L'infection chronique par le virus de l’hépatite B (HBV) est un problème de santé majeur dans le monde et peut conduire à la cirrhose et à l’hépatocarcinome. Des réponses défectives des cellules T ont été démontrées, mais les événements précis qui peuvent contribuer à l'insuffisance des réponses des cellules B restent à déterminer. L'expansion et la différenciation optimales des cellules B reposent sur l'activité du récepteur Toll Like Receptor 9 (TLR9) qui détecte l'ADNdb et est exprimée chez l'homme principalement par les cellules dendritiques plasmacytoïdes (pDC) et les cellules B. L'impact de HBV sur TLR9 dans les cellules B humaines reste à explorer. Dans cette étude, nous avons exploré les effets de HBV sur l’expression et les fonctions médiées par TLR9 dans les cellules B humaines en utilisant une lignée cellulaire de cellules B ainsi que des lymphocytes B primaires. De plus, on a corroboré nos résultats dans une cohorte de patients HBV chroniques. L'expression de TLR9 a été réduite chez les toutes les sous-populations de cellules B du sang périphérique, exposées au VHB. Les fonctions des cellules B médiées par TLR9 comme la prolifération et la sécrétion de cytokines pro-inflammatoires ont été abrogées en présence de HBV. Nos résultats montrent que l’antigène de surface HBsAg inhibe la phosphorylation du facteur de transcription CREB qui ne peut plus se lier sur son site CRE situé sur le promoteur TLR9 ce qui affecte la transcription de ce dernier. Enfin, nous avons corroboré nos résultats in vitro dans une cohorte de porteurs chroniques du HBV et constaté que l'expression et la fonction de TLR9 étaient significativement diminuées. Nos résultats révèlent le mécanisme qui induit une réponse immunosuppressive par HBV sur la fonction TLR9 dans les cellules B humaines, ce qui peut contribuer à la persistance de ce virus chez l'hôte et la complication de la phase chronique de la maladie / Chronic HBV infection is a major health problem worldwide. Ineffective T cell and antibody responses have been demonstrated, yet the precise events that may contribute to insufficient B cell responses remain to be determined. Optimal B cell function, expansion and differentiation rely on Toll Like Receptor 9 (TLR9) activity which senses dsDNA and is expressed in human mainly by plasmacytoid dendritic ( pDC) and B cells. The impact of HBV on TLR9 in human B cell subsets remains to be explored.Here, we investigated the effects of HBV on TLR9 function in human B cells. Both primary and B cell lines were used to analyze the effect of HBV on TLR9 expression and function. These results were corroborated in a cohort of chronically infected HBV patients. TLR9 expression was reduced in all peripheral blood B cells subsets exposed to HBV. B cell function mediated by TLR9, such as proliferation and pro-inflammatory cytokines secretion were abrogated in the presence of HBV. Our results show that the viral surface antigen HBsAg inhibited the phosphorylation of the transcription factor CREB which could no longer bind the CRE site located on the TLR9 promoter. Finally, we corroborated our in vitro findings in a cohort of chronic HBV carriers (CHB) and found that TLR9 expression and function were significantly suppressed.Our findings reveal the mechanism that induces an immunosuppressive response by HBV on TLR9 function in human B cells, which may contribute to HBV persistence in the host

The genome of hepatitis B virus (HBV) can be assessed by sequence analysis of HBV DNA in serum. In most chronic HBV infected patients treated with potent nucleos(t)ide analogues (NAs) this approach however is limited by the fast decrease of serum HBV DNA during NA treatment. In contrast, HBV RNA was shown to persist in serum of some HBV infected individuals receiving NA treatment. In this study, we established the sequencing of serum HBV RNA as a method for the monitoring of HBV variants during NA treatment after the decrease of serum HBV DNA to undetectable levels. Using this approach, we studied the evolution of HBV variants in follow-up serum samples (n=156) of 25 patients treated with the potent polymerase inhibitor tenofovir (TDF) as second or third-line treatment. In our cohort, specific reverse transcribed full-length and truncated HBV RNA remained detectable with real-time PCR for long periods in most serum samples, also after the decline of HBV DNA during treatment with TDF. The HBV genome could be analyzed based on serum HBV DNA sequencing in 61 serum samples for a mean duration of 6.0 ± 4.5 (0 – 13) months. After this, sequencing of reverse transcribed serum HBV RNA allowed the analysis of the HBV genome for an additional mean duration of 33.9 ± 12.7 (16 - 65) months in 68 serum samples. The comparison of serum HBV DNA and serum HBV RNA derived sequences showed a high homology. In most patients, acquired HBV resistance variants in the reverse transcriptase (rt) region of the polymerase gene were detectable on HBV DNA and HBV RNA basis. Serum HBV RNA sequencing further revealed a long persistence of these variants during TDF treatment (mean duration of 26.5 ± 15.8 (0 – 50) months), which indicates a high conservation in the cccDNA of the infected individuals. Also HBV stop mutations in the small surface (s) gene, which were discussed in the pathogenesis of hepatocellular carcinoma (HCC), were present at baseline in 5 patients and remained detectable on HBV RNA basis during follow-up. In this study, we demonstrated that sequencing of reverse transcribed HBV RNA from patient serum is a suitable method to assess HBV variants during NA treatment. We further provided insights into the evolution of HBV variants during strong suppression of the viral replication with the polymerase inhibitor TDF. Future studies should investigate more comprehensively the clinical application of the here presented method of serum HBV RNA sequencing for the early detection of resistant HBV variants during NA treatment and the observation of HBV s gene variants related to HCC development.:Table of content I Table of content 3 II Abbreviations 6 1 Introduction 10 1.1 HBV 11 1.1.1 Classification 11 1.1.2 HBV virion structure and genomic organization 11 1.1.3 HBV proteins 12 1.1.4 HBV replication cycle 15 1.2 Chronic HBV infection 17 1.2.1 Epidemiology 17 1.2.2 Natural course of chronic HBV infection 17 1.3 Treatment of chronic HBV infection with nucleos(t)ide analogues 18 1.3.1 Nucleos(t)ide analogues 18 1.3.2 Treatment goals 18 1.3.3 Response to nucleos(t)ide analogue treatment 20 1.3.4 Treatment with nucleos(t)ide analogues and liver disease 21 1.4 Evolution of HBV variants during antiviral treatment 22 1.4.1 HBV resistance mutations in the pol gene 22 1.4.2 Resistance rates to treatment with nucleos(t)ide analogues 25 1.4.3 HBV variants in the s gene 26 1.5 HBV RNA in serum of chronically infected patients 29 1.5.1 HBV RNA molecules 29 1.5.2 HBV RNA packaging and release 29 1.5.3 HBV RNA as serum marker 31 1.6 Aim of the study 31 2 Materials and methods 33 2.1 Materials 33 2.1.1 Chemicals 33 2.1.2 Devices 33 2.1.3 Laboratory materials 34 2.1.4 Cycler 34 2.1.5 Kits 35 2.1.6 Buffers and solutions 36 2.1.7 Primers 36 2.1.8 Data analysis 39 2.2 Methods 39 2.2.1 Patient set and sample selection 39 2.2.2 Extraction of nucleic acids from serum samples 40 2.2.3 Reverse transcription of HBV RNA 40 2.2.4 Quantification of HBV serum DNA and HBV RNA by real-time PCR 41 2.2.4.1 Real-time PCR 41 2.2.4.2 Quantification of serum HBV DNA 43 2.2.4.3 Quantification of serum HBV trRNA and HBV flRNA 45 2.2.5 Sequencing of serum HBV DNA and HBV RNA 47 2.2.5.1 Primer design 47 2.2.5.2 Amplification by PCR 48 2.2.5.3 Purification of amplification products 50 2.2.5.4 Sanger sequencing of PCR fragments 51 2.2.6 Quantification of serum HBsAg and HBeAg 53 2.2.7 Cloning of HBV variants 53 2.2.8 Data analysis 55 2.2.8.1 Serum HBV DNA and HBV RNA quantities 55 2.2.8.2 Analysis of HBV DNA and HBV RNA sequences 55 3 Results 59 3.1 Composition of the patient set 59 3.2 Quantification of HBV DNA and HBV RNA in serum samples 59 3.2.1 Quantitative courses of serum HBV DNA 60 3.2.2 Quantitative courses of serum HBV flRNA and HBV trRNA 61 3.3 Sequencing of HBV DNA and HBV RNA 64 3.3.1 Method 64 3.3.2 Follow-up with sequencing of HBV DNA and HBV RNA 67 3.3.3 Genotyping of baseline samples 67 3.4 Evolution of HBV variants in the rt region 68 3.4.1 HBV resistance mutations in the rt region at baseline 68 3.4.2 HBV resistance mutations in the rt region during antiviral treatment 70 3.5 HBV variants in the s gene 77 3.5.1 HBV s gene variants at baseline 77 3.5.2 HBV s gene variants during antiviral treatment 80 4 Discussion 82 4.1 Patient cohort 82 4.2 Quantification of serum HBV DNA, HBV flRNA and HBV trRNA 82 4.3 Quantitative courses of serum HBV DNA, HBV flRNA and HBV trRNA 83 4.4 Sequencing of serum HBV RNA as novel method for HBV genome analysis 85 4.5 Evolution of HBV variants in the rt region 89 4.6 Evolution of HBV stop mutations in the s gene 91 4.7 Conclusion 92 III Summary 94 IV References 96 V List of Figures 104 VI List of Tables 105 VII Supplement 106 VIII Erklärung über die eigenständige Abfassung der Arbeit 107 IX Curriculum Vitae 108 X Publications 110 XI Acknowledgment 114

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Inflow forecasting is important when planning the use of water in a hydropower plant. The process of making forecasts is characterized by using knowledge from previous events and occurrences to make predictions about the future. Traditionally, inflow is predicted using hydrological models. The model developed by the Hydrologiska Byråns Vattenbalansavdelning (HBV model) is one of the most widely used hydrological models around the world. Machine learning is emerging as a potential alternative to the current HBV models but needs to be evaluated. This thesis investigates machine learning for inflow forecasting as a mixed qualitative and quantitative case study. Interviews with experts in various backgrounds within hydropower illustrated the key issues and opportunities for inflow forecasting accuracy and laid the foundation for the machine learning model created. The thesis found that the noise in the realised inflow data was one of the main factors which affected the quality of the machine learning inflow forecasts. Other notable factors were the precipitation data from the three closest weather stations. The interviews suggested that the noise in the realised inflow data could be due to faulty measurements. The interviews also provided examples of additional data such as snow quantity measurements and ground moisture levels which could be included in a machine learning model to improve inflow forecast performance. One proposed application for the machine learning model was as a complementary tool to the current HBV model to assist in making manual adjustments to the forecasts when considered necessary. The machine learning model achieved an average Mean Absolute Error (MAE) of 1.39 compared to 1.73 for a baseline forecast for inflow to the Lake Kymmen river system 1-7 days ahead over the period 2015-2019. For inflow to the Lake Kymmen river system 8-14 days ahead the machine learning model achieved an average MAE of 1.68 compared to 2.45 for a baseline forecast. The current HBV model in place had a lower average MAE than the machine learning model over the available comparison period of January 2018. / Tillrinningsprognostisering är viktig vid planeringen av vattenanvändningen i ett vattenkraftverk. Prognostiseringsprocessen går ut på att använda tidigare kunskap för att kunna göra prediktioner om framtiden. Traditionellt sett har tillrinningsprognostisering gjorts med hjälp av hydrologiska modeller. Hydrologiska Byråns Vattenbalansavdelning-modellen (HBV-modellen) är en av de mest använda hydrologiska modellerna och används världen över. Maskininlärning växer för tillfället fram som ett potentiellt alternativ till de nuvarande HBV-modellerna men behöver utvärderas. Det här examensarbetet använder en blandad kvalitativ och kvantitativ metod för att utforska maskininlärning för tillrinningsprognostisering i en fallstudie. Intervjuer med experter med olika bakgrund inom vattenkraft påtalade nyckelfrågor och möjligheter för precisering av tillrinningsprognostisering och lade grunden för den maskininlärningsmodell som skapades. Den här studien fann att brus i realiserade tillrinningsdata var en av huvudfaktorerna som påverkade kvaliteten i tillrinningsprognoserna av maskininlärningsmodellen. Andra nämnvärda faktorer var nederbördsdata från de tre närmaste väderstationerna. Intervjuerna antydde att bruset i realiserade tillrinningsdatana kan bero på felaktiga mätvärden. Intervjuerna bidrog också med exempel på ytterligare data som kan inkluderas i en maskininlärningsmodell för att förbättra tillrinningsprognoserna, såsom mätningar av snömängd och markvattennivåer. En föreslagen användning för maskininlärningsmodellen var som ett kompletterande verktyg till den nuvarande HBV-modellen för att underlätta manuella justeringar av prognoserna när det bedöms nödvändigt. Maskininlärningsmodellen åstadkom ett genomsnittligt Mean Absolute Error (MAE) på 1,39 jämfört med 1,73 för en referensprognos för tillrinningen till Kymmens sjösystem 1–7 dagar fram i tiden under perioden 2015–2019. För tillrinningen till Kymmens sjösystem 8–14 dagar fram i tiden åstadkom maskininlärningsmodellen ett genomsnittligt MAE på 1,68 jämfört med 2,45 för en referensprognos. Den nuvarande HBV-modellen hade ett lägre genomsnittligt MAE jämfört med maskininlärningsmodellen under den tillgängliga jämförelseperioden januari 2018.

At present the origins of major human pathogens associated with hepatic disease are poorly understood. The absence of such information pertaining to the evolutionary history of hepatitis B virus (HBV) and hepatitis C virus (HCV) and its genetically related viruses impacts upon the development of vaccines and effective eradication strategies. Studies are currently limited by the absence of historical samples from which to date the emergence of human infections and therefore the evolution of human hepatic viruses relies on epidemiological studies and genetic analysis of contemporary virus populations worldwide. Approximately one third of the world’s population is infected with HBV, and despite the availability of a vaccine, the virus is attributed with over 1 million deaths per year through liver disease. HBV variants infecting humans show genetic and antigenic heterogeneity and are currently classified into 8 genotypes A-H with nucleotide divergence of between 9-13%. In addition to these variants, recombination has been detected between genotypes A and D, and B and C, which can generate novel variants. The past 10 years has seen the detection of HBV in chimpanzees, gorillas and other non-human primates (NHPs) at frequencies comparable to those observed in regions of endemic human HBV infection. Despite the genetic divergence between human and NHP HBV variants the detection of recombination between human genotype C and chimpanzee and gibbon variants suggests that HBV can share hosts in nature. The evolutionary process that may have given rise to the distinct species-specific variants of NHP HBV within overlapping geographical regions has not been reconciled, with evidence supporting both allopatric speciation and co-speciation. HCV a member of the Flaviviridae family currently infects approximately 3% of the world’s population and is one of the major causes of chronic liver disease, hepatocellular carcinoma and liver cirrhosis. Human pegivirus (HPgV) a member of the Pegivirus genus of the Flaviviridae family infects approximately 5% of the world’s population, although it is of unknown disease association. Very recently, several studies of wild rodent and bat populations have revealed much greater viral diversity of members of both Hepacivirus and Pegivirus genera. Homologues of HCV have been detected in a range of species including domestic dogs (canine hepacivirus [CHV]) and horses (non-primate hepaciviruses [NPHV]). Similarly, several new pegiviruses have been described in horses (equine pegivirus, [EPgV] and Theiler’s Disease Associated Virus [TDAV]), several species of rodents (rodent pegivirus [RPgV]), and further species of bats (bat pegivirus, [BPgV]). Despite the differences in pathogenicity between HCV and HPgV infections, they share similar genomic organisation and are capable of establishing persistent infections in humans. Studies into bat, horse and rodent homologs of HCV and HPgV have yet to determine disease associations, transmission routes and seroprevalence. Studies presented within this thesis broaden our understanding of the clinical presentations and host range of NPHV and EPgV. Screening to determine the level of active and past infection to both viruses provides novel insight into infection frequencies, host range, disease progression and examines the correlation between infections and the presence or absence of hepatic disease. Research examining HBV variants circulating in NHPs in Cameroon provides novel evidence for the occurrence of recombination and cross species transmission between NHP variants of HBV and examines the role these findings play in expanding our understanding of the evolution of HBV.

Hepatitis B virus is a member of the hepadnavirus family. The hepatitis B virus core gene codes for two proteins viz. core protein and pre-core protein. These proteins assemble to form particles viz. HBcAg and HBeAg respectively. The structure of the HBcAg has been widely studied but very little is known about the structure of HBeAg. Therefore, the aim of this study was to identify the disulfide bonding patterns in HBeAg. Recombinant HBeAg was isolated from E.coli and used for this study along with various mutants of HBeAg. There are four cysteines present in HBeAg each at position -7, 48, 61 and 107. From this study it can be inferred that the cysteine at 61 and 48 were found to be involved in inter-molecular disulfide bonds between the cysteine at 61 and 48 of other identical monomers. These di-mers were further inter-molecularly linked with cysteine at -7 to form chains. Moreover, the cysteine at -7 and cysteine at 107 were sometimes involved in intra-molecular disulfide bond formation. Thus, the HBeAg in a solution was found be particulate with a heterogeneous pattern of inter chain disulfide bonds.

Le virus de l'hépatite B (VHB) est un virus enveloppé composé d'un ADN partiellement double brin (ADNrc) contenu dans une capside icosahédrique. Le VHB est responsable d'infections aiguës et chroniques. VHB est non cytopathique mais l’inflammation chronique entraîne une fibrose hépatique, une cirrhose et un carcinome hépatocellulaire. Le VHB se réplique via un intermédiaire à ARN. La transcription nécessite que l'ADNrc soit convertit en un ADN circulaire clos de manière covalente (ADNccc). Cet ADNccc sert de matrice pour la transcription de l'ARN prégénomique (ARNpg), qui est spécifiquement encapsidé grâce aux interactions entre la polymérase virale, l'ARNpg et la protéine core (Cp) qui forme la capside. La polymérase rétrotranscrit l'ARNpg en ADN monocaténaire puis en ADNrc, conduisant à des matrices de capside matures (MatC). Cp avec 185 aa contient un domaine N-terminal structuré, et un domaine C-terminal (CTD) flexible. Le CTD comprend deux signaux de localisation nucléaire (NLS) et un domaine de liaison avec l’importin β (IBB). Le CTD est orienté vers l'intérieur de la capside de part son interaction avec les acides nucléiques simples brins tandis qu'il est exposé vers l'extérieur dans les capsides vides (EmpC) et les MatC. De plus Cp étant surexprimée, cela conduit à l'assemblage des EmpC. Le VHB doit délivrer son génome dans le noyau des cellules infectées pour sa réplication. Le transport nucléaire est médié par la capside qui interagit avec les récepteurs d'import. L’équipe a démontré préalablement que ce transport a besoin des récepteurs Importin α (Imp.α) et Importin β (Imp.β) en induisant le transport des capsides au panier nucléaire où elle est stoppée par l'interaction avec la nucléoporine 153 (Nup153). Nous avons démontré que l’Imp.α, mais pas l'Imp.β, se lie aux MatC suggérant que seule la partie du CTD qui contient les NLS est exposée à l’extérieur des MatC. En comparaison, nous avons analysé les EmpC en collaboration avec Adam Zlotnick (Université d'Indiana, États-Unis) et démontré que les EmpC sont capables de lier directement l'Imp.β. Cette interaction qui est plus forte que l’interaction avec l'Imp.α s'effectue via la reconnaissance du domaine IBB du CTD, ce qui implique une exposition totale du CTD à l'extérieur de la capside. Nous avons aussi montré que la liaison avec l'Imp.β à des concentrations très élevées fournit des forces de déstabilisation menant au désassemblage des EmpC. La libération du génome dans le panier nucléaire implique que l’interaction entre les MatC et Nup153 participe au désassemblage de la capside. Afin de valider cette hypothèse, nous avons exposé des MatC dont le génome est radiomarqué avec un fragment de Nup153 contenant le domaine clé, montré pour interagir avec la capside, en présence de nucléases. Nous avons mis en évidence qu'en présence de ce fragment, les MatC restent stables. Cela suggère la nécessité de facteurs cellulaires additionnels pour le désassemblage des MatC. Cette conclusion est conforme avec nos résultats sur noyaux isolés, dans lesquels nous avons observé une localisation nucléaire des capsides laissant supposer que les facteurs cellulaires nécessaires au désassemblage des MatC sont nucléaires. Afin d'étudier plus en détail l'étape de désassemblage et la libération du génome viral, nous avons mis au point un système permettant de suivre en temps réel le devenir du génome viral. / The Hepatitis B Virus (HBV) is an enveloped virus containing a partially double stranded DNA genome (rcDNA). HBV causes acute and chronic infections. HBV is not cytotoxic but chronic inflammation leads to liver fibrosis, cirrhosis and hepatocellular carcinoma. HBV replicates via an RNA intermediate, which is transcribed from a covalently closed circular form of the viral DNA (cccDNA). This pregenomic RNA is specifically encapsidated into the capsid by interaction with the viral polymerase, which also interacts with the core protein (Cp), forming the capsid. The polymerase retrotranscribes the pregenomic RNA into single stranded DNA and subsequently partially double stranded DNA resulting in mature capsids (MatC). Cp is an 185 aa long polypeptide comprising a N-terminal assembly domain, and a flexible C-terminal domain (CTD). The CTD includes two overlapping nuclear localization signals (NLS) of eight aa and an Importin ß Binding Domain (IBB) of 34 aa. The CTD is fixed in the interior of the capsid by interacting with single stranded nucleic acids but translocates to the exterior in MatC and empty capsids (EmpC). Cp is over expressed leading to assembly of EmpC. The virus has to deliver its genome into the nucleus of infected cells for replication. Nuclear transport is mediated by the capsid that interacts with nuclear import receptors. The group has recently shown that MatC need either both, importin  (Imp.) and importin ß (Imp.ß), or Imp.ß alone for transport of the capsids into the nuclear basket. In this structure where genome liberation likely occurs, the transport of the capsid is arrested by interaction between the capsid and the nucleoporin Nup153. In the thesis we demonstrate that MatC binds to Imp.α but not Imp.ß, suggesting that only the part of the CTD, which contains the NLSs is exposed on capsids’ surface. In collaboration with the Adam Zlotnick (Indiana University, U.S.A.) we showed that EmpC, in contrast, bind Imp.β directly without Imp.α acting as an adaptor. This interaction, which is stronger than the one of Imp. occurs needs IBB exposure, meaning that the entire CTD becomes externalized. Furthermore, exposure to very high Imp.ß concentration led to EmpC destabilization. The genome release within the nuclear basket implies that Nup153 is involved in genome liberation from MatC. To verify this hypothesis we used MatC with a radioactively labeled genome, which were exposed to the capsid binding-Nup153 fragment. Investigating the accessibility of the genome to nucleases we found that the Nup153 fragment had no impact on capsids stability, suggesting the need of cellular factors driving disassembly. This conclusion is in agreement with our observation that MatC added to isolated nuclei resulted in nuclear capsid entry, which requires disassembly. To further study the disassembly step and the consequent release of the viral genome, we developed a system to directly visualize the viral genome allowing investigations of genome uncoating in real time. The system is based on the cooperative binding of a fluorescent fusion protein between the bacterial protein OR with GFP to a double stranded DNA sequence called Anch. Using this model we showed that infection of OR-GFP-expressing hepatoma cells with HBV containing a modified Anch genome allowed monitoring genome release into the nucleus. In future, this system may help identifying factors involved in genome release and repair and to decipher their molecular interactions.

An effective and safe vaccine against Hepatitis B virus already exists, yet morbidity and mortality of this illness are still high. The key to developing a reliable treatment is a deep knowledge of the virus' life cycle and functions of all its components. In the presented work we explored an interactome of the Core protein of the Hepatitis B virus. Using proximity-dependent biotin identification technique (BioID) coupled to mass spectrometry we have identified a list of potential candidates that are either significantly enriched (in total 105 proteins) or less abundant in the presence of the HBV Core protein in the cell (40 proteins). The list also includes known HBV Core interacting proteins SRPK1 and SRPK2, and p53 protein whose expression is known to be repressed due to the HBV Core interaction with the E2F1 transcription factor. Many of the newly identified possible HBV Core interacting proteins are involved in biological processes already known or are suspected to be influenced by the HBV such as translational and transporting processes or gene expression and macromolecule production. Overall, this work comprehensively characterizes the interaction landscape of the HBV Core protein in the live cells and might thus serve as a reliable start for in depth HBV-host interaction analysis. Key...

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<p>Water is a basic necessity for sustaining life and development of society. Proper management, protection and exploitation of water resources are the challenges imposed by population growth, increasing pressure on the water and land resources by competing usage. A good amount of clean water exists on Earth although it is normally inadequate in supply because of anthropogenic activities such as deforestation and land use change. Like many other catchments that provide economic activities for the community’s livelihood, the Chalimbana river catchment in Zambia has been deforested heavily and most of the local communities believe that deforestation could be the main contributing factor to the drying up of Chalimbana River. The objective of this study was to analyse the impact of deforestation on the stream flow of Chalimbana River Catchment with the help of a conceptual hydrological model, HBV. There was a 24% reduction in the annual average rainfall amounts for the deforested period as compared to the period before deforestation. The Qrec/Qsim ratios had revealed that the annual stream flow generation for the period after deforestation (1987 to 1996) for the Chalimbana River had decreased by about 12% as compared to the period with enough forest cover (1975 to 1985). The ratio of annual Qrec/P had indicated that after a 30% forest loss in Chalimbana catchment, there was a 33% increase in the generation of the stream flow. Based on the results that were obtained, a number of recommendations aiming at improving the catchment management were made.</p>