Example of Scientific proposal

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Student:      Mr Ayman Johargy.
Supervisor: Dr Pamella Vallely.
Location:     Department of Virology, 3rd Floor
              Clinical Sciences Building
              Manchester Royal Infirmary.
              The University of Manchester.
              Oxford Road
              M13 9WL


The aims of the study are to:
   1) Determine whether B19 is involved in neurological illness.
   2) Determine what is the pathogenesis mechanism of it.
In order to achieve this aim I will:
   1) Test the presence of parvovirus B19 DNA in CSF samples taken from a large
       cohort of paediatric patients with un-explained neurological illness and
       samples taken from Malawi cohort, using a sensitive PCR assay.
   2) Test CSF and serum samples from all positive patients for specific
       immnunological IgG and IgM antibodies response against a variety of B19
   3) Examine a post morteum tissue for the presence of B19 DNA and antigen by
       in situ hybridisation.
   4) Try to isolate a B19 virus in various cell culture lines.
   5) Sequence the genome of B19 virus from infected patients and compare the
       sequence with referenced sequences of other B19 to detect any viral diversity.
   6) Review data collected from B19 positive cases in association with the clinical
       history and previous laboratory data.

History of Parvovirus:

Parvovirus B19, the only parovirus known to be pathogenic in human, was discovered
in 1974 by Cossart as a contaminant of human serum being screened for hepatitis B.

Classification, structure, and organisation:

The family Parvoviridea are divided into two subfamilies named Parvovirinae and
Densovirinae. Parvovirinae are subdivided into three genera termed: Parvovirus,
Dependovirus, and Erythrovirus. B19 classified as a member of the Erythrovirus
The B19 virion has a simple structure composed of two proteins and a linear, single-
strand DNA molecule. The non-enveloped icosahedra viral particles are 24 nm in
diameter with a molecular weight of 5.6 X 106. The absence of a lipid envelope makes
B19 extremely resistant to physical inactivation.
The genome of B19 contains approximately 5600 bases, encoding one major non-
structural protein (NS1), two capsid proteins (VP1 and VP2), and several small
peptides of unknown function. By multiple alignment of a large number of B19
isolates obtained from the serum of patient with sickle cell disease all reported
isolates intimately clustered and show only 6% divergence among themselves
particularly in VP1 and VP2 regions which show 2 to 3% variability. Recently, a B19
isolate, termed V9, was identified in a French child with transient aplastic anemia, and
on sequence analysis this isolate was seen to be markedly (>11%) different from other
B19 sequences. The prevalence of V9 and its association with clinical disease remains
Pathogenesis and immune response of B19 infection:

The cellular receptor for B19 has been identified as globoside or erythrocyte P
antigen, which is expressed not only on mature erythrocytes and erythroid
progenitors, but also on megakaryocytic, endothelial cells, placenta, and fetal liver
and heart. Individuals who genetically lack P antigen (P phenotype 1 in 200,000
individuals) are resistant to B19 infection.
Replication of the B19 is limited to human erythroid progenitor cells in the bone
marrow and blood, where it is directly cytotoxic to these cells.
The cytopathic effect of infection of erythroid progenitor cells is manifested as giant
pronormoblast (also known as lantern cells).

Both virus specific IgM and IgG antibodies are made following B19 infection. IgM
specific B19 antibodies appear 10-14 days after infection and last for several months
after exposure, while IgG antibodies against B19 infection appear two weeks after
infection and last for life. IgA antibodies to B19 can also be detected and play a role
in protection against infection by nasopharyngeal route.


B19 is a common and widespread infection. Seroprevalence in developed countries is
2-10% in children under 5 years, 40-60% in adults over than 20 years, and 85% or
more in those over 70 years. The main routes of transmission are: respiratory route,
vertical transmission from mother to enfant and transmission via blood products

Diseases associated with B19 infection:

Diseases associated with B19 infection can be classified into two main categories:
-Clinical syndromes commonly associated with B19 infection.
- Clinical syndromes less commonly associated with B19 infection.
Diseases classified among the first category are: erythema infectiosum (EI) also
known as Fifth Disease, transient aplastic crisis in those with an underlying
haemolytic disorder, chronic pure red cell aplasia, non-immune hydrops fetalis, and
arthropathy syndromes.
While, diseases classified among the second category are: skin rashes, haematological
disorders, hepatobiliary diseases, rheumatic disease, myocarditis, and neurological

Neurological diseases:

B19 is not typically considered as a virus causing neurological illness although there
have been isolated case reports describing neurological symptoms in patients during
or following EI. A recent retrospective study in this laboratory done by Barah et al
suggests that the virus is present in the CSF of almost 5% of undiagnosed paediatric
encephalitis/ meningitis cases. The association has not been proved to be casual
although in some cases it was possible to identify IgM antibody in the CSF suggesting
the viral infection was current or recent at the time of illness.

The aim of this study will be to determine whether B19 is a causative agent of
neurological illness. The data collected will be correlated with clinical symptoms and
patient outcome. If this virus is established as a cause of neurological infection,
appropriate diagnosis and clinical management regimens can be developed.


1) Subject of study:

CSF samples will be taken from:
   1) Paediatric patients presenting with neurological symptoms. Samples will be
        selected from those sent to Clinical Virology Laboratory at Manchester Royal
        Infirmary for testing for other viral infections (typically HSV, VZV,
   enteroviruses), from paediatric (>18 years) patients with neurological

   A true control sample group is not easy to obtain as CSF samples are rarely
   taken from healthy children. However, the laboratory also receives samples
   taken from children with acute lymphoblastic leukaemia who do not
   necessarily have neurological symptoms and these will be tested alongside the
   neurological samples.

2) Samples taken from Malawi patients where a group of individuals suffering
   from neurological diseases of unknown causative agents.

2) Detection of B19 DNA by nested PCR:

DNA will be extracted from CSF and will be uses as a template in nested PCRs
for B19 NS1 and VP1 gene using the primers shown in table 1.
For NS1 gene, first (40 cycles) and second (40 cycles) rounds consist of initial
denaturation of 95 C for 6 min, 95 C for 30 s, 55 C for 30 s, and 72 C for 30 s.
For VP1 gene, first (40 cycles) and second (35 cycles) rounds consist of 95 C for
6 min, 95 C for 60 s, 55 C for 60 s, and 72 C for 60 s.
Amplifications will be carried out using the GeneAmp PCR 9600 thermal cycler
(Perkin Elmer Cetus, Norwalk, CT, USA) in 50 μl PCR mix containing 10 mmol
tris-hydrochloride (Ph 8.3), 50 mmol potassium chloride, 1.5 mmol magnisum
chloride, 0.01% (weight/volume) gelatin, 200 μmol each deoxyribonucleotide
triphosphate, 0.5 μmol each primer, and 1.25 U Amplitaq polymerase (Perkin
Elmer Cetus, Norwalk, CT, USA). As positive controls, B19 viraemic serum at
dilutions of 10-3 and 10-6 will be used throughout. Sterile distilled water will be
used as a negative control.
Sensitivity of both nested PCRs will be established with ten-fold serial dilution of
plasmid pGEM-1 containing the complete B19 coding region (from J P Clewley,
Public Health Laboratory Service, Colindale, London).
3) IgG and IgM antibodies Testing in CSF and serum samples:

Anti-B19 IgG and IgM antibodies will be tested in CSF and serum samples by
ELISA and western blot analysis (Mikrogene, Martinsried, Germany).

4) Inoculation CSF samples onto various cell lines:

All CSF samples will be examined microscopically and will be inoculated onto
various cell monolayers for virus culture.

5) Sequencing B19 genome form positive cases:

B19 genome from positive cases will be sequenced and compared with other
reference sequences to detect any viral diversity.
Table 1: Sequence and position of primers, which will be used for in-house DNA
Primer name               Primer sequence               Nucleotide    Product
   (gene)                                               numbering       size
  P1 (NS1)     5’-AATACACTGTGGTTTTATGGGCCG-3’           1399-1422
  P6 (NS1)     5’-CCATTGCTGGTTATAACCACAGGT-3’           1659-1682       284
  P2 (NS1)     5’-GAAAACTTTCCATTTAATGATGTAG-3’          1498-1525
  P5 (NS1)     5’-CTAAAATGGCTTTTGCAGCTTCTAC-3’          1576-1600       103
Prm-4a (VP1)     5’-AACGCCTCAGAAAAATACCC-3’             3101-3120
Prm-4b (VP1)    5’-TAAGTGCTGAAACTCTAAAGG-3’             3425-3445       345
B19-1 (VP1)      5’-CAAAAGCATGTGGAGTGAGG-3’             3187-3206
B19-2 (VP1)      5’-ACCTTATAATGGTGCTCTGGG-3’            3271-3290       104

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