Acute respiratory tract infections

Health and economic impactHuman respiratory tract is prone to infection by different pathogens such as viruses, bacteria, and fungi.

Lower ARTIs: First cause of death in low-income countries.

Health impact:

US$ 600 million annually in 42 low-income countries WHO, 2011.

Fourth cause of death in middle-income countries.Fifth cause of death in high-income countries.Deaths (3.9 millions annually)

WHO, 2008

Hospitalization (HRSV responsible for 100,000 cases annually)

Deaths, hospitalizations, medications, and vaccination. Temporal loss of activity, sleepless nights, work / school absence.

Economic impact:

Viruses are considered the major cause of ARTIs.Viruses are responsible for one-third of the deaths caused by ARTIs in the developing countries. More than 200 viruses belonging to different families are incriminated as possible causes of ARTIs.

RhinovirusesCoronavirusesAdenoviruses

Human Respiratory Syncytial VirusInfluenzaPara-Influenza Viruses

URTI LRTI

Acute respiratory tract infections

Impact of viruses

HRSV is the primary cause of lower ARTIs in children.

HRSV is responsible for:64 million cases; 100,000 hospitalization; up to 1 million deaths every year worldwide

HRSV infects 50% of children during the first year of life. All children become infected by the end of second year.

HRSV is highly contagious, nosocomial pathogen, spread by close contact with infectious secretions.

Two major strain groups of HRSV, designated A and B, were found to circulate concurrently during epidemics.

Human Respiratory Syncytial Virus

HRSV

HRSV Classification

Fusion (F) protein

Attachment )G (protein

Small hydrophobic

)SH (protein-ss RNA

M2-1 & M2-2

Phosphoprotein

Polymerase

Matrix protein 3´ 5´

NS1 and NS2

HRSV Structure

HRSV in Saudi Arabia

Detection and typing of HRSV in clinical samples collected from KKUH, Riyadh during two winters (2007/08 and 2008/09).

Preparation and evaluation of polyclonal antibodies to HRSV for the development of in-house diagnostic kits.Sequence and phylogenetic analysis of the most variable and immunogenic protein genes (G and F) of HRSV Saudi strains.

Isolation and characterization of HRSV Saudi strains.

Aim of the Study

Nasopharyngeal Aspirate samples

HRSV detection(RT-PCR)

Positive Samples

Sequence analysis

HRSV Isolation(Cell

culture)

Typing(RT-PCR)

HRSV Characterization (RT-PCR, IFA, VNT

and ELISA).

Production of Hyper-immune

serum

HRSV Concentration(PEG-6000) and

titration

Phylogenetic

analysis.Part one

Part four

Part two

Part three

Part 1

Detection and

Typing of HRSV in

Clinical Samples

100 NPAs were collected from hospitalized children at KKUH during two winters

Detection and Typing of HRSV in Clinical Samples

Viral RNA was extracted from NPAs and used for detection and typing of HRSV by one-step RT-PCR assays.

Sex Age

HRSV Incidence and Risk Factors

The two types of HRSV were found to circulate concurrently with slight predominance of type A (16%) more than type B (10%).

Infants younger than 6 months of age (31.1%) are highly infected than any other age group.

Males (31.1%) are more prone to HRSV infection thanfemales (17.9%).

26 out of 100 NAPs were HRSV-positive (26%).

Conclusion (part 1)

Part 2Isolation and

Characterization of HRSV Saudi strains ?

Three passages (7-days each) were applied for each positive sample.

No CPE was developed on VERO cells (All samples).

Two types of permissive cell lines were used (VERO, HEp2).

Two samples (Code 38 and 91 ) developed HRSV characteristic CPE (Syncytia) on HEp2 cells – Both are type A viruses.

The CPE produced by the isolate 38 is more rapid than that of isolate 91.

HRSV Isolation

A B

C D

Progress of CPE on HEp2 cells (10X):

The recovered isolates were titrated using infectivity titration assay:

Isolate 38 = 108.5

TCID50/ml Isolate 91= 105.5

TCID50/ml

The recovered isolates were concentrated using PEG-6000 and were kept at -80oC for further use.

HRSV Titration and Concentration

Characterization of HRSV Saudi isolates

Two Saudi HRSV type A strains were isolated using cell culture for the first time in Saudi Arabia.The two isolates varied in their behavior on cell culture.

Both isolates were able to produce a significant titer of infective virus particles.

HRSV Saudi isolates share the common antigenic and genetic characteristics of international HRSV strains as confirmed by: RT-PCR, ELIZA, IFA and VNT.

Conclusion (part 2)

Part 3Production and Evaluation

of polyclonal antibodies

in Rats against Saudi HRSV strains

?

1 2 3 4Hyper immune serum Preparation

Chart

PEG-Concentrated antigens

(virus+Hep-2)

Preparation of antigen

and adjuvant mixture

Rat injection (3 Doses)

2 weeks each

Rat Bleeding and serum collection

5Evaluation of Serum

6Serum will be processed for diagnostic

uses.

Part 4Sequence and Phylogenetic

Analysis of G and F genes

?

Why G and F genes?

G is the most variable gene among HRSV strains.

HRSV is classified into two distinct groups based on the reactivity of the G protein with monoclonal antibodies.

Due to its highly conserved nature, the fusion protein induces protective immunity against different HRSV strains.

The molecular changes of G and F genes affect the functional and antigenic stability of HRSV.

Cytoplasmic tail (37 aa)Hydrophobic

domain

NH2COOH

Strain specific epitopes

Conserved epitopes

Group specific epitopes

Structure of G protein

Structure of the F protein

SــــــS69ــــــ212

Antigenic Site IAntigenic Site IV, V, VI

Antigenic Site II

F1F2

Cleavage site I Cleavage site II

1 2 3 4Sequence and Phylogenetic

Analysis Chart

Selection of strain

candidatesSequencing Strategy and

primer design

Amplification of fragments cover the G

gene(RT-PCR)

Sequencing of amplified fragments(Gene Art, Germany)

5Sequence

editing and assembly(Bioedit)

6Retrieval of complete gene sequence and

GenBank submission

7Sequence Analysis(MegAlign)

Sequence and Phylogenetic Analysis Chart

Premature stop codon was formed in Riyadh strain 91/2009.

Replacement of serine by threonine or phenylalanine was reported to abrogate antibody binding. Amino acids at positions 269 and 270 were changed from serine to threonine and phenylalanine, respectively (Thr269Ser and Phe270Ser) .

Replacement of a hydrophilic amino acid by a hydrophobic one was reported to abrogate antibody binding. Amino acids at positions 106 and 133 were changed from hydrophilic to hydrophobic (Gly106Glu and Ile133Thr).

No changes were recorded in the conserved region.

COOHNH2

The effect of aa changes on the antigenicity of G protein

The effect of glycosylation on the antigenicity of G protein

1 2 3 4Sequence and Phylogenetic

Analysis Chart

Selection of strain

candidatesSequencing Strategy and

primer design

Amplification of fragments cover the G

gene(RT-PCR)

Sequencing of amplified fragments(Gene Art, Germany)

5Sequence

editing and assembly(Bioedit)

6Retrieval of complete gene sequence and

GenBank submission

7Sequence Analysis(MegAlign)

8Phylogenetic analysis

(MegAlign)

Phylogenetic Analysis

Mutation rate in G gene of HRSV type A strains is higher than type B strains. This may explain the predominance of type A over type B.

Conclusion (part 4)

In G protein, -FOUR amino acid changes were recorded to have a possible impact on epitope integrity.- SIX amino acid changes, FOUR in type A and

TWO in type B were predicted as highly potential sites for O-glycosylation.

- TWO amino acid changes in type B were predicted as highly potential sites for N-glycosylation.

American and Argentinian strains appear to be the most relevant to Saudi HRSV type B strains.

Asian strains are the most relevant to Saudi HRSV type A strains. They may be originated from the same ancestor.

A set of objective and comprehensive studies is required for better understanding of HRSV circulation in Saudi Arabia.HRSV Saudi isolates, antigens and antibodies produced in this study will be fundamental for:

Recommendations

Sequence analysis of more HRSV strains on temporal and spatial basis will be helpful for understanding HRSV circulation pattern in Saudi Arabia. Complete genome sequencing of Saudi HRSV strains will be helpful to determine their ancestral origin and evolutionary pathway.

- Further basic and applied studies on HRSV.- Development of improved diagnostic tools.

- Vaccine production.

Milestone Achievements

Isolation, characterization and sequence analysis of two HRSV Saudi isolates.

Eight genes were deposited at GenBank Database.

“Polyclonal Antibodies against Saudi strains of Human Respiratory Syncytial Viruses”. A Patent was submitted to (Intellectual Property and

Technology Licensing Program, KSU, KSA)

Two papers in preparation for submission.