Jannick Verbeeck

Evaluation of Versant Hepatitis C Virus Genotype Assay (LiPA) 2.0

ABSTRACT Hepatitis C virus (HCV) genotyping is a tool used to optimize antiviral treatment regimens. The newly developed Versant HCV genotype assay (LiPA) 2.0 uses sequence information from both the 5′ untranslated region and the core region, allowing distinction between HCV genotype 1 and subtypes c to l of genotype 6 and between subtypes a and b of genotype 1. HCV-positive samples were genotyped manually using the Versant HCV genotype assay (LiPA) 2.0 system according to the manufacturers instructions. For the comparison study, Versant HCV genotype assay (LiPA) 1.0 was used. In this study, 99.7% of the samples could be amplified, the genotype of 96.0% of samples could be determined, and the agreement with the reference method was 99.4% when a genotype was determined. The reproducibility study showed no significant differences in performance across sites (P = 0.43) or across lots (P = 0.88). In the comparison study, 13 samples that were uninterpretable or incorrectly genotyped with Versant HCV genotype assay (LiPA) 1.0 were correctly genotyped by Versant HCV genotype assay (LiPA) 2.0. Versant HCV genotype assay (LiPA) 2.0 is a sensitive, accurate, and reliable assay for HCV genotyping. The inclusion of the core region probes in Versant HCV genotype assay (LiPA) 2.0 results in a genotyping success rate higher than that of the current Versant HCV genotype assay (LiPA) 1.0.

Investigating the Origin and Spread of Hepatitis C Virus Genotype 5a

ABSTRACT Epidemiological and phylogenetic studies of hepatitis C virus (HCV) have identified six major HCV genotypes and have attempted to characterize their origin and spread worldwide. Putative regions of endemic infection have been identified for all HCV genotypes except HCV genotype 5a. Although HCV genotype 5a was previously thought to be largely restricted to the northern part of South Africa, this study reports an unexpected cluster of the genotype in West Flanders Province in Belgium. To investigate the molecular epidemiology of this cluster and of HCV genotype 5a in general, a rigorous phylogenetic analysis of Belgian and South African HCV genotype 5a samples was performed. Remarkably, the Belgian and South African strains form two distinct clusters of similar diversity. We used a Bayesian coalescent method to estimate the rate of virus spread through time for HCV genotype 5a in both regions. Our results indicate that HCV genotype 5a strains have been spreading independently in Belgium and South Africa for more than 100 years, with a rate of spread characteristic of an epidemic genotype. These findings have major implications for tracing the origin of HCV genotype 5a. Here, we speculate about the possible origins of these clusters.

JC viral loads in patients with Crohn’s disease treated with immunosuppression: can we screen for elevated risk of progressive multifocal leukoencephalopathy?

Background and aims: Anti-α4 integrin therapy with natalizumab is efficacious in refractory Crohn’s disease and in multiple sclerosis, but carries an estimated 1/1000 risk of progressive multifocal leukoencephalopathy (PML) caused by reactivation of latent JC virus infection. Athough anti-α4 integrin therapies are likely to be introduced in the clinic, screening for the risk of PML has not been developed. Methods: We prospectively collected urine, serum, plasma and buffy coats from 125 patients with Crohn’s disease, 100 control subjects with gastrointestinal (GI) disease, and 106 healthy volunteers. Four to eight weeks after this first sample collection, we additionally collected a set of urine, serum, plasma and buffy coat samples from the 125 patients with Crohn’s disease, and a next set of samples was collected 12–16 weeks after the first collection. JC viral loads were determined with quantitative real-time polymerase chain reaction (PCR), and JC virus seroprevalence with a specific enzyme-linked immunosorbant assay (ELISA). Results: The overall JC virus seroprevalence was 65%. JC virus DNA copies were detected in the urine from 29–44% of subjects, both those with Crohn’s disease and controls. Median viral loads were significantly higher in patients with Crohn’s disease who were immunosuppressed (7.36×106 copies/ml) compared to healthy volunteers (2.77×105 copies/ml) and compared to GI controls (1.8×106 copies/ml). Clearance at any time point occurred in 4/107 (3.7%) subjects only. JC viraemia was found in two patients with Crohn’s disease. Conclusions: The natural history of JC virus in patients with Crohn’s disease is still unknown. Our study results show that JC virus latency and urine viral shedding is frequent in immunosuppressed patients with Crohn’s disease. More prospective studies are needed in order to agree on possible recommendations concerning the exclusion of patients with JCV viraemia from anti-α4 integrin treatment.

Molecular Evolutionary Analysis and Mutational Pattern of Full-Length Genomes of Hepatitis B Virus Isolated From Belgian Patients With Different Clinical Manifestations

Molecular evolutionary patterns of 62 HBV full‐length genomes obtained from Belgian patients were characterized. Phylogenetic analysis revealed diverse HBV subgenotypes including A2 and A6 (46.8%), D1–D4 (38.8%), E (9.7%), C1 (1.6%), and B2 (1.6%). The study population consisted of patients with different ethnic origin (Caucasian, Turkish, Asian, Arab, and African). One HBV D/C recombinant isolate was identified, which encoded subtype adw2. An HBV subgenotype D4 with an aberrant subtype ayw4 was detected. Although none of the genotypes was associated with a specific disease outcome, several nucleotide substitutions, deletions and insertions were observed within the HBV preS1/S and X genes, particularly among patients with active chronic hepatitis B infection and patients with cirrhosis. Within the immunological domain of the HBsAg gene, the most frequent substitutions were sT125M and sT118A. High rates of precore and basal core promoter mutations were detected in patients infected with genotype D of HBV. Almost half of the patients who received lamivudine therapy for at least 1 year had HBV variants associated with lamivudine drug resistance. In conclusion, the most common HBV genotypes in West Europe (A and D) also prevail in Belgium. The highest degree of genetic diversity was detected in HBV genotype D. In addition, this study reveals the circulation of exotic HBV genotypes B, C, and E in Belgium. J. Med. Virol. 82:379–389, 2010.

An outbreak of hepatitis A associated with the consumption of raw beef

BACKGROUND In July 2004, a sharp increase of hepatitis A, a notifiable disease in Belgium, was detected. OBJECTIVES We investigated the outbreak in order to identify the source and take appropriate action. STUDY DESIGN We conducted an outbreak investigation which included a matched case-control study to analyse the association with a range of food items and food providers. A phylogenetic analysis was used to study the relation between the outbreak cases and the identified source. RESULTS We registered 269 cases of hepatitis A. Consumption of raw beef (OR 16.0; 95% CI 2.1-120.7) was the most probable way of infection. A food handler working at an epidemiologically linked meat distribution plant had contracted hepatitis A 1 month before the start of the outbreak. HAV strains from the food handler and the patients involved in the outbreak were monophyletically related. CONCLUSIONS Since serological immunity in Belgium is decreasing over time, foodborne outbreaks of hepatitis A are a substantial risk. In this outbreak, a single food handler, at the level of the distribution chain, has been identified as the most likely source, through cross-contamination of raw beef. This outbreak investigation suggests the need to consider vaccination against hepatitis A in food handlers.

Phylogenetic analysis of hepatitis B virus full-length genomes reveals evidence for a large nosocomial outbreak in Belgium

BACKGROUND Hepatitis B virus (HBV) is primarily transmitted from mother to child, by sexual contact, intravenous drug abuse, or unsafe health care-related injection practices. Despite increased safety efforts, nosocomial acquired hepatitis B infection remains problematic. OBJECTIVES A large HBV outbreak was investigated comprising 36 patients with acute HBV infection in a primary care physicians practice. STUDY DESIGN In a retrospective study (2003-2008), 36 serum samples from patients with acute HBV infection were collected. They had received several injections by the same physician at least 3 months before the onset of clinical symptoms. As a control group, sera were collected from HBV patients from other physicians from the same province. Full-length HBV genomes were amplified and were phylogenetically analysed. RESULTS HBV complete genomes of 32 patients were successfully amplified and sequenced, and clustered together with the reference genotype A, subgenotype A2 strains. We also analysed 26 control HBV genotype A samples. All 32 HBV strains from the patient group clustered in a monophyletic branch with a bootstrap value of 100, whereas the control samples branched separately in another clade. The genetic distance value showed small differences within the patients group, whereas the rate within the control group was seven times higher. These observations confirm that the source of transmission was clearly different in both groups. CONCLUSION Maximum likelihood analysis and genetic distance calculations based on the full-length genomes of HBV strains isolated from patients and controls provided strong evidence for a common nosocomial source of infection for all 32 patient cases.

Molecular epidemiology of hepatitis C among drug users in Flanders, Belgium: association of genotype with clinical parameters and with sex- and drug-related risk behaviours

The aim of this study was to determine the genotypic variation of hepatitis C among drug users in Flanders and to relate the distribution of genotypes to the characteristics of the population. Hepatitis C virus RNA (HCV-RNA) quantification and genotyping was performed on stored samples from 161 anti-HCV-positive injecting and non-injecting drug users. Information on sociodemographic status, drug-related risk behaviour and sexual risk behaviour was available for each drug user. HCV-RNA was present in 152 of 161 samples (94.4%). Genotype 1 was predominant (48.7%), followed by genotype 3 (41.2%), genotype 4 (8.8%) and genotype 2 (1.4%). In the multivariate analysis, lack of a history of injecting drug use was confirmed as a statistically significant predictor for infection with genotype 1. Predictors for infection with genotype 3 were the presence of anti-HBc antibodies and a history of injecting drug use. Being tattooed emerged as a statistically significant predictor for infection with genotype 4. The 94.4% prevalence of HCV-RNA among anti-HCV-positive drug users was considerably higher than the 54–86% chronicity rate found globally among HCV-infected patients. The results of this study suggest the existence of separate transmission networks for injecting drug users and non-injecting drug users. Finally, the results suggest that tattooing practices play a role in the spread of HCV among drug users.

Antiviral Activity of Chloroquine against Human Coronavirus OC43 Infection in Newborn Mice

ABSTRACT Until recently, human coronaviruses (HCoVs), such as HCoV strain OC43 (HCoV-OC43), were mainly known to cause 15 to 30% of mild upper respiratory tract infections. In recent years, the identification of new HCoVs, including severe acute respiratory syndrome coronavirus, revealed that HCoVs can be highly pathogenic and can cause more severe upper and lower respiratory tract infections, including bronchiolitis and pneumonia. To date, no specific antiviral drugs to prevent or treat HCoV infections are available. We demonstrate that chloroquine, a widely used drug with well-known antimalarial effects, inhibits HCoV-OC43 replication in HRT-18 cells, with a 50% effective concentration (± standard deviation) of 0.306 ± 0.0091 μM and a 50% cytotoxic concentration (± standard deviation) of 419 ± 192.5 μM, resulting in a selectivity index of 1,369. Further, we investigated whether chloroquine could prevent HCoV-OC43-induced death in newborn mice. Our results show that a lethal HCoV-OC43 infection in newborn C57BL/6 mice can be treated with chloroquine acquired transplacentally or via maternal milk. The highest survival rate (98.6%) of the pups was found when mother mice were treated daily with a concentration of 15 mg of chloroquine per kg of body weight. Survival rates declined in a dose-dependent manner, with 88% survival when treated with 5 mg/kg chloroquine and 13% survival when treated with 1 mg/kg chloroquine. Our results show that chloroquine can be highly effective against HCoV-OC43 infection in newborn mice and may be considered as a future drug against HCoVs.

Molecular Characterization of Hepatitis B Virus Strains Circulating in Belgian Patients Co-Infected With HIV and HBV: Overt and Occult Infection

Hepatitis B virus (HBV) and human immunodeficiency virus (HIV) have similar transmission routes, implying that patients infected with HIV are at particular risk for HBV infection. Patients who are co‐infected with HIV and HBV progress more rapidly to end‐stage liver disease and different HBV genotypes may have a distinct impact on disease progression. One hundred ninety‐one anti‐HBc‐positive sera from Belgian patients co‐infected with HIV and HBV were collected during 1998–2008. Full‐length HBV genomes as well as large S or partial S genes were amplified and their molecular evolutionary history was analyzed. Clinically, 30 (65.8%) patients were categorized as “overt infection” and 16 (34.7%) cases were categorized as “occult infection.” Five distinct HBV genotypes comprising A (69.6%), E (19.6%), followed by D, C, and G were detected. HBV genotype A was observed in all clinical groups and in patients with varying ethnical background. HBV genotype E could be detected in African patients who were mostly infected by heterosexual contacts. Several clinically important mutations at the HBs major hydrophilic region were detected in the new isolates but with no significant difference between occult and overt infection. The high prevalence of HBV genotype A in overt and occult cases, and in particular the detection of certain HBV subgenotypes in patients co‐infected with HIV and HBV that carry diagnostic escape mutations, may provide useful information for national guidelines for prophylaxis and treatment. J. Med. Virol. 83:1876–1884, 2011.

Evidence for a Complex Mosaic Genome Pattern in a Full-length Hepatitis C Virus Sequence

The genome of the hepatitis C virus (HCV) exhibits a high genetic variability. This remarkable heterogeneity is mainly attributed to the gradual accumulation of mutational changes, whereas the contribution of recombination events to the evolution of HCV remains controversial so far. While performing phylogenetic analyses including a large number of sequences deposited in the GenBank, we encountered a full-length HCV sequence (AY651061) that showed evidence for inter-subtype recombination and was, therefore, subjected to a detailed analysis of its molecular structure. The obtained results indicated that AY651061 does not represent a “simple” HCV 1c isolate, but a complex 1a/1c mosaic genome, showing five putative breakpoints in the core to NS3 regions. To our knowledge, this is the first report on a mosaic HCV full-length sequence with multiple breakpoints. The molecular structure of AY651061 is reminiscent of complex homologous recombinant variants occurring among other members of the flaviviridae family, e.g. GB virus C, dengue virus, and Japanese encephalitis virus. Our finding of a mosaic HCV sequence may have important implications for many fields of current HCV research which merit careful consideration.