Phylogenetic and virulence analysis of tick-borne encephalitis virus field isolates from Switzerland

Tick-borne encephalitis (TBE) is an endemic disease in Switzerland, with about 110-120 reported human cases each year. Endemic areas are found throughout the country. However, the viruses circulating in Switzerland have not been characterized so far. In this study, the complete envelope (E) protein sequences and phylogenetic classification of 72 TBE viruses found in Ixodes ricinus ticks sampled at 39 foci throughout Switzerland were analyzed. All isolates belonged to the European subtype and were highly related (mean pairwise sequence identity of 97.8% at the nucleotide and 99.6% at the amino acid level of the E protein). Sixty-four isolates were characterized in vitro with respect to their plaque phenotype. More than half (57.8%) of isolates produced a mixture of plaques of different sizes, reflecting a heterogeneous population of virus variants. Isolates consistently forming plaques of small size were associated with recently detected endemic foci with no or only sporadic reports of clinical cases. All of six virus isolates investigated in an in vivo mouse model were highly neurovirulent (100% mortality) but exhibited a relatively low level of neuroinvasiveness, with mouse survival rates ranging from 50% to 100%. Therefore, TBE viruses circulating in Switzerland belong to the European subtype and are closely related. In vitro and in vivo surrogates suggest a high proportion of isolates with a relatively low level of virulence, which is in agreement with a hypothesized high proportion of subclinical or mild TBE infections.

Compartmentalization of small ruminant lentivirus between blood and colostrum in infected goats

The compartmentalization of small ruminant lentivirus (SRLV) subtype A (Maedi-Visna virus) and B (caprine arthritis-encephalitis virus) variants was analyzed in colostrum and peripheral blood mononuclear cells of four naturally infected goats. Sequence analysis of DNA and RNA encompassing the V4-V5 env regions showed a differential distribution of SRLV variants between the two compartments. Tissue-specific compartmentalization was demonstrated by phylogenetic analysis in three of the four cases. In these animals colostrum proviral sequences were clustered relative to the blood viral sequences. In one goat, the blood and colostrum-derived provirus sequences were intermingled, suggesting trafficking of virus between the two tissues or mirroring a recent infection. Surprisingly, the pattern of free virus variants in the colostrum of all animals corresponded only partially to that of the proviral form, suggesting that free viruses might not derive from infected colostral cells. The compartmentalization of SRLV between peripheral blood and colostrum indicates that lactogenic transmission may involve specific viruses not present in the proviral populations circulating in the blood.

Orosomucoid (alpha1-acid glycoprotein) plasma concentration and genetic variants: effects on human immunodeficiency virus protease inhibitor clearance and cellular accumulation

BACKGROUND AND OBJECTIVE: Protease inhibitors are highly bound to orosomucoid (ORM) (alpha1-acid glycoprotein), an acute-phase plasma protein encoded by 2 polymorphic genes, which may modulate their disposition. Our objective was to determine the influence of ORM concentration and phenotype on indinavir, lopinavir, and nelfinavir apparent clearance (CL(app)) and cellular accumulation. Efavirenz, mainly bound to albumin, was included as a control drug. METHODS: Plasma and cells samples were collected from 434 human immunodeficiency virus-infected patients. Total plasma and cellular drug concentrations and ORM concentrations and phenotypes were determined. RESULTS: Indinavir CL(app) was strongly influenced by ORM concentration (n = 36) (r2 = 0.47 [P = .00004]), particularly in the presence of ritonavir (r2 = 0.54 [P = .004]). Lopinavir CL(app) was weakly influenced by ORM concentration (n = 81) (r2 = 0.18 [P = .0001]). For both drugs, the ORM1 S variant concentration mainly explained this influence (r2 = 0.55 [P = .00004] and r2 = 0.23 [P = .0002], respectively). Indinavir CL(app) was significantly higher in F1F1 individuals than in F1S and SS patients (41.3, 23.4, and 10.3 L/h [P = .0004] without ritonavir and 21.1, 13.2, and 10.1 L/h [P = .05] with ritonavir, respectively). Lopinavir cellular exposure was not influenced by ORM abundance and phenotype. Finally, ORM concentration or phenotype did not influence nelfinavir (n = 153) or efavirenz (n = 198) pharmacokinetics. CONCLUSION: ORM concentration and phenotype modulate indinavir pharmacokinetics and, to a lesser extent, lopinavir pharmacokinetics but without influencing their cellular exposure. This confounding influence of ORM should be taken into account for appropriate interpretation of therapeutic drug monitoring results. Further studies are needed to investigate whether the measure of unbound drug plasma concentration gives more meaningful information than total drug concentration for indinavir and lopinavir.

Estimating the net contribution of interleukin-28B variation to spontaneous hepatitis C virus clearance

The identification of associations between interleukin-28B (IL-28B) variants and the spontaneous clearance of hepatitis C virus (HCV) raises the issues of causality and the net contribution of host genetics to the trait. To estimate more precisely the net effect of IL-28B genetic variation on HCV clearance, we optimized genotyping and compared the host contributions in multiple- and single-source cohorts to control for viral and demographic effects. The analysis included individuals with chronic or spontaneously cleared HCV infections from a multiple-source cohort (n = 389) and a single-source cohort (n = 71). We performed detailed genotyping in the coding region of IL-28B and searched for copy number variations to identify the genetic variant or haplotype carrying the strongest association with viral clearance. This analysis was used to compare the effects of IL-28B variation in the two cohorts. Haplotypes characterized by carriage of the major alleles at IL-28B single-nucleotide polymorphisms (SNPs) were highly overrepresented in individuals with spontaneous clearance versus those with chronic HCV infections (66.1% versus 38.6%, P = 6 × 10(-9) ). The odds ratios for clearance were 2.1 [95% confidence interval (CI) = 1.6-3.0] and 3.9 (95% CI = 1.5-10.2) in the multiple- and single-source cohorts, respectively. Protective haplotypes were in perfect linkage (r(2) = 1.0) with a nonsynonymous coding variant (rs8103142). Copy number variants were not detected. CONCLUSION: We identified IL-28B haplotypes highly predictive of spontaneous HCV clearance. The high linkage disequilibrium between IL-28B SNPs indicates that association studies need to be complemented by functional experiments to identify single causal variants. The point estimate for the genetic effect was higher in the single-source cohort, which was used to effectively control for viral diversity, sex, and coinfections and, therefore, offered a precise estimate of the net host genetic contribution.

Genome-wide association study identifies variants associated with progression of liver fibrosis from HCV infection

Polymorphisms in IL28B were shown to affect clearance of hepatitis C virus (HCV) infection in genome-wide association (GWA) studies. Only a fraction of patients with chronic HCV infection develop liver fibrosis, a process that might also be affected by genetic factors. We performed a 2-stage GWA study of liver fibrosis progression related to HCV infection.

Liver kidney microsomal type 1 antibodies reduce the CYP2D6 activity in patients with chronic hepatitis C virus infection

Liver kidney microsomal type 1 (LKM-1) antibodies have been shown to decrease the CYP2D6 activity in vitro and are present in a minority of patients with chronic hepatitis C infection. We investigated whether LKM-1 antibodies might reduce the CYP2D6 activity in vivo. All patients enrolled in the Swiss Hepatitis C Cohort Study and tested for LKM-1 antibodies were assessed (n = 1723): 10 eligible patients were matched with patients without LKM-1 antibodies. Patients were genotyped for CYP2D6 variants to exclude individuals with a poor metabolizer genotype. CYP2D6 activity was measured by a specific substrate using the dextromethorphan/dextrorphan metabolic ratio to classify patients into four activity phenotypes. All patients had a CYP2D6 extensive metabolizer genotype. The observed phenotype was concordant with the CYP2D6 genotype in most LKM-negative patients, whereas only three LKM-1 positive patients had a concordant phenotype (six presented an intermediate and one a poor metabolizer phenotype). The median DEM/DOR ratio was sixfold higher in LKM-1 positive than in LKM-1 negative patients (0.096 vs. 0.016, P = 0.004), indicating that CYP2D6 metabolic function was significantly reduced in the presence of LKM-1 antibodies. In chronic hepatitis C patients with LKM-1 antibodies, the CYP2D6 metabolic activity was on average reduced by 80%. The impact of LKM-1 antibodies on CYP2D6-mediated drug metabolism pathways warrants further translational studies.

Design, expression, and processing of epitomized hepatitis C virus-encoded CTL epitopes

Hepatitis C virus (HCV) vaccine efficacy may crucially depend on immunogen length and coverage of viral sequence diversity. However, covering a considerable proportion of the circulating viral sequence variants would likely require long immunogens, which for the conserved portions of the viral genome, would contain unnecessarily redundant sequence information. In this study, we present the design and in vitro performance analysis of a novel "epitome" approach that compresses frequent immune targets of the cellular immune response against HCV into a shorter immunogen sequence. Compression of immunological information is achieved by partial overlapping shared sequence motifs between individual epitopes. At the same time, sequence diversity coverage is provided by taking advantage of emerging cross-reactivity patterns among epitope variants so that epitope variants associated with the broadest variant cross-recognition are preferentially included. The processing and presentation analysis of specific epitopes included in such a compressed, in vitro-expressed HCV epitome indicated effective processing of a majority of tested epitopes, although re-presentation of some epitopes may require refined sequence design. Together, the present study establishes the epitome approach as a potential powerful tool for vaccine immunogen design, especially suitable for the induction of cellular immune responses against highly variable pathogens.

Increased cytotoxic T-lymphocyte epitope variant cross-recognition and functional avidity are associated with hepatitis C virus clearance

Hepatitis C virus (HCV) clearance has been associated with reduced viral evolution in targeted cytotoxic T-lymphocyte (CTL) epitopes, suggesting that HCV clearers may mount CTL responses with a superior ability to recognize epitope variants and prevent viral immune escape. Here, 40 HCV-infected subjects were tested with 406 10-mer peptides covering the vast majority of the sequence diversity spanning a 197-residue region of the NS3 protein. HCV clearers mounted significantly broader CTL responses of higher functional avidity and with wider variant cross-recognition capacity than nonclearers. These observations have important implications for vaccine approaches that may need to induce high-avidity responses in vivo.

Origin of Minority Drug-Resistant HIV-1 Variants in Primary HIV-1 Infection

Background. Drug-resistant human immunodeficiency virus type 1 (HIV-1) minority variants (MVs) are present in some antiretroviral therapy (ART)–naive patients. They may result from de novo mutagenesis or transmission. To date, the latter has not been proven. Methods. MVs were quantified by allele-specific polymerase chain reaction in 204 acute or recent seroconverters from the Zurich Primary HIV Infection study and 382 ART-naive, chronically infected patients. Phylogenetic analyses identified transmission clusters. Results. Three lines of evidence were observed in support of transmission of MVs. First, potential transmitters were identified for 12 of 16 acute or recent seroconverters harboring M184V MVs. These variants were also detected in plasma and/or peripheral blood mononuclear cells at the estimated time of transmission in 3 of 4 potential transmitters who experienced virological failure accompanied by the selection of the M184V mutation before transmission. Second, prevalence between MVs harboring the frequent mutation M184V and the particularly uncommon integrase mutation N155H differed highly significantly in acute or recent seroconverters (8.2% vs 0.5%; P < .001). Third, the prevalence of less-fit M184V MVs is significantly higher in acutely or recently than in chronically HIV-1–infected patients (8.2% vs 2.5%; P = .004). Conclusions. Drug-resistant HIV-1 MVs can be transmitted. To what extent the origin—transmission vs sporadic appearance—of these variants determines their impact on ART needs to be further explored.

Molecular detection of hepatitis E virus in German domestic pigs

In industrial countries, Hepatitis E virus (HEV) transmission to humans is predominantly assumed to be a zoonotic infection. Recently, it has been demonstrated that about 50% of domestic pigs in Germany carry HEV-specific antibodies. However, further investigations concerning the distribution of HEV in different age groups of German domestic pigs, phylogenetic analyses and the viral load in the porcine liver are still pending. Liver samples of all age groups from herds in a pig-dense region in north-western Germany were investigated for the presence and quantity of HEV RNA and subsequently genotyped. Out of 251 liver samples, 34 contained ORF2-specific RNA, whereas 19 samples were positive using ORF1-specific primers, resulting in an overall detection rate of 13.5% and 7.6%, respectively. Especially nursery pigs and growers were tested positive for viral RNA. Furthermore, determination of the HEV copy numbers revealed high replication levels. Up to 10(9) genome copies per g of liver tissue could be detected suggesting a likely high degree of viral spread to the environment. In the HEV-positive liver samples we found no hints for pathohistological changes reflecting the HEV status.The HEV sequences showed marked diversity but could be assigned to HEV genotype 3 without exception. However, by comparing two different genomic fragments, we found indications for infections with two different HEV variants in domestic pigs. Apart from this, the current study confirms the outcome of our recent serological HEV survey and for the first time gives direct proof of HEV infections in the German domestic pig population.

Generation of a recombinant Gag virus-like-particle panel for the evaluation of p24 antigen detection by diagnostic HIV tests.

BACKGROUND Detection of HIV-1 p24 antigen permits early identification of primary HIV infection and timely intervention to limit further spread of the infection. Principally, HIV screening should equally detect all viral variants, but reagents for a standardised test evaluation are limited. Therefore, we aimed to create an inexhaustible panel of diverse HIV-1 p24 antigens. METHODS We generated a panel of 43 recombinantly expressed virus-like particles (VLPs), containing the structural Gag proteins of HIV-1 subtypes A-H and circulating recombinant forms (CRF) CRF01_AE, CRF02_AG, CRF12_BF, CRF20_BG and group O. Eleven 4th generation antigen/antibody tests and five antigen-only tests were evaluated for their ability to detect VLPs diluted in human plasma to p24 concentrations equivalent to 50, 10 and 2 IU/ml of the WHO p24 standard. Three tests were also evaluated for their ability to detect p24 after heat-denaturation for immune-complex disruption, a pre-requisite for ultrasensitive p24 detection. RESULTS Our VLP panel exhibited an average intra-clade p24 diversity of 6.7%. Among the 4th generation tests, the Abbott Architect and Siemens Enzygnost Integral 4 had the highest sensitivity of 97.7% and 93%, respectively. Alere Determine Combo and BioRad Access were least sensitive with 10.1% and 40.3%, respectively. Antigen-only tests were slightly more sensitive than combination tests. Almost all tests detected the WHO HIV-1 p24 standard at a concentration of 2 IU/ml, but their ability to detect this input for different subtypes varied greatly. Heat-treatment lowered overall detectability of HIV-1 p24 in two of the three tests, but only few VLPs had a more than 3-fold loss in p24 detection. CONCLUSIONS The HIV-1 Gag subtype panel has a broad diversity and proved useful for a standardised evaluation of the detection limit and breadth of subtype detection of p24 antigen-detecting tests. Several tests exhibited problems, particularly with non-B subtypes.

Performance of HBsAg point-of-care tests for detection of diagnostic escape-variants in clinical samples

BACKGROUND Hepatitis B viruses (HBV) harboring mutations in the a-determinant of the Hepatitis B surface antigen (HBsAg) are associated with reduced reactivity of HBsAg assays. OBJECTIVES To evaluate the sensitivity and specificity of three HBsAg point-of-care tests for the detection of HBsAg of viruses harboring HBsAg mutations. STUDY DESIGN A selection of 50 clinical plasma samples containing HBV with HBsAg mutations was used to evaluate the performance of three HBsAg point-of-care tests (Vikia(®), bioMérieux, Marcy-L'Étoile, France. Alere Determine HBsAg™, Iverness Biomedical Innovations, Köln, Germany. Quick Profile™, LumiQuick Diagnostics, California, USA) and compared to the ARCHITECT HBsAg Qualitative(®) assay (Abbott Laboratories, Sligo, Ireland). RESULTS The sensitivity of the point-of-care tests ranged from 98% to 100%. The only false-negative result occurred using the Quick Profile™ assay with a virus harboring a D144A mutation. CONCLUSIONS The evaluated point-of-care tests revealed an excellent sensitivity in detecting HBV samples harboring HBsAg mutations.

Efficacy of a live attenuated vaccine in classical swine fever virus postnatally persistently infected pigs

Classical swine fever (CSF) causes major losses in pig farming, with various degrees of disease severity. Efficient live attenuated vaccines against classical swine fever virus (CSFV) are used routinely in endemic countries. However, despite intensive vaccination programs in these areas for more than 20 years, CSF has not been eradicated. Molecular epidemiology studies in these regions suggests that the virus circulating in the field has evolved under the positive selection pressure exerted by the immune response to the vaccine, leading to new attenuated viral variants. Recent work by our group demonstrated that a high proportion of persistently infected piglets can be generated by early postnatal infection with low and moderately virulent CSFV strains. Here, we studied the immune response to a hog cholera lapinised virus vaccine (HCLV), C-strain, in six-week-old persistently infected pigs following post-natal infection. CSFV-negative pigs were vaccinated as controls. The humoral and interferon gamma responses as well as the CSFV RNA loads were monitored for 21 days post-vaccination. No vaccine viral RNA was detected in the serum samples and tonsils from CSFV postnatally persistently infected pigs for 21 days post-vaccination. Furthermore, no E2-specific antibody response or neutralising antibody titres were shown in CSFV persistently infected vaccinated animals. Likewise, no of IFN-gamma producing cell response against CSFV or PHA was observed. To our knowledge, this is the first report demonstrating the absence of a response to vaccination in CSFV persistently infected pigs.

A frequent hypofunctional IRAK2 variant is associated with reduced spontaneous hepatitis C virus clearance.

UNLABELLED Patients carrying very rare loss-of-function mutations in interleukin-1 receptor-associated kinase 4 (IRAK4), a critical signaling mediator in Toll-like receptor signaling, are severely immunodeficient, highlighting the paramount role of IRAK kinases in innate immunity. We discovered a comparatively frequent coding variant of the enigmatic human IRAK2, L392V (rs3844283), which is found homozygously in ∼15% of Caucasians, to be associated with a reduced ability to induce interferon-alpha in primary human plasmacytoid dendritic cells in response to hepatitis C virus (HCV). Cytokine production in response to purified Toll-like receptor agonists was also impaired. Additionally, rs3844283 was epidemiologically associated with a chronic course of HCV infection in two independent HCV cohorts and emerged as an independent predictor of chronic HCV disease. Mechanistically, IRAK2 L392V showed intact binding to, but impaired ubiquitination of, tumor necrosis factor receptor-associated factor 6, a vital step in signal transduction. CONCLUSION Our study highlights IRAK2 and its genetic variants as critical factors and potentially novel biomarkers for human antiviral innate immunity.

Distribution and variability of deformed wing virus of honeybees (Apis mellifera) in the Middle East and North Africa.

Three hundred eleven honeybee samples from twelve countries in the Middle East and North Africa (MENA) (Jordan, Lebanon, Syria, Iraq, Egypt, Libya, Tunisia, Algeria, Morocco, Yemen, Palestine and Sudan) were analyzed for the presence of deformed wing virus (DWV). The prevalence of DWV throughout the MENA region was pervasive, but variable. The highest prevalence was found in Lebanon and Syria, with prevalence dropping in Palestine, Jordan and Egypt before increasing slightly moving westwards to Algeria and Morocco Phylogenetic analysis of a 194 nucleotide section of the DWV Lp gene did not identify any significant phylogenetic resolution among the samples, although the sequences did show consistent regional clustering, including an interesting geographic gradient from Morocco through North Africa to Jordan and Syria. The sequences revealed several clear variability hotspots in the deduced amino acid sequence, that furthermore showed some patterns of regional identity. Furthermore, the sequence variants from the Middle East and North Africa appear more numerous and diverse than those from Europe. This article is protected by copyright. All rights reserved.