Multiple human herpesvirus-8 infection

In Malawian patients with Kaposi sarcoma (KS) and their relatives, we investigated nucleotide-sequence variation in human herpesvirus-8 (HHV-8) subgenomic DNA, amplified from oral and blood samples by use of polymerase chain reaction. Twenty-four people had amplifiable HHV-8 DNA in >1 sample; 9 (38%) were seropositive for human immunodeficiency virus type 1, 21 (88%) were anti-HHV-8-seropositive, and 7 (29%) had KS. Sequence variation was sought in 3 loci of the HHV-8 genome: the internal repeat domain of open-reading frame (ORF) 73, the KS330 segment of ORF 26, and variable region 1 of ORF K1. Significant intraperson/intersample and intrasample sequence polymorphisms were observed in 14 people (60%). For 3 patients with KS, intraperson genotypic differences, arising from nucleotide sequence variations in ORFs 26 and K1, were found in blood and oral samples. For 2 other patients with KS and for 9 people without KS, intraperson genotypic and subgenotypic differences, originating predominantly from ORF K1, were found in oral samples; for the 2 patients with KS and for 4 individuals without KS, intrasample carriage of distinct ORF K1 sequences also were discernible. Our findings imply HHV-8 superinfection

Hepatitis B virus genotype assignment using restriction fragment length polymorphism patterns

AbstractHepatitis B virus (HBV) is classified into genotypes A–F, which is important for clinical and etiological investigations. To establish a simple genotyping method, 68 full-genomic sequences and 106 S gene sequences were analyzed by the molecular evolutionary method. HBV genotyping with the S gene sequence is consistent with genetic analysis using the full-genomic sequence. After alignment of the S sequences, genotype specific regions are identified and digested by the restriction enzymes, HphI, NciI, AlwI, EarI, and NlaIV. This HBV genotyping system using restriction fragment length polymorphism (RFLP) was confirmed to be correct when the PCR products of the S gene in 23 isolates collected from various countries were digested with this method. A restriction site for EarI in genotype B was absent in spite of its presence in all the other genotypes and genotype C has no restriction site for AlwI. Only genotype E is digested with NciI, while only genotype F has a restriction site for HphI. Genotype A can be distinguished by a single restriction enzyme site for NlaIV, while genotype D digestion with this enzyme results in two products that migrates at 265 and 186 bp. This simple and accurate HBV genotyping system using RFLP is considered to be useful for research on HBV

A functional polymorphism in the epidermal growth factor gene predicts hepatocellular carcinoma risk in Japanese hepatitis C patients

Background: A single nucleotide polymorphism (SNP) in the epidermal growth factor (EGF) gene (rs4444903) has been associated with increased risk of cancer, including hepatocellular carcinoma (HCC). The aim of this study was to examine the relationship between the EGF SNP genotype and the development and prognosis of HCC, in a Japanese population. Methods: Restriction fragment-length polymorphism was used to determine the presence of the EGF SNP genotype in 498 patients, including 208 patients with HCC. The level of EGF messenger ribonucleic acid (mRNA) expression in cancerous tissues was measured by quantitative reverse transcription polymerase chain reaction. The correlation between the EGF SNP genotype and prognosis was statistically analyzed in the patients with HCC. Results: The proportion of the A/A, A/G, and G/G genotypes were 5.3%, 42.8%, and 51.9%, respectively, in the patients with HCC, whereas in those without HCC, they were 8.6%, 35.9%, and 55.5%, respectively, revealing that the odds ratio (OR) of developing HCC was higher in patients with a G allele (OR =1.94, P=0.080 for A/G patients and OR =1.52, P=0.261 for G/G patients, as compared with A/A patients). In particular, when the analysis was limited to the 363 patients with hepatitis C, the OR for developing HCC was 3.54 (P=0.014) for A/G patients and was 2.85 (P=0.042) for G/G patients, as compared with A/A patients. Tumoral EGF mRNA expression in G/G patients was significantly higher than that in A/A patients (P=0.033). No statistically significant differences were observed between the EGF SNP genotype and diseasefree or overall survival. Conclusion: The EGF SNP genotype might be associated with a risk for the development of HCC in Japanese patients but not with prognosis. Of note, the association is significantly stronger in patients with hepatitis C, which is the main risk factor for HCC in Japan

Clinical application of high throughput molecular screening techniques for pharmacogenomics.

Genetic analysis is one of the fastest-growing areas of clinical diagnostics. Fortunately, as our knowledge of clinically relevant genetic variants rapidly expands, so does our ability to detect these variants in patient samples. Increasing demand for genetic information may necessitate the use of high throughput diagnostic methods as part of clinically validated testing. Here we provide a general overview of our current and near-future abilities to perform large-scale genetic testing in the clinical laboratory. First we review in detail molecular methods used for high throughput mutation detection, including techniques able to monitor thousands of genetic variants for a single patient or to genotype a single genetic variant for thousands of patients simultaneously. These methods are analyzed in the context of pharmacogenomic testing in the clinical laboratories, with a focus on tests that are currently validated as well as those that hold strong promise for widespread clinical application in the near future. We further discuss the unique economic and clinical challenges posed by pharmacogenomic markers. Our ability to detect genetic variants frequently outstrips our ability to accurately interpret them in a clinical context, carrying implications both for test development and introduction into patient management algorithms. These complexities must be taken into account prior to the introduction of any pharmacogenomic biomarker into routine clinical testing

Association of IL-10 polymorphisms with hepatitis B virus infection and outcome in Han population

BACKGROUND: This study evaluated the correlation of single nucleotide polymorphisms interleukin (IL)-10-592 and -1082 with hepatitis B virus (HBV) susceptibility and recovery. METHODS: Total 190 chronic hepatitis B (CHB) patients, 81 individuals with self-limited HBV infections and 81 normal controls from the first Hospital of Jilin University were recruited. The IL-10 polymorphisms were detected by polymerase chain reaction with restriction fragment length polymorphism (PCR–RFLP). The χ(2) test (p < 0.05) and Fisher’s exact test were separately performed to analyze and compare the genotype frequencies of IL-10-592 and -1082 among different groups. Furthermore, logistic regression analysis (p < 0.05) was conducted to determine the correlation of genotypes with HBV infection and recovery. Genotype A/A, A/C and C/C of IL-10-592 had been detected in the three groups. RESULTS: The frequencies of -592A separately were 55.56, 64.67 and 55.33 % in the three groups. Genotypes of IL-10-592 only had significant difference among the patients and normal controls (p = 0.021). Genotypes A/A, A/G and G/G of IL-10-1082 were detected in CHB patients and individuals with self-limited HBV infection; however, genotype G/G had not been detected in normal controls. The frequencies of -1082G separately were 3.68, 6.17 and 11.11 % in the three groups. Genotypes of IL-10-1082 only had no significant difference among the patients and individuals with self-limited HBV infections (p = 0.130). We found that their risks of HBV infection existed significant difference. CONCLUSION: The IL-10-592 and -1082 polymorphisms might be associated with HBV infection, but not with the recovery after HBV infection

Relating the outcome of HCV infection and different host SNP polymorphisms in a Majorcan population coinfected with HCV–HIV and treated with pegIFN-RBV

Hepatitis C virus (HCV) is one of the major causes of chronic hepatitis, cirrhosis, and hepatocellular carcinoma, and the development of HCV-related disease is accelerated in individuals coinfected with human immunodeficiency-1 virus (HIV). In the present study, we correlated different host single-nucleotide polymorphisms (SNPs) in the IL28B, CTLA4, LDLr, and HFE genes and mitochondrial DNA (mtDNA) haplogroups with the outcome of HCV infection and the response to pegylated-interferon plus ribavirin (pegIFN-RBV) treatment. Our study population consisted of 63 Majorcan patients coinfected with HCV and HIV and 59 anonymous unrelated controls. Whereas the population frequency of IL28B alleles was similar to that found in a North-American cohort of European descent, the frequency of the rs12979860 C allele was lower than that determined in other cohorts from Spain. The frequencies of CTLA4 and LDLr polymorphisms were comparable to those reported in other populations. Significant differences between cases and control cohorts occurred only for the H63D mutation of the HFE gene. There were no other differences in the frequencies of other polymorphisms or mtDNA haplogroups. The IL28B rs12979860 CC genotype was shown to be associated with a rapid virological response, and the spontaneous viral clearance rate for HCV was higher in patients with the CTLA4+49 G allele. There was no relationship between SNPs in the LDLr and HFE genes and mtDNA haplogroups and the response to treatment. Our results suggest that the host genetic background plays a significant role in the pegIFN-RBV response of patients coinfected with HCV and HIV. [Int Microbiol 2014; 17(1):11-20]Keywords: HCV&ndash;HIV co-infection &middot; mtDNA haplogroups &middot; SNP polymorphism

Expert Rev Mol Diagn

Mass spectrometry (MS) has found numerous applications in life sciences. It has high accuracy, sensitivity and wide dynamic range in addition to medium- to high-throughput capabilities. These features make MS a superior platform for analysis of various biomolecules including proteins, lipids, nucleic acids and carbohydrates. Until recently, MS was applied for protein detection and characterization. During the last decade, however, MS has successfully been used for molecular diagnostics of microbial and viral infections with the most notable applications being identification of pathogens, genomic sequencing, mutation detection, DNA methylation analysis, tracking of transmissions, and characterization of genetic heterogeneity. These new developments vastly expand the MS application from experimental research to public health and clinical fields. Matching of molecular techniques with specific requirements of the major MS platforms has produced powerful technologies for molecular diagnostics, which will further benefit from coupling with computational tools for extracting clinical information from MS-derived data.CC999999/Intramural CDC HHS/United States2018-03-01T00:00:00Z23638820PMC58310797469vault:2743

Geographical distribution of hepatitis C virus genotypes in blood donors:an international collaborative survey

The frequency of infection with the six classified major genotypes of hepatitis C virus (HCV) was investigated in 447 infected volunteer blood donors from the following nine countries: Scotland, Finland, The Netherlands, Hungary, Australia, Egypt, Japan, Hong Kong, and Taiwan. Viral sequences in plasma from blood donors infected with HCV were amplified in the 5'-noncoding region and were typed by restriction fragment length polymorphism analysis. Electrophoresis of DNA fragments produced by cleavage with HaeIII-RsaI and ScrFI-HinfI allowed HCV types 1 (or 5), 2, 3, 4, and 6 to be identified. Further analysis with MvaI-HinfI allowed sequences of the type 5 genotype to be distinguished from sequences of type 1 genotype. Types 1, 2, and 3 accounted for almost all infections in donors from Scotland, Finland, The Netherlands, and Australia. Types 2 and 3 were not found in the eastern European country (Hungary), where all but one of the donors were infected with type 1. Donors from Japan and Taiwan were infected only with type 1 or 2, while types 1, 2, and 6 were found in those from Hong Kong. HCV infection among Egyptians was almost always by type 4. Donors infected with HCV type 1 showed broad serological reactivity with all four antigens of the second generation Chiron RIBA-2 assay (Chiron Corporation, Emeryville, Calif.), while infection with divergent HCV genotypes elicited antibodies mainly reactive to c22-3 and c33c. Reactivities with antibodies 5-1-1 and c100-3 were infrequent and were generally weak, irrespective of the geographical origin of the donor. Because the envelope region of HCV is even more variable than the NS-4 region, it is likely that vaccines based on these proteins need to be multivalent and perhaps specifically adapted for different geographical regions.link_to_subscribed_fulltex

Molecular and Cellular Studies of Human Hepatitis B Virus Variants

Despite unceasing efforts of the medical community, hepatitis B remains, besides hepatitis C, the most serious type of viral hepatitis and one of the major problems of global public health. According to the latest World Health Organization fact sheets (2000), of the 2 billion people who have been infected with the hepatitis B virus (HBV), more than 350 million have chronic infections. These chronically infected persons are at high risk of death from cirrhosis of the liver and liver cancer, diseases that kill about 1 million persons each year (WHO, 2000). The prevalence of HBV varies tremendously in different part of the world, with a much higher incidence in the Eastern than in the Western Hemisphere (WHO, 2001). High prevalence areas have been identified in Southeast Asia, China and Africa (reviewed by Lee, 1997). About 100 million carriers, making up 75% of the world’s HBV carriers living in Asia, are from China (Tandon and Tandon, 1997). In Malaysia, voluntary testing carried out on 17 048 healthy volunteers indicated a HBsAg seropositivity of 5.24% (Merican et al., 2000). xxv HBV belongs to the Orthohepadnavirus genus of the Hepadnaviridae family, which is related to the large order of Retroid viruses (Kann and Gerlich, 1998). Within a size of only about 3.2 kb, its compact, partially double-stranded DNA genome is extremely small, bearing four highly overlapping open reading frames (ORFs), which encode at least seven proteins (Kann and Gerlich, 1998; Nassal, 1999; Seeger and Mason, 2000). Due to the use of a viral RNA-dependent polymerase without proofreading function, HBV has a higher mutational rate than other DNA viruses (Blum1995; Petzold et al., 1999). Thus, it is generally assumed that this reverse transcription step accounts for the majority of point mutations and deletions or insertions that can be observed in the HBV genome. There are 2 major types of mutations in HBV. Firstly, there are genotype-specific mutations that allow the distinction of currently eight genotypes (A-H) (Norder et al., 1993; Stuyver et al., 2000; Arauz-Ruiz et al., 2002). These genotypes cluster geographically. Genotype A seems to represent the main European inland strain; genotype B and C, the Asian strain; genotype D, the Mediterranean basin strain; genotype E, the African strain; and genotype F, the New World strain (Norder et al., 1994; Magnius and Norder, 1995; Kidd Ljunggren, 1996). Genotype G was identified in France and United States (Stuyver et al., 2000) and genotype H was recently encountered in Nicaragua, Mexico and California (Arauz-Ruiz et al., 2002). xxvi The second type of HBV variability concerns mutations that emerge in an individual during chronic infection. Several specific mutations of this type have been identified by a large number of longitudinal as well as cross-sectional studies conducted during the past decade (reviewed in Gunther et al., 1999). Most of the corresponding variants accumulate during infection and persist as a dominant population until the late phase. These mutants are clinically important. It is learned that the presence or emergence of specific mutations is associated with particular stages of chronic infections (Gunther et al., 1999). In general, the enhancer II/core promoter and precore stop codon mutants appear to be associated with disease severity and progression (Lindh et al., 1999; Scaglioni et al., 1997; Pult et al., 1997; Takahashi et al., 1999). Mutations in the core antigen contribute strongly to immune escape at the T helper and cytotoxic T lymphocyte (CTL) level (Wakita et al., 1991; Chisari and Ferrari, 1995). Recent reports also revealed that mutations at basal core promoter (BCP) and precore/core (preC/C) mutations may influence the response rate to interferon-alpha (IFN-α) therapy (Fattovich et al., 1995; Zhang et al., 1996; Erhardt et al., 2000). Surface antigen mutants allow for escape from humoral immune responses and reduce the effectiveness of diagnostic tests and vaccination (Waters et al., 1992; Karthigesu et al., 1994; Carman et al., 1995,; Wallace and Carman, 1997; Hsu et al., 1999a). xxvii HBV is a typical non-cytopathic virus that can induce tissue damage of variable severity by stimulating a protective immune response that can simultaneously cause damage and protection, by resolving intracellular virus through the destruction of virus infected cells (Ferrari et al., 2003). Therefore, immune elimination of infected cells can lead to the termination of infection when it is efficient, or to a persistent necroinflammatory disease when it is not. Destruction of infected cells, however, is not the only mechanism implicated in the elimination of intracellular virus, as demonstrated by studies carried out in human hepatitis B showing the importance of cytokine-mediated, non-cytolytic mechanisms of antiviral protection. The first experimental evidence in favour of such mechanisms derives from studies performed in the transgenic mouse model (Guidotti and Ferrari, 2001). These studies showed that single stranded and relaxed circular double stranded HBV DNA replicative intermediates can be eliminated from the cytoplasm of HBV transgenic hepatocytes as a result of the antiviral effect of the interferon-gamma (IFN-γ) and tumour necrosis factor-alpha (TNF-α) released within the transgenic liver primarily by infiltrating HBV-specific CD8+ cells (Guidotti et al., 1996; Heise et al., 1999b) but also CD4+ T cells (Franco et al., 1997). Although the existence of genotypes is known for a long period of time, only very recently an association of genotype and clinical outcome was proposed (Kao et al., 2000a; Lindh et al., 1999). Recently, HBV genotypes have been partially clarified as xxviii influencing the clinical manifestation of chronic liver disease in hosts. A higher disease-inducing capability of genotype C than genotype B has been observed in Asia (Orito et al., 2001a; Kao et al., 2000a; Lindh et al., 1999). Several studies, mostly from Taiwan and Japan, have shown that HBV genotype C is associated with the development of hepatocellular carcinoma (HCC) (Kao et al., 2000a; Ding et al., 2001; Fujie et al., 2001) and has a lower response rate to interferon therapy as compared to genotype B (Kao et al., 2000b). As for other HBV genotypes, most patients in Europe with genotype A have chronic hepatitis, whereas most patients with genotype D have acute hepatitis (Mayerat et al., 1999) and may predict the occurrence of HCC in young Indian patients (Thakur et al., 2002). The genotype-related differences in HBV pathogenesis have been associated with the HBeAg/anti-HBe status. In the natural course of chronic HBV infection, early HBeAg/anti-HBe seroconversion usually associated with the cessation of virus replication and thus a favourable outcome (Chen, 1993). In contrast, late seroconversion of HBeAg after multiple episodes of reactivation and remission may accelerate the progression of chronic hepatitis B and thus have a poor clinical outcome (Perillo, 2001). Reports have revealed that the prevalence of HBeAg is more common in HBV genotype C than B. The reverse held true for the prevalence of anti-HBe, in that it is less common in genotype C than B (Ding et al., 2002; Chu et al., 2002; Kao et al., 2002; Orito et al., 2001a; Kobayashi et al., 2002; Yuen et al., 2003; Akuta et al., 2003). xxix Taken together, these data from different parts of the world have lent strong support to possible pathogenic differences among HBV variants. At present, those findings have been reported only in a few Asian countries. Moreover, the molecular virologic mechanisms that contribute to these clinical differences among HBV genotypes remain to be explored. The major limitation of previous studies is the lack of simple and efficient genotyping methods. Genotyping of viruses by sequencing and subsequent homology comparison or phylogenetic tree analysis is tedious and labour intensive and, therefore, not practical for diagnostic purposes. With the recent advances in molecular techniques, several novel genotyping methods, including polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) (Lindh et al., 1997; Mizokami et al., 1999), PCR with type-specific primers (Naito et al., 2001), commercial hybridization assay (Hou et al., 2001) and serologic genotyping assay (Usuda et al., 1999) have been introduced. In Malaysia, where the incidence rate of HBV was 12.19 per 100,000 population in 2001 (Ministry of Health Malaysia, 2001), limited information on the molecular biology of the HBV is available. The prevalence of HBV genotypes and the clinical relevance of HBV variants have not been discussed. The studies from other areas may not apply worldwide because the HBV strains in various parts of the world are different, and thus the clinical outcome and the mechanisms responsible may be different in this country. This provided a strong motivation to investigate the molecular variants of HBV in our population and the immune response evoked by these HBV variants

Gene polymorphisms in primary biliary cirrhosis: association with the disease and hepatic osteopathy

Genetic factors have been implicated in the pathogenesis of osteoporosis, a common disorder in primary biliary cirrhosis (PBC). Estrogen receptor-alpha gene (ER-�), vitamin-D-receptor gene (VDR) and IL-1-receptor-antagonist gene (IL-1RN) are all attractive candidates for osteoporosis susceptibility. Furthermore insulin-like growth factor-I (IGF-I) gene microsatellite repeat polymorphism was found to be associated with osteoporosis in some studies and collagen-I�1 (COLIA1) Sp1 s allele was associated with lower bone mineral density (BMD) in one study in PBC. IGF-I treatment restored osteopenia and reduced fibrogenesis in experimental cirrhosis. In this study we summarize our results on polymorphisms of the above genes and bone disease in Hungarian PBC patients. Patients and methods: 70 female patients with PBC were enrolled (age:57.6yrs, range:37-76yrs, each AMA-M2 positive, stage II-IV). 139 age-matched female subjects served as controls (age: 55.9 yrs, range:43-72 yrs). COLIA1 Sp1 and IGF-I microsatellite polymorphisms were determined by PCR in all patients and controls. VDR BsmI, IL-1RN variable-number tandem repeat (VNTR) and ER-� PvuII and XbaI polymorphisms were detected in 33 patients and controls. BMD was measured by dual energy x-ray absorptiometry (Lunar,Prodigy,USA) in lumbar spine (LS) and femoral neck (FN). Results: There was no difference in IGF-I microsatellite repeat polymorphism (192/192=34.2%, 194/192=28.6%, other=37.2%) and COLIA1 Sp1 polymorphism (SS=72.9%, Ss=22.8% and ss=4.3%) and IL-1 VNTR polymorphism between PBC patients and controls, however, the COLIA1 Sp1 s allele was significantly less frequent in patients with PBC (p=0.038). The genotype frequency of VDR BsmI (BB=57.5%, Bb=33.3%, bb=9.1%, p=0.01) and ER-a PvuII (PP=18.2%, Pp=75.6%, pp=6.2%, p=0.03) and XbaI (XX=9.1%, Xx=90.9%, xx=0%, p=0.0003) of the patients was different from that of the control group, with higher frequency of the BB, Pp and Xx genotypes in PBC. Osteoporosis (t score<-2.5) was detected in 22 patients (31.4%). Osteoporotic patients were elder and had longer disease history (p=0.01 for both). An association was found between the IGF-I genotypes and ODM data, the 192/192 genotype was associated with higher FN Z-score compared to other genotypes (p=0.036). Conclusions: In contrast to previous studies the COLIA1 Sp1 s allele was less frequent in patients with PBC, and its presence was not associated with BMD. We confirmed previous findings on higher frequency of VDR BsmI BB genotype in patients with PBC. The ER-α PvuII and XbaI Pp and Xx genotypes were more frequent in PBC patients, while IL-1RN VNTR and IGF-I microsatellite repeat polymorphism was not different. Since IGF-I polymorphism was associated to BMD, it may be hypothesized that not COLIA1 but IGF-I together with other genetic and environmental factors may be involved in the complex regulation of BMD in PBC