Viral Ribonucleic Acid Research Articles

Liver histology in patients with sporadic acute hepatitis E: a study of 11 patients from South-West France

Hepatitis E virus is a ribonucleic acid (RNA) enterically transmitted virus that causes both epidemics and sporadic cases of acute hepatitis E in many countries of Asia and Africa. Domestically acquired (non-travel-associated) hepatitis E has been reported recently in many industrialized countries including the USA, Europe, and Japan. There is little information available on liver histology in these patients. We report a series of 11 patients with sporadic acute hepatitis E and needle liver histology in South-West France. Hepatitis E was diagnosed based on elevated transaminases (>10 upper limit normal) and the presence of specific serum antibodies (immunoglobulin-G class, present in all 11 patients) and/or viral RNA detection in serum and/or stools. Acute hepatitis lesions were observed in all cases with marked necro-inflammatory activity in nine patients. Confluent necrosis was present in five cases. Anisocaryosis and Kupffer's cell aggregates with siderosis were observed in most of the 11 patients. Cholangitis was frequent (9/11 cases). Cholestasis was observed in eight cases. Pseudo-glandular pattern was present in only one case but without zonal repartition. Characteristic pathological signs of acute hepatitis E were severe intralobular necrosis, polymorph inflammation, and acute cholangitis with numerous neutrophils.

Integrated reverse transcription polymerase chain reaction systems for virus detection☆

The current study reports on an integrated microreverse transcription polymerase chain reaction (RT-PCR) system for molecular diagnosis of microorganisms automatically. By using antibodies-conjugated superparamagnetic beads, the developed system can detect viruses with higher sensitivity and specificity when compared with traditional biological diagnosis methods using a ribonucleic acid (RNA) extraction kit. The target viruses were first captured by the conjugated antibodies on the magnetic beads, and were enriched using a magnetic field generated by micro-electromagnets or permanent magnets. With this approach, the virus can be purified and concentrated first, then the virus RNA was extracted and transcripted to complementary deoxyribonucleic acid (cDNA), followed by a nucleic acid amplification process using a micro-RT-PCR module. The integrated microfluidic chip can perform the whole process automatically with the aid of integrated micropumps and microvalves. This study successfully performs the specific detection of two different types of viruses, Dengue virus serotype 2 and enterovirus (EV) 71 using this developed integrated system. Comparable to a large-scale apparatus, the integrated microsystem can perform mixing, incubation, purification, transportation, and nucleic acid amplification of virus, possibly making it a crucial platform for future diagnosis applications.

Different viral kinetics between hepatitis C virus genotype 1 and 2 as on-treatment predictors of response to a 24-week course of high-dose interferon-alpha plus ribavirin combination therapy

To elucidate the genotype-specific virus-host-drug interaction and the on-treatment viral kinetics in predicting sustained virologic response (SVR), serial serum hepatitis C virus (HCV) ribonucleic acid (RNA) levels at baseline, treatment week 2 (W2), treatment week 4 (W4), and treatment week 12 (W12) were measured in 199 chronic HCV-infected Taiwanese patients receiving interferon-alpha (INF-alpha) 6 million units (MU) three times weekly plus 1000 to 1200 mg/day of ribavirin for 24 weeks. The SVR rate was 90.5% (95/105) for HCV genotype 2 (HCV-2) patients and 47.9% (45/94) for HCV-1 patients (P < 0.0001). HCV-2 patients had a significantly higher rate of rapid virologic response (RVR) at W2 than HCV-1 patients. HCV RNA negativity at W4 had the highest accuracy of prediction (80%) of SVR with sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of 81%, 79%, 78%, and 82%, respectively, for HCV-1 patients. HCV RNA negativity or 2 logs drop at W4 had the highest accuracy of prediction (92%) with sensitivity, specificity, PPV, and NPV of 100%, 20%, 92%, and 100%, respectively, for HCV-2 patients. In multivariate analysis, the significant factors associated with SVR in HCV-1 patients were HCV RNA negativity at W12 and W4. HCV RNA negativity or 2 logs drop was the only significant factor associated with SVR in HCV-2 patients. In conclusion, a RVR at W4 could predict an SVR with a high degree of accuracy to a 24-week course of high-dose IFN/ribavirin for both HCV-1 patients and HCV-2 patients. With respect to each HCV genotype, the on-treatment virologic responses are the most important factors associated with SVR.

Microfluidic systems for extracting nucleic acids for DNA and RNA analysis

This paper is to review the differences in the developments of microfluidic chips for extracting genomic deoxyribonucleic acid (DNA) and viral ribonucleic acid (RNA) from blood by the Biosensor Focus Interest Group (BFIG) in Singapore. DNA was extracted in a multi-step process by isolating and lysing white blood cells (WBC), typically ∼10 μm in diameter. Viral RNA was extracted directly from the submicron viruses in the blood. In terms of basic microfluidic components required, both DNA and RNA extractions used similar mixers for mixing reagents, filters for capturing or separating the blood cells, and a binder for capturing and purifying the DNA/RNA molecules. The designs of the filters were adapted to either capture WBC for DNA isolation or capture all virus particles for RNA isolation. The designs of these two kinds of filters had to be different. Besides the differences in the sizes of WBC and viruses, the concentration of the virus particles is usually much lower than WBC. Thus, a much higher volume of blood for filtering would be required for extracting viral RNA, especially for the intention to detect the viruses at early onset of infection. With proper modifications of the protocols, it has been demonstrated that both genomics DNA and viral RNA could be extracted successfully in these microfluidic chips. The quality of the extracted samples was verified by polymerase chain reaction (PCR) and gel-electrophoresis after the extractions.

Diagnostic tests in HIV management: a review of clinical and laboratory strategies to monitor HIV-infected individuals in developing countries

We conducted a systematic review on the performance of diagnostic tests for clinical and laboratory monitoring of HIV-infected adults in developing countries. Diagnostic test information collected from computerized databases, bibliographies and the Internet were categorized as clinical (non-laboratory patient information), immunologic (information from immunologic laboratory tests), or virologic (information from virologic laboratory tests). Of the 51 studies selected for the review 28 assessed immunologic tests, 12 virologic tests and seven clinical and immunologic tests. Methods of performance evaluation were primarily sensitivity and specificity for the clinical category and correlation coefficients for immunologic and virologic categories. In the clinical category, the majority of test performance measures was reported as >70% sensitive and >65% specific. In the immunologic category, correlation coefficients ranged from r=0.54 to r=0.99 for different CD4 count enumeration techniques, while correlation for CD4 and total lymphocyte counts was between r=0.23 and r=0.74. In the virologic category, correlation coefficients for different human immunodeficiency virus (HIV) ribonucleic acid (RNA) quantification techniques ranged from r=0.54 to r=0.90. Future research requires consensus on designing studies, and collecting and reporting data useful for decision-makers. We recommend classifying information into clinically relevant categories, using a consistent definition of disease across studies and providing measures of both association and accuracy.

A Case of Dengue from Pune, India

Dengue is an acute infectious disease, caused by a single stranded ribonucleic acid (RNA) virus of the family of Flaviviridae, genus Flavivirus, transmitted by Aedes mosquitos, the most important vectors being Aedes aegypti and Aedes albopictus. There are four serotypes, DEN-1, DEN-2, DEN-3, DEN-4 that are nearly human specific. The geographical distribution of dengue is pantropical, except for Madagascar and some African regions. It is endemo-epidemic in tropical and subtropical countries included between 25 degrees north latitude and 25 degrees south latitude, particularly in southeast Asia and epidemic in the Caribbean, West-Africa, tropical American and Pacific Islands. Further spread will depend on the invasion of Aedes aegypti into new areas in South America. Maintenance of dengue is supported by an increasing demography, uncontrolled urbanization and climatic conditions favorable to the vectors. Spread of dengue is primarily caused by modern transportation, especially aircraft. No vaccine and no specific treatment exist so that mosquito control is the only way to reduce the incidence of dengue around the world. Dengue is, obviously, an important risk for travelers going to endemic areas.

ARE ELDERLY PATIENTS POOR CANDIDATES FOR PEGYLATED INTERFERON PLUS RIBAVIRIN IN THE TREATMENT OF CHRONIC HEPATITIS C?

ARE ELDERLY PATIENTS POOR CANDIDATES FOR PEGYLATED INTERFERON PLUS RIBAVIRIN IN THE TREATMENT OF CHRONIC HEPATITIS C?

Progress in Anti-SARS Coronavirus Chemistry, Biology and Chemotherapy

Proteolytic processing of the coronavirus replicase polyproteins is essential for ongoing viral ribonucleic acid (RNA) synthesis. Therefore, the severe acute respiratory syndrome (SARS)-coronaviruses (SARS-CoV) proteases are attractive targets for the development of antiviral drugs to reduce viral replication and pathogenicity. The structure and activity of the coronavirus 3C-like protease (3CLpro) has already been elucidated, and the design of inhibitors to 3CLpro as therapeutics has been proposed. The chapter discusses SARS-CoV 3CLpro inhibitors that include covalent inhibitors, noncovalent inhibitors, and inhibitors from screening. SARS-CoV papain-like protease (PLpro) is considered an equally viable target to 3CLpro for drug design because both are essential for viral replication. However, PLpro has likely not been pursued because of the paucity of structural information. Several compounds have been identified that have shown inhibitory activity against SARS-CoV. However, no information regarding their mechanism of action or the corresponding target is known. Glycyrrhizin showed inhibitory activity for SARS-CoV replication with EC50 = 300 mg/L after virus absorption in Vero cells. Some glycyrrhizin acid derivatives were found to inhibit SARS-CoV replication in vitro with EC50 values ranging from 5 to 50 μM. Unfortunately, these compounds show high cytotoxity.

Serogrouping of United States and some African serotypes of bluetongue virus using RT-PCR

The diagnostic potential of RT-PCR for detection of bluetongue virus (BTV) ribonucleic acid (RNA) sequence in cell culture and tissue samples from infected ruminants from United States, Sudan, South Africa and Senegal, was evaluated. The non structural protein 1 (NS1) gene of North American BTV serotype 11 was targeted for PCR amplification. The United States BTV serotypes 2, 10, 11, 13 and 17 and the Sudanese BTV serotypes 1, 2, 4 and 16 and BTV serotype 4 from South Africa and BTV serotype 2 from Senegal were studied. RNAs from all BTV field isolates used in this study, propagated in cell cultures, were detected by the described RT-PCR-based assay. The first specific 790bp BTV PCR products were amplified using a pair of outer primers (BTV1 and BTV2). Specificity of the PCR products was confirmed by a nested amplification of a 520bp PCR product using a pair of internal (nested) primers (BTV3 and BTV4). The BTV PCR products were visualized on ethidium bromide-stained agarose gels. Amplification products were not detected when the RT-PCR-based assay was applied to RNAs from closely related orbiviruses including, epizootic hemorrhagic disease virus (EHDV) prototypes serotypes 1, 2, 4; RNA from Sudanese isolate of palyam orbiviruses serogroup and total nucleic acid extracts from uninfected Vero cells. Application of the nested BTV RT-PCR to clinical samples resulted in amplification of BTV RNA from blood and serum samples from goats experimentally infected with BTV4 and from naturally infected sheep, goats, cattle and deer. The results of this study indicated that this RT-PCR assay could be applied for rapid detection of BTV, in cell culture and clinical samples from susceptible ruminants during an outbreak of the disease, in the United States and African.

Peginterferon Alpha/Ribavirin Combination Therapy for the Treatment of Hepatitis C Infection

Chronic infection with the hepatitis C virus is widespread in the United States. This disease is associated with a progression of fibrosis of the liver leading to liver cirrhosis in as many as 20% of cases. The current standard of care for the treatment of chronic hepatitis C infection is combination therapy with pegylated interferon alpha plus ribavirin. In more than 50% of patients, this regimen has been shown to induce a sustained viral response, defined as undetectable hepatitis C viral ribonucleic acid (RNA) for 6 months after the end of treatment. Typically, patients are treated for 24 or 48 weeks. A number of possible adverse events are associated with combination therapy, and patient empowerment through supportive nursing care is critical to facilitating patient adherence to treatment. This article provides an update on information concerning the diagnosis and treatment of chronic hepatitis C infection. This information can be tailored to provide patient-focused assessment, education, and treatment.

Peginterferon Alfa-2a Does Not Alter the Pharmacokinetics of Methadone in Patients with Chronic Hepatitis C Undergoing Methadone Maintenance Therapy*

Our objective was to quantify the pharmacokinetics of methadone and the pharmacokinetics and pharmacodynamics of peginterferon alfa-2a (40 kd) in patients with chronic hepatitis C undergoing methadone maintenance therapy. Adults with chronic hepatitis C who had been receiving a consistent methadone maintenance regimen for at least 3 months were eligible for this open-label, multicenter, nonrandomized drug interaction study. All patients received 180 microg subcutaneous peginterferon alfa-2a once weekly for 4 weeks and continued their methadone regimen. Serial blood samples were collected at baseline and immediately before and for up to 168 hours after study drug administration for the purposes of quantifying methadone and peginterferon alfa-2a serum concentrations, measuring serum 2',5'-oligoadenylate synthetase activity, and determining hepatitis C virus ribonucleic acid levels. Twenty-four patients were enrolled. Methadone exposure, as measured by maximum serum concentration (C(max)) and area under the concentration-time curve (AUC) normalized to a 100-mg/d dose, after 4 doses of peginterferon alfa-2a increased by 10% to 15% when compared with baseline. The week 4/baseline ratio of the mean C(max) was 1.11 (90% confidence interval [CI], 1.02-1.22), and for AUC from time 0 to 24 hours, the week 4/baseline ratio was 1.15 (90% CI, 1.08-1.23). The mean accumulation ratios (week 4/first dose) for C(max) and AUC from time 0 to 168 hours of peginterferon alfa-2a were 2.1 and 2.3, respectively. Peginterferon alfa-2a does not appreciably alter the pharmacokinetics of methadone.

Evidence of viral infection in the myocardium of American and Japanese patients with idiopathic dilated cardiomyopathy

Enteroviruses have been implicated in the pathogenesis of idiopathic dilated cardiomyopathy (IDC). Recently, the association of adenovirus or parvovirus with IDC has been reported. Viral infection in the myocardium of American and Japanese patients with end-stage IDC was evaluated. Myocardial specimens from 30 American patients with IDC and 47 Japanese patients with IDC were analyzed for the presence of cardiotropic viruses. The strand-specific detection of enteroviral ribonucleic acid (RNA) was performed to determine viral activity in hearts with IDC. Established reverse transcription-polymerase chain reaction (PCR) or PCR techniques were used to detect genomic sequences of influenza viruses, mumps virus, adenovirus, parvovirus, herpes simplex viruses, varicella-zoster virus, and Epstein-Barr virus. Enteroviral RNA was detected in 7 of the 30 American patients (23%) and in 15 of the 47 Japanese patients (32%). Minus-strand enteroviral RNA, an indicator of active enteroviral RNA replication, was demonstrated in 5 of 7 plus-strand-positive American patients (71%) and in 12 of 15 plus-strand-positive Japanese patients (80%). Sequence analysis revealed that the viruses detected were Coxsackie B viruses. No genomic sequences of other viruses were detected in the myocardium of either American or Japanese patients with IDC. Therefore, active group B Coxsackie virus RNA replication in the myocardium was demonstrated in a significant proportion of American and Japanese patients with end-stage IDC. There was no evidence of persistent infection by other viruses in hearts with IDC. Specific therapy should be designed for Coxsackie virus positive patients with IDC.

Modelling HIV‐RNA viral load in vertically infected children

Human immunodeficiency virus (HIV) ribo-nucleic acid (RNA) viral load is a measure of actively replicating virus and is used as a marker of disease progression. For a thorough understanding of the dynamics of the evolution of the virus in the early life of HIV-1 vertically infected children, it is important to elucidate the pattern of HIV-RNA viral load over age. An aspect of assay systems used in the quantification of RNA viral load is that they measure values above particular cut-off values for detection, below which the assays used are not sufficiently sensitive. In this way, measurements are potentially left-censored. Recent adult studies suggest that to adequately model RNA pattern over age, it is necessary to account for within-subject correlation, due to repeated measures, and censoring. The aim of this study, therefore, was to establish whether it is necessary to use complex methods to allow for repeated measures within individuals and censoring of the HIV-RNA viral load in children enrolled in a cohort study. The approach involved the identification of an appropriate model for the basic pattern of RNA viral load by age and subsequent assessment of various estimation procedures accounting for repeated measures and censoring in different ways. Methods developed by Hughes involving the expectation-maximization (EM) algorithm and the Gibbs sampler were taken as the benchmark for comparison of simpler alternatives. Other approaches considered involve linear mixed-effects and ordinary least squares in which censoring is dealt with informally by taking the cut-off value as absolute or taking the mid-point between cut-off and zero. Fractional polynomials provided a substantially superior approach for modelling the dynamics of viral load over age compared to conventional polynomials or change-point models. Allowing for repeated measures was necessary to improve the power of the likelihood ratio tests required to establish the final model, but methods beyond taking the mid-point for censored values did not further improve the fit. Although Hughes' methodology is the best approach, its implementation is not necessary for the identification of the optimal model.

SARS: Challenge of the New Century

SARS: Challenge of the New Century

Type C chronic hepatitis with the discovery of a small hepatocellular carcinoma 7 years after successful interferon therapy

The patient, a 61-year-old man, had sustained injuries in a traffic accident at the age of 26, for which he received a blood transfusion. Since 1988 (age, 49 years), abnormal hepatic function had been detected, and, because of the presence of hepatitis C virus antibodies, he was diagnosed as having type C chronic hepatitis. Based on a liver biopsy that was conducted in July 1992 (age, 53), a histological diagnosis of chronic active hepatitis (F(1)/A(2)) was made. Over a period of 6 months, starting in 1992, the patient was treated with interferon (IFNalpha-2a; total dosage, 720 MU). At the end of this regimen, the alanine aminotransferase level was normalized and serum hepatitis C virus-ribonucleic acid was negative. This condition was maintained until August 1996 (age, 57), after which the patient stopped reporting to our hospital. In June 2000 (age, 61) when he was hospitalized for an adhesive ileus, a small hepatocellular carcinoma (a solitary lesion measuring 18 mm in diameter) at S(8) was found, and it was extirpated by a segmental excision in July. The case is introduced to call attention to the need for longterm follow-up observation, even after effective IFN therapy.

Intracranial calcification with congenital rubella syndrome in a mother with serologic immunity.

We report a case of an infant with congenital rubella. The mother had received rubella vaccine at the age of 13 years. Rubella serology was performed on day 34 of pregnancy and the result was interpreted as being a positive titer. The patient was a girl born by cesarean section owing to intrauterine growth retardation and fetal distress after 37 weeks' gestation. A computed tomographic scan at 4 days of age showed several cortical low-density areas and calcifications of the periventricular area and basal ganglia. Magnetic resonance imaging (MRI) performed at 4 weeks of age showed almost similar findings. The infant had serum IgG and IgM antibodies against rubella. Rubella virus ribonucleic acid (RNA) was detected from the serum, urine, and cerebrospinal fluid of the infant. At 2 months of age, the patient showed severe bilateral hearing loss. At 12 months of age, she had mild mental retardation and developmental delay.

Hepatitis C virus: prevalence and routes of infection among blood donors of West Mexico

In Latin America few studies have explored frequency and risk factors predicting hepatitis C virus (HCV) infection in blood donors. In this study we determined the prevalence of HCV infection in blood donors from West Mexico. Potential risk factors, clinical, histological and virologic characteristics presented in this group were also evaluated. Methods: HCV antibodies were evaluated in 57 108 blood donors with commercial second-generation enzyme immunoassays. Positive results were confirmed by a recombinant immunoblot assay. Repeatedly seropositive donors were further studied for risk factors, history for hepatitis, hepatic enzymes (aspartate aminotransferase and alanine aminotransferase (AST and ALT)), liver histology and hepatitis C virus–ribonucleic acid (HCV–RNA) detection. Results: A total of 499 blood donors were initially tested positive doubtful for antibodies to HCV Ab (0.8%). While there was no difference in HCV prevalence with respect to age or gender, the most frequent risk factors identified were transfusion (42%), household exposure (14.8%), multiple sex partner (6.8%) and intranasal cocaine use (2.3%). Also, we found that from a subgroup of donors tested for histological analysis, 19% presented abnormal ALT levels and 90% showed abnormal liver histology. No correlation was found between abnormal ALT levels and the presence of HCV–RNA in serum. Conclusion: These results demonstrate a low prevalence (0.8%) of HCV infection among Western Mexican blood donors, which was comparable to those established for Western countries, but in contrast in our study the most frequent risk factor continues being transfusion followed by household exposure.

Clinical trial results of peginterferons in combination with ribavirin.

Of the large number of patients chronically infected with hepatitis C virus (HCV), only about one third have progressive liver disease, and will eventually develop cirrhosis and hepatocellular carcinoma. These are the patients for whom effective antiviral treatment is most needed. Therapy is currently recommended for patients with chronic hepatitis C who have abnormal alanine aminotransferase (ALT) levels, detectable hepatitis C virus ribonucleic acid (HCV RNA) in the blood, and significant necroinflammatory changes and/or fibrosis on liver biopsy. The current gold standard in terms of treatment efficacy is the combination of peginterferon (PEG-IFN) and ribavirin. The overall sustained virological response rate (SVR) with these regimens is 54 to 61% following 48 weeks of therapy. Patients with genotype 1 infection have a 42 to 51% likelihood of response to 48 weeks of therapy. Those with genotypes 2 or 3 infection will respond to 24 weeks of therapy in 78 to 82% of cases. These SVR rates are 5 to 10 percentage points higher in all patient groups than in those obtained with standard doses of interferon (IFN) and ribavirin. Retreatment of nonresponders to standard IFN monotherapy using PEG-IFN and ribavirin has achieved SVR rates of 34 to 40%. Retreatment of patients who relapsed after IFN monotherapy has resulted in an SVR rate of about 60%. A SVR after retreatment of relapsers and nonresponders with PEG-IFN and ribavirin is more likely in patients previously treated with IFN monotherapy, those with HCV genotypes 2 or 3, patients with low viral load (<2 million copies/mL), and individuals who had a significant decrease in HCV RNA levels during the initial treatment. The potential benefits of long-term anti-HCV suppressive therapy in nonresponders are currently under investigation.

Viral RNA in nerve tissues of patients with hepatitis C infection and peripheral neuropathy.

To assess the presence of viral ribonucleic acid (RNA) in nerve tissues of 15 patients with hepatitis C virus (HCV) infection and peripheral neuropathy with (11) or without (4) mixed cryoglobulinemia, nested reverse transcription-polymerase chain reaction (RT-PCR) was performed. Amplification of HCV-RNA was successful in 7 patients with and 3 without mixed cryoglobulinemia. This study demonstrates that the nested RT-PCR technique is a sensitive method to detect viral RNA in nerve tissue, and offers further evidence that in patients with HCV infection peripheral neuropathy can occur in the absence of mixed cryoglobulinemia.

Interferon alpha and ribavirin for membranoproliferative glomerulonephritis and hepatitis C infection

Cryoglobulinemic membranoproliferative glomerulonephritis, an extrahepatic manifestation of chronic hepatitis C virus infection, often poses a therapeutic challenge (1). Although glucocorticosteroids and interferon alpha are effective in controlling the acute phase of the disease, a sustained response is unusual after discontinuation of treatment (2,3). The combination of interferon alpha and ribavirin, a nucleoside analog with broad activity against both deoxyribonucleic acid and ribonucleic acid (RNA) viruses, may improve the prognosis of hepatitis C virus infection in patients without renal disease (4,5). The few studies of this combination in patients with hepatitis C virus–associated cryoglobulinemic glomerulonephritis have had variable results (6-8). We report two additional patients who achieved a long-term response with early combination antiviral treatment.