Class 10 - Warszawski Uniwersytet Medyczny

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Katedra i Zakład Mikrobiologii Lekarskiej
Warszawski Uniwersytet Medyczny
English Division, 6-year programme
Class 10
DNA viruses
I.
Seminar: General properties, pathogenesis and clinial features of selected DNA
viruses from Herpesviridae, Papillomaviridae and Adenoviridae family
II.
Assays to be performed:
1. Continuation of Class 9: Haemagglutination assay and haemagglutinin titration
2. Laboratory diagnosis of infectious mononucleosis
2a. Paul-Bunnel-Davidsohn assay (detection of non-specific heterophile
antibodies)
2b. ELISA assay for the detection of specific humoral response against various
antigens of EBV - interpretation
3. ELISA assay for the detection of specific humoral response against various
antigens of EBV in cases of EBV reactivation - interpretation
4. Identification of adenovirus based on nucleotide sequence comparison using blastn
software
III.
Demonstrations:
1. Laboratory diagnosis of viral meningoencephalitis – application of multiplex-PCR
method.
2. ELISA: detection of antibodies and viral antigen in patient’s serum in suspected
infection with parvovirus B19.
3. Viral CPE: adenovirus, herpes simplex virus
Copyright © 2015/2016 Warszawski Uniwersytet Medyczny/Medical University of Warsaw
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1. Haemagglutination assay and haemagglutinin titration
1. Mix one drop of hen’s egg allantoic fluid containing virus with one drop of 1% RBC on microscopic slide,
along with the controls (positive and negative)
2. Perform hemagglutinin titration:
a. Add 50 µl of PBS (phosphate-buffered saline) to the wells 1-7 and to cell control (CC)
b. Add 50 µl of virus suspension to first well in the row (1:2) and mix with PBS. Add 50 µl of virus to
the virus control well (VC)
c. Transfer 50 µl of diluted virus from first well to the second, mix. Repeat for wells 3-7
d. Discard 50 µl of diluted virus from well 7 (1:128) to the jar with disinfectant
e. Add 50 µl of 1% RBC to all wells (1-7, CC, VC)
f. Mix and incubate for at least 1 hour at room temperature
2. Serological diagnosis in infectious mononucleosis (IM)
Case report (patient 1):
18-year-old male presented to the physician because of sore throat, malaise and high fever. On physical
examination enlarged tonsils and neck lymph nodes, splenomegaly and liver tenderness were noticed. Blood
picture presented mild leukocytosis (12 500/mm3) and appearance of atypical lymphocytes. After initial
recognition of infectious mononucleosis, physician decided to order serological diagnostic tests which should
confirm his diagnosis.
2a. Monospot test (modified Paul-Bunnel-Davidsohn assay) – non-specific serological test
In the course of infectious mononucleosis, increase of the level of heterophile antibodies often appears in the
first week after first manifestations of the infection.
To detect heterophile antibodies, agglutination assays are used. In these assays, stabilized horse erythrocytes
containing heterophile antigens (Paul-Bunnel-Davidsohn assay – PBD, „monospot” test) or latex particles
covered with heterophile antigen are used. Assay is performed with patient’s serum.
To be performed:
On microscope slides check the reaction of standard sera (positive and negative) with erythrocytes. Next,
perform the test with patient’s serum.
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Specific humoral response in IM:
In the course of infectious mononucleosis, first anti-VCA antibodies arise (against viral capsid antigen), a little
bit later – anti-EA (against early antigen), but it’s worth stressing that these antibodies don’t appear in all
patients with IM symptoms. Finally, anti-EBNA antibodies appear in the patients’ sera (against nuclear antigen).
High titer of anti-EBNA antibodies persisting for a long time is characteristic for chronic infections and
lymphoproliferative changes as a result of EBV infection.
Clinical meaning of anti-EBV antibodies
VCA IgM: indicative for acute phase of infection, detectable as a first type of specific antibodies in primary
infection, and also during latent virus reactivation
VCA IgG: indicator of past infection, detectable in high titers for years
EA IgM: like VCA IgM, marker of active infection, they arise in course of primary infection, rarely in
reactivations. Response in EA IgM is much less often than in VCA IgM
EA IgG: characteristic for convalescent phase. Observed in small percent of infected individuals
EBNA IgM: meaning is not fully explained. Probably correspond with establishing of viral latency
EBNA IgG: they appear relatively late after convalescence (3-4 months after infection), detectable for years
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2b. ELISA assay in the diagnosis of infectious mononucleosis – detection of specific anti-EBV antibodies
Detection of specific antibodies against EBV antigens is a good method for infection confirmation. Comparison
of the results of specific serological tests, clinical symptoms, blood tests and non-specific tests (PBD, latex test)
is helpful in making a correct unambiguous diagnosis of infectious mononucleosis (to be performed: ELISA
interpretation)
3. ELISA assay for detection of specific humoral response (IgM, IgG) against EBV antigens in cases of
latent virus reactivation (patients 2 and 3)
Besides diagnostics in suspected infectious mononucleosis, which is the most popular form of EBV infection in
immunocompetent population, specific serological assays can be helpful in determination of humoral response in
patients from risk groups for severe forms of EBV infections. In immunosuppressed patients, individuals with
inherited and acquired immunodeficiencies, and in patients with neoplasms, both primary infections and latent
virus reactivations may lead to a variety of clinical syndromes: limphoproliferative diseases, chronic active EBV
infections, or syndromes resembling mononucleosis, but with more severe symptoms.
Patient 2.
37 year-old man reported to a family physician because of general malaise, physical weakness, loss of apetite,
fatiguability and increased body temperature (38 – 39ºC). Symptoms appeared few days ago. Patient
demonstrated skin paleness, enlargement of cervical and submandibular lymph nodes and hepatomegaly. Blood
examination revealed slightly decreased number of RBCs, thrombocytopaenia and increased number of
leukocytes. Liver aminotransferases and billirubin were at increased levels. Patient was redirected to a hospital
for further diagnosis and treatment. After admission, examination for specific EBV antibodies was performed, as
well as serological testing for CMV and toxoplasma infection. (to be performed: ELISA interpretation)
Patient 3
Woman, 43 y.o., was hospitalised because of acute lymphoblastic leukemia. At day 42 after allogeneic
umbillical cord blood stem cells transplantation, patient developed high fever. Among series of microbiological
examinations, PCR for detection of EBV DNA in whole blood gave positive result. Detection of specific antiEBV antibodies was performed with serum sample obtained at the same day (ELISA plate: sample 3-a).
Introduced treatment (vidarabine, cytostatics and steroids) lead to viraemia negativization after 11 days. Next 10
days later, another examination of anti-EBV antibodies was performed (sample 3-b) (to be performed: ELISA
interpretation)
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TO BE PERFORMED – ELISA tests interpretation:
Pattern of calibrators and tested serum samples in ELISA microplate:
Please, interprete the results:
VCA IgM
Patient 1
AB level
VCA IgG
EA IgM
EA IgG
EBNA-1
EBNA-1
IgM
IgG
78.9
11.7
32.3
26.9
1.0
1.1
32.7
118.7
24.3
2.3
2.3
80.5
43.3
32.0
18.6
0.9
64.1
32.2
2.9
5.3
1.3
0.9
1.0
11.3
Interpretation
Patient 2
AB level
Interpretation
Patient 3
AB level
sample a
Interpretation
Patient 3
AB level
sample b
Interpretation
Analytical interpretation:
Positive results for 10 U/ml and more; negative results for less than 10 U/ml
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4. Virus identification based on nucleotide sequence comparison using BLASTN
software
1. In the class facility computer: using notebook windows program open the file
“virus.fas” (located on desktop) containing sequence of PCR product. Primers
used for PCR were specific for human enteroviruses.
2. In Windows notebook select entire sequence and copy it (ctrl+c).
3. Open web browser window. NCBI BLAST (basic local alignment search tool)
interface should be visible.
4. In “Basic BLAST” section choose “nucleotide blast” program
5. Paste copied sequence to “Enter accesion number, gi or FASTA sequence”
window (ctrl+v)
6. Check if:
a. “nucleotide collection (nr/nt)” database is chosen
b. “highly similar sequence” option is marked
c. “Show results in a new window” box is checked
7. Click “BLAST” button and wait for the results
8. Identify adenovirus type based on its similarity to other sequences in database.
Check complementarity of the most similar database sequence (%/number of
nucleotide mismatches)
Identified adenovirus type: _____________________________________
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Demonstrations:
1. Diagnosis of viral meningoencephalitis – application of multiplex PCR technique
Case report:
A 37-year-old male with temperature of 39ºC, chills, intense headaches, nausea and vomits was transported to
the admission room of a regional hospital.
Physical examination revealed confusion, dyssynergia, balance disorders, slight hemiparesis and tachycardia.
Blood and cerebro-spinal fluid were taken for microscopic, biochemical and microbiological examinations.
Laboratory examination of cerebro-spinal fluid revealed mild pleocytosis, glucose and protein levels normal.
Based on clinical examination and laboratory results, multidirectional microbiological examination was
performed, including virological testing of CSF using multiplex-PCR technique for herpes simplex viruses.
Test procedure:
I.
DNA extraction from the clinical sample (200 µl of cerebro-spinal fuid) using alkaline lysis method
II.
Evaluation of quantity and purity of obtained DNA
III.
DNA amplification
a.
Preparation of reaction mixture and working mixtures from sample and control
b.
Samples/controls placing in the thermocycler, setting the reaction conditions, amplification
IV.
Detection of amplified products
a.
Preparation of agarose gel (with ethidium bromide) in proper electrophoretic buffer
b.
Moving the samples to the gel
c.
Electrophoresis of the PCR products
d.
Visualization in UV light
Picture of photographed gel:
Legend:
100 bp M: product length marker;
100 basepairs
K (-): negative control
HSV 1 TK-, HSV 2 TK-: standard
viral strains with altered TK gene,
resistant to acyclovir
HSV 1 TK+, HSV 2 TK+: standard
viral strains with normal TK gene,
sensitive to acyclovir
CSF: examined cerebro-spinal fluid
Products length :
HSV-1: 469 pz
HSV-2: 390 pz
Thymidine kinase: 1200 pz
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2. ELISA assay in the diagnosis of aplastic crisis bound to the parvovirus B19 infection
Case report:
A mother decided to take her 8-year-old boy to paediatric clinic, because of reported episodes of nose bleeding
in he past couple of days. Patient’s medical history revealed in the past two weeks progressing decrease of
motoric activity, drowsiness, lose of appetite and sustained elevation of body temperature (37,5°C). On physical
examination, skin paleness and tiny haemorrhagic effusions on mucosa of oral cavity and throat were observed.
The results of laboratory blood tests were as follows: significantly decreased number of reticulocytes,
normocytic anaemia, granulocytopaenia with relative lymphocytosis and thrombocytopaenia as well as
prolonged bleeding time, elevated level of Fe2+ in serum and a high level of C-reactive protein
Based on the results of clinical and laboratory examination, decreased haemopoietic ability of the bone marrow
was diagnosed. As one from possible reasons, infection with parvovirus B19 was recognized. Physician
commited serological assays: detection of anti-B19 antibodies in IgM and IgG classes and detection of viral
antigen in patient’s serum.
Course of primary infection with parvovirus B19
*)TAC – transient aplastic crisis
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Class 10 - Warszawski Uniwersytet Medyczny

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