---

Hepatitis B virus (HBV) is one of the causes of viral hepatitis, and its worldwide burden is enormous. HBV ranks ninth among all causes of mortality, and is the fifth leading cause of death due to infectious disease . Genotypes, subgenotypes, and HBsAg subtypes, represent genetically stable viral populations that share a common evolutionary history. Additional stable changes are originated from mutations and mutant selection. These viral subpopulations are known as the HBV variants, and some of them display medical and public health relevance. The emergence of some specific variants has a major impact on disease evolution. Drug resistance variants, HBV immunoglobulins (HBIg) escape variants, HBV vaccines escape variants and diagnostic assay escape variants, potentially lead to serious problems in therapy, prophylaxis and diagnosis of HBV infections. In order to understand the mechanism of disease progression and conversion, it is of great interest to study the molecular evolution of HBV by using phylogenetics and bioinformatic tools. Through a well organized archive, we have access to 3000 serial patient samples, some of whom even date back to the 1970s. In this research project, we are interested to investigate the within-host and between-host evolution of HBV by applying molecular biology and bioinformatics techniques. We strongly believe that these results will lead to a better understanding of HBV disease progression and optimal prophylactic, diagnostic, and treatment strategies of hepatitis B virus infections.

---

The Hepatitis C Virus (HCV) is a blood-borne pathogen that already infected at least 170 million people worldwide. Our research project focuses on the epidemiology and evolution of HCV. In a first part, the origin, endemic source and epidemic spread of HCV genotype 5a is studied. We investigated the molecular epidemiology of HCV genotype 5a in Belgium and South Africa, and we estimated the rate of virus spread over time using a phylogenetic analysis and a Bayesian coalescent method (Verbeeck et al., 2006). Our results show that the Belgian HCV genotype 5a clade has an age similar to that of the South African clade, indicating that the ancestor might be harboured outside of South Africa. This finding sheds new light on the hypothesis concerning the emergence of HCV genotype 5a. In order to trace the origin of this unfamiliar genotype, we already collected additional samples from France, the Netherlands, Somalia, Syria and the United Kingdom, and we are collaborating with several research groups to collect samples from different regions in Africa. Since most Belgian HCV 5a infected patients do not know how they got infected, it is interesting to investigate this small cluster in detail to find out more about the possible transmission events. In a second part, we are studying the HCV genotype 1b full-genome evolution in responders and non-responders by using the pyrosequencing technique. The samples will also be cloned, and the sequencing results after cloning will be compared with the results after pyrosequencing. Our aim is to identify specific genome regions and mutations that might explain (non-)response to therapy.

---

Enteroviruses comprise a large genus within the family of the picornaviridae. Human enterovirus B species are the most common cause of aseptic meningitis worldwide. The 5' noncoding region (5'NCR) is extremely conserved among all enteroviruses and therefore used extensively in diagnostic RT-PCR assays. This genetic conservation in the 5'NCR does not allow to accurately identify the enterovirus type. By establishing a database of matching 5'NCR and VP1 sequences, we can successfully deduce the correct enterovirus genotype of novel clinical isolates by using phylogenetic analysis based on the 5'NCR sequence. Every year the positive samples are molecularly typed for epidemiological surveillance in Belgium.

---

Hantaviruses cause two frequently fatal human diseases, hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). HFRS-associated hantaviruses cause vascular hemorrhage and kidney dysfunction while HPS-associated hantaviruses cause acute pulmonary edema. Both diseases are associated with acute thrombocytopenia and changes in vascular permeability, and either disease may have pulmonary or renal components. The means by which specific hantaviruses cause pulmonary or renal diseases and increase vascular permeability is just beginning to be investigated. Hantaviruses cause disease in humans but not in their animal hosts, and both pathogenic and non-pathogenic hantaviruses have the same tissue tropism, replicating predominantly in endothelial cells and macrophages. Infected endothelial cells are not lysed by hantaviruses, and although immune cells are recruited to the infected endothelium, it is unclear to what extent these contribute to vascular disease. The genome of hantaviruses consists of three segments of a negative-sense single stranded RNA. The large (L) segment codes for the RNA polymerase, the medium (M) segment for two glycoproteins G1 and G2, and the small (S) segment for the nucleocapsid (N) protein. Hantaviruses carried by Cricetidae rodents (Rodentia subfamilies Arvicolinae, Neotominae, and Sigmodontinae) have in their S segment an additional open reading frame (ORF) for the nonstructural protein NSs. It has been shown recently that this NSs protein is an inhibitor of the interferon (IFN) response which can be an explanation for the high pathogenicity of some of these hantaviruses. Interestingly, hantaviruses carried by Muridae rodents (Rodentia subfamily Murinae), do not posses this NSs ORF, even though these viruses can show high pathogenicity. To try to better understand the role of this nonstructural NSs protein, we will use reverse genetics to express recombinant hantaviruses containing the complete deletion of the NSs protein and we will compare these recombinant viruses with wild-type hantaviruses in IFN-competent cells and IFN-deficient cells in vitro, and in different animal models in vivo. By incorporating a reporter moiety into these recombinant viruses, we will try to simultaneously develop live virus research tools that may be useful for the rapid screening of antiviral therapeutic compounds.

---

Noroviruses are the most common pathogens of nonbacterial gastroenteritis. They are often associated with epidemic disease in families, schools, nursing homes and other institutional settings. The disease is very contagious and characterized by nausea, vomiting, diarrhoea and abdominal pain. The prototype of norovirus is Norwalk virus, classified as a member of Genogroup I (GI). Genogroup II (GII) contains most of the strains infecting humans, and the GII genetic cluster 4 (GII.4) noroviruses have been the predominant circulating strains detected since the 90s. In our lab studies the molecular epidemiology in Belgium.

---

Papillomaviruses form a large group of species-specific pathogens that can cause epithelial proliferations in a wide spectrum of vertebrate hosts. To date more than 100 different types have been isolated from humans. For animals, this number is markedly lower, but nevertheless papillomaviruses have been detected in most vertebrate species where an investigational effort has been made. These types cover a wide diversity of host species, mostly mammals (domestic and wild animals) and some birds. In our research project we study the molecular evolution of  the Papillomaviridae. The general consensus on papillomavirus evolution is that different ancient PV lineages have co-evolved and co-speciated with their vertebrate host species during vertebrate evolution. Such a co-phylogenetic descent would imply that papillomaviruses of closely related host species show a close phylogenetic relationship themselves. Furthermore, the papillomavirus phylogenetic tree has to mirror the host species tree, with dating of the divergence of the papillomaviruses coinciding with the host species divergence time points. We aim to obtain a more complete and well-funded picture of the Papillomaviridae genetic diversity, and to investigate whether the hypothesis of co-phylogenetic descent holds.

---

Respiratory syncytial virus (RSV) infections are the primary cause of hospitalization of children during their first year of life, and nearly all children have experienced an RSV infection by the age of two to three years. Outbreaks are seasonal occurring from late fall through spring (November to May). The clinical manifestations range from mild upper respiratory tract infections, croup and middle ear infections to apnea, bronchiolitis and pneumonia. Two major antigenic groups of RSV have been identified: subgroup A (RSV-A) and subgroup B (RSV-B). Monitoring the genetic diversity of RSV can provide important information on the distribution and prevalence of different lineages or genotypes. Moreover, it can permit the identification of outbreak strains and trace their dissemination. To investigate the molecular epidemiology and evolution of RSV viruses, we studie the attachment G glycoprotein, which is one of the main antigens responsible for inducing a neutralizing immune response and shows the highest genetic and antigenic diversity between and within the two RSV subgroups. More and more, the interest grows to expand phylogenetic analyses to all the surface proteins and even to the entire RSV genome. Therefore, we are also investigating the genetic variability of the F protein through the collection of sequence data over several epidemic seasons. This can lead to a better understanding of the evolutionary relationship between surface proteins and might help explain the ability of RSV to evade the immune system. Because of its more conserved nature, the F protein has also been selected by researchers as an interesting mark for antiviral compounds. In that point of view, the collection of sequence data of the F gene is essential to detect mutations leading to therapy resistant viruses.

---

Rotaviruses cause diarrhoea in human children and young animals, and every year approximately 500.000 children die as a result of rotavirus infection. Rotaviruses have a segmented genome, and when 2 different rotaviruses infect one host, these genes can reassort to form new rotavirus strains with novel characteristics, as with Influenza viruses. In figure 1 an example of such reassortments is shown as was found in rotavirus samples from the Democratic Republic of Congo. Reassortment can also occur between rotavirus from different host species, such as humans, pigs, cattle, dogs, etc., resulting in the introduction of animal rotavirus genes in the human rotavirus population. The rotavirus research in our group focuses on the molecular diversity and epidemiology of complete rotavirus genomes from human and animal origin.