

Properties of hepatitis B virus genome recovered from Vietnamese patients with fulminant hepatitis in comparison with those of acute hepatitis

Among the many mutations found in the hepatitis B virus (HBV) genome, some have been associated with fulminant hepatitis, as exemplified by precore-defective mutations. The aim of this study was to determine whether such mutations also are found in Vietnamese cases of fulminant hepatitis B. The full-genome nucleotide sequence of HBV in three patients with fulminant hepatitis (F-2, F-3, and F-6) and one with acute hepatitis (A-3), who were admitted to Cho Ray Hospital, Ho Chi Minh City, Vietnam was ascertained. Additionally, two patients with fulminant hepatitis (F-1 and F-7) and three with acute hepatitis (A-1, A-2, and A-5) were examined only for the precore/core region of HBV. Remarkably, the nonsense mutation at precore codon 28 (Trp82Stop) was found in four of the five patients with fulminant hepatitis, while all the acute hepatitis patients harbored wild type (one had a mixture of wild and mutant types). The missense mutations within the core region, Ile97Leu and Pro130Ile/Thr/Ser, were also remarkable in fulminant hepatitis. Only F-2 was free from these precore/core mutations, but F-2 was unique in that it possessed a chimeric genotype: it could be classified into genotype C as a whole, but its X region was of genotype B, like the other four fulminant hepatitis isolates (F-1, F-3, F-6, and F-7). The codon 41 of the X protein was Pro in all three fulminant hepatitis cases examined for this region, while it was Ser in the wild-type isolates of genotype B. Of note as negative data, the mutations C1653T and T1753M of the enhancer II (Enh II) and A1762T and G1764A of the precore/core promoter regions, once reported to be relevant to severe or fulminant hepatitis, were not found in the present cases. The results with the Vietnamese cases of fulminant hepatitis corroborated results of previous studies with respect to the mutations Trp28Stop of precore and Ile97Leu and Pro130Ile/Thr/Ser of core, but not for the mutations within Enh II and precore/core promoter region. Whether the Ser41Pro mutation in the X region of genotype B HBV is Vietnam-specific or disease-specific deserves further investigation.     

Analysis of the full-length genome of hepatitis A virus isolated in South America: heterogeneity and evolutionary constraints

Hepatitis A virus (HAV) is a hepatotropic member of the family Picornaviridae. Currently, the entire nucleotide sequence is available for only 26 HAV isolates. The complete genome sequence of genotype IA HAV from strains isolated in South America, where genotype IA is the most prevalent genotype, remains unknown. In this study, the complete nucleotide sequence was determined for a genotype IA HAV isolate recovered from a Uruguayan patient (HAV5). Phylogenetic analysis performed using HAV5 and all available full-length IA genotype HAV strains revealed a high synonymous substitution rate throughout the HAV polyprotein. The results of these studies revealed strong selection against amino acid replacements along the HAV polyprotein and may explain, at least in part, the presence of a single HAV serotype.     

Mutations within the hepatitis B virus genome among chronic hepatitis B patients with hepatocellular carcinoma

Chronic hepatitis B infection is a major cause of hepatocellular cancer (HCC). The pathogenesis of the carcinogenesis is not fully understood. Viral proteins such as the X protein and the truncated middle S protein have been implicated to be transactivators. In order to investigate whether any mutations within relevant parts of the hepatitis B virus (HBV) genome could be associated with the development of HCC, the genomes of 16 HBV strains from chronic HBV carriers with HCC were studied. Serum samples were subjected to PCR and the HBV DNA sequenced subsequently. Genotypes A-D were represented. The sequence analysis showed that an especially high proportion, 50% (CI 95%, 25-75%), of the patients with HCC carried HBV mutants with deletions or insertions in the N-terminal half of the pre-S2 region or had a point mutation in the start codon of pre-S2 compared with controls with chronic HBV infection, 21% (CI 95%, 3-39%). A high proportion (69%) also had mutations at position 1762 (A --> T) and/or 1764 (G --> A) in the core promoter region, but the proportion of core promoter mutations was no different from what was found in a control group of HBV carriers without HCC (68%). The pre-S2 variants, which involve deletions of immunogenic regions, may have a survival advantage as they are mostly found in long-standing HBV infection. There were no other mutations found frequently within the region coding for the X protein.     

Serum hepatitis B virus DNA in acute hepatitis B

With the use of the dot blot hybridization technic, sera from 30 consecutive patients with acute hepatitis B were examined for the presence of hepatitis B virus (HBV)-DNA. In an additional five patients with acute hepatitis B, serial serum samples, beginning before the serum alanine amino transferase elevation to the clearance of hepatitis B surface antigen, also were tested for various hepatitis B virus markers, including HBV-DNA. Thirteen of the 30 patients (43%) had circulating HBV-DNA and HBeAg at the time of their first clinic visit. However, 11 additional patients with HBeAg did not have HBV-DNA in their sera. In the 13 patients with HBV-DNA, there was no correlation between the titers of HBeAg and the levels of HBV-DNA. Examination of the serial serum samples from the additional five patients showed HBV-DNA and HBeAg to be present several days before the peak of serum alanine amino transferase. In all five patients, HBV-DNA was present in the serum before the appearance of IgM anti-HBc. However, HBsAg was present in all these patients' sera at the time of HBV-DNA positivity. None of the patients in this study became chronic carriers of hepatitis B virus.     

Analysis of antibody reactivity in paired cerebrospinal fluid and serum of a relapsing remitting multiple sclerosis patient

Increasing evidence indicates an involvement of B cells in multiple sclerosis (MS). However, little is known about antigenic targets recognized by antibodies present in blood and cerebrospinal fluid (CSF) of MS patients. This study was therefore aimed at identifying the antigen reactivity of antibodies present in CSF and compares the identified antibody profile with that of the serum of the same patient using cDNA phage display. Selection rounds on paired CSF and serum of this patient identified 13 antigenic targets of which 5 were enriched by serum antibodies and 2 were identified by CSF antibodies. Interestingly, the six remaining antigenic targets were shown to be recognized by both CSF and serum antibodies. These findings point towards both common as well as distinct antibody profiles in CSF and serum of MS patients.     

Delta virus superinfection in a patient with chronic viral hepatitis B

Hepatitis-Delta virus is a defective RNA virus enclosing the Delta antigen and genome within a coat of hepatitis B surface antigen (HBsAg). Prevalence of Delta virus is limited to individuals with hepatitis B infection. Two modes of infection have been identified: coinfection with hepatitis virus B of healthy individuals and superinfection of HBsAg carriers. In a prospective study of 485 liver biopsies from HBsAg seropositive patients, 320 non-replicative HBsAg carriers (without evidence of core antigen) were discovered. In only one of these cases could a Delta virus superinfection be confirmed by immunohistology. It caused a shift from a preexisting chronic persistent to a chronic active hepatitis. This is the first case of chronic Delta virus infection confirmed by immunhistology in the G.D.R.