- Open Access
Measles viral load may reflect SSPE disease progression
© Simmonds M et al; licensee BioMed Central Ltd. 2006
- Received: 14 January 2006
- Accepted: 21 June 2006
- Published: 21 June 2006
Subacute sclerosing panencephalitis (SSPE) is a rare, slowly progressive neurological disorder caused by the persistent infection with measles virus (MV). Despite much research into SSPE, its pathology remains obscure. We examined autopsy tissues of eight SSPE patients by real time quantitative PCR, immunohistochemistry and immunoblotting to determine viral load. MV N, M and H gene RNA could be detected in the central nervous system (CNS) of all patients and in two non-CNS tissues of one patient. The viral burden between patients differed up to four-fold by quantitative PCR and corresponded with detection of MV protein. The level of both viral RNA and antigen in the brain may correlate with disease progression.
- Viral Load
- Measle Virus
- Central Nervous System Tissue
- Subacute Sclerosing Panencephalitis
- Viral Burden
Subacute sclerosing panencephalitis (SSPE) is a rare, slowly progressive neurological disorder of children and young adults, caused by the persistent infection with measles virus (MV). After acute infection with MV, typically at a very young age, the patients appear to recover normally. Following a period of asymptomatic persistence, usually 3 to 10 years, neural dysfunctions develop and progress over months to years, resulting eventually in coma and death. Although much effort has gone into researching SSPE, its pathology remains largely obscure. The progression of SSPE greatly differs in individual patients . What causes a rapid deterioration within months or slow progression over several years (4+) remains an unresolved question.
Viral load has been correlated to disease development, progression and outcome in several infections, including HIV, SIV, cytomegalovirus [2, 3] and Epstein-Barr virus . Occurrence of cognitive impairment in HIV infected patients and development of SIV encephalitis in macaques was linked to the level of viral RNA in the cerebrospinal fluid (CSF). In contrast, CNS disease did not correlate with plasma viral load in either HIV or SIV [5–7]. No studies have been carried out so far trying to relate the progression of SSPE with viral load. Due to the rarity of the condition and the invasiveness of removing biopsy samples, obtaining information on viral load during the course of disease is problematic. However, the use of autopsy samples may provide some insight into this relationship.
As MV can usually be detected in brain tissue from biopsy or autopsy by RT-PCR and/or immunohistochemistry, it is thought to persist and spread within the CNS. Although the brain appears to be the main location of persistent MV in SSPE, it is not entirely clear if this is also the site of persistence and the only target during disease. Viral antigen or RNA have been detected outside the CNS in SSPE cases [8–10]. To address the question of MV persistence outside the CNS in SSPE, we included available peripheral tissues of two SSPE patients in our analysis.
This study was carried out to determine whether differences in viral load caused diversity of disease progression. Autopsy tissues of brain and non-brain origin of eight confirmed cases of SSPE were analysed by quantitative PCR to determine amounts of the MV RNAs encoding the nucleoprotein (N), matrix protein (M) and haemagglutinin (H) and by immunological techniques to detect the N protein.
Patients and samples
Age at disease onset
Duration of persistence (acute MV to SSPE onset)
Duration of disease (SSPE onset to death)
Detection of MV RNA
MV N RNA positive samples were also used for detection of MV M and H gene RNA. The H gene PCR lead to very similar results as the N gene PCR, both in copy numbers and %GAPDH. Occipital brain tissue of UK111 contained just under 2 × 106 copies of H RNA per ng total RNA. Again the weakest positive CNS sample was temporal brain of UK585 with just under 2 × 103 copies/ng RNA. This is approximately 9 × 103 %GAPDH and 3% GAPDH, respectively. The M gene signal was approximately ten-fold weaker, but the different cases were similar in relation to each other as with the N and H genes. Occipital brain of UK111 contained 4 × 105 copies M gene/ng RNA or 3 × 102 %GAPDH. This gene could not be detected in temporal brain tissue of patient UK585. In PCRs with MV N, M and H gene targets, occipital brain of UK111 was the strongest positive when looking at copies/ng RNA, while the brain of UK85 contained most MV RNA when expressed as a function of GAPDH. The efficiency of all three PCR reactions was found to be similar when titrations of p(+)MV, a MV genome containing plasmid, were used as template (data not shown), which indicates that the numbers obtained with the different PCRs can be compared. The M and H gene results confirm the relative findings with the N gene. All quantities are illustrated in Figure 1.
Detection of MV protein
Disease progression, viral burden and MV M gene expression
To investigate a possible link between virus integrity and viral spread, MV loads detected here were compared with the extent of MV M gene and protein mutation reported previously . In cases UK111 and UK85 the MV M proteins contained few substitutions and a high virus load of 6 × 103 and 1 × 105 %GAPDH, respectively, was detected. The M gene of virus in case UK585, on the other hand, was strongly mutated and low virus levels of <1% GAPDH were measured. In case UK83, however, viral load was high (1 × 103 %GAPDH), despite presence of virus with a highly mutated M protein.
By Spearman's ranked correlation, no statistically significant links were found between viral load, duration of disease or infection and number of total or expressed mutations in the M gene of the isolated viruses.
Great variation in levels of MV RNA and protein were detected in different SSPE patients, while findings with quantitative PCR and immunological methods are in agreement. In the occipital brain of UK111, 2 × 106 copies of MV N RNA could be detected per ng total RNA, approximately 100 times more than in the brain samples of UK98. The brain of UK111 was the only analysed tissue positive by immunohistology and was a much stronger positive by immunoblotting than specimens from UK98. In the brain of UK585, which was negative by immunoblotting, only 4 × 102 to 2 × 103 copies could be detected. Thymus and appendix of UK98 were the only non-CNS samples positive for the N gene by real time PCR. GAPDH normalisation of these samples may be less reliable due to greater heterogeneity of tissue origin. However, taking into account exclusively the copy numbers per total RNA, they were weak positives, contained fewer than 102 copies per ng RNA.
A weak correlation between the amount of MV RNA and the duration of SSPE was observed. As some CNS samples contained relatively little MV RNA, high viral load alone did not seem to be responsible for death, but may have accelerated destruction of tissue. MV specific interferon-gamma release by host immune cells may determine disease progression . It is feasible that release of high levels of interferon restricts viral growth and so reduces viral load and the speed of disease progression. Additionally, treatment with interferon or other antiviral agents during the course of SSPE may influence viral replication. Unfortunately, treatment history of the patients was not known and could therefore not be taken into account.
As the MV genome is encoded in RNA, PCR detection does not distinguish between genomic and mRNA. It is therefore not known if the identified virus is actively transcribed, but presence of MV protein indicates that the virus is transcribed. However, it is not clear whether a larger number of MV RNA copies indicate a greater number of infected cells or greater number of MV RNA copies per infected cell. Increased MV RNA levels per cell may indicate a more actively replicating virus, which may causes faster progressing disease. Alternatively, infection of a greater number of cells may lead to destruction of more tissue, which may also lead to accelerated disease.
Of patient UK98, tissues from five brain areas, frontal, parietal, occipital, temporal lobes and cerebellum, were available for analysis. Virus levels in the cerebrum – frontal, parietal, occipital and temporal lobes – were within a factor of 3.5, which may indicate that the virus did not favour any one particular area. Alternatively this may be an indication of very slow disease progression, as is the case in this patient, during which the virus had sufficient time to spread into all regions. In contrast, infection of the cerebellum was 30 to 100 times lower. The cerebellum is a very extensively connected part of the brain, which integrates information from the cerebrum as well as the periphery. Both efferent and afferent pathways link it to the cerebrum. Lack of axonal connection does therefore not cause this lower level of infection. It may rather be the different cell types present within this organ. The cerebellar neurons, the granule cells, may be less susceptible to MV infection.
The detection of MV RNA in non-CNS tissues was very low, which indicated that these tissues were not reservoirs of infection. The detected RNA may have derived from infiltrating lymphocytes that were infected within the CNS. Peripheral lymphocytes of individuals with SSPE have been shown to contain MV . Alternatively, the source of MV sequences may have been contamination during autopsy. No information was available on the exact autopsy procedures, contamination could therefore not be ruled out.
In summary, the amount of MV protein and RNA differs greatly in different cases of SSPE, which may influence progression of the disease. Further studies of greater numbers of cases are required to confirm these findings.
Real time PCR
Primer sequences used for the amplification of MV and housekeeping genes with amplicon length.
Gene (GenBank accession number)
Primer sequence 5'-3'
Expected amplicon length
CAA GAC CCT GAG GGA TTC AA
GTT CCT CAC CAC ATC CAA CC
TGT TAC CCG CTG ATG GGT AT
TTC TGG CTG TCA TTG TGA GG (M3'-1R, Jin 2002)
ATC AGG GAT CAG GGA AAG GT
CAT TCG CAA CTT GTC ATC TG (Mh4, Jin 1998)
GAG TCA ACG GAT TTG GTC GT
TTG ATT TTG GAG GGA TCT CG
GGA CTT CGA GCA AGA GAT GG
AGC ACT GTG TTG GGC GTA CAG
18S rRNA (X03205)
CAT GGC CGT TCT TAG TTG GT
CGC TGA GCC AGT CAG TGT AG
Expression of housekeeping genes in tissues of SSPE patients.
(104 copy eqivalents/5 ng RNA)
(104 copy eqivalents/5 ng RNA)
(104 copy eqivalents/5 ng RNA)
Real time PCR controls
DNA and RNA standards were used for qPCR and RT-qPCR, respectively. The concentration of a plasmid containing the entire MV genome, p(+)MV (kindly provided by M. Billeter, University of Zürich), was determined by spectrophotometry. A ten-fold dilution series was made and used as DNA standard. From this plasmid, the whole N, M and H genes were amplified by PCR and cloned into pGEM (Promega, UK). The pGEM plasmid was linearised with SpeI and the MV fragments transcribed in vitro using the T7 Riboprobe System (Promega, UK) according to the manufacturer's instructions. The resulting RNA was purified and the concentration determined by spectrophotometry. Ten-fold dilution series were made and included in the RT reactions as RNA standards.
In order to compare results from different tissues and correct for use of different numbers of cells in each sample, two normalisation methods were employed. First, the same concentration of total isolated RNA was used for each RT-PCR. This lead to results expressed as copies of MV RNA per ng total RNA input. Alternatively, a housekeeping gene was employed to compare RNA levels between different samples. To find an appropriate gene to normalise sample input, 5 ng RNA from nine samples were used for qPCR with primers against all three housekeeping genes, beta-actin, GAPDH and 18S rRNA. Results are shown in Table 3. Both beta-actin and GAPDH were found to be suitable for normalisation, GAPDH was used in subsequent experiments. Results are expressed as units MV RNA/units GAPDH mRNA × 100 = amount MV RNA in %GAPDH.
Formalin fixed, paraffin embedded tissues were analysed by immunohistochemistry. The method used by McQuaid et al.  was modified as follows. Formalin fixed, paraffin embedded 7 μm thick tissue sections on silane-coated slides were hydrated in xylene and alcohol. Endogenous peroxidase activity was blocked by 10 min incubation in 3% H2O2 in methanol. Tissues were washed in water and blocked in blocking solution (1:20 dilution of normal rabbit serum in PBS-T (0.1% Tween-20 in PBS)) for 10 min. Sections were incubated with a monoclonal mouse antibodies against the N protein (clone 7C11, ) at a dilution of 1:2000 in blocking solution overnight at 4°C, washed in PBS-T and incubated with a biotin conjugated secondary antibody (Dako, UK) at 1:400 in blocking solution for 1 hr. After further washing, streptavidin coupled HRP (Dako, UK) was added for 30 min, tissues washed and incubated with DAB, followed by haematoxylin counterstaining.
Tissues were homogenised in lysis buffer (50 mM Tris, 150 mM NaCl, 2 mM EDTA, 2 mM EGTA, 1% Triton X-100 and added protease inhibitors) and protein concentrations of whole lysates, without removal of cell debris, determined by spectrophotometry. Equal amounts of total protein of each sample were analysed by SDS-PAGE and immunoblotting. The 7C11 MV N specific antibody was used to probe the blots, followed by detection with an anti-mouse-HRP antibody (Dako, UK) and ECL plus reagents (Amersham Bioscience, UK).
We would like to thank Dr Dhan Samuel for helpful discussion on this work. This study was supported by a grant from the Health Protection Agency.
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