Uwe G. Liebert

Sequence divergence of measles virus haemagglutinin during natural evolution and adaptation to cell culture

Phylogenetic analysis of the sequence of the H gene of 75 measles virus (MV) strains (32 published and 43 new sequences) was carried out. The lineage groups described from comparison of the nucleotide sequences encoding the C-terminal regions of the N protein of MV were the same as those derived from the H gene sequences in almost all cases. The databases document a number of distinct genotype switches that have occurred in Madrid (Spain). Well-documented is the complete replacement of lineage group C2, the common European genotype at that time, with that of group D3 around the autumn of 1993. No further isolations of group C2 took place in Madrid after this time. The rate of mutation of the H gene sequences of MV genotype D3 circulating in Madrid from 1993 to 1996 was very low (5 x 10(-4) per annum for a given nucleotide position). This is an order of magnitude lower than the rates of mutation observed in the HN genes of human influenza A viruses. The ratio of expressed over silent mutations indicated that the divergence was not driven by immune selection in this gene. Variations in amino acid 117 of the H protein (F or L) may be related to the ability of some strains to haemagglutinate only in the presence of salt. Adaptation of MV to different primate cell types was associated with very small numbers of mutations in the H gene. The changes could not be predicted when virus previously grown in human B cell lines was adapted to monkey Vero cells. In contrast, rodent brain-adapted viruses displayed a lot of amino acid sequence variation from normal MV strains. There was no convincing evidence for recombination between MV genotypes.

Restricted expression of measles virus proteins in brains from cases of subacute sclerosing panencephalitis

The presence of five structural proteins of measles virus in brain material obtained at autopsy from four patients with subacute sclerosing panencephalitis (SSPE) was examined by immunofluorescence employing monoclonal antibodies. In addition, the humoral immune response against measles virus antigens in serum and cerebrospinal fluid was analysed by immunoprecipitation in combination with gel electrophoresis, revealing a reduced response mainly to the matrix (M) protein. In none of the brain material were all five structural proteins simultaneously detected. Nucleocapsid protein and phosphoprotein were found in every diseased brain area, whereas haemagglutinin (H) protein was detected in two, fusion (F) protein in three and M protein only in one SSPE case. In two cases, variations in the occurrence of H and F proteins could be observed between regions displaying different degrees of neuropathological changes. No correlation was observed between the humoral immune response and the immunohistological findings. These data support the hypothesis of a restricted synthesis of measles virus proteins, in particular the envelope and M proteins, in SSPE.

Virological aspects of measles virus-induced encephalomyelitis in Lewis and BN rats

Lewis and Brown Norway (BN) rats which are susceptible or resistant to autoimmune reactions against brain antigen, respectively, were inoculated intracerebrally with a neurotropic measles virus. Suckling rats died from a rapidly fatal acute encephalopathy (AE). With increasing age Lewis rats developed a subacute measles encephalomyelitis (SAME) whereas BN rats showed a clinically silent encephalitis (CSE). Infectious virus could occasionally be recovered from SAME animals using cocultivation techniques but not from BN rats with CSE. With monoclonal antibodies against measles virus, viral proteins were localized in brain tissue. Nucleocapsid and phosphoprotein were detected in infected brain cells of all animals with AE, SAME and CSE, whereas measles virus haemagglutinin, fusion and matrix proteins were either reduced or absent, suggesting a restricted synthesis of measles virus envelope proteins. These data suggest that the different diseases of the two rat strains are related to the immunogenetic background rather than to the replication of measles virus in the central nervous system. This animal model provides the opportunity to investigate further the events occurring during establishment of measles virus persistence in the brain, and the genetic control of associated immunological and immunopathological reactions.

Induction of autoimmune reactions to myelin basic protein in measles virus encephalitis in Lewis rats.

Abstract Intracerebral inoculation of weanling Lewis rats with measles virus led to the development of subacute measles encephalomyelitis (SAME) 4–8 weeks after infection. The disease is characterized pathologically by an intense inflammatory infiltration within both the white and grey matter of the central nervous system (CNS) without apparent demyelination. Both during and after SAME splenic lymphocytes from these animals could be restimulated in vitro to proliferate in the presence of myelin basic protein (MBP). MBP-specific class II MHC-restricted T cell lines were isolated from this cell population. They were shown to exhibit no cross-reactivity with measles virus and to induce experimental allergic encephalitis (EAE) in naive syngeneic recipients following adoptive transfer. The clinical and histopathological signs of this T cell-mediated disease were identical to that seen in classical T cell-mediated EAE. A humoral immune response to MBP was only detected in a limited number of those rats with SAME. These results indicate that autoimmune reactions to brain antigen can arise during measles virus infection which may contribute to the pathogenesis of measles virus-associated encephalomyelitis.

Restriction of measles virus gene expression in acute and subacute encephalitis of Lewis rats

Abstract Measles virus (MV) replication in brain tissue of Lewis rats with acute (AE) and subacute (SAME) encephalitis was characterized by biochemical techniques. Messenger RNAs specific for measles virus nucleocapsid (N), phospho (P)-, matrix (M), fusion (F), and haemagglutinin (H) protein were detected in all brain extracts examined. The quantity of the individual MV mRNA species was quite different in comparison to lytically infected Vero cells. A steep gradient of MV transcripts was found in brain tissue which is most likely due to strongly attenuated transcription of mRNAs along the viral genome, representing particularly low transcription of the glycoprotein genes. In addition, in vitro translation assays only revealed synthesis of N and P protein in consistent fashion. The mRNAs for the glycoproteins did not direct the synthesis of detectable viral proteins whereas the M mRNA revealed some activity in animals with AE. The data indicate a strong restriction of the MV envelope gene expression in infected brain tissue, which is independent of the incubation time and type of the central nervous system (CNS) disease. This phenomenon which is similar to the findings observed in measles inclusion body encephalitis and subacute sclerosing panencephalitis suggest that host factors may initially be responsible for the initiation of transcriptional and translational alterations.

Measles Virus Infections of the Central Nervous System

Acute measles is a classic infectious disease of childhood with worldwide distribution. Its causative agent, measles virus (MV), is an efficient pathogen, persisting in nature in populations large enough to support it, even though it is able to cause an acute infection in any individual only once in his lifetime. The characteristic clinical hallmarks of measles, fever and rash, coincide with antiviral immune response. MV-specific T lymphocyte and antibody responses contribute to virus clearance and protection from reinfection, respectively. Concomitant with immune activation immunologic abnormalities arise during MV infection. The ensuing suppression of cellular immune responses is presumably responsible for increased susceptibility to other infections. Additionally, central nervous system (CNS) complications of MV infection with different pathogenesis occur. Autoimmune disease may appear in the form of acute measles encephalomyelitis. Furthermore, MV may persist in the CNS in rare cases without periodic shedding of infectious virus. Measles inclusion body encephalitis can develop on the basis of inadequate virus-specific cell-mediated immune responses and subacute sclerosing panencephalitis occurs many years after primary measles as a slow virus infection. Host cell factors operating in cells of the CNS together with mutations particularly in genes coding for viral envelope proteins initiate and maintain the persistent state of infection with a viral replication cycle that is attenuated at the transcriptional and translational level.

P2X7 receptor-mRNA and -protein in the mouse retina; changes during retinal degeneration in BALBCrds mice

A combined real-time PCR/immunohistochemistry study was carried out to investigate whether P2X(7) receptors, known to induce apoptosis and necrosis, may be causally related to the process of retinal degeneration in BALBCrds mice. In the retinae of BALBCrds mice, P2X(7) receptor-mRNA was the highest at an age of 20-40 days, and declined afterwards. At the same time, the P2X(7) receptor-message was constantly low in the retina of control BALBC mice until postnatal day 100. The receptor-mRNA in total brain tissue of both strains of mice was comparable with that of BALBCrds retinae. Double immunofluorescence in combination with laser scanning microscopy was used to study the distribution of P2X(7) receptor-immunoreactivity (IR) on neurons and different glial cell types of the retina. An exclusively neuronal localization of P2X(7)-IR in the ganglion cell layer was found by using either anti-neuronal nuclei or microtubule associated protein-2 as neuronal markers. There was a slight age-dependent decrease in the abundance of neuronal P2X(7)-IR both in BALBCrds or BALBC mice. P2X(7)-IR failed to co-localize with any of the non-neuronal markers used to stain microglial or Müller glial cells. No P2X(7) receptor-IR was found in the retinal ganglion cell layer of P2X(7)(-/-) animals, when compared with the control littermates. Hence, we suggest that, in BALBCrds mice, an early up-regulation of neuronal P2X(7) receptors may cause injury of retinal neurons and thereby functionally contribute to the retinal damage.

Efficacy of individual measles virus structural proteins in the protection of rats from measles encephalitis.

Lewis rats were immunized with recombinant vaccinia virus (VV) expressing the nucleocapsid (N), phospho (P), matrix (M), fusion (F), and haemagglutinin (H) proteins of measles virus (MV). Animals developed humoral as well as cell-mediated immune (CMI) responses to the corresponding MV proteins. Rats immunized with recombinants VVN, VVF or VVH survived a MV challenge infection whereas VVP- and VVM-immunized rats were only partially protected. In vivo depletion of CD8+ T lymphocytes did not prevent the protective effect of the N, F or H protein-specific CMI response in rats. VVH and VVF immunization induced neutralizing antibodies, but no such antibodies were detected after VVN immunization. Further investigation of the temporal occurrence of the antiviral antibodies indicated that the observed protection provided by VVN and VVF immunization depends on CD4+ N- or F-specific T cells in the absence of neutralizing antibodies and CD8+ T cells. A role for neutralizing antibodies induced by VVH cannot be ruled out.

Restricted expression of measles virus in primary rat astroglial cells

Persistent infection of the central nervous system (CNS) with measles virus (MV) is associated with characteristic restrictions of viral envelope gene expression as documented in subacute sclerosing panencephalitis (SSPE), measles inclusion body encephalitis (MIBE), or subacute measles encephalitis (SAME) in rats. To determine whether these restrictions are the result of a long lasting virus-host cell interaction or primarily based on intrinsic brain cell factors MV gene expression was analyzed in primary rat astroglial cultures. It could be shown that MV infection of these cells led to a defective replication cycle with a reduced synthesis of viral envelope proteins and a steep expression gradient of the monocistronic viral mRNAs similar to the findings in brain tissue of SSPE, MIBE, and SAME. This restriction of MV gene expression has not been observed in cells of nonneural origin. We suggest that this cell-type specific regulation of MV gene expression contributes to early events in the establishment of MV persistent infection in CNS tissue.

Human Infection with G12 Rotaviruses, Germany

Rotavirus group A G12 genotypes were detected in 3 (1.5%) of 198 stool samples positive for human rotavirus. G12P[6] was present in 2 samples, and a mixed G3G12P[8] was found in 1 sample. Phylogenetic analysis of complete open reading frames of all 11 genomic RNA segments proved their Wa-like genogroup affiliation.