Download MolecularCharacterization of theSurface Glycoproteins of Influenza B

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.
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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
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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
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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
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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
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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 25l of Phosphate Buffered Solution added to
all wells followed by 25l of the isolate in the column one well and mixed. Serial two-fold
dilutions of the isolates were carried out by transferring 25l of the mixture well to well. Another
25l of PBS was added to all wells. 50l 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. 25l PBS was added to all wells, and then 25l 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 25l from well 1 to well 11. 25l 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. 50l 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. Since reassortment was observed in the Kenyan influenza B viruses within the NA
gene segment, full 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.
66
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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