Acute hepatitis C virus infection induces anti-host cell receptor antibodies with virus-neutralizing properties.

UNLABELLED: Hepatitis C virus (HCV) causes persistent infection in the majority of infected individuals. The mechanisms of persistence and clearance are only partially understood. Antibodies (Abs) against host cell entry receptors have been shown to inhibit HCV infection in cell culture and animal models. In this study, we aimed to investigate whether anti-receptor Abs are induced during infection in humans in vivo and whether their presence is associated with outcome of infection. We established an enzyme-linked immunosorbant assay using a recombinant CD81-claudin-1 (CLDN1) fusion protein to detect and quantify Abs directed against extracellular epitopes of the HCV CD81-CLDN1 coreceptor complex. The presence of anti-receptor Abs was studied in serum of patients from a well-defined cohort of a single-source HCV outbreak of pregnant women and several control groups, including uninfected pregnant women, patients with chronic hepatitis B and D virus (HBV/HDV) infection, and healthy individuals. Virus-neutralizing activity of Abs was determined using recombinant cell culture-derived HCV (HCVcc). Our results demonstrate that HCV-infected patients have statistically significantly higher anti-CD81/CLDN1 Ab titers during the early phase of infection than controls. The titers were significantly higher in resolvers compared to persisters. Functional studies using immunoadsorption and HCV cell culture models demonstrate that HCV-neutralizing anti-receptor Abs are induced in the early phase of HCV infection, but not in control groups. CONCLUSION: The virus-neutralizing properties of these Abs suggest a role for control of viral infection in conjunction with antiviral responses. Characterization of these anti-receptor Abs opens new avenues to prevent and treat HCV infection.

NKT cells determine titer and subtype profile of virus-specific IgG antibodies during herpes simplex virus Infection.

Invariant NKT cells (iNKT cells) are innate lymphocytes that recognize lipid-derived Ags presented by the MHC class I-related protein CD1d. In this study, we analyzed the role of iNKT cells in the generation of Abs against HSV type 1 (HSV-1). In sera from healthy hman donors, we found a correlation between HSV-1-specific IgG titers and proportions of CD4(+) iNKT cells. In HSV-1-infected iNKT cell-deficient mice, the amount of specific IgM and IgG Abs were significantly reduced compared with wild-type mice. Moreover, iNKT cell-deficient mice were unable to upregulate CD1d on B cells and failed to establish an IFN-γ-driven subtype profile of HSV-1-specific IgG Abs. In spleens of HSV-1-infected wild-type mice, the percentage of iNKT cells expressing CCR6, a marker for inflammatory iNKT cells secreting IFN-γ, was significantly decreased at 6 mo postinfection, suggesting that these cells were released from the spleen to other tissues. Finally, in vitro experiments showed that in the absence of CD1d-restricted cells, HSV-1 induced markedly lower IFN-γ production in splenocytes from naive mice. Taken together, our results indicate that iNKT cells shape the Ab response to HSV-1 infection and provide a basis for rational development of antiviral vaccines.

Hantavirus infections by Puumala or Dobrava-Belgrade virus in pregnant women.

BACKGROUND: Hantavirus infection in humans usually occurs via inhalation of infectious aerosolized excreta of rodents. Horizontal human-to-human transmission was reported only for the highly virulent Andes virus. The likelihood of vertical transmission and the clinical outcome of hantavirus infections in pregnancy is still unpredictable. OBJECTIVES: Very few data were published about the impact of hantaviruses in pregnancy. Here we present four cases of pregnant women infected by European hantaviruses. The risk of vertical virus transmission was investigated. STUDY DESIGN: Four pregnant women with clinical signs of acute hantavirus disease were investigated for hantavirus IgM and IgG after onset of clinical symptoms. Furthermore, the newborns were tested for presence of viral RNA and antibodies in cord blood and, if any parameter was found positive, 8-12 months after delivery. RESULTS: Four women suffered from a hantavirus infection, two of them due to infection by Puumala virus and two by Dobrava-Belgrade virus. Three women delivered healthy babies by vaginal route and one woman by Caesarean section (week 28). In no case hantavirus RNA was detected in cord blood after delivery or in the 8-12 month old babies. Hantavirus IgG was detectable in the cord blood of 3 babies (but not in the preterm child); these antibodies disappeared after 8-12 months indicating a passive transfer of immunoglobulins. No child had any clinical sign of hantavirus infection. CONCLUSIONS: In this study, the absence of vertical hantavirus transmission was demonstrated for pregnant women with onset of hantavirus disease between gestation weeks 14 and 28.

Human papillomavirus E6/E7 mRNA testing has higher specificity than liquid-based DNA testing in the evaluation of cervical intraepithelial neoplasia.

OBJECTIVE: To examine the specificity of human papillomavirus (HPV) E6/E7 mRNA testing for intraepithelial precursor lesions and invasive carcinoma of the uterine cervix in 358 women and compare the results with those of the most widely used DNA technique. STUDY DESIGN: For HPV E6/E7 mRNA testing an amplification assay was used. For DNA determination a hybridization assay was applied. Both techniques were used simultaneously in patients with normal morphology (150), cervical intraepithelial neoplasia (173) and invasive carcinoma of the cervix (35). RESULTS: HPV DNA positivity rates were significantly higher than E6/E7 mRNA in women with normal morphology (21-7%), cervical intraepithelial neoplasia (CIN) 1 and 2 (75-43%), and CIN 3 (93-63%). In invasive cervical carcinoma, both methods tested equally high (94% vs. 97%). Considering that E6/E7 up-regulation represents the initial step in cervical carcinogenesis, it can be assumed that this test allows a more specific detection of lesions with a potential for progression. CONCLUSION: HPV E6/E7 mRNA may serve as a more specific discriminator between transient cervical dysplasias and potentially progressive lesions. Accordingly, testing for high-risk HPV E6/E7 mRNA might reduce the psychologic burden associated with HPV-DNA testing.

Quantitative detection and typing of hepatitis D virus in human serum by real-time polymerase chain reaction and melting curve analysis.

Hepatitis D virus (HDV) infection is an important etiologic agent of fulminant hepatitis and may aggravate the clinical course of chronic hepatitis B infection resulting in cirrhosis and liver failure. This report describes the establishment of a real-time reverse transcriptase polymerase chain reaction method that allows the quantitative detection of HDV-1 and HDV-3 with a sensitivity in a linear range of 2 x 10(3) to 10(8) copies/mL. Additionally, the new assay provides the opportunity to distinguish HDV-1 from HDV-3 by a subsequent melting curve analysis, an important option because these HDV types are highly associated with severe clinical outcome. The results of the melting curve analysis of 42 HDV sequences obtained in this study and the phylogenetic analysis based on 139 full-length sequences from GenBank were consistent and showed that all sequences described here cluster within the HDV-1 clade. Therefore, this assay is useful for monitoring of antiviral treatment and molecular epidemiologic studies of HDV distribution.

High HEV presence in four different wild boar populations in East and West Germany.

Swine Hepatitis E virus (HEV) can be transmitted from pigs to humans causing hepatitis. A high prevalence of HEV in wild boar populations is reported for several European countries, but actual data for Germany are missing. During the hunting season from October to December 2007 liver, bile and blood samples were collected from wild boars in four different German regions. The samples were tested for HEV RNA by quantitative PCR (qPCR) and anti-HEV IgG antibodies by two different ELISAs and a Line immunoassay. A seroprevalence of 29.9% using ELISA and 26.2% in the Line immunoassay was determined. The seroprevalence rate varied greatly within the analyzed regions. However, qPCR analysis revealed a higher prevalence of 68.2% positive animals with regional differences. Surprisingly, also adult wild sows and wild boars were highly HEV positive by qPCR. Compared to liver and serum samples, bile samples showed a higher rate of positive qPCR results. Sequencing and phylogenetic analysis of a 969nt fragment within ORF 2 revealed that all isolates clustered within genotype 3 but differed in the subtype depending on the hunting spot. Isolates clustered within genotypes 3i, 3h, 3f and 3e. Within one population HEV isolates were closely related, but social groups of animals in close proximity might be infected with different subtypes. Two full-length genomes of subtypes 3i and 3e from two different geographic regions were generated. The wild boar is discussed as one of the main sources of human autochthonous infections in Germany.

Co-replication analyses of naturally occurring defective hepatitis B virus variants with wild-type.

Complex hepatitis B virus (HBV) variants with mutations in core promoter (Cp) plus deletions in the C gene and/or preS region--that are associated with development of liver cirrhosis in renal transplant recipients--show a drastically changed phenotype with altered transcription and disturbed surface and core protein expression. Here, we analyzed the replication phenotype of six different defective variant genomes, isolated from two patients, after co-transfection with HBV wild-type (wt) in varying proportions. Both in HuH7 and HepG2 cells, the variants showed enhanced replication and enrichment in the different transfected variant-wt mixtures. Contrary to artificial variants with only C gene deletions in wt context, the original complex variants as well as wt genomes carrying C gene and Cp mutations of the variants did mostly not suppress wt replication. Thus, the Cp mutations compensate the suppression of wt by C gene deletions and furthermore enhance the replication level.

Detection and typing of human pathogenic hantaviruses by real-time reverse transcription-PCR and pyrosequencing.

BACKGROUND: Because the clinical course of human infections with hantaviruses can vary from subclinical to fatal, rapid and reliable detection of hantaviruses is essential. To date, the diagnosis of hantavirus infection is based mainly on serologic assays, and the detection of hantaviral RNA by the commonly used reverse transcription (RT)-PCR is difficult because of high sequence diversity of hantaviruses and low viral loads in clinical specimens. METHODS: We developed 5 real-time RT-PCR assays, 3 of which are specific for the individual European hantaviruses Dobrava, Puumala, or Tula virus. Two additional assays detect the Asian species Hantaan virus together with Seoul virus and the American species Andes virus together with Sin Nombre virus. Pyrosequencing was established to provide characteristic sequence information of the amplified hantavirus for confirmation of the RT-PCR results or for a more detailed virus typing. RESULTS: The real-time RT-PCR assays were specific for the respective hantavirus species and optimized to run on 2 different platforms, the LightCycler and the ABI 7900/7500. Each assay showed a detection limit of 10 copies of a plasmid containing the RT-PCR target region, and pyrosequencing was possible with 10 to 100 copies per reaction. With this assay, viral genome could be detected in 16 of 552 (2.5%) specimens of suspected hantavirus infections of humans and mice. CONCLUSIONS: The new assays detect, differentiate, and quantify hantaviruses in clinical specimens from humans and from their natural hosts and may be useful for in vitro studies of hantaviruses.

Hantavirus disease outbreak in Germany: limitations of routine serological diagnostics and clustering of virus sequences of human and rodent origin.

In Europe, hemorrhagic fever with renal syndrome results mainly from infection with Puumala virus (PUUV) or Dobrava virus. For 31 patients from a hantavirus disease outbreak in Lower Bavaria, a district in southeast Germany, serodiagnosis was undertaken by enzyme-linked immunosorbent assay, immunofluorescence assay, and immunoblot analysis. In a few of these cases, however, PUUV-specific typing of antibodies by these standard assays failed and a virus neutralization assay under biosafety level 3 conditions was required to verify the infection by this virus type. PUUV RNA was amplified by reverse transcription-PCR from acute-phase sera of three patients and was found to be very closely related to virus sequences obtained from bank voles (Clethrionomys glareolus) trapped in the same area. These findings link the outbreak with a novel PUUV lineage, "Bavaria," circulating in the local rodent population. The Bavaria lineage associated with the outbreak is only distantly related to other PUUV lineages from Germany.

Proteasomal degradation of core protein variants from chronic hepatitis B patients.

The accumulation of complex hepatitis B virus (HBV) variants with internal in-frame deletions in the C gene in immunosuppressed renal transplant recipients is associated with a severe course of the infection leading to end-stage liver disease (ESLD). A set of six HBV C genes with internal in-frame deletions corresponding to the pattern of HBV population in immunosuppressed patients has been expressed in two different eukaryotic cell lines. Synthesis and proteasomal degradation of HBV core (HBc) protein variants were compared with those of the wild-type HBc. In all cases, the steady-state level of internally deleted HBc proteins, predominantly with longer deletions, were considerably lower and turnover was significantly higher in comparison with those of the wild-type HBc, since all deletion variants were degraded rapidly via the proteasome pathway. Involvement and consequences of the proteasomal degradation machinery in the HBc protein turnover during HBV infection with complex HBV variants in the immunosuppressed patients are discussed.

Rapid induction of virus-neutralizing antibodies and viral clearance in a single-source outbreak of hepatitis C.

In contrast to a detailed understanding of antiviral cellular immune responses, the impact of neutralizing antibodies for the resolution of acute hepatitis C is poorly defined. The analysis of neutralizing responses has been hampered by the fact that patient cohorts as well as hepatitis C virus (HCV) strains are usually heterogeneous, and that clinical data from acute-phase and long-term follow-up after infection are not readily available. Using an infectious retroviral HCV pseudoparticle model system, we studied a cohort of women accidentally exposed to the same HCV strain of known sequence. In this single-source outbreak of hepatitis C, viral clearance was associated with a rapid induction of neutralizing antibodies in the early phase of infection. Neutralizing antibodies decreased or disappeared after recovery from HCV infection. In contrast, chronic HCV infection was characterized by absent or low-titer neutralizing antibodies in the early phase of infection and the persistence of infection despite the induction of cross-neutralizing antibodies in the late phase of infection. These data suggest that rapid induction of neutralizing antibodies during the early phase of infection may contribute to control of HCV infection. This finding may have important implications for understanding the pathogenesis of HCV infection and for the development of novel preventive and therapeutic antiviral strategies.

Development of novel immunoglobulin G (IgG), IgA, and IgM enzyme immunoassays based on recombinant Puumala and Dobrava hantavirus nucleocapsid proteins.

Human infections with Asian and European hantaviruses can result in hemorrhagic fever with renal syndromes of differing severities characterized by renal dysfunction and sometimes by pulmonary symptoms. For the serological detection of human infections by hantaviruses relevant for Europe, we developed monoclonal antibody capture immunoglobulin G (IgG) and IgA enzyme-linked immunosorbent assays (ELISAs) based on yeast-expressed nucleocapsid proteins of Puumala and Dobrava hantaviruses. Moreover, for diagnosis of acute infections, mu-capture IgM ELISAs were established with nucleocapsid proteins expressed in Drosophila melanogaster Schneider S2 cells. The cutoff values of the ELISAs were determined by investigation of up to 500 human anti-hantavirus-negative serum samples. The specificities of the Puumala and Dobrava virus-specific IgM, IgA, and IgG ELISAs were found to be 100%. The sensitivities of these ELISAs were determined to be 100% with panels of characterized anti-Puumala or anti-Dobrava virus-positive human serum samples. In most cases, Puumala and Dobrava virus infections could be differentiated by ELISA reactivity alone, i.e., endpoint titration with homologous and heterologous antigens.

Functional analysis of complex hepatitis B virus variants associated with development of liver cirrhosis.

BACKGROUND & AIMS: Development of cirrhosis in renal transplant recipients with chronic hepatitis B is associated with the accumulation of complex hepatitis B virus (HBV) variants carrying deletions in the C gene and/or preS region and deletions/insertions in the core promoter. Here, we characterized for the first time the phenotype of these complex HBV variants. METHODS: Representative full-length genomes of the HBV variants that were isolated and cloned from serum and liver of an immunosuppressed renal transplant recipient before and during end-stage liver disease were transfected into the human hepatoma cell line HuH7 and functionally analyzed. RESULTS: The variant genomes showed considerably reduced levels of precore and surface messenger RNA (mRNA) and of the major spliced pregenomic RNA, an increased level of pregenomic RNA, and a partial or complete defect in hepatitis B e antigen, core, and surface protein expression/secretion. Very low amounts of variant core protein with internal deletion were detectable. Reduced hepatitis B surface antigen secretion of some variants correlated with aberrant localization of surface proteins in endoplasmic reticulum. Despite the defects in viral protein expression, enhanced replication and enrichment in competition to wild-type HBV were observed. Enhanced reverse transcription and possibly increased levels of pregenomic RNA seem to be responsible for this effect. CONCLUSIONS: Development of cirrhosis is associated with accumulation of complex variants, which exhibit a drastically altered phenotype combining enhanced replication with defects in protein expression. This phenotype appears to be based on the major mutations in the core promoter and C gene but is considerably influenced by additional mutations throughout the genome.

Hantavirus in African wood mouse, Guinea.

Hantaviruses are rodent-borne, emerging viruses that cause life-threatening human diseases in Eurasia and the Americas. We detected hantavirus genome sequences in an African wood mouse (Hylomyscus simus) captured in Sangassou, Guinea. Sequence and phylogenetic analyses of the genetic material demonstrate a novel hantavirus species, which we propose to name "Sangassou virus."

Serological assays for the detection of human andes hantavirus infections based on its yeast-expressed nucleocapsid protein.

BACKGROUND: The objective of the study was to develop and evaluate IgM and IgG ELISAs and an IgG Western blot test for the serological detection of human infections with Andes virus (ANDV), the major cause of hantavirus cardiopulmonary syndrome (HCPS) in South America. METHODS: The entire nucleocapsid (N) protein-encoding sequence of ANDV (strain AH-1) was cloned and expressed in the yeast Saccharomyces cerevisiae. The polyhistidine-tagged recombinant N (rN) protein of ANDV was purified by nickel-chelation chromatography and characterized by its reactivity with different N-specific monoclonal antibodies. To detect an antibody response directed against ANDV in humans, indirect IgM and IgG ELISAs and an IgG Western blot test based on ANDV rN antigen were developed. The evaluation of the tests was performed using a negative serum panel and 63 blinded sera from Argentina and Chile, containing acute-phase and convalescent sera from HCPS patients. RESULTS: The specificities and sensitivities for the IgM and IgG ELISAs were demonstrated to be very high. The IgG ELISA data were confirmed by the IgG Western blot assay based on the same rN antigen. Almost all anti-ANDV-positive sera reacted to higher endpoint titers with N protein of ANDV than with those of Sin Nombre, Laguna Negra or Puumala virus. The cross-reactivity of anti-ANDV-N IgG-positive sera to rN proteins of other hantaviruses was found to be increased with time after the onset of HCPS. CONCLUSION: The high sensitivity of the novel assays should facilitate early diagnosis of ANDV infections and might contribute to a successful treatment of HCPS patients.

Monitoring of human cytomegalovirus, HHV-6 and HHV-7 infection in kidney transplant recipients by molecular methods to predict HCMV disease after transplantation: a prospective study.

OBJECTIVES: Recently, highly sensitive molecular assays to detect HCMV, HHV-6 and HHV-7 have been developed but their ability to detect patients at high risk for disease is unclear. METHODS: The positive predictive values (PPV) of pp65-antigenemia, quantitative plasma DNA and pp67-mRNA for CMV-disease were prospectively compared in 82 transplant recipients (72 renal, 10 pancreas-kidney) without CMV-prophylaxis. In addition, the prevalence of HHV-6 and HHV-7 infection were assessed using qualitative PCR. The assays were performed weekly. RESULTS: Three patients (3,7%) developed CMV-disease and were effectively treated. They were positive in all three CMV-assays. The PPVs of pp65-Ag, DNA viral load and pp67-mRNA were 33%, 20% and 25% in CMV-positive and 100%, 67% and 50% in seronegative recipients. Sensitivity and negative predictive value were 100% for all assays. Using cut-offs, PPVs were 75% (pp65-Ag > or = 20/200.000 cells) and 100% (PCR > or =30.000 copies/ml). Transfusion of >2 packed red cells, rejection and non-functioning graft were risk factors for CMV Five patients and one patient were positive for HHV-6 and HHV-7 resp.; both were symptomless and did not have a HCMV infection. CONCLUSIONS: Therefore, pp65-antigenemia and plasma PCR with a cut-off could be useful for monitoring preemptive therapy.