Release of virus from lymphoid tissue affects human immunodeficiency virus type 1 and hepatitis C virus kinetics in the blood

Kinetic parameters of human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) infections have been estimated from plasma virus levels following perturbation of the chronically infected (quasi-) steady state. We extend previous models by also considering the large pool of virus localized in the lymphoid tissue (LT) compartment. The results indicate that the fastest time scale of HIV-1 plasma load decay during therapy probably reflects the clearance rate of LT virus and not, as previously supposed, the clearance rate of virus in plasma. This resolves the discrepancy between the clearance rate estimates during therapy and those based on plasma apheresis experiments. In the extended models plasma apheresis measurements are indeed expected to reflect the plasma decay rate. We can reconcile all current HIV-1 estimates with this model when, on average, the clearance rate of virus in plasma is approximately 20 day(-1), that of LT virus is approximately 3 day(-1), and the death rate of virus-producing cells is approximately 0.5 day(-1). The fast clearance in the LT compartment increases current estimates for total daily virus production. Because HCV is produced in the liver, we let virus be produced into the blood compartment of our model. The results suggest that extending current HCV models with an LT compartment is not likely to affect current estimates for kinetic parameters and virus production. Estimates for treatment efficacy might be affected, however.     

A multiplexed protein microarray for the simultaneous serodiagnosis of human immunodeficiency virus/hepatitis C virus infection and typing of whole blood

All donor blood samples must be tested pretransfusion to determine the donor blood type. Standard testing protocols require that assays be performed for important bloodborne pathogens such as hepatitis C, syphilis, hepatitis B, and human immunodeficiency virus. We have demonstrated proof of the concept that a protein microarray can type whole blood and detect antibody to significant pathogens simultaneously from the same donor blood sample. The data collected demonstrate the ability of the array to accurately type blood samples while also detecting the presence of antibodies against both human immunodeficiency virus and hepatitis C virus. In conclusion, we have successfully developed a platform capable of typing human whole blood samples, while at the same time testing for the presence of antibodies specific for human immunodeficiency virus/hepatitis C virus. The major benefits of this system are its amenability to expansion with additional assays, for example, rhesus typing and syphilis and/or hepatitis B virus detection, and also the adaptability of the assay to higher-throughput analysis, currently 16 individual samples per slide, but readily expandable to a 96-well format.     

Archived Guthrie blood spots as a novel source for quantitative DNA methylation analysis

Sodium bisulfite treatment followed by PCR and DNA sequencing is widely considered the gold standard for the analysis of DNA methylation patterns. However, this technique generally requires substantial quantities of genomic DNA as starting material and is often associated with degradation of DNA. Here, we assess the feasibility of performing bisulfite sequencing on DNA isolated from 3-mm diameter punches of dried blood Guthrie spots. We demonstrate that it is possible to perform bisulfite sequencing from both freshly prepared and archived dried blood spots, using a combination of high purity DNA extraction and efficient bisulfite conversion. With the number of new technologies available for DNA methylation studies, we have extended this analysis and have successfully used a high-throughput mass spectrometry method for DNA methylation analysis on these samples. This provides a new source of material for epigenetic analysis of birth samples and provides an invaluable reference point to track temporal change in epigenetic profiles possibly linked with health and disease.     

Molecular characterization of human immunodeficiency virus type 1 and hepatitis C virus in paid blood donors and injection drug users in china

China is facing a rapid upsurge in cases of human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) infection due to large numbers of paid blood donors (PBD), injection drug users (IDU), and sexual partners of infected individuals. In this report, a total of 236 HIV-1-positive blood samples were collected from PBD, IDU, and their sexual partners in the most severely affected provinces, such as Henan, Yunnan, Guangxi, and Xinjiang. PCR was used to amplify the p17 region of gag and the C2-V3 region of env of HIV-1 and the 5' noncoding region and a region of E1/E2 of HCV. Genetic characterization of viral sequences indicated that there are two major epidemics of HIV-1 and multiple HCV epidemics in China. The PBD and transfusion recipients in Henan harbored HIV-1 subtype B', which is similar to the virus found in Thailand, and HCV genotypes 1b and 2a, whereas the IDU in Yunnan, Guangxi, and Xinjiang carried HIV-1 circulating recombinant forms 07 and 08, which resemble those in India, and HCV genotypes 1b, 3a, and 3b. Our findings show that the epidemics of HIV-1 and HCV infection in China are the consequences of multiple introductions. The distinct distribution patterns of both the HIV-1 and HCV genotypes in the different high-risk groups are tightly linked to the mode of transmission rather than geographic proximity. These findings provide information relevant to antiviral therapy and vaccine development in China and should assist public health workers in implementing measures to reduce the further dissemination of these viruses in the world's most populous nation.     

Hepatitis A virus and hepatitis E virus prevalence relates to human immunodeficiency virus infection in Japanese male blood donors

Hepatitis A virus (HAV) has begun to spread globally among men who have sex with men (MSM). Hepatitis E virus (HEV) also may be transmitted through sexual contact among MSM. To assess the current status of these viruses among MSM in Japan, the seroprevalence of both viruses using 503 plasma samples collected between 2009 and 2018 from human immunodeficiency virus (HIV)-positive male donors who were presumed to be mainly MSM was investigated. Our results suggested that HAV may be spreading within this population, as reported elsewhere. By contrast, the spread of HEV was confirmed only among younger HIV-positive donors.     

Human immunodeficiency virus seroconversion and evolution of the hepatitis C virus quasispecies

When chronic hepatitis C virus (HCV) infections are complicated by acquisition of human immunodeficiency virus (HIV), liver disease appears to accelerate and serum levels of HCV RNA may rise. We hypothesized that HIV might affect the HCV quasispecies by decreasing both complexity (if HIV-induced immunosuppression lessens pressure for selecting HCV substitutions) and the ratio of nonsynonymous (d(N)) to synonymous (d(S)) substitutions, because d(N) may be lower (if there is less selective pressure). To test this hypothesis, we studied the evolution of HCV sequences in 10 persons with chronic HCV infection who seroconverted to HIV and, over the next 3 years, had slow or rapid progression of HIV-associated disease. From each subject, four serum specimens were selected with reference to HIV seroconversion: (i) more than 2 years prior, (ii) less than 2 years prior, (iii) less than 2 years after, and (iv) more than 2 years after. The HCV quasispecies in these specimens was characterized by generating clones containing 1 kb of cDNA that spanned the E1 gene and the E2 hypervariable region 1 (HVR1), followed by analysis of clonal frequencies (via electrophoretic migration) and nucleotide sequences. We examined 1,320 cDNA clones (33 per time point) and 287 sequences (median of 7 per time point). We observed a trend toward lower d(N)/d(S) after HIV seroconversion in 7 of 10 subjects and lower d(N)/d(S) in those with rapid HIV disease progression. However, the magnitude of these differences was small. These results are consistent with the hypothesis that HIV infection alters the HCV quasispecies, but the number of subjects and observation time may be too low to characterize the full effect.     

Impact of highly active antiretroviral therapy and immunologic status on hepatitis C virus quasispecies diversity in human immunodeficiency virus/hepatitis C virus-coinfected patients

This study analyzes the effect of highly active antiretroviral therapy (HAART), and thus immunologic status, on hepatitis C virus (HCV) load and quasispecies diversity in patients coinfected with the human immunodeficiency virus (HIV) and HCV. Three cohorts of coinfected patients were analyzed retrospectively over a period of 7 to 10 months: group A was antiretroviral drug na?ve at baseline and then on HAART for the remainder of the study, group B did not receive antiretroviral therapy at any point, and group C was on HAART for the entire study. HCV quasispecies diversity was analyzed by sequencing hypervariable region 1. In a longitudinal analysis, there was no significant change from baseline in any immunologic, virologic, or quasispecies parameter in any of the three groups. However, in comparison to groups A and B, group C had significantly higher CD4+- and CD8+-cell counts, a trend toward a higher HCV load, and significantly increased number of HCV clones, entropy, genetic distance, and ratio of nonsynonymous substitutions per nonsynonymous site to synonymous substitutions per synonymous site (Ka/Ks). In addition, CD4+-cell count was positively correlated with HCV load, genetic distance, and Ka. Interestingly, patients infected with HCV genotype 2 or 3 had a significantly higher CD4+-cell count, HCV load, genetic distance, and Ka/Ks than those infected with genotype 1. These results suggest that there is no immediate effect of HAART on HCV but that, with prolonged HAART, immune restoration results in an increase in HCV load and quasispecies diversity.     

Persistently infected cultures as a source of hepatitis A virus

Primary African green monkey kidney, continuous African green monkey kidney cell line BS-C-1, and buffalo green monkey kidney cultures were infected with a uniform inoculum of hepatitis A virus (HAV). Although both the cell line BS-C-1 and primary African green monkey kidney cultures produced useful amounts of virus, HAV was detected earlier and in greater quantities in primary African green monkey kidney cultures. A persistently infected primary African green monkey kidney culture was developed. The influence of incubation time (4 to 40 days) and concentration (2 to 15%) of fetal calf serum in the maintenance medium on production of HAV by this culture was examined. An incubation period of 24 to 28 days was found to be optimal; reducing this period led to decreased yields of HAV. No significant difference in the amount of HAV produced was observed with differing concentrations of fetal calf serum. Three different methods of extraction and the effect of multiple extractions on the recovery of HAV from cell lysates were examined. Sonication was a critical factor. Two extractions yielded more than 90% recoverable virus. Yields in excess of 10(11) physical particles of HAV per 850-cm2 roller bottle were routine. The total yield could be increased by concentrating the HAV present in spent maintenance medium by using bentonite or organic flocculation.     

Host sphingolipid biosynthesis as a target for hepatitis C virus therapy

An estimated 170 million individuals worldwide are infected with hepatitis C virus (HCV), a serious cause of chronic liver disease. Current interferon-based therapy for treating HCV infection has an unsatisfactory cure rate, and the development of more efficient drugs is needed. During the early stages of HCV infections, various host genes are differentially regulated, and it is possible that inhibition of host proteins affords a therapeutic strategy for treatment of HCV infection. Using an HCV subgenomic replicon cell culture system, here we have identified, from a secondary fungal metabolite, a lipophilic long-chain base compound, NA255 (1), a previously unknown small-molecule HCV replication inhibitor. NA255 prevents the de novo synthesis of sphingolipids, major lipid raft components, thereby inhibiting serine palmitoyltransferase, and it disrupts the association among HCV nonstructural (NS) viral proteins on the lipid rafts. Furthermore, we found that NS5B protein has a sphingolipid-binding motif in its molecular structure and that the domain was able to directly interact with sphingomyelin. Thus, NA255 is a new anti-HCV replication inhibitor that targets host lipid rafts, suggesting that inhibition of sphingolipid metabolism may provide a new therapeutic strategy for treatment of HCV infection.     

A combined oligonucleotide and protein microarray for the codetection of nucleic acids and antibodies associated with human immunodeficiency virus, hepatitis B virus, and hepatitis C virus infections

A multiplexed assay based on the codetection of nucleic acids and antibodies in human serum infected by human immunodeficiency virus (HIV), hepatitis B virus (HBV) or hepatitis C virus was proposed. The combined immuno- and oligosorbent array (CombOLISA) microarray is prepared in 96-well standard microplates by spotting (1). nucleic probes specific for a virus genome, (2). viral proteins for the capture of serum antibodies, and (3). nonspecific proteins for verifying specificity. Experimental assay conditions were optimized so that both DNA hybridization and immunological reactions can be achieved simultaneously in the same well and buffer and all at the same temperature. A generic detection system based on the precipitation of an insoluble colorimetric substrate in the presence of enzyme-labeled antibodies or streptavidin was proposed. The optical density of each spot was correlated to the corresponding analyte concentration. The influence of critical parameters on CombOLISA performance such as serum concentration was studied. Calibration curves and sensitivity thresholds were established for each parameter. Serial dilutions of serum were correlated to results obtained with validated immunoassay platforms such as a microplate enzyme-linked immunosorbent assay or the VIDAS automat. Also, several HIV- and HBV-infected serum samples were tested independently by CombOLISA and VIDAS. Coefficients of variation for genomic and proteomic parameters vs spot density were below 15%.     

Persistently infected cultures as a source of hepatitis A virus.

Primary African green monkey kidney, continuous African green monkey kidney cell line BS-C-1, and buffalo green monkey kidney cultures were infected with a uniform inoculum of hepatitis A virus (HAV). Although both the cell line BS-C-1 and primary African green monkey kidney cultures produced useful amounts of virus, HAV was detected earlier and in greater quantities in primary African green monkey kidney cultures. A persistently infected primary African green monkey kidney culture was developed. The influence of incubation time (4 to 40 days) and concentration (2 to 15%) of fetal calf serum in the maintenance medium on production of HAV by this culture was examined. An incubation period of 24 to 28 days was found to be optimal; reducing this period led to decreased yields of HAV. No significant difference in the amount of HAV produced was observed with differing concentrations of fetal calf serum. Three different methods of extraction and the effect of multiple extractions on the recovery of HAV from cell lysates were examined. Sonication was a critical factor. Two extractions yielded more than 90% recoverable virus. Yields in excess of 10(11) physical particles of HAV per 850-cm2 roller bottle were routine. The total yield could be increased by concentrating the HAV present in spent maintenance medium by using bentonite or organic flocculation.     

Microarray multiplex assay for the simultaneous detection and discrimination of hepatitis B, hepatitis C, and human immunodeficiency type-1 viruses in human blood samples

Hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus type-1 (HIV-1) are transfusion-transmitted human pathogens that have a major impact on blood safety and public health worldwide. We developed a microarray multiplex assay for the simultaneous detection and discrimination of these three viruses. The microarray consists of 16 oligonucleotide probes, immobilized on a silylated glass slide. Amplicons from multiplex PCR were labeled with Cy-5 and hybridized to the microarray. The assay detected 1 International Unit (IU), 10 IU, 20 IU of HBV, HCV, and HIV-1, respectively, in a single multiplex reaction. The assay also detected and discriminated the presence of two or three of these viruses in a single sample. Our data represent a proof-of-concept for the possible use of highly sensitive multiplex microarray assay to screen and confirm the presence of these viruses in blood donors and patients.     

Shared alterations in NK cell frequency, phenotype, and function in chronic human immunodeficiency virus and hepatitis C virus infections

Human immunodeficiency virus (HIV) and hepatitis C virus (HCV) cause clinically important persistent infections. The effects of virus persistence on innate immunity, including NK cell responses, and the underlying mechanisms are not fully understood. We examined the frequency, phenotype, and function of peripheral blood CD3⁻ CD56⁺ NK subsets in HIV⁺ and HCV⁺ patients and identified significantly reduced numbers of total NK cells and a striking shift in NK subsets, with a marked decrease in the CD56^dim cell fraction compared to CD56^bright cells, in both infections. This shift influenced the phenotype and functional capacity (gamma interferon production, killing) of the total NK pool. In addition, abnormalities in the functional capacity of the CD56^dim NK subset were observed in HIV⁺ patients. The shared NK alterations were found to be associated with a significant reduction in serum levels of the innate cytokine interleukin 15 (IL-15). In vitro stimulation with IL-15 rescued NK cells of HIV⁺ and HCV⁺ patients from apoptosis and enhanced proliferation and functional activity. We hypothesize that the reduced levels of IL-15 present in the serum during HIV and HCV infections might impact NK cell homeostasis, contributing to the common alterations of the NK pool observed in these unrelated infections.     

Inactivation of human immunodeficiency virus type 1 in blood samples stored as high-salt lysates

Blood samples to be tested for the presence of parasite DNA by using specific DNA probes are routinely stored in our laboratory as high-salt lysates (HSL). To safeguard against the risk of accidental infection with etiological agents such as the human immunodeficiency virus type 1 (HIV-1) while manipulating large numbers of blood samples in preparation for DNA probing, we determined the residual infectivity of HIV-1 after exposure to HSL components. Both high-titer virus stocks or provirus-carrying cells, suspended either in tissue culture medium or freshly drawn blood, were completely inactivated upon contact with the HSL components. This was verified by the absence of any detectable HIV-1-specific antigen in the supernatants of long-term cultures and the absence of virus-specific DNA fragments after amplification by polymerase chain reaction with DNA from such cultures as target DNA. These results support the conclusion that the virus is in fact completely inactivated by contact with the HSL components, rendering blood specimens stored as HSL noninfectious in regard to HIV-1.     

Structural biology of human CD81, a receptor for hepatitis C virus

Human CD81, which is belonged to tetraspanin family, has been previously identified as a receptor for the hepatitis C virus envelope E 2 glycoprotein. The crystal structure of the human CD81 long extracellular domain, binding site for E 2 glycoprotein, is presented here at 1.6 A resolution. The tertiary structure of CD81-LEL, which is composed of five alpha-helices, is resemble for a mushroom-shaped molecules (stalk and head subdomains) and forms a dimer in the crystallographic asymmetric unit. The two disulfide bridges, which are conserved all the tetraspanin and are necessary for CD 81-HCV interaction, are stabilizing the conformation of the head domain. This head domain is solvent exposed surface region and is locating the amino acid residues which are essential for the E 2 binding. The hydrophobic cluster in this head domain may suggest that the presence of a docking site for a low complementary surface cavity in the partner E 2 glycoprotein. We proposed that the dimer structure may be important in the interactions of HCV E 2 glycoprotein and also the viral protein may occur in dimeric aggregation on the HCV envelope. This common structural motif of the tetraspanin provides the first insight onto the mechanism of HCV binding to human cell and may be targets for structure-based antiviral drug.