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University of Nairobi MolecularCharacterization of theSurface Glycoproteins of Influenza B Viruses Isolated in Kenya from 2011-2012 MSc Thesis By: JUMBA GWEYANI GLORIA Reg No: W64/70261/2011 University of Nairobi Institute of Tropical and Infectious Diseases (UNITID) College of Health Sciences, University of Nairobi A thesis submitted in partial fulfillment of the requirement for the award of the degree of Masters of Science in Tropical and Infectious Diseases at University of Nairobi ©2015 DECLARATION This project is my original work and has not been presented for a degree in any other university. JumbaGweyani Gloria W64/70261/2011 M. Sc. Tropical and Infectious diseases University of Nairobi School of medicine Signed …………………………………………………………. Date ………………………... Supervisors: Prof. Wallace Bulimo Department of Biochemistry University of Nairobi Signed …………………………………………………………. Date ………………………... Dr. George Gachara Department of Medical Laboratory Sciences Kenyatta University Signed …………………………………………………………. i Date ………………………... DEDICATION This degree is dedicated to my siblings who have supported me endlessly in my academic work and to my mother who has mentored me in science. To my father who has supported me tirelessly in my studies. ii ACKNOWLEDGEMENT To Prof Bulimo, I am very thankful and I appreciate your support and mentorship as I undertook my project. I am very grateful for your time and assistance in my laboratory experiments and data analyses which eventually formed this thesis. To Dr. George Gachara, I am very grateful for your guidance and mentorship to me while carrying out this work. I appreciate and am very thankful to Global Emerging Infectious surveillance (GEIS) for generously funding this project. To the entire staff of National Influenza Center laboratory; for your much appreciated assistance and kindness as I carried out the laboratory experiments and procedures. I thank God for His grace and favor for me to finish this work. iii TABLE OF CONTENTS DECLARATION ....................................................................................................................... I DEDICATION ........................................................................................................................ II ACKNOWLEDGEMENT ..................................................................................................... III LIST OF FIGURES ............................................................................................................... IX LIST OF APPENDICES .......................................................................................................... X LIST OF ABBREVIATIONS ................................................................................................ XI ABSTRACT ......................................................................................................................... XII CHAPTER ONE: INTRODUCTION ......................................................................................1 1.0 BACKGROUND ...............................................................................................................1 CHAPTER TWO: LITERATURE REVIEW ..........................................................................3 2.1 THE INFLUENZA VIRUS ........................................................................................................3 2.1.1 Classification...............................................................................................................3 2.1.2 Virus morphology .......................................................................................................4 2.1.3 Influenza Virus Replication Cycle ...............................................................................5 2.2 THE INFLUENZA DISEASE.....................................................................................................6 2.2.1 Clinical presentation of influenza and its determinants ................................................7 2.2.2 Epidemiology ..............................................................................................................7 2.2.3 Influenza virus host range and pathogenesis ................................................................8 2.2.3.1 Host range.............................................................................................................8 2.2.3.2 Pathogenesis .........................................................................................................9 iv 2.3 GENOME STRUCTURE OF INFLUENZA VIRUSES AND PROTEINS THEY ENCODE .........................9 2.3.1 Internal proteins ..........................................................................................................9 2.3.2 External Protiens ....................................................................................................... 12 2.3.2.1 Neuraminidase and NB ....................................................................................... 12 2.3.2.2 Hemagglutinin .................................................................................................... 14 2.4 PROBLEM STATEMENT ............................................................................................... 18 2.5 JUSTIFICATION .................................................................................................................. 18 2.6 RESEARCH QUESTION ....................................................................................................... 18 2.7 GENERAL OBJECTIVE ........................................................................................................ 19 2.7.1 Specific Objectives .................................................................................................... 19 CHAPTER THREE: MATERIALS AND METHODS ......................................................... 20 3.0 STUDY SITES .................................................................................................................... 20 3.1 STUDY DESIGN ................................................................................................................. 21 3.2 STUDY POPULATION .......................................................................................................... 22 3.2.1 Inclusion criteria........................................................................................................ 22 3.2.2 Exclusion criteria....................................................................................................... 22 3.3 SAMPLE SIZE/STATISTICS .................................................................................................. 23 3.4 EXPERIMENTAL PROCEDURES: ........................................................................................... 23 3.4.1 Revival of stored isolates ........................................................................................... 23 3.4.2. Virus Isolation: ......................................................................................................... 23 3.4.3. Haemagglutination test (HA) and titration of the virus .............................................. 24 3.4.3 Haemagglutination inhibition (HAI) assay ................................................................. 25 3.4.4 RNA extraction ......................................................................................................... 25 v 3.4.5 Conventional RT-PCR to amplify the HA and NA genes ........................................... 26 3.4.6 Agarose Gel electrophoresis of the amplicons............................................................ 27 3.4.7 Purification of the PCR amplicons and sequencing .................................................... 27 3.4.8. Sequencing by dideoxy Chain termination method ................................................... 28 3.4.10 Nucleotide sequence assembly ................................................................................. 28 3.4.11 Phylogenetic reconstruction, positive selection and evolutionary analyses ............... 29 3.4.12 Genetic antigenicity analyses ................................................................................... 30 3.4.13 Genetic drug-susceptibility analyses ........................................................................ 31 3.4.14 Neuraminidase phenotyping assays.......................................................................... 31 RESULTS ................................................................................................................................ 33 4.1 STUDY SUBJECTS .............................................................................................................. 33 4.3 ANTIGENIC CHARACTERIZATION OF THE INFLUENZA B ISOLATES USING SEROLOGY ............. 36 4.3.1 Analysis of Antigenic drift on HA ............................................................................. 38 4.3.2 Results on Antigenic drift on NA............................................................................... 41 4.3.4 Glycosylation analysis ............................................................................................... 43 4.3.5 Analysis of Selection Pressure ................................................................................... 46 4.4 EVOLUTIONARY RATES ..................................................................................................... 49 4.5 PHYLOGENETIC ANALYSES................................................................................................ 49 4.7 ANTIVIRAL SUSCEPTIBILITY .............................................................................................. 57 DISCUSSION .......................................................................................................................... 59 CONCLUSION AND RECOMMENDATIONS .................................................................... 65 CONCLUSIONS ..................................................................................................................... 65 vi RECOMMENDATIONS ........................................................................................................ 65 REFERENCES ........................................................................................................................ 67 vii List of Tables Table 1: Reagents used for sequencing PCR .......................................................................................... 28 Table 2: Demography and Clinical Presentation of the study subjects .................................................... 34 Table 3: HA and HAI titers of the 24 Influenza B isolates tested in the study. ........................................ 35 Table 4: Haemagglutination Inhibition Assay titers of the isolates compared to the two 2011-2012 southern and northern hemisphere reference strains ............................................................................... 37 Table 5: Amino acid substitutions in the HA of the Kenyan strains relative to the 2011-2012 WHO reference strain (B/Brisbane/08) ............................................................................................................ 40 Table 6: Amino acid changes in the NA as compared to the B/Brisbane/60/08 reference strain............... 42 Table 7: Hemagglutinin Glycosylation Sites .......................................................................................... 44 Table 8: Neuraminidase Glycosylation sites ........................................................................................... 45 Table 9: This table shows negative selection in the HA at various positions ........................................... 47 Table 10: This table shows negative selection in the NA at various positions. ........................................ 48 viii List of Figures Figure 1: Diagram of Influenza B virion structure.. .................................................................................. 4 Figure 2: Influenza Virus Replication. ..................................................................................................... 6 Figure 3: Major epitopes of influenza B virus ....................................................................................... 17 Figure 4: Sentinel sites of the Walter Reed Influenza Surveillance Network in Kenya ............................ 21 Figure 5: Phylogenetic analysis of Kenyan influenza B viruses using the HA amino acid sequences ...... 50 Figure 6: Phylogenetic analysis on HA Nucleotide sequences ................................................................ 51 Figure 7: Phylogenetic Analysis of NA nucleotide sequences................................................................. 52 Figure 8: Phylogenetic Analysis of NA amino acid ................................................................................ 53 Figure 9: Multiple sequence alignment of NA amino acid sequences of Kenyan Influenza B isolates with reference strains to identify loci involved in NAI resistance ................................................................... 56 ix List of Appendices Appendix 1: Isolates sampled for this study ........................................................................................... 74 Appendix 2: Ethical approval from the scientific steering committee ..................................................... 75 Appendix 3: Ethical approval Walter Reed Army Institute of Research .................................................. 76 Appendix 4: Ethical approval Ethical Review Committee ...................................................................... 77 x List of Abbreviations B/Vic/2/87 – B/Victoria/2/87 B/Yam/16/88 – B/yamagata/16/88 cDNA – complementary DNA DNA – Deoxyribonucleic acid ELISA – Enzyme-linked immunosorbent assay HA – Haemagglutinin IgA – Immunoglobulin A NA – Neuraminidase NAI – Neuraminidase Inhibitor NP - Nucleoprotein NS – Non structural protein PA – Acidic polymerase PB1 – Polymerase Basic protein PB2 - Polymerase Basic protein PCR – Polymerase chain reaction RNP - Ribonucleoprotein RNA – Ribonucleic acid RT-PCR – Reverse transcriptase polymerase chain reaction vRNA – viral RNA WHO – World Health Organization xi ABSTRACT Influenza B viruses can cause severe respiratory disease and occasionally epidemic outbreaks. Vaccination is the mainstayof prevention and reduces disease impact. Vaccine efficacy is determined mainly by the degree of haemagglutinin (HA) antigen matching between the vaccine and circulating strains. Neuraminidase inhibitor (NI) antivirals are important for prophylaxis and treatment of severe cases of influenza disease. However, mutations in the two major surface proteins, hemagglutinin (HA) and neuraminidase (NA) can cause the virus to escape host defenses leading to failure of the host immune system to recognize the viruses as well as failure to antiviral therapy. Due to these mutations, antibodies produced against these viruses may become ineffective against anew emergent viral strain or the changes may lead to antiviral therapy failure. This is further complicated by the existence of the two distinct lineages.B/Victoria/2/87-like viruses and B/Yamagata/16/88-like viruses- that continue to co-circulate globally in the human population. These viruses exist as independent lineages and antibodies against one lineage are generally not cross-protective. The World Health Organization (WHO)Strategic Advisory Group of Experts which issues vaccine component recommendations to be included in the annual influenza vaccine formulations recently revised the seasonal vaccine composition from atrivalent to an alternative containing quadrivalent components. Thus, information about lineage of circulating influenza B viruses is important for determination of the appropriateness of either a trivalent or a quadrivalent vaccine composition. The objective of this study wasto investigatethe molecular characteristicsof influenza B viruses that circulated in Kenya from 2011-2012 and the appropriateness of antiviral therapies including the WHO vaccine recommendationsusing bioinformatic analyses on nucleotide sequences of NA and HA glycoproteins of selected virus isolates. Nasopharyngeal swab specimens obtained from patients meeting WHO case definition for influenza-like-illness (ILI) were screened by real-time PCR for influenza B viruses. Positive samples were inoculated onto monolayers of Madin-Darby Canine Kidney (MDCK) cells and the lineages of the isolates determined by hemagglutination inhibition assay (HAI). To confirm the lineages and susceptibility to neuraminidase inhibitor drugs, HA and NA gene segments of selected isolates were amplified by conventional PCR, sequenced and analyzed using bioinformatic tools. The isolates were also tested for phenotypic antiviral susceptibility. The findings of this study showed that influenza B viruses that circulated in the study period were B/Brisbane/60/2008-like belonging to the B/Victoria/2/87 lineage. Thus the WHOrecommended vaccine for both the Southern and Northern hemispheres was appropriately matched in the influenza B component and was protective in Kenya. In addition, the influenza B viruses in circulation were all susceptible to Oseltamivir and Zanamivir drugs thus indicating the appropriateness of these drugs used in the treatment of Influenza B in Kenya. No positive selection was observed amongst the codons of HA and NA genes in the Kenyan influenza B viruses. Reassortment was observed in the Kenyan influenza B viruses within the NA gene segment. This study has underscored the importance of sustained monitoring of drug susceptibility as well as the antigenic characteristics of the influenza viruses as a measure of epidemic preparedness. It has also shown that the WHO recommendations for vaccine and antiviral usage cover Kenya well and theMinistry of health should continue to implement these recommendations. Full xii genome sequencing of the Kenyan influenza B viruses that circulated during the study period is recommended to unravel the extent and probable consequences of the reassortments amongst these viruses. xiii CHAPTER ONE: INTRODUCTION 1.0 BACKGROUND There are frequent influenza epidemics due to both influenza A and B viruses. Since 1977, Influenza B virus strains cause annual epidemics in humans together with the H1 and H3 subtype strains of influenza A virus(Cox and Subbarao, 2000). Influenza B viruseshave the capacity to undergo antigenic drift which is a gradual alteration by point mutations of theirmajor glycoproteins namely haemagglutinin (HA) and neuraminidase (NA).This resultsin the inefficiency of antibodies to previous strains to neutralize the mutant virus strains. This can lead to periodic epidemics and individuals can have many influenza infections over a lifetime due to this antigenic drift. The emergence of these new strains necessitates the frequent updating of influenza vaccine virus strains (Kanegae et al., 1990). Influenza A and B viruses are similar in a number of ways both biologically and biochemically. They both consist of a segmented genome comprising eight negative senseRNA segments (Lindstrom et al., 1999). This segmented genome of the influenza virus can lead to reassortment whereby different influenza viruses can exchange gene segments during co infection of a cell. Influenza B viruseshave a single haemagglutinin (HA) and neuraminidase (NA) type but the different lineages may reassort(McCullers et al., 1999)like influenza A viruses which are subdivided into multiple subtypes. The main difference between the two viruses isthat in terms of evolutionary patterns, influenza A has a higher evolutionary rate of the HA gene while influenza B evolves at a much slower rate (Murphy and Webster, 1990). Influenza disease occurs throughout the year in tropical areas while in the Northern Hemisphere, the influenza season starts in the fall and ends in late spring(Viboud et al., 2006). Influenza 1 epidemics also peak in the winter season in the Southern Hemisphere, however the severity of influenza B disease is less than that of influenza A (Webster et al., 1992). Since the mid-1980s, through a mechanism of systematic insertion and deletion of nucleotides, the HA gene of influenza B viruses has been shown to have evolved into two genetically distinct lineages which are named the B/Yamagata/16/88 and B/Victoria/2/87 lineages(Rota et al., 1992). Studies have proven that there is reassortment between the HA and NA of distinct lineages and sublineages thus highlighting the significance of frequent and detailed molecular analyses of their surface glycoproteins in understanding the evolution of influenza B viruses (Puzelli et al., 2004). Influenza B virus is able to escape host defense mechanisms by altering its antigenic character by molecular mechanisms (Nakajima, 2003). The main protection against influenza viruses is by antibody formation by the host immune system. The anti-HA antibody prevents the binding of the virus to the host cell receptor and is the most important while the anti-NA antibody prevents the release of newly formed virus from the host cells. The IgA antibody is the most significant in preventing infection because it acts at the mucous membranes of the respiratory tract where the virus first attaches (Webster, 1968). The HA and NA are thus of main immunological importance when the immune system mounts an immune response to the virus (Webster et al., 1968). In order to control epidemics of influenza by maximizing vaccine efficacy, frequent and continuous monitoring of the virus antigenicity changes is very important (Schweiger et al., 2002, Rebmann and Zelicoff, 2012). 2 CHAPTER TWO: LITERATURE REVIEW 2.1 The Influenza virus 2.1.1 Classification The influenza viruses together with the Thogoto-like viruses (Thogoto, Batken and Dhori viruses) belong to the family Orthomyxoviridae(Cox, 2000). Myxo is the Greek word for mucus, which means that members of the family have a strong affinity for and possess an enzyme capable of removing chemical side chains from mucoproteins. These properties facilitate infection of cells of the mucous membranes in the respiratory tract (Metselaar and Simpson, 1982). The family Orthomyxoviridae is divided into five genera; Influenzavirus A, Influenzavirus B, Influenzavirus C, Isavirus and Thogotovirus, based on antigenic differences in two of the major structural proteins of the virus, the nucleoprotein (NP) and the matrix protein (M)(Taubenberger and Kash, 2010). Two glycoproteins on the surface of the virus particle, the Neuraminidase or NA protein and the Haemagglutinin or HA (Haemagglutinin-esterase in influenza C) protein are involved in the interaction between virus and host cells. Influenza A viruses are classified into subtypes based on the antigenic differences between these two glycoproteins. Currently there are 18 (H1-H18) distinct HA subtypes and 9 (N1-N9) NA subtypes (Luke and Subbarao, 2006);(Tong et al., 2012). However, historically human type A influenza virus subtypes have been limited to H1, H2, and H3 and in the past 100 years to N1 and N2. In recent years, H5N1, H7N7, H9N2 and H7N9 continue to pose a pandemic threat due to their sporadic interspecies transmission into humans. There is only one HA and one NA subtype that has been identified among type B influenza viruses (Nicholson et al., 2003) but with two distinct lineages, the Victoria and Yamagata lineages (Rota et al., 1992); (Chen and Holmes, 2008a) 3 2.1.2 Virus morphology The mature influenza A virion is 100-200nm in diameter. Influenza viruses are more or less spherical, with about 500 projecting spikes covering the lipid envelope. The HA has a mushroom-like shape and is a rod-shaped glycoprotein with a triangular cross section (Lamb and Choppin, 1983).Approximately 80 percent of the viral envelope protein is the haemagglutinin while the neauraminidase constitutes about 17 percent (Palermo et al., 2007). The HA forms spikes on the lipid membrane of which binds to sialic acid on the host cell’s surface membrane (Skehel and Wiley, 2000). Eight discrete fragments of the negative-sense ssRNA genome, approximately 17 kb in size are complexed with proteins (NP, PA, PB1, and PB2) to form a ribonucleoprotein (RNP) arranged in a helix (Fig. 1). The 5’ and 3’ ends of each of the genome segments contain sequences that are complementary and each genome segments bind through these complementary ends to form a pan handle. Figure 1: Diagram of Influenza (http://viralzone.expasy.org/all_by_species/80.html). 4 B virion structure.Source: 2.1.3 Influenza Virus Replication Cycle When the HA, which is the ligand, binds to the host cell’s sialic acid residues (the receptors), endocytosis occurs and the virus gains entry into an endosome. The low pH in the endosome triggers the fusion of the endosomal and viral membranes. To accomplish this, aconformational change at the HA precursor is induced by the low pH exposing the HA2 fusion peptide (Huang et al., 2003). This fusion peptide inserts itself into the endosomal membrane, bringing both the viral and endosomal membranes into contact with each other. When the viral ribonucleoproteins (vRNP) are released into the cytoplasm they are then localized into the host cell nucleus where transcription and replication take place. The influenza virus uses its endonuclease enzyme to cleave about 10-13 nucleotides and the 5 methyl guanosine cap from the normal nuclear RNA of the host cell (Plotch et al., 1979). Protein synthesis and cellular mRNA transport are inhibited by the virus. The viral replicase cannot cap mRNA and thus the cellular mRNA cap is used as a primer to generate new viral mRNAs (Dhar et al., 1980). The snatched cap and associated nucleotides are then used as a primer for transcription of each of the eight negative sense RNA gene segments. The genome is first converted to a positive sense RNA by the viral RNA dependent RNA polymerase to serve as a template for the production of viral genomic RNAs (Bouloy et al., 1978). The influenza viral mRNA synthesis requires the activity of at least two influenza virus polymerase subunits, PB1 and PB2. PB1 has active sites that bind the conserved 32 and 52 sequences of vRNA, as well as having endonuclease activity. PB2 has cap binding activity and it is to this subunit that the host pre-mRNA binds (Boulo et al., 2007). The newly replicated genomes are transported to the cytoplasm for maturation of new virus particles (Shapiro et al., 1987). The new influenza virions are assembled at the host-cell surface 5 membrane and released by a process of budding in which both HA and NA are involved. Viral NA has the important function of cleaving sialic acid from viral and cellular glycoproteins, thus preventing virus aggregation and allowing individual virions to be released from the cell (Nayak et al., 2009). Figure 2: Influenza Virus Replication Cycle. Source: Cambridge University Press, 2001: Expert Reviews in Molecular Medicine. (http://varuncnmicro.blogspot.com/2012/05/influenzahush-bush.html). 2.2 The Influenza disease Influenza, also known as "the flu", is an infectious disease of birds and mammals caused by RNA viruses of the family Orthomyxoviridae, the influenza viruses. 6 2.2.1 Clinical presentation of influenza and its determinants In humans, the most common symptoms are chills, fever, runny nose, sore throat, muscle pains, headache (often severe), coughing, weakness/fatigue and general discomfort. The incubation period for influenza ranges from 2-3 days after which there is an onset of shivering, malaise, headache and aching in the limbs and back. Unlike common cold infections, influenza is not characterized by runny noses or sore throat at the beginning. Body temperature rises rapidly to about 39 degrees Celsius. Influenza is more severe among the very young (under five years old) and the elderly (above sixty five years of age). Non pulmonary complications include myositis, cardiac complications and encephalopathy (Walker et al., 1994). Influenza pneumonia can occur due to viral replication in the epithelial cells of the alveoli leading to rupture of walls of alveoli and bronchioles causing exudation into the air sacs. Secondary bacterial infection often leads to influenza pneumonia leading to respiratory distress, cyanosis and collapse within 2-3 days of the onset of infection(Rello and Pop-Vicas, 2009). The most common co-infecting bacterial species are Streptococcus pneumoniae and Staphyloccocus aureus. 2.2.2 Epidemiology Seasonal influenza is often recognized as a disease of the elderly, the very young, and people with high-risk chronic illnesses such as chronic obstructive pulmonary disease (COPD) and diabetes (Cox et al., 2000, Kaji et al., 2003). While this is true in relation to the likelihood of death and hospitalization, from another perspective this is totally incorrect. Large cohort studies of respiratory virus infections in families, which took place in the late 1960s and 1970s in the USA have established that the highest annual attack rates for influenza occur in children and teenagers (Leung et al., 2013). This is especially true for influenza B, which affects adults far 7 less frequently than children (Aymard et al., 2003). While attack rates in the young are highest, infection generally carries fewer consequences in children over 2 years old, teenagers and healthy adults, with correspondingly low levels of morbidity, mortality and hospitalization (Thompson et al., 2003). Thus, seasonal influenza is somewhat paradoxical with the highest attack rates in the young but the greatest public health impact in the elderly. In temperate climates, influenza infections at whatever level of intensity are characterized by a flu season. In these areas, the disease is thought to exist at a low level throughout the year but exhibit a marked seasonal increase, typically during the winter months. Influenza peak seasons happen between November-March in the Northern Hemisphere and during April-October in the Southern Hemisphere. In these regions, influenza-related deaths contribute ~5% of all winter mortality in persons over 65 years of age (Shaman and Kohn, 2009). Influenza epidemics and outbreaks occur in tropical areas as well, although the timing and impact is not as well defined (Shek and Lee, 2003). Most studies suggest that influenza in tropical regions circulate throughout the year with slight increases during the colder months (Freedman and Leder, 2005). 2.2.3 Influenza virus host range and pathogenesis 2.2.3.1 Host range Influenza A viruses have a greater host range than influenza B viruses. Thus, whereas influenza A viruses infects many avian (swans, ducks, chicken, mallards, quail etc.) and mammalian species (humans, horses, swine, minks, whales etc.), influenza B viruses predominantly infects humans. However, it may also infect seals and ferrets (Jakeman et al., 1994, Osterhaus et al., 2000). Influenza C viruses only infect humans (Muraki and Hongo, 2010). 8 2.2.3.2 Pathogenesis In humans, seasonal influenza is generally an infection of the upper respiratory tract. It is transmitted by contact with saliva or other respiratory secretions from an infected individual or by aerosol spread. Once the virus attaches to the epithelial cells, it infects and multiplies inside cells lining the mucosa. The virus multiplies in both upper and lower respiratory tract and destroys the cilia in the nose and sinus passages. The cilia are part of the innate immune system which is the first line of defense against respiratory pathogens. Thus, the pathogenesis of influenza viruses earlier on during the infection may be due to destruction on the innate (barrier) first-line defense of the host(Cox et al., 2004). 2.3 Genome structure of Influenza Viruses and proteins they encode As stated above, influenza A and B viruses have eight genomic segments while type C has seven segments (Palese, 1977). The first three segments encode the subunits of the viral polymerase complex. These segments are polymerase basic protein 2 (PB2) containing 2.4kb nucleotides, polymerase basic protein 1 (PB1) with 2.3kb nucleotides and polymerase acidic protein (PA) containing 2.2kb nucleotides. These three are the largest gene segments and are known for transcribing messenger RNAs and for synthesizing positive sense antigenomic template RNAs (cRNAs) which are transcribed into genomicsegments (vRNAs). These gene segments are expressed in two types of proteins: Internal and external proteins. 2.3.1Internal proteins Basic Polymerase Protein 2 9 The first segment of Influenza B viruses encodes the PB2protein which is 2.4kb in size. The PB1, PB2 and PA form the polymerase complex which carries out virus transcription and replication. The PB2 contains binding sites for both the PB1 subunit and the nucleoprotein (NP) and is also responsible for cap-binding during viral mRNA synthesis. This leads to creation of primers for viral mRNA synthesis by binding to the 5’ methylated cap of the host cell premRNAs before they are cleaved (Shi et al., 1996). Basic Polymerase Protein 1 The second segment encodes the PB1 protein whose function is for polymerase activity.ThePB1 subunit contains binding sites for both the PA and PB2 thus plays a key role in both the assembly of the three polymerase protein subunits and serves the catalytic function of RNA polymerization. It has been proposed that the catalytic specificity of PB1 subunit is modulated to the transcriptase by binding PB2 or the replicase by interaction with PA (Kosik et al., 2013). The PB1 segment encodes an additional protein PB1-F2 which has been implicated in regulation of polymerase activity, immunopathology, susceptibility to secondary bacterial infection, and induction of apoptosis (McAuley et al., 2007, Mitzner et al., 2009). Acidic Polymerase Protein (PA) The third segment encodes the acidic polymerase protein (PA). It contains nuclear localization signals (Nieto et al., 1994) requiredfor transport into the nucleus. Recent studies have shown that the PA subunit contains the endonuclease active site which synthesizes the viral messenger RNAs. In addition, the PA contains residues important for protein stability, promoter binding and cap-binding. PA-X is a fusion protein incorporating the N-terminal endonuclease domain of the 10 PA protein with a short C-terminal domain encoded by an overlapping ORF (‘X’) in segment 3 (Jagger et al., 2012). This protein functions to repress cellular gene expression especially those genes involved in regulating the initiation of the cellular immune response which results in host cell shut off (Weber et al., 1999). Nucleoprotein The product of the fifth segment is the Nucleoprotein (NP) which is 1.5kb in size. The NP mediates the transport of the viral ribonucleoproteins(RNP) from the viral particles to the nucleus and also plays a role in assembly and budding of the Influenza virus. It encapsidates the negative strand viral RNA for it to be recognized as templates for the viral polymerase. Matrix proteins The seventh segment encodes two gene products, the matrix 1 (M1) and matrix 2 (M2) proteins (BM2 in influenza B viruses) which is 1.027 kb in length. The M1 interacts with both the viral RNP and surface glycoproteins. The M1 mRNA product after transcription is responsible for virus assembly and budding after viral replication while the M2 is an integral membrane protein which forms an ion channel on the viral envelope(Rossman and Lamb, 2011). The M2 protein is only present in Influenza A viruses and form a drug target for the anti-influenza drug called adamatanes. This class of drugs belongs to the M2 ion channel blocker group and includes Rimantidine and Amantadine. Amantadine is more commonly used and has more side effects than rimantadine. The adamatanes are ineffective against Influenza B virus since these viruses lack the M2 protein in their viral structure but has a substitute called the NB that is not affected by amantadine (Brassard et al., 1996) Non Structural Proteins 11 The eighth segment is 890 bases long and encodes two proteins: Nonstructural protein1 (NS1) and Nonstructural protein 2 (NS2). The NS1 protein is a multifunctional protein involved in nuclear exportation of mRNA, posttranscriptional regulation, and inhibition of cellular interferon response while the NS2 protein mediates the nuclear export of virion RNAs by acting as an adaptor between viral ribonucleoprotein complexes and the nuclear export machinery of the cell (Steinhauer and Skehel, 2002b). The NS1 has been shown to block the innate host immune response by interfering with the signaling pathway of retinoic acid-inducible gene-1 (RIG-1) which together with the toll-like receptor 7 (TLR 7) activate antiviral host responses and lead to production of type 1 interferons (Kumagai et al., 2008). The NS1 also attaches to the antiviral protein, protein kinase R (PKR) whose function is to suppress the translation of viral mRNA in the host cell. When the NS1 binds to the PKR, there is inhibition of antiviral function of PKR(Min et al., 2007). 2.3.2External Proteins 2.3.2.1 Neuraminidase and NB The sixth segment which is 1.4kb encodes neuraminidase (NA) which destroys sialic acid containing inhibitors for the virus in the mucus secretions of the respiratory tract thus allowing mature virions to be released. The NA is a homotetramer and consists of a globular head, a thin stalk, a transmembrane domain and a cytoplasmic domain(Neumann and Kawaoka, 2011). The NB protein is only found inInfluenza B virus and is also encoded by segment 6. The NB protein possesses ion channel activity and is thought to function as an ion channel protein. The NB has been shown to initiate efficient replication in vivo but not in cell culture(Hatta and Kawaoka, 2003). 12 The neuraminidase active site is a major drug target against influenza viruses. The neuraminidase is a viral enzyme that has three functions in initiating virus spread(Gubareva et al., 2000). Firstly, neuraminidase digests neuraminic (sialic) acid in the hemagglutinin receptors thus releasing the virus particles that bud off the host cell membrane. Secondly, the virus particles released from the host cell have hemagglutinin receptors from the host’s cell membrane coating them. Thesenewly released virus particles bind to the hemagglutinins of other newly released viruses causing them to clump. The NA cleaves these residues allowing the virus to disperse and infect other cells. Thirdly, NA also digests neuraminic acid in respiratory mucus. Currently, two classes of influenza antivirals namely Adamantanes and neuraminidase inhibitors (NAI) are available to manage influenza. Each class inhibits a different step in the viral replication cycle. The NAI drugs block the activity of the neuraminidase by binding to the NA active site. However, a mutation at the NA active site leads to resistance to the anti-NA drugs(Samson et al., 2013). The two Neuraminidase inhibitors that are in current use in the treatment of influenza are Oseltamivir (Tamiflu) and Zanamivir (Relenza). Zanamivir is used by inhalation while Oseltamivir is administered orally. The two antivirals reduce the duration of illness by about 1.5 to 2.5 days and also the severity of disease is modified (Stiver, 2003). Recently, three new neuraminidase inhibitors have been developed and are in various stages of development. These include Laninamivir which has been approved for use in Japan, Favipiravir and Peramivir. Both Favipiravir and Peramivir are in various stages of clinical trials(Furuta et al., 2013). In neuraminidase inhibition by antivirals, inhibitor molecules mimic NA’s natural substrate and bind to the active site thus preventing NA from cleaving host cell receptors and releasing 13 progenyvirus. A rearrangement of amino acids in the active site is necessary to accommodate oseltamivir’s hydrophobic side chain; mutations that prevent this rearrangement may lead to resistance to oseltamivir. Zanamivir is more structurally similar to the natural substrate of NA enabling it to fit directly into the active site. Mutations that prevent the rearrangement would therefore not bring about resistance to zanamivir (Moscona, 2008). An amino acid substitution at the conserved NA residues decreases NA enzymatic activity (Yen et al., 2006). The drug will thus no longer be able to bind to the NA active site becoming less effective. The sites where these mutations have been known to occur in influenza B virus NA are E119G, E119A, D198N, D198Y, D198E, I222V/I, I222T, H274Y, N294S, R371K, G402S and R152K. 2.3.2.2 Hemagglutinin The fourth gene segment encodes the haemagglutinin glycoprotein (HA) which is 1.7kb in size. It is responsible for binding to the monosaccharide sialic acid which is present on the surface of its target host cells. The virus is then engulfed by the host cell through endocytosis forming an endosome (Murphy and Webster, 1990). The HA is synthesized as a precursor polypeptide, HA0 (Carr and Kim, 1993). This precursor polypeptide is post-translationally cleaved into two disulphide-linked subunits, HA1 and HA2 (See figure 3). The cleavage of the HA0 is a prerequisite for viral infectivity. This is followed by membrane fusion at the amino terminus of the HA2. This cleavage also allows the native HA molecule to undergo a conformational change, a process which is triggered by an acidic environment and is essential for membrane fusion (Carr et al., 1997). In general, the HA0 is believed to be cleaved by trypsin-like proteases extracellularly. However, the presence of multiple basic amino acid residues within the cleavage site allow the protein to be cleaved by intracellular proteases, e.g. furin(Chen et al., 1998). 14 There are various strategies employed by Influenza virus to ensure immune evasion from the host (Alcami and Koszinowski, 2000). Adaptive immune response includes generation of quasi viral species formed from accumulating amino acid substitutions in the antigenic sites of HA that are recognized by virus-neutralizing antibodies. Commonly known as antigenic drift, this phenomenon has been shown to allow the virus to evade recognition by antibodies and to cause recurrent influenza epidemics yearly (Steinhauer and Skehel, 2002a). There are various strategies used by viruses to evade recognition by virus-specific T cells. For example, encoding proteins that interfere with various steps in the antigen processing and presentation pathways is a mechanism commonly employed by viruses with large genomes. Most RNA viruses with smaller genomes and limited coding capacity, employ high mutation rates and subsequent selective pressure as a means of evading recognition by T cells (Reanney, 1982). The haemagglutinin being a major surface glycoprotein on influenza virus is very significant in vaccine design. Whereas resistance due to NA mutations can be detected by phenotypic enzyme inhibition assays, there is as yet no validated assay for identifying HA mutations that confer resistance to antivirals in humans (Moscona, 2005)(Varghese et al., 1998). Antigenic variation in influenza B virus is mostly caused by amino acid substitutions at four major antigenic epitopes (120-loop, 150-loop, 160 loop and 190- helix). These epitopes have been identified in previous studies (Pechirra et al., 2005). Thus, mutations in the HA antigenic region (positive selection) can result in vaccine failure due to the virus not being recognized by the immune system (Shen et al., 2008). The 120 loop and its surrounding regions (This loop is located at position 116-137 on the HA structure). The 120 loop appears to be one of the most frequently mutated regions in field 15 isolates (Wang et al., 2008). Indeed, virus variants of a B/Victoria like strain with specific substitutions at HA1 residues 129 and 137, produced by using reverse genetics, were found to cause altered antigenicity(Shen et al., 2009)(Wang et al., 2008). The 150 loop (This loop is located at position 141-150 on the HA structure).The 150 loop is an unusually long protruding loop, the tip of which (Thr1471) is pointed away from the main body of the structure by 9 Å. This site is one of the most obvious antibody-binding sites onB/HongKong/8/73 HA(Wang et al., 2008). The 160 loop (This loop is located at position 162-167 on the HA structure). The 160 loop is the only region in influenza B virus HAs where insertion and deletion have repeatedly been detected in field isolates from different epidemic seasons. The protruding nature of the 160 loop may make it easy to accommodate even multiple-residue insertions or deletions(Wang et al., 2008). The 190 helix and its surrounding regions (This loop is located at position 194-202 on the HA structure). All of the residues at the external face of the 190 helix have important antigenic roles. The location on the 190 helix HA1 (194–196), is a potential glycosylation site. A single mutation of Ala19613Thr, which potentially creates a new glycosylation site at HA1 (194–196), rendered the virus epidemic(Krystal et al., 1983). 16 Figure 3: Major Epitopes of influenza B virus. Trimeric structure shown with one monomer highlighted in color: Pink forHA1 and yellow for HA2. Mutations in four regions, the 120-loop (cyan), 150-loop (green), 160-loop (blue), and 190-helix (red), have been found to cause antigenicity variation(Shen et al., 2009). 17 2.4 Problem Statement Influenza B viruses have evolved into two main antigenically distinct lineages, the B/Yamagata/16/88 (Yamagata) lineage and the B-Victoria/2/87 (Victoria) lineage (Yamashita et al., 1988). Strains of one lineage can predominate during one season and virtually disappear from the population in the subsequent year. At the same time, it is well established that both lineages of influenza B viruses may co-circulate, thus providingan opportunity for reassortment leading to emergence of new variantswith unique biological and antigenic properties. Whereas influenza B viruses have been detected in Kenya since surveillance began in 2003, information on the genetic and antigenic characteristics of Kenyan influenza B viruses and their antiviral susceptibility is sparse. Neither have the appropriateness of the vaccine components of this influenza type been comprehensively characterized in Kenya. 2.5 Justification The need to characterize influenza B viruses in Kenya is imperative because it will provide information and understanding to policy makers regarding antiviral susceptibility, the effectiveness of theWHO-recommended vaccine, strains/lineages in circulation and evolution of influenza B viruses. This will assist in preparedness in case of outbreaks due to influenza B viruses as well as provide the clinicians with rational approaches for patient management. 2.6 Research Question What were the antigenic and molecular characteristics of influenza B viruses that circulated in Kenya in 2011-2012 in regard to their surface glycoproteins? 18 2.7 General Objective To investigate the molecular characteristics of the surface glycoproteins of Influenza B viruses isolated in Kenya from 2011-2012. 2.7.1 Specific Objectives 1. To antigenically characterize the influenza B viruses isolated in Kenya between 2011 and 2012 using the hemagglutinationinhibition assay and HA gene analyses. 2. To determine the antiviral susceptibility of Influenza B viruses in Kenya using phenotypic and molecular analyses of the NA genes 3. To determine the lineages of the Kenyan influenza B viruses using Bioinformatic tools for phylogenetic reconstruction. 4. To analyze the evolution of the influenza B viruses using bioinformatic approaches. 19 CHAPTER THREE: MATERIALS AND METHODS 3.0 Study Sites Between July 2006 and October 2013, the United States Army medical Research Unit-Kenya (USAMRU-K) carried out nationwide influenza surveillance in Kenya as part of the WHO Global Influenza Surveillance and Response System (GISRS) under the protocol WRAIR # 1267 /KEMRI SSC # 981. The surveillance network consisted of sites located at seven district hospitals and one provincial hospital representing the whole country. The sites included Malindi District hospital, Alupe Sub-District Hospital, Port Reitz District Hospital, New Nyanza Provincial hospital;Isiolo District Hospital, Kisii District Hospital, Kericho District hospital and Mbagathi District hospital (see Fig 4). The study sites were selected based on representation of the major population centers, frequency of international movement through the site or other characteristics of interest, e.g., anecdotal reports of frequent respiratory disease or representation of a different apparent region or disease situation. Samples that were used in this study were obtained from these sites and were collected between January 2011 and December 2012. 20 Figure 4: Sentinel sites of the Walter Reed Influenza Surveillance Network in Kenya.Adopted from (Bulimo et al., 2008). 3.1 Study Design This was a cross sectional laboratory-based study design thatutilized archivedinfluenza B isolates collected from 2011 – 2012 during active influenza surveillance across Kenya. 21 3.2 Study population This surveillance project’s study population consisted of persons of both sexes from two months of age onwards who presented to participating outpatient and inpatient clinics with acute upper respiratory illnesses either as influenza-like-illness (ILI) or severe acute respiratory illness (SARI) with no obvious cause on initial clinical examination. Subjects were recruited by a clinical officer assigned to the project based on the inclusion criteria detailed below. 3.2.1Inclusion criteria The inclusion criteria used was based on WHO case definitions of ILI and SARI(WHO, 2012). The WHO case definition forILI involves an acute respiratory infection withfever >38º C (oral or equivalent) and cough. The onset of ILI must have been within the previous 7 days because influenza virus cannot be routinely isolated from clinically ill individuals after approximately 72 hours due to the reduced level of viremia associated with the appearance of cytokines and anti-influenza immunoglobulins. For Severe acute respiratory illness (SARI) casesan acute respiratory infection with history of fever or measured fever of ≥ 38 C° and cough with onset within the last seven days, requiring hospitalization. 3.2.2 Exclusion criteria The exclusion criteria used was: children under 2 months of age, a refusal to participate or unwillingness to be sampled, children without available parent or legal guardian, no more than two individuals from any given household presenting with similar symptoms during the same timeframe and samples werenot to be taken from an individual who hadobvious exudative pharyngitis or tonsillitis. 22 All symptoms of the above case definition was to be present when collecting samples for this protocol, but the final decision on whether to collect a sample was left to the clinical judgment of the attending physician or clinical officer. 3.3 Sample Size/Statistics Because the samples were obtained from a surveillance project, sample size was determined by the laboratory’s capability to process samples, not the number needed to answer a specific statistical question. The initial maximum was established as 24 samples per week from each site. 3-4 influenza B virus isolates obtained from each of the eight sites during the specified period were randomly selected and then analyzed. These numbers, though not statistically determined, provided a good representation of genetic diversity of influenza B viruses that circulated in Kenya during the study period. 3.4 Experimental procedures: 3.4.1 Revival of stored isolates The influenza B virus isolates had previously been stored at -80OC in cryovials. The isolates were thawed and kept on ice throughout all the procedures. 3.4.2. Virus Isolation: Virus isolation was performed in Madin-Darby canine kidney (MDCK) cells. The MDCK cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 4.5g/L glucose and 10% Foetal Bovine Serum(FBS), 1% sodium pyruvate, 1% penicillin-streptomycin solution and 1% glutamine (Invitrogen, Germany). The cells were grown as monolayers in tissue culture (TC) tubes (Nuclon, Denmark). 100 µl of each sample was inoculated onto 70-90% confluent 23 MDCK cells in the flat side of the tubes after pre-treatment with TPCK trypsin in order to facilitate virus entry into the cells. Tubes were then incubated with the cap loose in a tissue culture incubator at 37°C with 5% CO2. Tubes wereexamined daily for 10 days for visual cytopathic effect (CPE) by light microscopy using an inverted microscope (Olympus, Tokyo). When clear CPE (i.e. >3+) was observed, supernatant fluid was collected and tested for hemagglutination titer, and if sufficient, hemagglutination inhibition (HAI) testing was conducted in accordance with CDC protocols using guinea pig red blood cells and the 2008 and 2009 reference reagents for influenza virus diagnosis from a WHO kit provided by the Center for Disease Control (CDC), Atlanta. 3.4.3. Haemagglutination test (HA) and titration of the virus Hemagglutination assay was carried out using 1% guinea pig red blood cells to check for the titre of the virus in the isolates in a 96 U-well microtitre plate using the WHO protocol (Freedman and Leder, 2005). The isolates were thawed and 25l of Phosphate Buffered Solution added to all wells followed by 25l of the isolate in the column one well and mixed. Serial two-fold dilutions of the isolates were carried out by transferring 25l of the mixture well to well. Another 25l of PBS was added to all wells. 50l of 1% guinea pig red blood cells was then added to all wells and the plate incubated at room temperature for one hour. Isolates causing agglutination of red blood cells were noted and their titers recorded. The titer of the virus was expressed as the reciprocal of the dilution that produced complete agglutination. For example, if the last dilution showing complete agglutination is 1: 64, then the HA titers was64. Isolates with no hemagglutination and those with a HA titre less than 4 were passaged once more. When the HA titer increased to more than 4, HAI was then performed. 24 3.4.3Haemagglutination inhibition (HAI) assay Identification and antigenic characterization was performed on HA positive isolates by the HAI test using the protocol and reference reagents prepared by the Centers for Disease control and Prevention (CDC)(Freedman and Leder, 2005). First, the positive HA isolates and the reference strains were adjusted to a titer of 4HA units using PBS. A 96 U-well plate was labeled with the reference antisera. 25l PBS was added to all wells, and then 25l of the reference antiserum was added to the well 1 of the appropriate row. Serial two-fold dilution of the antisera was carried out by transferring and mixing 25l from well 1 to well 11. 25l of the 4HA units of the isolates was added to all wells of the diluted set of antisera, wells 1 to 11. After mixing, the plate was incubated at room temperature for 30 minutes. 50l of 1% guinea pig red blood cells was added to all the wells and after incubation for 1 hour at room temperature, the HAI titre which is the last dilution of the antiserum that completely inhibitedhemagglutination was noted and recorded. HAI of the reference strains against the reference antisera wasalso performed. To identify and characterize the influenza B virus isolates, HAI titers obtained with the isolates were compared with those obtained with reference antigens. An isolate reacting at the same titers as the reference strain was characterized as being like that strain. 3.4.4 RNA extraction The virus RNA was extracted from the influenza B isolates using the QIAamp Viral RNA extraction kit (Qiagen, Inc., USA) following the manufacturer’s protocol. Briefly, 100µl of the virus was added to 500µl of lysis buffer per tube and allowed to incubate at room temperature for 10 minutes to allow for lysis. 500µl of ethanol was added and pulse vortexing performed for 15 seconds to give a homogeneous solution. 630µl of the lysed solution was applied to the spin columns and centrifuged at 6000 x g for 1 minute and column placed in a clean collection tube. 25 500µl of Buffer AW1 (wash buffer 1) was added to the spin column and centrifuged at 6000 x g for 1 minute in anEppendorff 5415R centrifuge (Eppendorf AG, Barkhausenweg, Hamburg, Germany) and the column placed in a clean collection tube. Then the column was washed with 500µl of Buffer AW2 (wash buffer 2) and centrifuged at 9750x g for 3 minutes. The spin column was then placed in a 1.5 ml micro centrifuge tube and 60µl of Buffer AVE (elution buffer) added to the column and allowed to incubate at room temperature for 1 minute. The column was then centrifuged at 6000 x g in anEppendorff 5415Rcentrifuge (Eppendorf AG, Barkhausenweg, Hamburg, Germany) at room temperature for 1 minute and the filtrate (RNA) stored at -800C. 3.4.5 Conventional RT-PCR to amplify the HA and NA genes The RT-PCR to amplify the HA and NA genes was performed using the Superscript III One-Step RT-PCR system (Invitrogen Corporation, Carlsbad, CA, USA). The reaction mix was prepared by mixing 12.5µl of the 2x reaction mix, 0.5 µl of the forward primer (20 µM), 0.5 µl of the reverse primer (20 µM), 1.0 µl Superscript III RT/Platinum Taq mix and this mixture was then topped using 7.5 µl of distilled water to make a total of 22 µl. 3 µl of the RNA template was then added. A list of the primer sequences used in this study is attached in appendix 1. Thermocycling was carried out on a 9700 ABI Thermal Cycler (Applied Biosystems, Lincoln Centre Drive Foster City, CA, USA) using the following conditions; 1 cycle of reverse transcription at 500C for 30 minutes followed by an initial denaturation of 940C for 2min, 35 cycles of 940C for 30 seconds, 550C for 30 seconds and 680C for 1 minute and a final extension of 680C for 1min. followed by storage of amplicons at 40C. 26 3.4.6 Agarose Gel electrophoresis of the amplicons 1.5% Agarose was prepared using 1x TBE buffer. The solution was mixed by swirling gently and then heating in a microwave until all the agarose was completely dissolved. The gel was then left to cool for a few minutes and then ethidium bromide was added to a final concentration of 0.5µg/ml. The gel was then poured onto an electrophoretic tank (Sigma-Aldrich Co., USA) containing combs and left to set for 30 minutes. The combs were then carefully removed. 3µl of the PCR products were mixed with 2µl of the loading dye (Sigma-Aldrich Co., USA) and then loaded into the wells. A 1kb DNA marker (Promega., Switzerland)was loaded on the first lane of each of the wells. The tank was then connected onto an electric current and run at 150 volts for about 30-45 minutes. The gel was visualized and the gel photo printed using the AlphaImager gel documentation system (Alpha Innotech, California, and USA) 3.4.7 Purification of the PCR amplicons and sequencing Briefly, the PCR tubes containing the PCR amplicons to be purified were spun after which 3µl of using Exonuclease I/Shrimp Alkaline Phosphatase (ExoSap-IT) enzyme (Affymetrix, Santa Clara, California,USA) on ice was added to each of the PCR tube. The mixture was vortexed followed by brief spinning. The PCR tubes were placed in the 9700 FAST thermoycler and incubated and sequenced directly on both strands with the same primers used in the PCR, on an automated ABI 3500XL Genetic Analyzer (Applied Biosystems, town, state, USA). Cycle sequencing was performed using the Big Dye Terminator v3.1 sequencing kit (Applied Biosystems, Massachusetts, USA), which incorporates fluorescent-labeled dideoxy-chain terminators and normal deoxynucleotides. 27 3.4.8. Sequencing by dideoxy Chain termination method Sequence template of the PCR amplicons was synthesized using the ABI BigDye Terminator version 3.1 cycle sequencing Kit (Applied Biosystems, Massachusetts, USA) which incorporates fluorescent-labeled dideoxy-chain terminators. The master mix composition for 1X reaction for each gene, fragment was prepared according to the protocol by the manufacturer (Applied Biosystems, Massachusetts, USA) shown in table 1: Table 1: Reagents used for sequencing PCR Reagent Vol. per test (µl) BigDye 3.1 2 BigDye 5X buffer 2 ddH2O 3 dsDNA 2 Total 9 3.4.10 Nucleotide sequence assembly The nucleotide sequences generated using ABI PRISM 3500XL genetic analyzer was assembled prior to analyses. Contiguous nucleotide sequence (contigs) assembly from the reverse and forward sequence runs were carried out using the DNA Baser Sequence Assembler v3 (HeracleBioSoft SRL Romania, (http://www.DnaBaser).The success of the nucleotide sequencing procedure was assessed by carrying out a similarity search against sequences in GenBank(http://www.ncbi.nlm.nih.gov/genbank/)using the basic local alignment search tool (BLAST) with the default parameters of the program. 28 3.4.11 Phylogenetic reconstruction, positive selection and evolutionary analyses Multiple sequence alignment was carried out using MUSCLE (Edgar, 2004a, Edgar, 2004b). Phylogenetic analyses was performed using MrBayes 3.2(Ronquist et al., 2005). The generalized time-reversible model parameters and priors were incorporated into the nexus file for execution in MrBayes. The data was executed in MrBayes by running 1 million Monte Carlo Markov chains with a sampling frequency of 1000. The phylogenetic tree was then visualized using FigTree, version 1.3.1 (http://tree.bio.ed.ac.uk/software/figtree/). Analysis for positive selection at individual codon sites was performed by two methods; singlelikelihood ancestor counting (SLAC), fixed effects likelihood (FEL) and Internal branches fixed effects likelihood (IFEL) implemented in Data monkey tool (http://datamonkey.org/). Statistical significance was defined as p-value ≤ 0.05. The mean dN/dS ratio and 95% confidence interval were computed based on Neighbor-Joining (NJ) trees under the HKY85 substitution model(Delport et al., 2010). The ratio of the nonsynonymous substitution rate (dN) to the synonymous rate (dS) was interpreted as follows: When the ratio dN/dS is zero, it is interpreted as natural selection; When the ratio of dN/dS is greater than 1, it is interpreted as positive selection, while if the dN/dS ratio is less than 1, it is interpreted as negative selection. To determine rates of evolution, analyses was carriedbased on the HKY85 substitution modelby running twenty million Monte Carlo Markov Chains (MCMC) implemented in BEAST (Drummond and Rambaut, 2007). For amino acid based phylogenetic analyses, aligned fasta amino acid data was converted into the Nexus file format using the Concatenator program (Pina-Martins and Paulo, 2008). The nexus formatted data was executed in MrBayes by running one million Monte Carlo Markov Chains (MCMC) with a sampling frequency of 1000 trees from which a consensus tree for each 29 of the NA and HA proteins was generated and visualized using FigTree version 1.3.1 (http://tree.bio.ed.ac.uk/software/figtree/). The phylogenetic tree for each of the NA and HA proteins of influenza B isolates showing presence or absence of markers of drug resistance was used to infer phylogenetic relationship of the isolates relative to antiviral susceptibility. 3.4.12 Genetic antigenicity analyses The HA nucleotide sequences were translated into amino acid code and analyzed for antigenic character focusing on the globular head of the hemagglutinin (HA1) protein. Nucleotide sequences of the gene segments were translated into protein code using Discovery studio gene (DS Gene) version 1.5 (Accelrys Inc.) and applying the universal genetic code. The isolates’amino acid codes were compared with those of vaccine strains using multiple sequence alignments. Thus, the HA1 proteins of B/Wisconsin/1/2010–like virus (the recommended vaccine strain for use in the 2013 influenza season in southern hemisphere winter), B/Massachusetts/2/2012-like virus (the recommended vaccine strain for use in the 2013-14 influenza season in the southern and northern hemisphere winter) andB/Brisbane/60/2008-like virus (the recommended vaccine strain for use in the 2011 and 2012 influenza seasons in the southern hemisphere winters) were included in the analyses. Substitutions and all known amino acid residues involved at the 120-loop, 150-loop, 160-loop and the 190-helix antigenic sites (Tumpey et al., 2007) were determined. Single alterations in the glycosylation patterns on the HA1 protein often times results in antigenic changes; any gain or loss of N-glycosylation site(s) at or close to the antigenic sites were also determined using the CountGS application tool of BioEdit(Hall, 2011)as implemented in the CDC Utility Bioinformatics Toolkit (cubit) package by Dr. James Smagala. The success of the nucleotide 30 sequencing procedure was assessed by carrying out a similarity search against sequences in GenBank using the basic local alignment search tool (BLAST) with the default parameters of the program. 3.4.13 Genetic drug-susceptibility analyses The amino acid sequences of the NA proteins were obtained after translation of the nucleotide sequences. Nucleotide sequences of the gene segments were translated into protein code using discovery studio gene (DS Gene) version 1.5 (Accelrys Inc.) and applying the universal genetic code. To correlate the phenotypic findings about drug susceptibility by the viruses, multiple sequence alignments were carried out using Muscle version 3.8 (Edgar, 2004a, Edgar, 2004b). Reference amino acid sequences from susceptible and oseltamivir/zanamivir-resistant influenza B viruses were then included in these analyses. The amino acid changes in the NA protein that have been associated with resistance to these drugs and hence were analyzed for include E119G/A, D198N/Y/E, I222V/T, H274Y, N294S, R371K, G402S and R152K. 3.4.14 Neuraminidase phenotyping assays Phenotypic characterization of the neuraminidase activity by enzyme inhibition tests was performed with virus isolates to confirm observations from the genotypic data, monitor natural variation in drug susceptibility and identify antiviral resistance from unknown mutations in the viral genome. Phenotypic characterization by these assays determined the concentration of neuraminidase inhibitor (NI) drug that results in a 50% inhibition of neuraminidase enzyme activity (IC50 value). The assays were used to measure susceptibility to both oseltamivir and zanamivir NI drugs. Susceptibility of viruses to the NAIs was assessed using fluorescent NI assays, (Sheu et al., 2008, Shinya and Kawaoka, 2006) which incorporates methyl umbelliferone N-acetylneuraminic acid (MUNANA) derivative of sialic acid (Buxton et al., 2000) as the 31 substrate. Briefly, the 50% inhibitory concentration (IC50) value, which is the concentration of drug that is required to inhibit the enzyme activity by 50%, was determined by assaying the NA activity of virus in the presence of serial half-log dilutions (from 10µM to 0.01 nM) of each NA inhibitor. After equal volumes of virus and inhibitor were mixed and incubated at room temperature for 30 min, fluorogenic substrate (2-(4-methylumbelliferyl)-D-N-acetylneuraminic acid; Sigma, St. Louis, MO) was added at a final concentration of 100 nM, and the reaction mixture then incubated at 37°C for 1 hr. The reaction was stopped by addition of the stop solution (150 µl of 0.5 M NaOH, pH 10.7, containing 25% ethanol), and fluorescence measured with the use of Tecan Infinite M1000 (Tecan, Mannedorf, Switzerland). The excitation wavelength was set at 365 nm, and the emission wavelength at 460 nm respectively. The activity of each virus sample was titrated, by assaying serial two-fold dilutions, and virus suspensions were adjusted to equivalent NA activities within the linear portion of the activity curve. NA inhibitors Zanamivir were acquired from GlaxoSmithKline (Uxbridge, United Kingdom), while oseltamivir carboxylate, the active compound of the ethyl ester prodrugoseltamivir phosphate, was supplied by Hoffman-La Roche (Basel, Switzerland). Calculation of 50% inhibitory concentration (IC50) values and curve fitting was performed by Robosage version 7.31 software (Glaxo- SmithKline, Research Triangle Park, NC), an add-in for Microsoft Excel (Microsoft Corp., Redmond, WA), using the equation where y = V max[X]/ (K+X) is the response being inhibited, X is the inhibitor concentration, K is the IC50 for the inhibition curve, and Vmax is the maximum rate of metabolism. 32 CHAPTER FOUR RESULTS 4.1Study subjects The study population included pediatric patients consisting of 18 males and 6 females with an average age of 2 years (Table 2). The clinical presentations included fever (100%), coughing (100%), runny nose (96%), nasal stuffiness (58%), malaise (50%)and vomiting (37.5%) amongst others (Table 2). 33 Bleeding Diarrhea Fatigue pain Joint Headache Sputum production nose Runny Nasal stuffines Abdominal pain Neurological Vomiting Malaise STRAIN FluBI07/11 2 7 M Y N U U U FluBK05/11 1 4 M Y N N U U FluB01/11 3 11 M Y N Y Y U FluB09/11 4 8 M Y N Y Y U FluB13/11 2 10 M Y N U U U FluB15/11 1 4 F Y N U U U FluB22/12 3 11 F Y Y N N N FluB23/12 1 1 F Y N U U U FluB24/12 0 8 M Y N U U U FluBM03/11 1 5 M Y Y N U U FluBM04/11 0 9 M Y Y N U U FluB06/11 0 6 M Y Y N U U FluBM08/11 5 6 M Y N N N N FluB10/11 4 4 F Y N N N N FluB11/11 4 6 M Y N N N N FluB12/11 5 3 F Y N N Y N FluB18/12 3 0 M Y Y N N N FluB19/12 4 0 M Y N N N N FluB20/12 0 6 M Y N N N N FluB16/12 1 1 M Y N U U U FluB14/11 1 11 M Y N U U U FluB17/12 0 10 M Y N U U N FluB21/12 0 6 M Y N U U N FluBK02/11 2 9 F Y N U U U % 100% 21% 8% 12.50% 0% M=male; F=female; Y=yes; N=no; U=undetermined Retroorbital pain Muscle Aches throat Sore Chills Difficulty breathing Cough Sex Age (month) Age (year) Table 2: Demography and Clinical Presentation of the study subjects U Y Y N N Y Y N N U U N N Y N N N N N Y U U U Y N N N Y N N Y N Y U Y U Y N N N Y Y N N N Y Y Y Y Y N N N Y N N Y N Y U U U Y N N N Y Y N U N Y U U U Y N N N N N N N Y Y N N N N N N N Y N N U N Y U U U Y N N N Y N N U N Y U U U Y N N U U Y Y U Y Y Y U U U N N U U Y Y U Y Y Y U U U N N U U Y Y U Y Y Y U U U N N N Y Y N Y Y Y N N N N N N N Y N N Y Y Y N N N N Y N N Y Y N Y Y Y N N N N N N N Y N N N Y Y N N Y N N N N N N N N Y Y N N N N N N N N N N N Y Y N N N N N N N N Y N Y Y Y N N N N Y N U U N N U N Y U U U U N N U U N U U Y N U U U U Y N N N N N N Y Y N U U U U U N N N N N N Y N N N N N N N Y N N U N Y U U U Y N N 4% 50% 37.50% 12.50% 25% 58% 96% 17% 8% 8% 33% 12.50% 0% 34 4.2HA Assay Titers of the Isolates A total of 3688 samples were collected during the 2011-2012 period by the USAMRU-K laboratory. Real Time PCR screening revealed that 212(6%) of the samples had Influenza B virus and from which virus isolates were obtained. When 24(11%) of these isolates were screened by HA, the HA titersobtained after the first passage ranged from 4-64 units, with majority having a titre of 16 (Table 3). When the 3 isolates with HA titres of less than 16 units were re-passaged, their titres increased to 16 (Table 3). Table 3: HA and HAI titers of the 24 Influenza B isolates tested in the study. P1 titres were obtained from the 1st passage of the viruses while P2 titres were those obtained using the second passage of the same virus. ND = not done. Serial 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Sample ID HA (P1) FluB17/12 8 FluB18/12 16 FluB20/12 16 FluB23/12 32 FluB22/12 16 FluB21/12 16 FluB24/12 16 FluB11/11 32 FluBI07/11 16 FluB09/11 16 FluBM03/11 8 FluBM04/11 16 FluB06/11 4 FluBM08/11 16 FluB10/11 16 FluB12/11 16 FluB16/12 32 FluBK05/11 64 FluB13/11 64 FluB15/11 64 FluB14/11 16 FluBK02/11 16 FluB01/11 16 FluB19/12 16 Titer HA Titer (P2) 16 64 32 64 32 32 32 32 ND ND 16 ND 16 ND ND ND ND ND ND ND 32 64 32 16 35 4.3 Antigenic characterization of the influenza B isolates using serology Determination of the lineages of the influenza B virus isolates using Haemagglutination inhibition (HAI) assays indicatedthat all the 24 isolates obtained during the study period were B/Brisbane/60/2008-like with HAI B/Brisbane/60/2008 (Table 4). titers of 640, equivalent to that of reference Since B/Brisbane/60/2008 belongs to the B/Victoria/2/87 lineage, therefore all the Kenyan influenza B isolates were characterized as belonging to the B/Victoria/2/87 lineage. 36 Table 4: Haemagglutination Inhibition Assay titers of the isolates compared to the two 2011-2012 southern and northern hemisphere reference strains Reference antisera Reference antigens B/Brisbane/60/ 2008 B/Massachusetts/02/2012 B/Brisbane/60/2008 640 <20 B/Massachusetts/02/2012 <20 1280 FluB12/11 640 <20 FluB11/11 640 <20 FluB10/11 640 <20 FluBM08/11 640 <20 FluB06/11 640 <20 FluBM04/11 640 <20 FluBM03/11 640 <20 FluB09/11 640 <20 FluBK02/11 640 <20 FluB01/11 640 <20 FluBI07/11 640 <20 FluB14/11 640 <20 FluB15/11 640 <20 FluB13/11 640 <20 FluBK05/11 640 <20 FluB24/12 640 <20 FluB21/12 640 <20 FluB22/12 640 <20 FluB23/12 640 <20 FluB20/12 640 <20 FluB19/12 640 <20 FluB18/12 640 <20 FluB16/12 640 <20 FluB17/12 640 <20 37 4.3.1 Analysis of Antigenic drift on HA The HA nucleotide sequence data of this study were deposited in GenBank and GISAID databases under accession numbers:JQ396198 - JQ396212 and EPI401426 - EPI441063 respectively. When the antigenic drifts in HAI region of the local influenza B isolates were analyzed bycomparing with the B/Brisbane/60/2008 vaccine reference strain, 19 amino acid substitution loci were discovered (Table 5). All the Kenyan isolates had a D197N mutation. In addition, the majority (87.5%) of the Kenyan isolates also had a mutation at position 146. Amongst these, 20 (83.3%) had I146V whereas a single isolate designated FluB22/12,from Kisii district hospital from a patient who presented on 13/08/12 had an I146A substitution. Two other mutations were also observed in four of the Kenyan isolates. These include FluB06/11 and FluBK02/11 which displayed a K397Q whereas FluB20/12 and FluB19/12 had D527YA parallel mutations respectively. A single isolate, FluB20/12, isolated from Mbagathi District Hospital on 11/05/2012 had the largest (6) number of amino acids substitutions within the HA protein. These changes included, V124I, I146V, A159G, P161S, D197N and D527Y. Two isolates had four substitutions each. Thisincluded FluBM04/11 isolated in a patient from Mbagathi district hospital who presented with symptoms on 04/02/11 and FluB16/12 isolated from a patient from Malindi district hospital on 03/01/12. Isolate FluBM04/11 had I76T, D197N, V225I and G256E substitutions, whereas isolate FluB16/12 had I146V, D197N, N233D and V379I changes respectively. Eight isolates had three substitution each in the HA protein. These isolates consisted of FluB06/11, FluBI07/11, FluBM08/11, FluB10/11, FluB13/11, FluB17/12, FluB23/12 and FluB24/12. FluB06/11 was isolated from Mbagathi District hospital in a patient seen on 05/02/11 and had the substitutions D197N, G229Sand K397Q. FluBI07/11 was from 38 Isiolo district hospital from a patient seen on 10/02/11 and had substitutions V90I, I146V, D197N. Others included FluBM08/11 in a patient from Mbagathi district hospital seen on 10/02/11 with substitutions R118K, I146V &D197N; FluB10/11 from a Mbagathi district hospital patient seen on 22/02/11 with substitutions I146V, D197N and T327A; FluB13/11 from Kisii district hospital patient seen on 13/04/11 with L58F, I146Vand D197N changes; FluB17/12 from Port Reitz district hospital patient seen on 05/01/12 with I146V, D197N and V379I mutations; FluB23/12 from a Kisii district hospital patient seen on 18/08/12 with I146V, D197Nand I261L substitutions; and finally FluB24/12 from a Kisii district hospital patient seen on 23/08/12 with I146V, D197N and E423K mutations. Thus, the two major parallel mutations were fixed at important antigenic sites, indicating the evolution of the Kenyan influenza B viruses to escape host 39 immune pressure. Table 5: Amino acid substitutions in the HA of the Kenyan strains relative to the 2011-2012 WHO reference strain (B/Brisbane/08) Amino acid at position Virus Strains 58 76 90 118 124 B.Brisbane.60.2008 L I V R V FluBM04/11 . T . . . FluB06/11 . . . . . FluBK02/11 . . . . . FluB24/12 . . . . . FluB22/12 . . . . . FluB20/12 . . . . I FluB19/12 . . . . . FluB13/11 F . . . . FluB16/12 . . . . . FluB18/12 . . . . . FluBI07/11 . . I . . FluB14/11 . . . . . FluBM03/11 . . . . . FluB10/11 . . . . . FluB12/11 . . . . . FluBK05/11 . . . . . FluB17/12 . . . . . FluB11/11 . . . . . FluBM08/11 . . . K . FluB09/11 . . . . . FluB01/11 . . . . . FluB15/11 . . . . . FluB21/12 . . . . . FluB23/12 . . . . . 146 I . . . V A V V V V V V V V V V V V V V V V V V V 159 A . . . . . G . . . . . . . . . . . . . . . . . . 161 P . . . . . S . . . . . . . . . . . . . . . . . . 197 D N N N N N N N N N N N N N N N N N N N N N N N N 40 225 V I . . . . . . . . . . . . . . . . . . . . . . . 229 G . S . . . . . . . . . . . . . . . . . . . . . . 233 N . . . . . . . . D . . . . . . . . . . . . . . . 256 G E . . . . . . . . . . . . . . . . . . . . . . . 261 I . . . . . . . . . . . . . . . . . . . . . . . L 327 T . . . . . . . . . . . . . A . . . . . . . . . . 379 V . . . . . . . . I . . . . . . . I . . . . . . . 397 K . Q Q . . . . . . . . . . . . . . . . . . . . . 423 E . . . K . . . . . . . . . . . . . . . . . . . . 527 D . . . . . Y Y . . . . . . . . . . . . . . . . . 4.3.2 Results on Antigenic drift on NA Those for the NA nucleotide sequences were deposited in GenBank under accession numbers JQ396213 - JQ396216 and KT266882- KT266893.Comparison of the NA amino acid sequences of influenza B viruses with the reference vaccine strain revealed substitutions at 19 amino acid positions when compared to B/Brisbane/60/2008 (Table 6). Twenty three of the twenty four (96%) isolates analyzed had a mutation at position 340. Among these, the vast majority (22 isolates; 96%) had a N340D change and a single isolate (4%) had a N340Y substitution.Fifteen isolates from the year 2011 were included in this study. Out of these 2011 isolates, 12 (80%) had a T49A substitution, while a single isolate (FluB23/12) obtained in late 2012 had the same T49A mutation. Six isolates (25%) had two parallel mutations of K107R and A389T. Amongst the six, the majority (67%) were isolated in 2012 and only 2 isolates in this category belong to the year 2011. 4(17%) of the isolates had a substitution at position 295. Amongst these, 3 had S295Rmutations whereas a single isolate had S295Kmutation. 50% of the isolates had parallel mutations involving 3 disparate substitutionsat positions358, 392, 397and 427. Thus isolates FluB24/12, FluB18/12 and FluB21/12 had the E358K substitution, FluB16/12, FluB17/12 & FluB01/11 had a D392E substitution, FluB11/11, FluB19/12 & FluB20/12 had S397G and finally isolates FluB01/11, FluB10/11 and FluBK02/11showed I427V change. The isolates FluB23/12 and FluB15/11 had two substitutions at V271I and L73F. Isolate FluB01/11 had the most number (6) of mutations amongst all the isolates. These mutations included K107R, N340T, A389T, D392E, I427V and E436K. 41 Table 6: Amino acid changes in the NA as compared to the B/Brisbane/60/08 reference strain Virus Strains B/Brisbane/60/08 FluB01/11 FluB10/11 FluBK02/11 FluB21/12 FluB11/11 FluB19/12 FluB20/12 FluB16/12 FluB17/12 FluB06/11 FluB22/12 FluB24/12 FluB18/12 FluB23/12 FluB15/11 FluB13/11 FluB14/11 FluBK05/11 FluB09/11 FluBM08/11 FluBI07/11 FluBM03/11 FluBM04/11 FluB12/11 2 L . . . . P . . . . . . . . . . . . . . . . . . . 49 T . A A . . . . . . . . . . A A A A A A A A A A A 50 M . . . . . . . . . . . . . . . I . . . . . . . . 73 L . . . . . . . . . . . . . F . . . . F . . . . . 107 K R . . . R R R R R . . . . . . . . . . . . . . . Amino Acid at Position: 148 157 271 295 324 G I V S D . . . . . E . . . . . . . . . . . . K . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . R . . . . R E . . . R . . . I . . . . I . . . . . . . . T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42 329 N . . . . . . . . . . D . . . . . . . . . . . . . 340 N D D D D D D D D D . D Y D D D D D D D D D D D D 358 E . . . K . . . . . . . K K . . . . . . . . . . . 389 A T . . . T T T T T . . . . . . . . . . . . . . . 392 D E . . . . . . E E . . . . . . . . . . . . . . . 395 A . . . . . . . . . . . . . . . . . V . . . . . . 397 S . . . . G G G . . . . . . . . . . . . . . . . . 427 I V V V . . . . . . . . . . . . . . . . . . . . . 436 E K . . . . . . . . . . . . . . . . . . . . . . . 4.3.4 Glycosylation analysis When N-glycosylation on the surface proteins were analyzed, it was revealed that only one of the 24 isolates, FluB/16/12, had lost a glycosylationsite in the HA protein at position 248relative to B/Brisbane/60/2008 (Table 7). Interestingly, this glycosylation site is also absent in HA of B/Victoria/02/1987 prototype strain. When the NA protein was analyzed for glycosylation potential, isolate FluB21/12 was shown to have gained a glycosylation site at position 329 (Table 8). This glycosylation site is absent in both B/Brisbane/60/2008 and B/Victoria/02/1987 reference strains. 43 Table 7: Hemagglutinin Glycosylation Sites Amino acid position No. of Strain glycosylation sites B/Brisbane/60/2008 12 B/Victoria/02/1987 10 FluB12/11 12 FluB11/11 12 FluB10/11 12 FluBM08/11 12 FluB06/11 12 FluBM04/11 12 FluBK02/11 12 FluB09/11 12 FluBK02/11 12 FluB01/11 12 FluBI07/11 12 FluB14/11 12 FluB15/11 12 FluB13/11 12 FluBK05/11 12 FluB24/12 12 FluB21/12 12 FluB22/12 12 FluB23/12 12 FluB20/12 12 FluB19/12 12 FluB18/12 12 FluB16/12 11 FluB17/12 12 40 X X X X X X X X X X X X X X X X X X X X X X X X X X 74 X X X X X X X X X X X X X X X X X X X X X X X X X X 160 X X X X X X X X X X X X X X X X X X X X X X X X X X 181 X X X X X X X X X X X X X X X X X X X X X X X X X X 44 212 248 319 X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X 348 X X X X X X X X X X X X X X X X X X X X X X X X X X 507 X X X X X X X X X X X X X X X X X X X X X X X X X X 533 X X X X X X X X X X X X X X X X X X X X X X X X X X 546 X X X X X X X X X X X X X X X X X X X X X X X X X X 578 X X X X X X X X X X X X X X X X X X X X X X X X X X Table 8: Neuraminidase Glycosylation sites # glycosylation Strain sites B/Brisbane/60/08 4 B/Victoria/2/87 4 FluB01/11 4 FluB10/11 4 FluBK02/11 4 FluB21/12 5 FluB11/11 4 FluB19/12 4 FluB20/12 4 FluB16/12 4 FluB17/12 4 FluB06/11 4 FluB22/12 4 FluB24/12 4 FluB18/12 4 FluB23/12 4 FluB15/11 4 FluB13/11 4 FluB14/11 4 FluBK05/11 4 FluB09/11 4 FluBM08/11 4 FluBI07/11 4 FluBM03/11 4 FluBM04/11 4 FluB12/11 4 X=glycosylation sites present Amino acid position 56 64 144 X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X 45 284 X X X X X X X X X X X X X X X X X X X X X X X X X X 329 X 4.3.5Analysis of Selection Pressure When global selective pressure on HA and NA gene segments of the Kenyan influenza B viruses was estimated using the dN/dS ratio (ω), the mean ω for HA gene was found to be = 0.561627 while that of NAwas 0.328863.A single codon site at position 69 was identified as being negatively selected in the HA when the SLAC method was used. However, this was insignificant because the P-value was 0.0825 which is greater than 0.05 (Table 9). 15 negatively selectedcodons were detected by the FELmethod. Amongst these, only three at codons positions 69, 85 and 460 showed significant negative evolution because their P-values were below 0.05 (Table 9). When IFEL method was used on the data set, two negatively selected sites were detected but the evolution was not significant within the 95% confidence interval (Table 9). Analysis of the NA genes by the SLAC method revealed no negatively or positively selected codons. However, the FEL method detected 14 negatively selected codon sites. Of these 14 codons, only 2 at positions 192 and 218 showed significance within the 95% confidence interval (Table 10). The IFEL method detected one negatively selected site that was insignificant within the 95% confidence limits 46 (Table 10). Table 9: This table shows negative selection in the HA at various positions Amino acid Residue changes From To Amino Codon acid Codon SLAC 69 TGC Cys TGT FEL 61 GGA Gly GGG 69 TGC Cys TGT 85 CCA Pro CCG 103 AGA Arg AGG 109 TGC Cys TGT 115 GAT Asp GAC 230 TCA Ser TCT 280 AGA Arg AGG 317 GGA Gly GGG 353 AGA Arg AGG 398 GCA Ala GCT 429 AGC Ser AGT 459 GAG Glu GAA 460 CTC Leu CTT 473 GAA Glu GAG IFEL 69 TGC Cys TGT 85 CCA Pro CCG The figures in bold represent the P-values of statistical significance Analysis Method Residue position Normalized dNP-value dS Amino acid Cys Gly Cys Pro Arg Cys Asp Ser Arg Gly Arg Ala Ser Glu Leu Glu Cys Pro 47 -47.8063 -558.119 -955.948 -1023.74 -665.41 -441.533 -474.581 -586.163 -488.338 -449.846 -521.56 -510.88 -388.131 -574.627 -765.951 -605.796 -955.948 -1023.74 Type selection 0.0825 Negative 0.070038 Negative 0.0185701 0.0197843 0.0542636 0.0997286 0.0869836 0.0941519 0.0778539 0.0844879 0.0734865 0.0782812 0.0905931 0.0674694 0.0336433 0.0718649 0.0721601 Negative 0.076115 of Table 10: This table shows negative selection in the NA at various positions. Analysis Method SLAC FEL Residue position 56 64 67 181 192 198 201 218 238 293 327 328 385 473 Amino acid Residue changes From To Amino Codon acid Codon Amino acid AAC AAC GCA GCG GGA AAT TTG GCA CTT GAT AGA CCA GGA GGA Asn Asn Ala Ala Gly Asn Leu Ala Leu Asp Arg Pro Gly Gly Asn Asn Ala Ala Gly Asn Leu Ala Leu Asp Arg Pro Gly Gly AAT AAT GCT GCA GGC AAC TTA GCG CTG GAC CGA CCT GGG GGG Normalized dNP-value dS IFEL 192 GGA Gly GGC Gly The figures in red and bold represent the P-values of statistical significance 48 Type selection -324.99 -322.087 -369.547 -360.735 -843.088 -483.203 -329.846 -359.702 -633.407 -403.238 -466.045 -407.503 -352.837 -335.34 0.0918385 Negative 0.0915505 0.0897187 0.0506372 0.0145176 0.0646472 0.0967468 0.0245721 0.0913246 0.0754648 0.0767115 0.0849401 0.093326 0.0971651 -843.088 0.0896179 Negative of 4.4 Evolutionary Rates TheBEAST analysis revealed that the rate of nucleotide substitution of the HA of the Kenyan influenza B virus isolateswas 3.66 X 10-3, ranging from 1.04 X 10-3 to 6.47 X 10-3 substitutions/ site/year, indicating that 6 out of 10 progeny genomes will contain a mutation in the HA. The rate of nucleotide substitution in the NA genes was estimated at 0.682 X 10-3 to 1.71 X 10-3 substitutions/ site/year in the NA with a mean of 1.18 X 10-3 translating to mutations in 2 out of 10 progeny genomes harboring a mutation. 4.5Phylogenetic Analyses Phylogenetic analyses using the HA amino acid sequences revealed that all the 24 influenza B viruses that circulated between 2011- 2012 belonged to the B/Victoria lineage and were closely related to the B/Brisbane/60/2008 vaccine strain (Fig. 5). Four main clusters were observed on the phylogram. All the Kenyan strains isolated in period 2011-2012 clustered on the same branch with B/Brisbane/60/2008, which was the WHO-recommended vaccine strain for use in the 2011 and 2012 influenza seasons in the southern hemisphere (Fig. 5). The general topology of the phylogenetic tree did not change when HA nucleotide sequences were used in the phyologenetic analyses (Fig. 6). Phylogenetic analyses using the NA amino acid revealed that all 24 influenza B Kenyan strains belonged to the Yamagata lineage (Fig 7). 49 Figure 5: Phylogenetic analysis of Kenyan influenza B viruses using the HA amino acid sequences. The tree was generated by Bayesian methods using MrBayes. The reference strains are B-Victoria/02/87, B-Yamagata 16/88, B-Wisconsin 01/2010 and BMassachusetts 02/2012. The horizontal bar scale represents the number of nucleotide changes per 100 amino acids. Bayesian posterior probabilities generated using MrBayes are indicated as percentages at the nodes. 50 Figure 6: Phylogenetic analysis on HA Nucleotide sequences.The tree was generated by Bayesian methods using MrBayes (Ronquist and Huelsenbeck, 2003). The reference strains are B-Victoria/02/87, B-Yamagata 16/88, B-Wisconsin 01/2010 and BMassachusetts 02/2012. The horizontal bar scale represents the number of nucleotide changes per 100 nucleotides. Bayesian posterior probabilities are indicated as percentages at the nodes. 51 Figure 7:Phylogenetic Analysis of NA nucleotide sequences.The tree was generated by Bayesian methods using MrBayes (Ronquist and Huelsenbeck, 2003). The reference strains are B-Victoria/02/87, B-Yamagata 16/88, B-Brisbane/60/2008, B-Wisconsin 01/2010 and B-Massachusetts 02/2012. The horizontal bar scale represents the number of nucleotide changes per 100 nucleotides. Bayesian posterior probabilities generated using MrBayes are indicated as percentages at the nodes. 52 Figure 8: Phylogenetic Analysis of NA amino acid: The tree was generated by Bayesian methods using MrBayes (Ronquist and Huelsenbeck, 2003). The reference strains are B-Victoria/02/87, B-Yamagata 16/88, B-Wisconsin 01/2010 and B-Massachusetts 02/2012. The horizontal bar scale represents the number of nucleotide changes per 100 nucleotides. Bayesian posterior probabilities generated using MrBayes are indicated 53 as percentages at the nodes. 4.6Drug Susceptibility genetic markers When the known resistant marker mutation in the NA were analyzed for previously associated with resistance or reduced susceptibility to Oseltamivir and/or Zanamivir, none of the 24 isolates of this studyshowed mutationsE119, R152, D198, I222, S250, H274, R371, and G402 (universal A/N2 numbering; Figure 9) that are associated with Oseltamivir or Zanamivir resistance. 54 A_H3N2_Tex B.Yamagata B.Victoria B.Massachu B.Wisconsi B.Brisbane FluB01.11 FluB11.11 FluB06.11 FluB15.11 FluB09.11 FluB10.11 FluB13.11 FluBK02.11 FluB14.11 FluBK05.11 FluBM08.11 FluBI07.11 FluBM03.11 FluBM04.11 FluB12.11 FluB21.12 FluB16.12 FluB17.12 FluB19.12 FluB20.12 FluB22.12 FluB24.12 FluB18.12 FluB23.12 : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : 120 * 140 * 160 * 180 * 200 * 220 * 240 * 260 WVTRVPYVSCDPDKCYQFALGQGTTLNNVHSNDTVRDRTPYRTLLMNELG-VPFHLGTKQVCIAWSSSSCHDGKAWLHVCITGDDKNATASFIYNGRLVDSIVSWSKEILRTQESECVCINGTCTVVMTDGSASGKADTKILFIEEGKIV LIIREPFIACGPKECKHFALTHYAAQPGGYYNGTREDRNKLRHLISVKLGKIPTVENSIFHMAAWSGSACHDGREWTYIGVDGPDSNALIKIKYGEAYTDTYHSYANNILRTQESACNCIGGDCYLMITDGSASGISKCRFLKIREGRII LIIREPFIACGPKECKHFALTHYAAQPGGYYNGTREDRNKLRHLISVNLGKIPTVENSIFHMAAWSGSACHDGREWTYIGVDGPDSNALIKIKYGEAYTDTYHSYANNILRTQESACNCIGGDCYLMITDGSASGISKCRFLKIREGRII LIIREPFIACGPTECKHFALTHYAAQPGGYYNGTREDRNKLRHLISVKLGKIPTVENSIFHMAAWSGSACHDGKEWTYIGVDGPDSNALLKIKYGEAYTDTYHSYAKNILRTQESACNCIGGDCYLMITDGPASGVSECRFLKIREGRII LIIREPFIACGPKECKHFALTHYAAQPGGYYNGTREDRNKLRHLISVKLGKIPTVENSIFHMAAWSGSACHDGREWTYIGVDGPDSNALLKIKYGEAYTDTYHSYAKNILRTQESACNCIGGDCYLMITDGPASGISECRFLKIREGRII LIIREPFIACGPNECKHFALTHYAAQPGGYYNGTRGDRNKLRHLISVKLGKIPTVENSIFHMAAWSGSACHDGKEWTYIGVDGPDNNALLKVKYGEAYTDTYHSYANKILRTQESACNCIGGNCYLMITDGSASGVSECRFLKIREGRII LIIREPFIACGPNECKHFALTHYAAQPGGYYNGTRGDRNKLRHLISVKLGKIPTVENSIFHMAAWSGSACHDGKEWTYIGVDGPDNNALLKVKYGEAYTDTYHSYANKILRTQESACNCIGGNCYLMITDGSASGVSECRFLKIREGRII LIIREPFIACGPNECKHFALTHYAAQPGGYYNGTRGDRNKLRHLISVKLGKIPTVENSIFHMAAWSGSACHDGKEWTYIGVDGPDNNALLKVKYGEAYTDTYHSYANKILRTQESACNCIGGNCYLMITDGSASGVSECRFLKIREGRII LIIREPFIACGPNECKHFALTHYAAQPGGYYNGTRGDRNKLRHLISVKLGKIPTVENSIFHMAAWSGSACHDGKEWTYIGVDGPDNNALLKVKYGEAYTDTYHSYANKILRTQESACNCIGGNCYLMITDGSASGVSECRFLKIREGRII LIIREPFIACGPNECKHFALTHYAAQPGGYYNGTRGDRNKLRHLISVKLGKIPTVENSIFHMAAWSGSACHDGKEWTYIGVDGPDNNALLKVKYGEAYTDTYHSYANKILRTQESACNCIGGNCYLMITDGSASGVSECRFLKIREGRII LIIREPFIACGPNECKHFALTHYAAQPGGYYNGTRGDRNKLRHLISVKLGKIPTVENSIFHMAAWSGSACHDGKEWTYIGVDGPDNNALLKVKYGEAYTDTYHSYANKILRTQESACNCIGGNCYLMITDGSASGVSECRFLKIREGRII LIIREPFIACGPNECKHFALTHYAAQPGGYYNGTREDRNKLRHLISVKLGKIPTVENSIFHMAAWSGSACHDGKEWTYIGVDGPDNNALLKVKYGEAYTDTYHSYANKILRTQESACNCIGGNCYLMITDGSASGVSECRFLKIREGRII LIIREPFIACGPNECKHFALTHYAAQPGGYYNGTRGDRNKLRHLISVKLGKIPTVENSIFHMAAWSGSACHDGKEWTYIGVDGPDNNALLKVKYGEAYTDTYHSYANKILRTQESACNCIGGNCYLMITDGSASGVSECRFLKIREGRII LIIREPFIACGPNECKHFALTHYAAQPGGYYNGTRGDRNKLRHLISVKLGKIPTVENSIFHMAAWSGSACHDGKEWTYIGVDGPDNNALLKVKYGEAYTDTYHSYANKILRTQESACNCIGGNCYLMITDGSASGVSECRFLKIREGRII LIIREPFIACGPNECKHFALTHYAAQPGGYYNGTRGDRNKLRHLTSVKLGKIPTVENSIFHMAAWSGSACHDGKEWTYIGVDGPDNNALLKVKYGEAYTDTYHSYANKILRTQESACNCIGGNCYLMITDGSASGVSECRFLKIREGRII LIIREPFIACGPNECKHFALTHYAAQPGGYYNGTRGDRNKLRHLISVKLGKIPTVENSIFHMAAWSGSACHDGKEWTYIGVDGPDNNALLKVKYGEAYTDTYHSYANKILRTQESACNCIGGNCYLMITDGSASGVSECRFLKIREGRII LIIREPFIACGPNECKHFALTHYAAQPGGYYNGTRGDRNKLRHLISVKLGKIPTVENSIFHMAAWSGSACHDGKEWTYIGVDGPDNNALLKVKYGEAYTDTYHSYANKILRTQESACNCIGGNCYLMITDGSASGVSECRFLKIREGRII LIIREPFIACGPNECKHFALTHYAAQPGGYYNGTRGDRNKLRHLISVKLGKIPTVENSIFHMAAWSGSACHDGKEWTYIGVDGPDNNALLKVKYGEAYTDTYHSYANKILRTQESACNCIGGNCYLMITDGSASGVSECRFLKIREGRII LIIREPFIACGPNECKHFALTHYAAQPGGYYNGTRGDRNKLRHLISVKLGKIPTVENSIFHMAAWSGSACHDGKEWTYIGVDGPDNNALLKVKYGEAYTDTYHSYANKILRTQESACNCIGGNCYLMITDGSASGVSECRFLKIREGRII LIIREPFIACGPNECKHFALTHYAAQPGGYYNGTRGDRNKLRHLISVKLGKIPTVENSIFHMAAWSGSACHDGKEWTYIGVDGPDNNALLKVKYGEAYTDTYHSYANKILRTQESACNCIGGNCYLMITDGSASGVSECRFLKIREGRII LIIREPFIACGPNECKHFALTHYAAQPGGYYNGTRGDRNKLRHLISVKLGKIPTVENSIFHMAAWSGSACHDGKEWTYIGVDGPDNNALLKVKYGEAYTDTYHSYANKILRTQESACNCIGGNCYLMITDGSASGVSECRFLKIREGRII LIIREPFIACGPNECKHFALTHYAAQPGGYYNGTRGDRNKLRHLISVKLGKIPTVENSIFHMAAWSGSACHDGKEWTYIGVDGPDNNALLKVKYGEAYTDTYHSYANKILRTQESACNCIGGNCYLLITVGSASGVSECRFLKIREGRII LIIREPFIACGPNECKHFALTHYAAQPGGYYNGTRGDRNKLRHLISVKLGKIPTVENSIFHMAAWSGSACHDGKEWTYIGVDGPDNNALLKVKYGEAYTDTYHSYANKILRTQESACNCIGGNCYLMITDGSASGVSECRFLKIREGRII LIIREPFIACGPNECKHFALTHYAAQPGGYYNGTRGDRNKLRHLISVKLGKIPTVENSIFHMAAWSGSACHDGKEWTYIGVDGPDNNALLKVKYGEAYTDTYHSYANKILRTQESACNCIGGNCYLMITDGSASGVSECRFLKIREGRII LIIREPFIACGPNECKHFALTHYAAQPGGYYNGTRGDRNKLRHLISVKLGKIPTVENSIFHMAAWSGSACHDGKEWTYIGVDGPDNNALLKVKYGEAYTDTYHSYANKILRTQESACNCIGGNCYLMITDGSASGVSECRFLKIREGRII LIIREPFIACGPNECKHFALTHYAAQPGGYYNGTRGDRNKLRHLISVKLGKIPTVENSIFHMAAWSGSACHDGKEWTYIGVDGPDNNALLKVKYGEAYTDTYHSYANKILRTQESACNCIGGNCYLMITDGSASGVSECRFLKIREGRII LIIREPFIACGPNECKHFALTHYAAQPGGYYNGTRGDRNKLRHLISVKLGKIPTVENSIFHMAAWSGSACHDGKEWTYIGVDGPDNNALLKVKYGEAYTDTYHSYANKILRTQESACNCIGGNCYLMITDGSASGVSECRFLKIREGRII LIIREPFIACGPNECKHFALTHYAAQPGGYYNGTRGDRNKLRHLISVKLGKIPTVENSIFHMAAWSGSACHDGKEWTYIGVDGPDNNALLKVKYGEAYTDTYHSYANKILRTQESACNCIGGNCYLMITDGSASGVSECRFLKIREGRII LIIREPFIACGPNECKHFALTHYAAQPGGYYNGTRGDRNKLRHLISVKLGKIPTVENSIFHMAAWSGSACHDGKEWTYIGVDGPDNNALLKVKYGEAYTDTYHSYANKILRTQESACNCIGGNCYLMITDGSASGVSECRFLKIREGRII LIIREPFIACGPNECKHFALTHYAAQPGGYYNGTRGDRNKLRHLISVKLGKIPTVENSIFHMAAWSGSACHDGKEWTYIGVDGPDNNALLKVKYGEAYTDTYHSYANKILRTQESACNCIGGNCYLMITDGSASGVSECRFLKIREGRII A_H3N2_Tex B.Yamagata B.Victoria B.Massachu B.Wisconsi B.Brisbane FluB01.11 FluB11.11 FluB06.11 FluB15.11 FluB09.11 FluB10.11 FluB13.11 FluBK02.11 FluB14.11 FluBK05.11 FluBM08.11 FluBI07.11 FluBM03.11 FluBM04.11 FluB12.11 FluB21.12 FluB16.12 FluB17.12 FluB19.12 FluB20.12 FluB22.12 FluB24.12 FluB18.12 FluB23.12 : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : * 280 * 300 * 320 * 340 * 360 * 380 * 400 * HTSTLSGSAQHVEECSC-YPRYPGVRCVCRDNWKGSNRPIVDINIKDHSIVSSYVCSGLVGDTPRKNDSSSSSHCLDPNNEEGGHGVKGW----AFDDGNDVWMGRTISEKSRLGYETFKVIEGWSNPKSKLQINRQVIVDRGNRSGYS KEIFPTGRVEHTEECTCGFASNKTIECACRDNSYTAKRPFVKLNVETDTAEIRLMCTETYLDTPRPDDGSITGPC-ESNGDKGRGGIKGGFVHQRMASKIGRWYSRTMSKTERMGMELYVKYDGDPWTDSDALAPSGVMVSMKEPGWYS KEIFPTGRVEHTEECTCGFASNKTIECACRDNSYTAKRPFVKLNVETDTAEIRLMCTETYLDTPRPDDGSITGPC-ESNGEKGRGGIKGGFVHQRMASKIGRWYSRTMSKTERMGMELYVKYDGDPWTDSDALAPSGVMVSMKEPGWYS KEIFPTGRVKHTEECTCGFASNKTIECACRDNRYTAKRPFVKLNVETDTAEIRLMCTETYLDTPRPNDGSITGPC-ESDGDKGSGGIKGGFVHQRMASKIGRWYSRTMSKTKRMGMGLYVKYDGDPWTDSEALALSGVMVSMEEPGWYS KEIFPTGRVKHTEECTCGFASNKTIECACRDNSYTAKRPFVKLNVETDTAEIRLMCTETYLDTPRPNDGSITGPC-ESNGDKGSGGIKGGFVHQRMASKIGRWYSRTMSKTKRMGMGLYVKYDGDPWTDSEALALSGVMVSMEEPGWYS KEIFPTGRVKHTEECTCGFASNKTIECACRDNSYTAKRPFVKLNVETDTAEIRLMCTDTYLDTPRPNDGSITGPC-ESNGDKGSGGIKGGFVHQRMESKIGRWYSRTMSKTERMGMGLYVKYDGDPWADSDALAFSGVMVSMKEPGWYS KEIFPTGRVKHTEECTCGFASNKTIECACRDNSYTAKRPFVKLNVETDTAEIRLMCTDTYLDTPRPNDGSITGPC-ESDGDKGSGGIKGGFVHQRMESKIGRWYSRTMSKTERMGMGLYVKYDGDPWTDSEALAFSGVMVSMKEPGWYS KEIFPTGRVKHTEECTCGFASNKTIECACRDNSYTAKRPFVKLNVETDTAEIRLMCTDTYLDTPRPNDGSITGPC-ESDGDKGSGGIKGGFVHQRMESKIGRWYSRTMSKTERMGMGLYVKYDGDPWTDSDALAFGGVMVSMKEPGWYS KEIFPTGRVKHTEECTCGFASNKTIECACRDNSYTAKRPFVKLNVETDTAEIRLMCTDTYLDTPRPNDGSITGPC-ESNGDKGSGGIKGGFVHQRMESKIGRWYSRTMSKTERMGMGLYVKYDGDPWADSDALAFSGVMVSMKEPGWYS KEIFPTGRIKHTEECTCGFASNKTIECACRDNSYTAKRPFVKLNVETDTAEIRLMCTDTYLDTPRPNDGSITGPC-ESDGDKGSGGIKGGFVHQRMESKIGRWYSRTMSKTERMGMGLYVKYDGDPWADSDALAFSGVMVSMKEPGWYS KEIFPTGRVKHTEECTCGFASNKTIECACRDNSYTAKRPFVKLNVETDTAEIRLMCTDTYLDTPRPNDGSITGPC-ESDGDKGSGGIKGGFVHQRMESKIGRWYSRTMSKTERMGMGLYVKYDGDPWADSDALAFSGVMVSMKEPGWYS KEIFPTGRVKHTEECTCGFASNKTIECACRDNSYTAKRPFVKLNVETDTAEIRLMCTDTYLDTPRPNDGSITGPC-ESDGDKGSGGIKGGFVHQRMESKIGRWYSRTMSKTERMGMGLYVKYDGDPWADSDALAFSGVMVSMKEPGWYS KEIFPTGRVKHTEECTCGFASNKTIECACRDNSYTAKRPFVKLNVETDTAEIRLMCTDTYLDTPRPNDGSITGPC-ESDGDKGSGGIKGGFVHQRMESKIGRWYSRTMSKTERMGMGLYVKYDGDPWADSDALAFSGVMVSMKEPGWYS KEIFPTGRVKHTEECTCGFASNKTIECACRDNSYTAKRPFVKLNVETDTAEIRLMCTDTYLDTPRPNDGSITGPC-ESDGDKGSGGIKGGFVHQRMESKIGRWYSRTMSKTERMGMGLYVKYDGDPWADSDALAFSGVMVSMKEPGWYS KEIFPTGRVKHTEECTCGFASNKTIECACRDNSYTAKRPFVKLNVETDTAEIRLMCTDTYLDTPRPNDGSITGPC-ESDGDKGSGGIKGGFVHQRMESKIGRWYSRTMSKTERMGMGLYVKYDGDPWADSDALAFSGVMVSMKEPGWYS KEIFPTGRVKHTEECTCGFASNKTIECACRDNSYTAKRPFVKLNVETDTAEIRLMCTDTYLDTPRPNDGSITGPC-ESDGDKGSGGIKGGFVHQRMESKIGRWYSRTMSKTERMGMGLYVKYDGDPWADSDALVFSGVMVSMKEPGWYS KEIFPTGRVKHTEECTCGFASNKTIECACRDNSYTAKRPFVKLNVETDTAEIRLMCTDTYLDTPRPNDGSITGPC-ESDGDKGSGGIKGGFVHQRMESKIGRWYSRTMSKTERMGMGLYVKYDGDPWADSDALAFSGVMVSMKEPGWYS KEIFPTGRVKHTEECTCGFASNKTIECACRDNSYTAKRPFVKLNVETDTAEIRLMCTDTYLDTPRPNDGSITGPC-ESDGDKGSGGIKGGFVHQRMESKIGRWYSRTMSKTERMGMGLYVKYDGDPWADSDALAFSGVMVSMKEPGWYS KEIFPTGRVKHTEECTCGFASNKTIECACRDNSYTAKRPFVKLNVETDTAEIRLMCTDTYLDTPRPNDGSITGPC-ESDGDKGSGGIKGGFVHQRMESKIGRWYSRTMSKTERMGMGLYVKYDGDPWADSDALAFSGVMVSMKEPGWYS KEIFPTGRVKHTEECTCGFASNKTIECACRDNSYTAKRPFVKLNVETDTAEIRLMCTDTYLDTPRPNDGSITGPC-ESDGDKGSGGIKGGFVHQRMESKIGRWYSRTMSKTERMGMGLYVKYDGDPWADSDALAFSGVMVSMKEPGWYS KEIFPTGRVKHTEECTCGFASNKTIECACRDNSYTAKRPFVKLNVETDTAEIRLMCTDTYLDTPRPNDGSITGPC-ESDGDKGSGGIKGGFVHQRMESKIGRWYSRTMSKTERMGMGLYVKYDGDPWADSDALAFSGVMVSMKEPGWYS KEIFPTGRVKHTEECTCGFASNKTIECACRDNKYTAKRPFVKLNVETDTAEIRLMCTDTYLETPRPNGSIIRGPC-ASDGDKGSGGIKGGFVHQRMKSKIGRWYSRTMSKTERMGMGLYVKYDGDPWADSDALAFSGVMVSMKEPGWYS KEIFPTGRVKHTEECTCGFASNKTIECACRDNSYTAKRPFVKLNVETDTAEIRLMCTDTYLDTPRPNDGSITGPC-ESDGDKGSGGIKGGFVHQRMESKIGRWYSRTMSKTERMGMGLYVKYDGDPWTDSEALAFSGVMVSMKEPGWYS KEIFPTGRVKHTEECTCGFASNKTIECACRDNSYTAKRPFVKLNVETDTAEIRLMCTDTYLDTPRPNDGSITGPC-ESDGDKGSGGIKGGFVHQRMESKIGRWYSRTMSKTERMGMGLYVKYDGDPWTDSEALAFSGVMVSMKEPGWYS KEIFPTGRVKHTEECTCGFASNKTIECACRDNSYTAKRPFVKLNVETDTAEIRLMCTDTYLDTPRPNDGSITGPC-ESDGDKGSGGIKGGFVHQRMESKIGRWYSRTMSKTERMGMGLYVKYDGDPWTDSDALAFGGVMVSMKEPGWYS KEIFPTGRVKHTEECTCGFASNKTIECACRDNSYTAKRPFVKLNVETDTAEIRLMCTDTYLDTPRPNDGSITGPC-ESDGDKGSGGIKGGFVHQRMESKIGRWYSRTMSKTERMGMGLYVKYDGDPWTDSDALAFGGVMVSMKEPGWYS KEIFPTGRVKHTEECTCGFASNKTIECACRDNRYTAKRPFVKLNVETDTAEIRLMCTDTYLDTPRPDDGSITGPC-ESDGDKGSGGIKGGFVHQRMESKIGRWYSRTMSKTERMGMGLYVKYDGDPWADSDALAFSGVMVSMKEPGWYS KEIFPTGRVKHTEECTCGFASNKTIECACRDNRYTAKRPFVKLNVETDTAEIRLMCTDTYLETPRPNDGSITGPC-ESYGDKGSGGIKGGFVHQRMKSKIGRWYSRTMSKTERMGMGLYVKYDGDPWADSDALAFSGVMVSMKEPGWYS KEIFPTGRVKHTEECTCGFASNKTIECACRDNRYTAKRPFVKLNVETDTAEIRLMCTDTYLDTPRPNDGSITGPC-ESDGDKGSGGIKGGFVHQRMKSKIGRWYSRTMSKTERMGMGLYVKYDGDPWADSDALAFSGVMVSMKEPGWYS KEIFPTGRIKHTEECTCGFASNKTIECACRDNSYTAKRPFVKLNVETDTAEIRLMCTDTYLDTPRPNDGSITGPC-ESDGDKGSGGIKGGFVHQRMESKIGRWYSRTMSKTERMGMGLYVKYDGDPWADSDALAFSGVMVSMKEPGWYS 55 Figure 9: Multiple sequence alignment of NA amino acid sequences of Kenyan Influenza B isolates with reference strains to identify loci involved in NAI resistance.The E119, R152, D198, I222, S250, H274, R371, and G402 mutations known to conifer resistance to NAIs are highlighted in Red. 56 4.7 Antiviral susceptibility The phenotypic drug susceptibility assay showed that the mean IC50s obtained using fluorescent-labeled substrates ranged from 0.0M-12.6nM for Zanamivir and 17.1nM-70.1nM for Oseltamivir (Table 11). These mean IC50 values were all within the 2011 WHO range of 8128nM for oseltamivir carboxylate and 0.5-12nM for Zanamivir. 57 Table 11: Phenotypic Drug assay results for HAI and Drug Sensitivity tests ISOLATE ID HA TITER HAI ZANAMIVIR (IC50) DRUG ASSAY VALUES FluB17/12 FluB19/12 FluB21/12 FluB22/12 FluB20/12 FluB14/11 FluB09/11 FluBM08/11 FluB12/11 FluB10/11 FluBM03/11 FluB06/11 FluB16/12 FluBK05/11 FluB13/11 FluB15/11 FluB18/12 FluB23/12 FluB24/12 FluB11/11 FluBI07/11 FluBK02/11 32 16 64 32 32 16 16 16 16 16 16 16 32 64 64 64 64 64 16 32 16 16 B/Brisbane 60/2008-like B/Brisbane 60/2008-like B/Brisbane 60/2008-like B/Brisbane 60/2008-like B/Brisbane 60/2008-like B/Brisbane 60/2008-like B/Brisbane 60/2008-like B/Brisbane 60/2008-like B/Brisbane 60/2008-like B/Brisbane 60/2008-like B/Brisbane 60/2008-like B/Brisbane 60/2008-like B/Brisbane 60/2008-like B/Brisbane 60/2008-like B/Brisbane 60/2008-like B/Brisbane 60/2008-like B/Brisbane 60/2008-like B/Brisbane 60/2008-like B/Brisbane 60/2008-like B/Brisbane 60/2008-like B/Brisbane 60/2008-like B/Brisbane 60/2008-like 0.8 0.9 0.7 0.6 0.9 2.8 0.6 0.6 0.6 1.6 0.6 0.7 12.6 1.1 6.5 0.7 0.6 0.0 0.1 0.0 0.0 0.1 OSELTAMIVIR (IC50) DRUG ASSAY VALUES 70.1 39.4 21.2 35.8 35.3 41.7 23.8 23.9 27.8 13.3 16.0 15.0 23.3 17.1 24.1 26.6 18.0 20.6 22.3 25.4 21.6 22.1 FluBM04/11 FluB01/11 16 32 B/Brisbane 60/2008-like B/Brisbane 60/2008-like 0.0 0.5 24.1 26.0 58 DISCUSSION The study examined the genetic and phenotypic characteristics of influenza B viruses that circulated in Kenya in a 23-month period in 2011–2012, by analyzing itstwo surface glycoproteins. Historically,influenza B viruses have been shown to undergo a series of insertions and deletions on their surface glycoproteins which result in changes in the virus leading to disparate antigenic reactivity of the viruses(McCullers et al., 1999). These changes are advantageous to the virus because they aid the virus in infecting new hosts as well as facilitate inresistance to antiviral drugs.Previous studies with influenza B viruses have demonstrated discordant antigenic drift of HA and NA (McKimm-Breschkin et al., 2003), suggesting the virus can overcome host immune mechanisms by modifying either the HA or NA antigens. At present, vaccine strain-selection decisions are based on antigenic characterization of HA and NA genetic data, also taking into consideration epidemiologic and human serologic data (Ampofo et al., 2012). Here, the phenotypic serologic assay showed that all the Kenyan influenza B viruses had identical HAI titres with the 2011-2012 WHO-recommended vaccine strain and were thus Brisbane/60/2008-like. Because Brisbane/60/2008 virus belongs to the B/Victoria/2/87 lineage, all the Kenyan influenza B isolates examined in circulation in the country in the 2011-2012 period were of B/Victoria/2/87 lineage. Phylogenetic analysisusing both the nucleotide and amino acid codes of the HA genes of the viruses confirmed that the influenza B viruses that circulated during the study period were B/Brisbane/60/2008-like and of the B/Victoria/2/87 lineage. Therefore, the influenza B vaccine component recommended by WHO for the period 2011-2012 and represented most of the influenza B circulating in the world at that time (WHO, 2011) was protective and appropriate for Kenya and her visitors. 59 Whereas serological and phylogenetic analyses revealed that the influenza B viruses in Kenya during the study period were B/Brisbane/60/2008-like, these two assays did not necessarily prove that the influenza B viruses were not undergoing antigenic changes in the HA protein. It is well established that antigenic variation in influenza B virus is caused by amino acid substitutions at four domains that constitute the major antigenic epitopes designated 120-loop, 150-loop, 160 loop and 190- helix(Shen et al., 2009).Changes at these antigenic sites and their surroundings often lead to altered antigenic reactivities of the virus(Wang, 2010). When the amino acid codes for the HA1 domain were analyzed,19 amino acid loci in the HA1 protein had undergone variation compared to B/Brisbane/60/2008. However, only three residues within the major antigenic sites were amongst the 19. Thus, the isolate designated FluB20/12 had a V124I amino acid change in the 120-loop antigenic site relative to B/Brisbane/60/2008. However, the majority (87.5%) of our isolates had the I146V/A amino acid change which is located in the 150-loop antigenic site. Furthermore, all the Kenyan isolates had a D197N amino acid change in the 190-helix antigenic site.Taken together, all theseantigenic drift variations identified in the local isolates are not considered epidemiologically significant enough to allow escape from immune pressure because phenotypically, they did not lead to loss of antigenicity. It has been shown previously thateach new influenza virus drift variant of epidemiologic importance ought to generally have four or more amino acid substitutions located in two or more of the antigenic sitesfor the antigenic character of the virus to change (Wilson and Cox, 1990). This explains why the WHO-recommended vaccine was appropriate despite the genetic drifts described above. 60 Glycosylation patterns are another form of antigenic variation that causes masking or unmasking of antigenic sites on viral surface epitopes (Wilson and Cox, 1990). Amongst viruses, glycosylation is a posttranslational modification involving addition of sugar units mostly to an asparagine (N) amino acid residue in certain contexts and is known to affect the antigenic character of proteins (Wilson and Cox, 1990). When the loss or gain of glycosylation sites in the HA protein of the local influenza B isolates was examined, most of the isolates had the same numberand at the same glycosylation sitesas the B/Brisbane/60/2008. However, strain FluB16/12lost a glycosylation sitein at position 248 in the HA protein. This loss of a glycolsylation site did not unmask new antigenic sites because this site is not an epitope on the HA protein.Nonetheless, clear evidence for the importance of carbohydrates in modulating antigenicity was provided by selection of a mutant HA (D197N) in which a new glycosylation site prevented antibody binding and viral neutralization(Wilson and Cox, 1990). The B/Victoria2/87lineage was the only lineage isolated during the period 2011 to 2012 as determined by both the HAI assay and bioinformatics. Recent studies in Kenya (2008-2011 period) showed that B/Yamagata/16/88 dominated in the 2008 season while B/Victoria2/87 dominated from 2009-2011 (Majanja et al., 2013). The current study shows that there was no cocirculation/co-infection of both strains (B/Victoria and B/Yamagata) reported within the study period (2011 – 2012). However, there have been incidents around the world especially in Taiwan where both B/Victoria2/87 and B/Yamagata/16/88 have co-circulated simultaneously in the 2011–2012 season(WHO, 2011). The changes in the circulation of the two different lineages have been hypothesized to be caused by changes in herd immunity among the affected populations. In Kenya, the B/Victoria/2/87 lineage predominance may have been as a result of an accumulation in herd immunity to the 61 B/Yamagata/16/88 lineage which had circulated in the previous years(Chen and Holmes, 2008b).B/Yamagata/16/88-lineage viruses were found to uniquely emerge in Taiwan while B/Victoria2/87 predominated in China(WHO, 2011) Genetic characterization of NA in comparison to the vaccine strain established that none of the amino acid substitutions seen in the NA were positioned at the eight amino acid positionsinvolved in conferring antiviral resistance namely E119, R152, D198, I222, S250, H274, R371, and G402 (universal A/N2 numbering) in the neuraminidase (NA) active site.These sites have previously been found to be associated with resistance or reduced susceptibility to the antivirals Zanamivir and Oseltamivir. However, there were mutations seen in the surrounding regionsof the active site. The amino acid substitutions (V271I) on FluB23/12 and FlubB15/11 occurred near the known marker H274Y. FluB21/12, FluB22/12, FluB24/12 and FluB18/12 possessed a mutation S295K/Rnear N294S while FluB24/12 (D324E) mutated near T325I.These mutations being very close to the neuraminidase active site may influence susceptibility in the future.The genetic findings showing that there was no resistance to Oseltamivir and Zanamivir were supported by the phenotypic findings which also did not show any evident resistance to the antivirals amongst the Kenyan influenza B viruses during the study period. Surveillance reports worldwide show that antivirals against influenza viruses are not used routinely in the treatment of influenza infections. However, Oseltamivir (and more rarely Zanamivir) therapies are used as a treatment regime in hospitalized patients, and often immunocompromised cases. This has been found to be a contributing factor that can lead to the selection of antiviral-resistant strains (Lackenby et al., 2011) that could then circulate in the community. To date, only four reports have described the isolation of influenza B viruses with reduced susceptibility to NAIs after treatment with either oseltamivir(Ison et al., 2006) or 62 zanamivir(Gubareva et al., 1998). Antiviral surveillance of influenza B viruses collected in mainland China during 2010-2011 identified 0.7%of viral strains tested with reduced susceptibility to NAIs (Burnham et al., 2013). Influenza B viruses with reduced susceptibility to NAIs have been identified in patients before initiation of antiviral therapy and without any known exposure to the drug (Higgins et al., 2012). Therefore the resistance may be attributed to inherent causes present in the virus.Based on the phenotypic and genetic findings of this study, the NA inhibitors were effective against the Influenza B viruses that circulated in Kenya during the 2011-2012. Whereas all the Kenyan influenza B isolates were shown to belong to the Victoria lineage using phenotypic and genotypic analyses of the HA gene segment, phylogenetic analyses using the NA gene segment revealed that this segment was of the Yamagata lineage. This is an interesting observation indicating that the Kenyan viruses were reassortants at least in the NA gene segment. However, this observation was not unique to the Kenyan viruses because the NA of the prototype B/Brisbane/60/2008 virus also indicated that the NA gene segment was derived from a Yamagata lineage. It is therefore reasonable to assume that the influenza B viruses of both Yamagata and Victoria lineages have in the recent past co-circulated in Kenya leading to this reassortment. Reasortment between these two lineages is common when they co-circulate and can compromise the efficacy of an influenza vaccine with a monovalent influenza B component (Tewawong et al., 2015), supporting the recent recommendation by the WHO Strategic Advisory Group of Experts (SAGE) to include a second influenza B component to the seasonal vaccine so that it is quadrivalent with a A(H1N1), A(H3N2), B(Yamagata) and B(Victoria) components (WHO, 63 2013). To be able to evaluate the complete influence of this reassortment, it would be prudent to undertake full genome sequencing of Kenyan influenza B viruses. The evolution rate of the influenza B viruses in Kenya during the study period was 3.66 X 103 (range of1.04 X 10-3 to 6.47 X 10-3) in the HA and 1.18 X 10-3 (range0.682 X 10-3 to 1.71 X 10- 3 ) substitutions/ site/year in the NA. These values are in the same range for influenza B viruses found elsewhere where the rates of evolution were shown to be 3.32 X 10-3 for HA and1.29 X 10-3substitutions/ site/year for NA (Chen and Holmes, 2008b). The Vic/2/87 lineage has been proven to evolve at a much faster rate than the Yam/16/88 lineage possibly because it is more positively selected(Chen and Holmes, 2008b). The rate of evolution in both the NA and HA were within the range of most RNA viruses(Chen and Holmes, 2008b). In terms of selection, the study revealed significant negative selection in the NA at positions 192 and 218. The evolutionary process of influenza B virus has been proven to be caused by both positive and negative selection with negative selection acting on the whole gene and positive selection acting mostly on the HA1 domain(Weber et al., 1997). While positive selection has been shown to play a role in antigenic drift, negative selection reduces the viruses fitness thus this could cause more exposure of the virus to the immune system. The lower evolution rate of influenza B has been hypothesized to be as a result of interactions of genome segments and proteins are less tolerant to mutations (Weber et al., 1997). The study limitations included lack of availability to sequence the whole genome and lack of ability to molecularly characterize more isolates. However, this study was able to characterize influenza B virus at a much deeper level than has been done before in Kenya. 64 CONCLUSION AND RECOMMENDATIONS Conclusions 1. Influenza B viruses that circulated in Kenya between 2011 and 2012 were antigenically similar to B/Brisbane/60/2008, a Victoria/2/87 lineagevirus. 2. Since B/Brisbane/60/2008 virus was a component of the WHO-recommended vaccine for 2011-2012 influenza season for both the Southern and Northern hemispheres, the WHO recommended vaccine was appropriate for use in Kenya during the study period. 3. Kenyan influenza B viruses in circulation during the study period were sensitive to Oseltamivir and zanamivir and therefore these drugs were appropriate for treatment then. 4. The mean rates of evolution of the Kenyan influenza B viruses was 3.66 X 10-3 for HA and 1.18 X 10-3 substitutions/site/year in the NA. Both rates are very similar to those for influenza B reported elsewhere. 5. No positively selected sites were observed amongst the Kenyan influenza B viruses. Recommendations 1. Since one of the isolates had an 1C50 value higher than the maximum required for antiviral resistance to take place, this study underscores the importance of sustained monitoring of drug susceptibility as well as the antigenic characteristics of the influenza viruses as a component of epidemic preparedness due to influenza B viruses. 65 2. Based on the findings of this study, the WHO recommendations for vaccine and antiviral usage for prevention and treatment of influenza, which are based on global data, seem to cover Kenya very well and Kenyan Ministry of health should continue to trust and use these recommendations because they cover the country well. 3. 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Journal of virology, 80, 87878795. 73 Appendices Appendix 1: Isolates sampled for this study Date of Collection Sample IDs Site of Collection 05/01/12 FluB17/12 Port-Reitz District Hospital 06/05/12 FluB18/12 Mbagathi District Hospital 18/08/12 FluB23/12 Kisii District Hospital 10/02/11 FluBI07/11 Isiolo District Hospital 11/05/12 FluB20/12 Mbagathi District Hospital 04/04/11 FluBK05/11 Kericho District Hospital 18/04/11 FluB14/11 New-Nyanza-Provisional Hospital 25/02/11 FluB11/11 Mbagathi District Hospital 13/08/12 FluB22/12 Kisii District Hospital 03/08/12 FluB21/12 Port-Reitz District Hospital 02/02/11 FluBM03/11 Mbagathi District Hospital 03/01/12 FluB16/12 Malindi District Hospital 23/08/12 FluB24/12 Kisii District Hospital 10/02/11 FluBM08/11 Mbagathi District Hospital 04/02/11 FluBM04/11 Mbagathi District Hospital 16/02/11 FluB09/11 Kisii District Hospital 05/02/11 FluB06/11 Mbagathi District Hospital 13/04/11 FluB13/11 Kisii District Hospital 28/02/11 FluB12/11 Mbagathi District Hospital 02/02/11 FluBM03/11 Mbagathi District Hospital 10/05/12 FluB19/12 Mbagathi District Hospital 25/01/11 FluB01/11 Kisii District Hospital 22/02/11 FluB10/11 Mbagathi District Hospital 18/04/11 FluB15/11 Kisii District Hospital 74 Appendix 2: Ethical approval from the scientific steering committee 75 Appendix 3: Ethical approval Walter Reed Army Institute of Research 76 Appendix 4: Ethical approval Ethical Review Committee 77