Salmonella virchow meningitis in an adult.

We report here a case of meningitis caused by Salmonella virchow in a woman without signs or history of immunosuppression. Salmonella meningitis is a rare complication of human salmonellosis. The patient was successfully treated with ciprofloxacin. To our knowledge, this is the first reported case of confirmed meningitis in an adult caused by this serotype.

Intracellular processing and antigenic maturation of measles virus hemagglutinin protein.

The intracellular processing and antigenic maturation of the measles virus (MV) hemagglutinin (H) protein in virus infected cells were probed with murine monoclonal antibodies (Mabs) that reacted with continuous and discontinuous epitopes. The antibodies distinguished between the immature, cotranslational monomeric form of the protein and the mature, dimeric hemagglutinin structure. This was evidenced by testing of immunoreactivity of the Mabs with synthetic peptides, by in vitro synthesized H protein analysis, and by pulse-chase analysis of gel separated monomeric and dimeric forms of the H protein. Time kinetics analysis showed that the protein was synthesized as monomers and most of them were converted into dimers with t1/2 about 30 min. The H protein remained endoglycosidase H (Endo H) sensitive up to 30 min and started to acquire partial resistance to Endo H between 30 and 60 min (t1/2 about 60 min) after synthesis. Oligomerization of the H protein was unaffected in virus infected cells treated with a compound (carbonylcyanide m-chlorophenylhydrazone, CCCP) that blocks transport from the endoplasmic reticulum (ER) to the Golgi complex. These results suggest that the H protein dimerization takes place in the ER before its transport to the medial Golgi complex. The Mabs specific for discontinuous epitopes reacted with the H protein in cells treated with CCCP. Thus conformational antigenic epitope formation appears to take place in the ER.

The mumps virus V protein is unstable in virus infected cells.

The mumps virus (MuV) V protein was characterized in virus infected cells by the use of antipeptide sera. In radioimmune precipitation assay (RIPA), the sera reacted with the V protein and also immunoprecipitated the nucleocapsid (NP) and phospho (P) proteins. However, by depletion RIPA (in which either the NP and P proteins or the V protein were removed) and Western immunoblotting, it was demonstrated that the V protein was not associated with the NP and P proteins, but that the anti-V sera cross-reacted with the NP protein. Pulse-chase experiments demonstrated that the V protein was gradually decreased during the chase period and could not be detected by antibodies raised against peptides representing three different regions of the protein at the end of the chase, while the NP and P proteins were relatively stable during the chase period. These results suggest that the V protein is unstable and degraded gradually in virus infected cells.

Molecular characterization of epitopes on the measles virus hemagglutinin protein.

The measles virus (MV) hemagglutinin (H) gene nucleotide sequences of the LEC-WI strain and 11 branched sequential neutralization escape variants of the strain derived by selection with five monoclonal antibodies (Mabs) were determined by direct analysis of amplified polymerase chain reaction products. The parental LEC-WI strain isolated from a patient with subacute sclerosing panencephalitis exhibited H gene sequence characteristics similar to other persistent virus strains derived from brain materials. Mostly single-point H gene mutations, coding for single amino acid substitutions in the H protein, were found to provide explanations for the resistance to the individual Mabs. Resistance to Mabs 16-CD11 and I-41 resulted from changes of Gly-491 to Asp (or Val) and Phe-552 to Val, respectively. One variant (B89) selected by Mab 16-CD11 had a mutation introduced by a single nucleotide deletion and subsequent nucleotide insertion, which caused a shift in the open reading frame. The epitope of Mab I-29 was assigned to Ser-313 or Gly-314, which were changed to Leu and Arg, respectively. The variants subjected to the Mab I-44 selection exhibited change of Ser-189 to Pro. Radioimmunoprecipitation assay and endoglycosidase H (Endo H) treatment revealed that this change destroyed a potential N-linked glycosylation site, indicating that the carbohydrate chain participates in formation of the epitope or indirectly influences its properties. Resistance to Mab 16-DE6 involved three specific amino acid changes in three different places, Gly-211 to Ser, Gly-388 to Asp, and Ser-532 to Phe or Arg-533 to Gly, reflecting the occurrence of a conformational epitope. In conclusion, this study identifies the precise positions of several critical sites on the MV H protein which react with neutralizing antibodies.

Humanized animal viruses with special reference to the primate adaptation of morbillivirus.

This review article discusses the evolution of human viruses with special reference to paramyxoviruses. This family of viruses causes epidemics representing the dissemination of infection from one acutely infected host to the next. Since there is no repository for human paramyxoviruses in animals or in the form of persistent infections in man, the history of epidemics afflicting human civilization is short, presumably not exceeding 4000-5000 years. Evolutionary relationships can be deduced for comparison of nucleotide sequences of genes or even complete genomes. The present paramyxovirus genus will probably in the future be divided into two separate genera. In the genus morbillivirus, two pairs of more closely related virus types can be distinguished: canine and phocid viruses, and rinder-pest and measles viruses, respectively. It is speculated that recombination events may have occurred in the evolution of the morbillivirus archetype.

The nucleotide and deduced amino acid sequence of the M gene of phocid distemper virus (PDV). The most conserved protein of morbilliviruses shows a uniquely close relationship between PDV and canine distemper virus.

The nucleotide sequence of the matrix gene (M) of a recently identified morbillivirus, phocid distemper virus (PDV), was determined and the amino acid composition deduced. The M gene of PDV shared many characteristics with the corresponding gene in other morbilliviruses. The nucleotide homology with the closely related canine distemper virus (CDV) was maximum at 67% followed by measles virus (MV) (58%) and rinderpest virus (RPV) (56%). The length of the 5' long untranslated region of PDV (408) was similar to that of CDV (406) but was somewhat shorter than that of MV (425) and RPV (437). The deduced matrix protein of PDV showed structural characteristics similar to the corresponding proteins of other morbilliviruses. PDV and CDV M proteins showed a remarkably high amino acid homology of 90%. The percent amino acid homology among other morbilliviruses was between 73-77%. The M protein was the most highly conserved protein among all morbilliviruses viral components.

Sequence analysis of the genes encoding the nucleocapsid protein and phosphoprotein (P) of phocid distemper virus, and editing of the P gene transcript.

The nucleotide and deduced amino acid sequences of two genes of phocid distemper virus (PDV) were determined by cDNA cloning and sequencing. The long open reading frame of the gene encoding the nucleocapsid (N) protein is presented. As with other morbilliviruses, the phosphoprotein (P) gene of PDV was found to be located after the 5' end of the N gene and before the 3' end of the matrix protein gene. The P gene was shown to have the capacity to encode three distinct proteins, P, V and C, in analogy to other morbilliviruses. The results presented provide evidence for editing of the PDV P mRNA transcript by insertion of G residues. When the nucleotide and deduced amino acid sequences of the N, P, V and C genes were aligned with corresponding sequences of other established members of the morbillivirus genus, compelling homology was found between PDV and canine distemper virus (CDV), whereas there was markedly less similarity between PDV and measles virus or rinderpest virus. On the basis of the alignments presented, the estimated amino acid sequence similarity between the N and P genes of PDV and CDV was 84% and 76%, respectively. These differences at the genomic level indicate that the viruses are two separate entities.

The nucleotide sequence and deduced amino acid composition of the haemagglutinin and fusion proteins of the morbillivirus phocid distemper virus.

The amino acid composition of the two surface proteins of the recently isolated morbillivirus phocid distemper virus (PDV) were deduced from the nucleotide sequence. The fusion (F) protein of PDV exhibited characteristics similar to those of other morbillivirus F proteins. The overall amino acid similarity with its closest homologue, canine distemper virus (CDV), was 72%. From the context of the starting codons and the requirement for a hydrophobic signal peptide, it is likely that translation of the PDV F mRNA starts at the third AUG, corresponding to codon 95 in the long open reading frame of the PDV F gene. After removal of the signal peptide, F0 starts at amino acid 105. From this position the F protein of PDV and CDV exhibit 84% amino acid similarity. The PDV haemagglutinin (H) protein showed 74% amino acid similarity with CDV H protein and highly conserved features responsible for the tertiary structure. Despite these similarities, the two H proteins show marked antigenic differences when probed with monoclonal antibodies. Earlier studies have indicated that rinderpest virus (RPV) is the prototype virus of the morbillivirus genus, from which first CDV/PDV and later measles virus (MV) evolved. From the close relationship shown in this study, it is likely that the divergence of CDV and PDV occurred after MV evolved from RPV.

Brain-derived cells can be infected with HIV isolates derived from both blood and brain.

HIV type 1 and 2 isolates derived from brain and blood of infected individuals were used to infect astrocytic cells of tumor origin. Infection was monitored by polymerase chain reaction. The majority of the isolates infected the glioma cells, independently of the source of isolation. Added to the fact that the majority of primary HIV isolates infect cells of the monocyte/macrophage lineage, these results indicate that primary blood and brain HIV strains have similar target cells. The production of virus from infected astrocytes was detected only upon infection with two macrophage-adapted strains. Also in this case, the number of infected cells was very low and only one in 5000 cells carried the proviral HIV genome.

The nucleotide and predicted amino acid sequence of the attachment protein of canine distemper virus.

The nucleotide sequence of the gene coding for the attachment protein of the Convac strain of the canine distemper virus (CDV), corresponding to the haemagglutinin (H) gene of measles virus was determined using a mRNA-derived cDNA clone and genomic viral RNA. The mRNA transcribed from the CDV H gene is 1944 nucleotides long excluding the polyadenylated tail. Only one long open reading frame was found comprising nucleotides 21-1841. The predicted protein has a single hydrophobic region which can serve as a membrane anchoring domain. The deduced 607 amino acids would code for a protein of 68,247 Da, to be compared with an approximate protein molecular weight in SDS-PAGE of the glycosylated protein, which is 85,000 Da. The CDV H protein exhibited seven potential N-linked glycosylation sites. These were concentrated to the carboxyterminal part of the CDV H protein and differed markedly from measles virus (MV) and rinderpest virus (RPV) where the potential sites were mostly conserved and located in the amino-terminal half of the proteins. In spite of the differences in amino acid composition of these three H proteins their hydrophilicity/hydrophobicity plots were closely similar with the major hydrophobic region at an identical location. All the 12 cysteine residues found in the CDV H protein were conserved in MV and RPV. The amino acid homology between CDV and MV H protein was 37% and between CDV and RPV H protein 38%. The fact that the corresponding homology between the MV and RPV proteins is almost 60% shows that the evolutionary separation between CDV and RPV occurred at a much earlier time than the separation between RPV and MV.

Molecular cloning and sequence analysis of human parainfluenza type 2 virus mRNA encoding the fusion glycoprotein.

A library of cDNA clones from mRNA of human parainfluenza type 2 virus (PIV2) was constructed and the nucleotide sequence of the fusion (F) glycoprotein gene determined. The F gene boundaries were obtained by primer extension sequencing on F mRNA and on viral genomic RNA. The mRNA coding for the F glycoprotein is composed of 1918 nucleotides. It contains a single large open reading frame that encodes a protein of 551 amino acids with an Mr of 59586. The predicted PIV2 F protein contains the cleavage-activation site (amino acids 101 to 106) including two Arg and two Lys residues, where the protein is cleaved by host protease into F1 and F2 subunits by analogy with other paramyxoviruses. Three hydrophobic domains are recognized, the signal peptide (amino acids 1 to 21), that is cleaved off in the mature protein, the fusion peptide (amino acids 107 to 132) at the cleavage-generated N terminus of subunit F1 and the membrane anchorage region (amino acids 486 to 513) near the C terminus of the protein. The predicted F protein has six potential glycosylation sites and 10 of the 12 Cys residues present have conserved positions as compared with those of other paramyxovirus F proteins. Primer extension sequencing on viral RNA gave the 3' end sequence as UAAAUUCU6 followed by the 5' end of the haemagglutinin-neuraminidase (HN) gene thus establishing the order of genes coding for the viral glycoproteins of PIV2 as 5'-F-HN-3'.

Hemagglutinin-neuraminidase (HN) amino acid alterations in neutralization escape mutants of Kilham mumps virus.

The hemagglutinin-neuraminidase genes of the Kilham strain of mumps virus and three neutralization escape mutants (M11, M12 and M13) of this strain (Löve et al., 1985a) were sequenced using their genomes as template. The predicted amino acid sequences were compared. While one mutant had only one amino acid substitution the other two mutants had four and five respectively. A putative region for the epitope of the selected neutralizing monoclonal antibody was identified in a hydrophilic region encompassing amino acids 352-360, since the single amino acid substitution of one mutant occurred in this region and the other two mutants showed non-conserved amino acid changes in this part of the protein. The previously sequenced prototype strain RW, which lacks capacity to react with the selected neutralizing monoclonal antibody also has one non-conserved amino acid change in the region of the proposed neutralizing epitope. The three mutants showed different biological characteristics. These particular characteristics were therefore interpreted to be primarily associated with strain-specific amino acid changes outside the region of the presumed neutralizing epitope. The decrease in molecular weight in one mutant (M11) was shown to be due to a substitution in position 329 of an asparagine for an aspartic acid, leading to abolishment of a potential N-linked glycosylation site. In the other mutants, one substitution in position 239 of a lysine for a methionine was correlated with an increased neuraminidase activity of strain M12, while a substitution in position 360 of an arginine for a cysteine appeared to represent the most likely explanation for the reduced neurovirulence of strain M13.

The mumps virus fusion protein mRNA sequence and homology among the paramyxoviridae proteins.

The complete nucleotide sequence of the fusion protein (F) mRNA of the virulent SBL-1 strain of mumps virus has been determined by sequencing cDNA clones and mRNA and confirmed by partially sequencing the genomic RNA. The mRNA was 1721 nucleotides long excluding the poly(A) sequence and had one long open reading frame which encoded a protein of 538 amino acids with a calculated Mr of 58,791. The predicted amino acid sequence had a proteolytic cleavage/activation site, Arg Arg His Lys Arg, cleavage at which yields proteins F2 and F1. The uncleaved protein contained three highly hydrophobic regions: (i) the amino-terminal signal peptide, (ii) the amino-terminal region of F1 and (iii) the carboxy-terminal membrane anchorage domain. There were seven potential N-glycosylation sites, two in F2 and five in F1. Comparison of the virulent strain F protein sequence with that of an avirulent strain of mumps virus showed a difference of 14 amino acids. Among paramyxoviruses, mumps virus fusion protein shows the highest degree of homology with the fusion proteins of simian virus 5 and Newcastle disease virus.

mRNA sequence and deduced amino acid sequence of the mumps virus small hydrophobic protein gene.

The mRNA of a putative small hydrophobic protein (SH) of mumps virus was identified in mumps virus-infected Vero cells, and its complete nucleotide sequence was determined by sequencing the genomic RNA and cDNA clones and partial sequencing of mRNA. The SH mRNA is 310 nucleotides long excluding the poly(A) and contains a single open reading frame encoding a protein of 57 amino acids with a calculated molecular weight of 6,719. The predicted protein is highly hydrophobic and contains a stretch of 25 hydrophobic amino acids near the amino terminus which could act as a membrane anchor region. There is no homology between the putative SH protein of mumps virus and the SH protein of simian virus 5, even though the SH genes are located in the same locus in the corresponding genome. One interesting observation is that the hydrophobic domain of simian virus 5 SH protein is at the carboxyl terminus, whereas that of mumps virus putative SH protein is near the amino terminus.

Sequence analysis of the mumps virus mRNA encoding the P protein.

The nucleotide sequence of the mumps virus phospho- or polymerase-associated (P) protein mRNA has been determined by sequencing a full-length cDNA clone and confirmed by partially sequencing the mRNA and the genome. The mRNA contains 1311 nucleotides excluding the poly(A) and encodes a protein of 390 amino acids with a calculated molecular weight of 41,574. Three small polypeptides were seen in in vitro translation of viral mRNA and hybrid-selected P mRNA, possibly representing internal initiation in the same reading frame of the P protein. A second overlapping reading frame is predicted from the sequence which has a capacity to code for two polypeptides of 56 and 34 amino acids, respectively. Whether these two polypeptides are expressed in infected cells is not known. Comparison of the P protein sequence with that of Sendai virus, measles virus, parainfluenza virus type 3, and canine distemper virus (CDV) showed no distinct homology but comparison with the P protein of Newcastle disease virus (NDV) showed 25.6% homology.

Complete nucleotide sequence of the hemagglutinin-neuraminidase (HN) mRNA of mumps virus and comparison of paramyxovirus HN proteins.

The complete nucleotide sequence of the hemagglutinin-neuraminidase protein (HN) mRNA of the virulent SBL-1 strain of mumps virus has been determined. The mRNA contains 1887 nucleotides excluding the poly(A). The protein encoded by the mRNA has 582 amino acids and a membrane anchorage domain near the amino terminus. The calculated molecular mass (64 kDa) of the protein is in good agreement with that of the unglycosylated HN protein (63 kDa) identified in tunicamycin treated mumps virus infected cells (Herrler and Compans, 1983). The predicted sequence has nine potential N-glycosylation sites out of which two contain a cysteine residue and one has a proline residue as the variable amino acid X in the glycosylation site (Asn-X-Ser or Asn-X-Thr) and therefore, may not be utilized. One potential glycosylation site is in the cytoplasmic region which may not also be glycosylated. Comparison of the mumps HN protein sequence with the HN protein sequences of Sendai virus, simian-virus 5 (SV5), parainfluenza virus type 3 and Newcastle disease virus (NDV) shows two major homology regions, one region near the middle of the protein and the other in the second half of the molecule. In terms of percentage amino acid homology, the HN proteins of mumps virus, SV5 and NDV are closely related to each other but distinct from Sendai virus and parainfluenza virus type 3 HN proteins.

Molecular cloning and characterization of six genes, determination of gene order and intergenic sequences and leader sequence of mumps virus.

mRNA isolated from mumps virus-infected Vero cells was converted into cDNA and cloned into the PstI site of the plasmid pBR322. After screening with 32P-labelled cDNA synthesized from poly(A)+ RNA of uninfected or mumps virus-infected Vero cells, five different groups of virus-specific clones were obtained. The virus specificity of the clones was confirmed by Northern blot analysis, in which the cDNA inserts from the five different groups hybridized to mRNAs of about 2100, 1500, 1450, 2000 and 2200 nucleotides. By the use of oligonucleotides synthesized on the basis of sequences obtained from the five cDNA clones and mRNAs, the sequence of the intergenic and surrounding areas was determined. During genome sequencing, a separate gene was identified between the fusion protein (F) gene and the haemagglutinin-neuraminidase protein (HN) gene. Using oligonucleotides synthesized on the basis of the new gene sequence, cDNA clones with poly(A) were isolated from the cDNA library. The gene order was determined to be 3' NC-P-M-F-SH-HN-L 5' (where NC, P, M, SH, and L represent the genes for the nucleocapsid, phosphoprotein or polymerase-associated, matrix or membrane, small hydrophobic and large proteins respectively). There is one nucleotide between the P and M (A), M and F (A), and HN and L genes (G), two between the NC and P (AA) and SH and HN (3'-CG) genes, and seven between the F and SH genes (3' GAUUUUA) as intergenic sequence. The leader sequence at the 3' end of the genome has been determined by sequencing the dicistronic leader-NC mRNA using oligonucleotide primers. The sequence from the 3' terminus to the NC gene start of the mumps virus genome is similar in length (55 nucleotides) to that present in Sendai virus, Newcastle disease virus and parainfluenza virus type 3, and the first five nucleotides are conserved in all negative-stranded RNA virus genomes sequenced to date.

Accentuated antibody response to paramyxoviruses in individuals infected with human immunodeficiency virus.

Sera from 31 human immunodeficiency virus (HIV)-infected patients, representing different clinical stages of HIV infection, were assayed for antibodies against measles and mumps viruses by various serological tests and compared to 23 healthy controls. Sera from four patients (two primary, one asymptomatic, and one acquired immunodeficiency syndrome) exhibited a pronounced antibody response to measles as detected by haemagglutination inhibition and radioimmuno-precipitation assay. The RIPA-positive sera showed increased reactivity to all the viral components and in particular to the haemagglutinin (HA) protein of the virus (Fig. 1). Three of these positive patients also showed a similar response to mumps virus. One of the control sera also showed an increase in antibody titre in measles serological tests. The measles antibodies were shown not be anti-HIV antibodies crossreacting with paramyxoviruses. The reactivity to haemagglutinin was still present when using nonglycosylated measles virus antigen grown in the presence of tunicamycin. Whether the accentuated antibody response is due to polyclonal activation mediated by HIV or to reactivation of the viruses remains to be answered.