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A recent study reported the detection of a bat-derived virus (BatPV/Epo_spe/AR1/DCR/2009, batMuV) with phylogenetic relatedness to human mumps virus (hMuV). Since all efforts to isolate infectious batMuV have reportedly failed, we generated recombinant mumps viruses (rMuVs) in which the open reading frames (ORFs) of the fusion (F) and haemagglutinin-neuraminidase (HN) glycoproteins of an hMuV strain were replaced by the corresponding ORFs of batMuV. The batMuV F and HN proteins were successfully incorporated into viral particles and the resultant chimeric virus was able to mediate infection of Vero cells. Distinct differences were observed between the fusogenicity of rMuVs expressing one or both batMuV glycoproteins: viruses expressing batMuV F were highly fusogenic, regardless of the origin of HN. In contrast, rMuVs expressing human F and bat-derived HN proteins were less fusogenic compared to hMuV. The growth kinetics of chimeric MuVs expressing batMuV HN in combination with either hMuV or batMuV F were similar to that of the backbone virus, whereas a delay in virus replication was obtained for rMuVs harbouring batMuV F and hMuV HN. Replacement of the hMuV F and HN genes or the HN gene alone by the corresponding batMuV genes led to a slight reduction in neurovirulence of the highly neurovirulent backbone strain. Neutralizing antibodies inhibited infection mediated by all recombinant viruses generated. Furthermore, group IV anti-MuV antibodies inhibited the neuraminidase activity of bat-derived HN. Our study reports the successful generation of chimeric MuVs expressing the F and HN proteins of batMuV, providing a means for further examination of this novel batMuV.

HPIV3 is a respiratory virus causing airway diseases, including pneumonia, croup, and bronchiolitis, during infancy and childhood. Currently there is no effective vaccine or anti-viral therapy for this virus. Studies have suggested that poor T cell proliferation following HPIV3 infection is responsible for impaired immunological memory associated with this virus. We have previously demonstrated that NK cells mediate regulation of T cell proliferation during HPIV3 infection. Here we add to these studies by demonstrating that the regulation of T cell proliferation during HPIV3 infection is mediated via NK receptors NKp44 and NKp46 and involves the surface glycoprotein haemagglutinin-neuraminidase but not the fusion protein of the virus. These studies extend our knowledge of the regulatory repertoire of NK cells and provide mechanistic insights which may explain reoccurring failures of vaccines against this virus.

In spite of the success of the mumps vaccination, recent mumps outbreaks have been reported even among individuals with a history of mumps vaccination. For a better understanding of why the vaccination failed in cases of vaccinees who fell ill during recent mumps outbreaks, the immunological events during infection and/or vaccination should be better defined. In the work presented here we sought to identify new neutralization sites on the mumps virus surface glycoproteins. By using anti-mumps mAbs, three amino acid positions at residues 221, 323 and 373 in the F protein of mumps virus were shown to be located in at least two conformational neutralization epitopes. mAbs that specifically target these sites effectively neutralized mumps virus in vitro. The newly acquired glycosylation site at position 373 or loss of the existing one at position 323 was identified as the mechanism behind the escape from the specific mAbs. Based on the findings of this study, we suggest that the influence of the antigenic structure of the F protein should not be ignored in a thorough investigation of the underlying mechanism of the mumps vaccine failure or when making a strategy for development of a new vaccine.

A bat virus with high phylogenetic relatedness to human mumps virus (MuV) was identified recently at the nucleic acid level. We analyzed the functional activities of the hemagglutinin-neuraminidase (HN) and the fusion (F) proteins of the bat virus (batMuV) and compared them to the respective proteins of a human isolate. Transfected cells expressing the F and HN proteins of batMuV were recognized by antibodies directed against these proteins of human MuV, indicating that both viruses are serologically related. Fusion, hemadsorption, and neuraminidase activities were demonstrated for batMuV, and either bat-derived protein could substitute for its human MuV counterpart in inducing syncytium formation when coexpressed in different mammalian cell lines, including chiropteran cells. Cells expressing batMuV glycoproteins were shown to have lower neuraminidase activity. The syncytia were smaller, and they were present in lower numbers than those observed after coexpression of the corresponding glycoproteins of a clinical isolate of MuV (hMuV). The phenotypic differences in the neuraminidase and fusion activity between the glycoproteins of batMuV and hMuV are explained by differences in the expression level of the HN and F proteins of the two viruses. In the case of the F protein, analysis of chimeric proteins revealed that the signal peptide of the bat MuV fusion protein is responsible for the lower surface expression. These results indicate that the surface glycoproteins of batMuV are serologically and functionally related to those of hMuV, raising the possibility of bats as a reservoir for interspecies transmission. IMPORTANCE The recently described MuV-like bat virus is unique among other recently identified human-like bat-associated viruses because of its high sequence homology (approximately 90% in most genes) to its human counterpart. Although it is not known if humans can be infected by batMuV, the antigenic relatedness between the bat and human forms of the virus suggests that humans carrying neutralizing antibodies against MuV are protected from infection by batMuV. The close functional relationship between MuV and batMuV is demonstrated by cooperation of the respective HN and F proteins to induce syncytium formation in heterologous expression studies. An interesting feature of the glycoproteins of batMuV is the downregulation of the fusion activity by the signal peptide of F, which has not been reported for other paramyxoviruses. These results are important contributions for risk assessment and for a better understanding of the replication strategy of batMuV.

Immunization programs have implemented live attenuated mumps vaccines which reduced mumps incidence ≥97%. Some of the vaccine strains were abandoned due to unwanted side effects and the genetic marker of attenuation has not been identified so far. Our hypothesis was that non-infectious viral particles, in particular defective interfering particles (DIPs), contribute to neuroattenuation. We showed that non-infectious particles of the mumps vaccine L-Zagreb attenuated neurovirulence of wild type mumps virus 9218/Zg98. Then, we attenuated recent wild type mumps virus MuVi/Zagreb.HRV/28.12 in Vero cells through 16 passages but already the fifth passage (p5) showed accumulation of DIPs and attenuated neurovirulence in a newborn rat model when compared to the second passage (p2). Sequence analysis of the p2 and p5 revealed a single mutation in the 5′ untranslated region of the HN gene. Analysis of the expression level of the HN protein showed that this mutation does not affect the expression of the protein. We conclude that the passages of MuVi/Zagreb.HRV/28.12 in Vero cells for only three passages accumulated DIPs which attenuate neurovirulence. These findings reveal DIPs as a very promising and general neuroattenuating factor which should be considered in the rational design of the new mumps vaccine.

The paramyxovirus entry machinery consists of two glycoproteins that tightly cooperate to achieve membrane fusion for cell entry: the tetrameric attachment protein (HN, H, or G, depending on the paramyxovirus genus) and the trimeric fusion protein (F). Here, we explore whether receptor-induced conformational changes within morbillivirus H proteins promote membrane fusion by a mechanism requiring the active destabilization of prefusion F or by the dissociation of prefusion F from intracellularly preformed glycoprotein complexes. To properly probe F conformations, we identified anti-F monoclonal antibodies (MAbs) that recognize conformation-dependent epitopes. Through heat treatment as a surrogate for H-mediated F triggering, we demonstrate with these MAbs that the morbillivirus F trimer contains a sufficiently high inherent activation energy barrier to maintain the metastable prefusion state even in the absence of H. This notion was further validated by exploring the conformational states of destabilized F mutants and stabilized soluble F variants combined with the use of a membrane fusion inhibitor (3g). Taken together, our findings reveal that the morbillivirus H protein must lower the activation energy barrier of metastable prefusion F for fusion triggering.

It is unknown how receptor binding by the paramyxovirus attachment proteins (HN, H, or G) triggers the fusion (F) protein to fuse with the plasma membrane for cell entry. H-proteins of the morbillivirus genus consist of a stalk ectodomain supporting a cuboidal head; physiological oligomers consist of non-covalent dimer-of-dimers. We report here the successful engineering of intermolecular disulfide bonds within the central region (residues 91–115) of the morbillivirus H-stalk; a sub-domain that also encompasses the putative F-contacting section (residues 111–118). Remarkably, several intersubunit crosslinks abrogated membrane fusion, but bioactivity was restored under reducing conditions. This phenotype extended equally to H proteins derived from virulent and attenuated morbillivirus strains and was independent of the nature of the contacted receptor. Our data reveal that the morbillivirus H-stalk domain is composed of four tightly-packed subunits. Upon receptor binding, these subunits structurally rearrange, possibly inducing conformational changes within the central region of the stalk, which, in turn, promote fusion. Given that the fundamental architecture appears conserved among paramyxovirus attachment protein stalk domains, we predict that these motions may act as a universal paramyxovirus F-triggering mechanism.

Morbillivirus cell entry is controlled by hemagglutinin (H), an envelope-anchored viral glycoprotein determining interaction with multiple host cell surface receptors. Subsequent to virus-receptor attachment, H is thought to transduce a signal triggering the viral fusion glycoprotein, which in turn drives virus-cell fusion activity. Cell entry through the universal morbillivirus receptor CD150/SLAM was reported to depend on two nearby microdomains located within the hemagglutinin. Here, we provide evidence that three key residues in the virulent canine distemper virus A75/17 H protein (Y525, D526, and R529), clustering at the rim of a large recessed groove created by β-propeller blades 4 and 5, control SLAM-binding activity without drastically modulating protein surface expression or SLAM-independent F triggering.

Multipotent mesenchymal stromal cells (MSC) are used to improve the outcome of hematopoietic stem cell transplantation (HSCT) and in regenerative medicine. However, human MSC may harbor persistent viruses that may compromise their clinical benefit. Parvovirus B19, known to cause the childhood disease erythema infectiosum (“fifth disease”), can be hazardous to HSCT recipients due to development of a virus mediated severe pancytopenia. Retro spectively screened, 1 of 20 MSC from healthy donors contained B19 DNA. The donors exhibited a seroprevalence of anti-B19 IgG comparable to the reported normal level. Using flow cytometry, we found that human MSC (n=6) expressed the erythrocyte P antigen (also called globoside), reported as the B19 receptor, and Ku 80, a reported B19 co-receptor. After in vitro exposure to plasma derived from B19 viremic patients, MSC (n=3) were found positive for intracellular B19 proteins as determined by immunofluorescence. This demonstrates that MSC can be infected by B19. Further studies revealed that MSC could transmit B19 to bone marrow cells in vitro, suggesting that the virus can persist in the marrow stroma of healthy individuals. Two HSCT patients received the B19 positive MSC as treatment for graft-versus-host disease. Neither developed viremia nor symptomatic B19 infection, even though they where severely immunocompromised by means of subnormal Ig levels, low leukocyte counts and poor responses in mixed lymphocyte cultures. These results demonstrate for the first time that persistent B19 in MSC can infect hematopoietic cells and underscore the importance of monitoring B19 transmission by MSC products.

Genotyping of respiratory syncytial (RS) virus group A, by means of a novel method based on PCR, FRET (fluorescence resonance energy transmission) detection and two-dimensional melting curve analysis, was carried out on 80 RS virus samples of group A collected in Stockholm from 1976 to 2005. The Tm values were assessed for three different genotypes (GA2, GA5 and GA7) circulating in Sweden. Two pairs of probes were used and results of subsequent data analysis were plotted in a two-dimensional system. The results obtained were compared to genotyping using conventional nucleotide sequencing and phylogenetic tree analysis. It was found that the new assay was able to correctly identify genotype in about 89% of the isolates; it identified the remaining 11% as untypeable and as candidates for conventional nucleotide sequencing. The new method constitutes a complement to nucleotide sequencing and could be useful in studies of large numbers of samples in epidemiological studies.

Despite large vaccination campaigns, measles virus (MeV) and canine distemper virus (CDV) cause major morbidity and mortality in humans and animals, respectively. The MeV and CDV cell entry system relies on two interacting envelope glycoproteins: the attachment protein (H), consisting of stalk and head domains, co-operates with the fusion protein (F) to mediate membrane fusion. However, how receptor-binding by the H-protein leads to F-triggering is not fully understood. Here, we report that an anti-CDV-H monoclonal antibody (mAb-1347), which targets the linear H-stalk segment 126-133, potently inhibits membrane fusion without interfering with H receptor-binding or F-interaction. Rather, mAb-1347 blocked the F-triggering function of H-proteins regardless of the presence or absence of the head domains. Remarkably, mAb-1347 binding to headless CDV H, as well as standard and engineered bioactive stalk-elongated CDV H-constructs treated with cells expressing the SLAM receptor, was enhanced. Despite proper cell surface expression, fusion promotion by most H-stalk mutants harboring alanine substitutions in the 126-138 “spacer” section was substantially impaired, consistent with deficient receptor-induced mAb-1347 binding enhancement. However, a previously reported F-triggering defective H-I98A variant still exhibited the receptor-induced “head-stalk” rearrangement. Collectively, our data spotlight a distinct mechanism for morbillivirus membrane fusion activation: prior to receptor contact, at least one of the morbillivirus H-head domains interacts with the membrane-distal “spacer” domain in the H-stalk, leaving the F-binding site located further membrane-proximal in the stalk fully accessible. This “head-to-spacer” interaction conformationally stabilizes H in an auto-repressed state, which enables intracellular H-stalk/F engagement while preventing the inherent H-stalk’s bioactivity that may prematurely activate F. Receptor-contact disrupts the “head-to-spacer” interaction, which subsequently “unlocks” the stalk, allowing it to rearrange and trigger F. Overall, our study reveals essential mechanistic requirements governing the activation of the morbillivirus membrane fusion cascade and spotlights the H-stalk “spacer” microdomain as a possible drug target for antiviral therapy., Author Summary With the ultimate aim to develop pan-morbillivirus fusion inhibitors, we here characterized a potent neutralizing monoclonal antibody. The antibody recognizes the ectodomain of the membrane-bound tetrameric attachment (H) protein, which together with the fusion protein and a host cell receptor executes plasma membrane fusion to deliver the viral genetic information into the cell. The H-ectodomain consists of a short F-binding/activating stalk region supporting receptor-binding head domains. Molecular characterization of the identified mAb epitope (which locates in the membrane-distal stalk module called “spacer”), enabled us to unravel two sequential conformational changes occurring in CDV H-tetramers that stand at the core of the molecular mechanism translating receptor binding to F-triggering. We additionally propose that both rearrangements are triggered upon receptor-induced “de-activation” of an auto-repressed state assumed by H prior to receptor binding. This locked state enables H/F interaction while preventing premature F-activation. Furthermore, although paramyxovirus attachment proteins may fold into very similar “pre-receptor-bound” conformational states, the presence of the “spacer” module in the stalk emerges as a key determinant leading to distinct mechanisms of membrane fusion triggering.

Allogeneic stem cell transplantation is often complicated by reactivation of herpesviruses. Mesenchymal stem cells (MSC) are immunomodulatory and may be used to treat graft-versus-host disease. We investigated if herpesviruses infect and can be transmitted by MSC, and if MSC suppress immune responses to various infectious agents. Mesenchymal stem cells from healthy seropositive donors were evaluated with polymerase chain reaction for the most common herpesviruses: cytomegalovirus (CMV), herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2, Epstein-Barr virus (EBV) and varicella zoster virus. The cytopathological effect (CPE) was investigated and viral antigens analyzed by immunofluorescence after in vitro exposure to CMV, HSV-1 and EBV. We also studied MSC effect on lymphocyte stimulation induced by various infectious agents. No viral DNA could be detected in MSC isolated from healthy seropositive individuals. However, a CPE was noted and intracellular viral antigens detected after infection in vitro by CMV and HSV-1, but not by EBV. The CMV and HSV-1 infections were productive. Lymphocyte proliferation by herpesviruses, candida mannan and protein A from Staphylococcus aureus was suppressed by MSC. The data indicate that the risk of herpesvirus transmission by transplantation of MSC from healthy seropositive donors is low. However, MSC may be susceptible to infection if infused in a patient with CMV or HSV-1 viremia. MSC transplantation may compromise the host's defense against infectious agents.

Respiratory syncytial virus (RSV) causes severe respiratory diseases in infants and young children. Inappropriate immunity to the virus can lead to disease enhancement upon subsequent infection. In this study, we have characterized the antiviral immunity elicited by the recombinant Semliki Forest virus (SFV) encoding the RSV fusion (F) and attachment (G) protein, and compared with that induced by the immune-stimulating complex (ISCOM)-incorporated FG proteins. Antiviral immunity against RSV elicited nasally or parentally by either of the immunogen having divergent profiles could reduce lung RSV titers upon challenge. However, resistance to RSV without disease enhancement was only observed in those vaccinated with SFV recombinants via nasal route. Presence of postvaccination pulmonary IFN-γ response to the H-2Kd-restricted T cell epitope (F85–93; KYKNAVTEL) was found to be associated with absence of enhanced pulmonary disease and goblet cell hyperplasia as well as reduced Th2-cytokine expression. This result demonstrates that the SFV recombinants can result in enhanced clearance of RSV without enhancing the RSV-associated disease, and underlines the importance in priming pulmonary MHC class I-restricted T cells when RSV FG-based vaccines are used.

The level of replication and immunogenicity of recombinant parainfluenza virus type 3 (rHPIV3) bearing one, two, or three gene insertions expressing foreign protective antigens was examined. cDNA-derived recombinant HPIV3s bearing genes encoding the open reading frames (ORFs) of the hemagglutinin-neuraminidase (HN) of HPIV1, the HN of HPIV2, or the hemagglutinin (HA) of measles virus replicated efficiently in vitro, including the largest recombinant, which had three gene unit insertions and which was almost 23 kb in length, 50% longer than unmodified HPIV3. Several viruses were recovered from cDNAs whose genome length was not a multiple of six nucleotides and these contained nucleotide insertions that corrected the length to be a multiple of 6, confirming that the “rule of six” applies to HPIV3. Using a hemagglutination inhibition assay, we determined that the HPIV1 HN expressed by recombinant HPIV3 was incorporated into HPIV3 virions, whereas using this assay incorporation of the HPIV2 HN could not be detected. HPIV3 virions bearing HPIV1 HN were not neutralized by HPIV1 antiserum but were readily neutralized by antibodies to the HPIV3 HN or fusion protein (F). Viruses with inserts were restricted for replication in the respiratory tract of hamsters, and the level of restriction was a function of the total number of genes inserted, the nature of the insert, and the position of the inserted gene in the gene order. A single insert of HPIV2 HN or measles virus HA reduced the in vivo replication of rHPIV3 up to 25-fold, whereas the HPIV1 HN insert decreased replication almost 1000-fold. This indicates that the HPIV1 HN insert has an attenuating effect in addition to that of the extra gene insert itself, presumably because it is incorporated into the virus particle. Viruses containing two inserts were generally more attenuated than those with a single insert, and viruses with three inserts were over-attenuated for replication in hamsters. Inserts between the N and P genes were slightly more attenuating than those between the P and the M genes. A recombinant HPIV3 bearing both the HPIV1 and the HPIV2 HN genes (r1HN 2HN) was attenuated, immunogenic, and protected immunized hamsters from challenge with HPIV1, HPIV2, and HPIV3. Thus, it is possible to use a single HPIV vector expressing two foreign gene inserts to protect infants and young children from the severe lower respiratory tract disease caused by the three major human PIV pathogens.

An epidemic of mumps in Lithuania started in December 1998 and continued until May 2000. The total registered number of cases was about 11.000 of a total of 3,7 million inhabitants in Lithuania (29,7 cases/10,000). Virus-containing samples were collected from 80 patients treated at the hospital of Kaunas from October 1999 until the end of the epidemic. Out of the 80 patients with parotitis, meningitis was observed in 11 patients and orchitis in 22 of 69 male patients. Twenty-seven virus strains were genotyped by nucleotide sequencing of the small hydrophobic (SH) protein gene, and the 57 amino acid sequences of the gene were deduced. Twenty-five virus strains belonged to the C genotype and two were of the D genotype. By phylogenetic analysis the virus strains causing meningitis grouped in a separate cluster, designated C1, within the C genotype. Another group of ten of the 25 genotype C strains exhibited an amino acid triplet at amino acid positions 28 to 30 of the protein, consisting of valine, alanine and serine, instead of the previously recognised valine, valine and serine combination of genotype C. The amino acid alanine at position 29 was found in combination with the amino acid serine at position 48. This variant was designated C2 and it was associated with parotitis. The amino acid alanine at position 29 and serine in position 48 of the C2 genotype may constitute a marker of low neurovirulence compared to other genotype C strains.

Retrospective evaluation of medical history and 3635 anti-TBE (tick-borne encephalitis) serologies during the years 2003-2008 indicates that childhood TBE is characterized by vague symptoms. Clinical findings suggest a nonspecific inflammatory disease with restricted encephalitic profile compared with adult TBE. Childhood TBE might elude diagnosis, which is unsatisfactory because of potential long-term consequences.

Reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) is synthesized and packaged into the virion as a part of the GagPol polyprotein. Mature RT is released by the action of viral protease. However, unlike other viral proteins, RT is subject to an internal cleavage event leading to the formation of two subunits in the virion: a p66 subunit and a p51 subunit that lacks the RNase H domain. We have previously identified RNase H to be an HIV-1 protein that has the potential to be a substrate for the N-end rule pathway, which is an ubiquitin-dependent proteolytic system in which the identity of the N-terminal amino acid determines the half-life of a protein. Here we examined the importance of the N-terminal amino acid residue of RNase H in the early life cycle of HIV-1. We show that changing this residue to an amino acid structurally different from the conserved residue leads to the degradation of RT and, in some cases, integrase in the virus particle and this abolishes infectivity. Using intravirion complementation and in vitro protease cleavage assays, we show that degradation of RT in RNase H N-terminal mutants occurs in the absence of active viral protease in the virion. Our results also indicate the importance of the RNase H N-terminal residue in the dimerization of RT subunits. IMPORTANCE HIV-1 proteins are initially made as part of a polyprotein that is cleaved by the viral protease into the proteins that form the virus particle. We were interested in one particular protein, RNase H, that is cleaved from reverse transcriptase. In particular, we found that the first amino acid of RNase H never varied in over 1,850 isolates of HIV-1 that we compared. When we changed the first amino acid, we found that the reverse transcriptase in the virus was degraded. While other studies have implied that the viral protease can degrade mutant RT proteins, we show here that this may not be the case for our mutants. Our results suggest that the presence of active viral protease is not required for the degradation of RT in RNase H N-terminal mutants, suggesting a role for a cellular protease in this process.

The WHO recently proposed an updated nomenclature for mumps virus (MuV). WHO currently recognizes 12 genotypes of MuV, assigned letters from A to N (excluding E and M), which are based on the nucleotide sequences of small hydrophobic (SH) and haemagglutinin-neuraminidase (HN) genes. A total of 66 MuV genomes are available in GenBank, representing eight of the 12 genotypes. To complete this dataset, whole genomes of seven isolates representing six genotypes (D, H, I, J, K and L) and one unclassified strain were sequenced. SH and HN genes of other representative strains were also sequenced. The degree of genetic divergence, predicted amino acid substitutions in the HN and fusion (F) proteins and geographic distributions of MuV strains were analysed based on the updated dataset. Nucleotide heterogeneity between genotypes reached 20% within the SH gene, with a maximum of 9% within the HN gene. The geographic and chronologic distributions of the 12 genotypes were summarised. This review contributes to our understanding of strain diversity for wild type MuV, and the results support the current WHO nomenclature.

Background: In addition to earlier reports on the association between viral infections and intrahepatic neonatal cholestasis, in recent studies, investigators have suggested a similar link to extrahepatic biliary atresia. Methods: Fifty-nine cholestatic infants (mean age 8 weeks) were investigated for signs of infection with a large spectrum of viruses. Twenty-one infants had extrahepatic biliary atresia, 38 had intrahepatic cholestasis. The virologic methods included serologic investigation in 59 infants and 54 mothers, virus isolation from stools (49 infants). urine (58 infants) and liver biopsies (40 infants). Polymerase chain reaction was used to detect cytomegalovirus DNA in 25 of the liver biopsy specimens. Two control groups, one with 35 noncholestatic infants and one with [1] healthy, pregnant women were checked for serologic signs of cytomegalovirus. Results: Nineteen of 59 (32%) cholestatic infants. including 8 of 21 (38%) with extrahepatic biliary atresia, compared with 2 of 35 (6%) control infants had cytomegalovirus-immunoglobulin (Ig) M detected in serum (p < 0.01). Fifty-one of 54 (94%) tested mothers of cholestatic infants were sero-positive for cytomegalovirus, compared with 83 of 111 (75%) control mothers (p < 0.01). Cytomegalovirus DNA in liver specimens was detected by polymerase chain reaction in 9 of 18 (50%) analyzed patients with biliary atresia and in specimens from 3 of 7 patients with intrahepatic cholestasis. Conclusions: Cytomegalovirus infection may play a role, not only in intrahepatic neonatal cholestasis, as was suggested earlier, but also in extrahepatic biliary atresia. The pathogenetic mechanism for this link remains to be established. JPGN 27: 57-64, 1998.

Specific pathogen free (SPF) domestic cats were inoculated with tissue homogenate obtained from a Chinese leopard (Panthera pardus japonensis) that had died in a North American zoo from a natural infection with canine distemper virus (CDV). The cats developed a transient cell-associated CDV viraemia along with pronounced lymphopenia but did not show any clinical symptoms. Plasma neutralizing-antibody titres against the homologous CDV (A92-27/4, isolated from the Chinese leopard) were consistently higher than against the CDV vaccine strain 'Bussell'. The Chinese leopard CDV isolate showed in vitro biological properties reminiscent of virulent, wild-type CDV strains. Sequence analysis of the H gene of two large felid CDV isolates from the USA (A92-27/4 and A92-6) revealed up to 10% amino acid changes including up to four additional potential N-linked glycosylation sites in the extra-cytoplasmic domain as compared to CDV vaccine strains. Phylogenetic analysis was performed using the entire coding region of the H gene and a 388 bp fragment of the P gene of several morbillivirus species. Evidence was obtained that recent CDV isolates from different species in the United States (including isolates from large felids), Europe and Africa are significantly distinct from CDV vaccine strains. All wild-type CDV isolates analysed clustered according to geographical distribution rather than to host species origin. By sequence analysis a CDV epizootic among large felids in a Californian safari park was linked to a virus which most likely originated from feral non-felid carnivores.

Background Neurologic sequelae, including cognitive deficits, after childhood tick-borne encephalitis (TBE) and neuroborreliosis (NB) are not well-characterized. These infections are among the most common affecting the central nervous system in children and can be difficult to diagnose due to vague symptomatology. The aim of this study was to investigate long-term (>1 year) consequences of pediatric TBE and NB as well as the value of markers for brain damage and genetic susceptibility. Methods From a previous prospective study, children diagnosed with TBE (n = 8) and NB (n = 12) as well as pediatric controls (n = 15) were followed up by clinical examination, semistructured interview and screening for cognitive dysfunction by the Five-to-Fifteen Questionnaire. The follow-up also included detection of serum autoantibodies against the neural proteins; glial fibrillary acidic protein and myelin basic protein, as well as genotyping of a 32 basepair deletion in the chemokine receptor type 5 gene. Results Children diagnosed with TBE displayed significantly more long-term subjective complaints (ie, fatigue, headache and irritability) compared with the NB and control groups. Significantly higher frequency of disabilities was also detected by the Five-to-Fifteen Questionnaire in the TBE group. Both TBE and NB cause consequences (eg, prolonged convalescence, worries and financial loss) for the families. Markers for genetic susceptibility and brain damage had no prognostic values in this cohort. Conclusions Pediatric TBE results in long-lasting residual symptoms and neurologic deficits affecting daily life. Vigilance for TBE-related morbidity among pediatricians and long-term clinical follow-up with assessment of cognitive dysfunctions and appropriate interventions seems reasonable for these children.

The morbilliviruses measles virus (MeV) and canine distemper virus (CDV) both rely on two surface glycoproteins, the attachment (H) and fusion proteins, to promote fusion activity for viral cell entry. Growing evidence suggests that morbilliviruses infect multiple cell types by binding to distinct host cell surface receptors. Currently, the only known in vivo receptor used by morbilliviruses is CD150/SLAM, a molecule expressed in certain immune cells. Here we investigated the usage of multiple receptors by the highly virulent and demyelinating CDV strain A75/17. We based our study on the assumption that CDV-H may interact with receptors similar to those for MeV, and we conducted systematic alanine-scanning mutagenesis on CDV-H throughout one side of the β-propeller documented in MeV-H to contain multiple receptor-binding sites. Functional and biochemical assays performed with SLAM-expressing cells and primary canine epithelial keratinocytes identified 11 residues mutation of which selectively abrogated fusion in keratinocytes. Among these, four were identical to amino acids identified in MeV-H as residues contacting a putative receptor expressed in polarized epithelial cells. Strikingly, when mapped on a CDV-H structural model, all residues clustered in or around a recessed groove located on one side of CDV-H. In contrast, reported CDV-H mutants with SLAM-dependent fusion deficiencies were characterized by additional impairments to the promotion of fusion in keratinocytes. Furthermore, upon transfer of residues that selectively impaired fusion induction in keratinocytes into the CDV-H of the vaccine strain, fusion remained largely unaltered. Taken together, our results suggest that a restricted region on one side of CDV-H contains distinct and overlapping sites that control functional interaction with multiple receptors.

Tick-borne encephalitis (TBE) and neuroborreliosis (NB) are well-known central nervous system (CNS) infections in children. Childhood tick-borne CNS infections are generally described as mild conditions. However, this view has recently been challenged, and the natural course, including potential sequelae, has been debated. If the diseases present with nonspecific symptoms and signs, some children may elude diagnosis. This study estimates the incidence of symptomatic tick-borne CNS infections in children under medical care and describes the spectrum of manifestations. One hundred twenty-four children with neurologic symptoms attending the Pediatric Emergency Department were included prospectively. Anti-TBE virus and anti-Borrelia serology results were analyzed together with inflammatory parameters in the blood and cerebrospinal fluid. Nearly one fourth of the children with neurologic symptoms were diagnosed with a tick-borne CNS infection (TBE, n = 10 [8%] and NB, n = 21 [16.8%]). In general, these children displayed an indistinct medical history and presented with nonspecific signs such as malaise/fatigue and headache. Diagnosis was based on analysis of acute and convalescent sera. Blood inflammatory parameters were nonspecific and did not contribute to the diagnostics. Conclusion: Pediatric tick-borne CNS infections are unexpectedly common and should be considered in children with unspecific and unexplained acute CNS-related symptoms.

The conserved aspartic acid residue 488 in the RNase H domain of HIV-1 reverse transcriptase (RT) was mutated to alanine. RT was expressed in Escherichia coli alone or with the entire pol-gene polyprotein consisting of proteinase, RT, and integrase and processed by the HIV-1 proteinase in the bacterial cell. Expression of mutant RT together with the proteinase resulted in an overproduction of RT p51 vs p66. The mutation also altered the conformation of the RT p66/p51 heterodimer as shown by the loss of binding of monoclonal antibodies to mutant RT in ELISA. Crystallographic data shows that a salt bridge exists between Asp 488 and Lys 465 of RNase H which stabilizes the uncleavable form of RT p66, and that substitution of Asp for Ala would prevent the formation of this salt bridge. Our results indicate that disruption of this salt bridge through mutation of Asp 488 interferes with the conformational changes that regulate the limited processing of p66 to 51 by the virus proteinase. Homology data suggest that such a bridge may be present in other lentiviruses. The mutation introduced caused a moderate decrease in both the RNase H activity and the polymerase activity of RT, indicating that the proper folding of the RNase H domain of RT is necessary to achieve full polymerase activity.

Nucleotide sequencing of the fusion protein (F) gene of phocid distemper virus-2 (PDV-2), recently isolated from Baikal seals (Phoca sibirica), revealed an open reading frame (nucleotides 84 to 2075) with two potential in-frame ATG translation initiation codons. We suggest that the second in-frame ATG triplet at positions 264 to 266 initiates the translation, resulting in a protein of 537 amino acid residues with a calculated M(r) of 63,035. The putative F1/F2 cleavage site, located approximately 100 amino acid residues from the N terminus, is identical to those of the F proteins of phocid distemper virus-1 (PDV-1) isolated from European harbour seals (Phoca vitulina) and of canine distemper virus (CDV). A full scale comparison of morbillivirus F genes reveals that the conserved F0 extracellular protein-encoding region contains a large number of non-expressed mutations, suggesting that this part of the protein is under strong functional constraints. Phylogenetic analysis of morbillivirus F gene nucleotide sequences revealed a closer evolutionary relationship between PDV-2 and CDV than between PDV-1 and PDV-2. These data were supported by cross-reactivity patterns of PDV-2 and CDV obtained with monoclonal antibodies to structural proteins of PDV-1 and CDV, and suggest that PDV-2 is a strain of CDV, resulting from a trans-species infection.

Since 1987, morbillivirus infections have caused serious disease outbreaks with high mortality among aquatic mammals. Baikal seals (Phoca sibirica) in Siberia were involved in an outbreak caused by a virus closely related to canine distemper virus (CDV) in 1987. Harbour seals (Phoca vitulina) and grey seals (Halichoerus grypus) in north-western Europe were struck by a newly recognised morbillivirus of seals (PDV1). A serological survey has indicated that these morbillivirus infections frequently occur among several pinniped species. Besides pinnipeds, the presence of morbillivirus infections among cetaceans, including whales, dolphins and porpoises, has been demonstrated since the outbreak of PDV among seals in north-western Europe. Morbillivirus was isolated from several stranded harbour porpoises (Phocoena phocoena). This virus proved to be very similar to a virus which was isolated during a disease outbreak with high mortality among striped dolphins (Stenella coeruleoalba) in the Mediterranean area. The viruses isolated from these cetacean species were quite different from the viruses isolated from the seals. They proved more related to the ruminant morbilliviruses, peste des petits ruminants virus and rinderpest virus. The potential transmission of the dolphin morbillivirus to the endangered population of the Mediterranean monk seal (Monachus monachus) has been considered. Studies are presently being conducted into the possibility of inducing protection against morbillivirus infection in this species by vaccination with an immune stimulating complex (ISCOM) preparation based on CDV.

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, presumbly 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 pphocid viruses, and rinderpest and measles viruses, respectively. It is speculated that recombination events may have occurred in the evolution of the morbillivirus archetype.

The use of a monoclonal antibody defines a subset of Sendai virus M protein representing about 30% of total. This M protein acquires, during the hour following synthesis, an epitope not present on the bulk of M. This epitope maturation is observed in acutely as well as in persistently infected cells. It takes place in vivo in absence of other viral proteins, but it is not observed when the protein is synthesized in a reticulocyte lysate. Epitope maturation does not appear to result from phosphorylation, acylation or disulfide bond formation. If immunofluorescent staining seems to indicate a preferential association of this subset of M protein with nucleocapsids, this is not confirmed by immunogold staining or by nucleocapsid isolation. Incubation of cytoplasmic extracts or of purified M protein in conditions which do not favor M to M protein association results in a relative increase of M protein carrying the maturing epitope. It is concluded that M protein exists in two distinct isoforms.

Rat dorsal root ganglia in tissue culture, which contain an interferon-gamma (IFN-gamma)-like immunoreactive subpopulation of neurons, were infected with paramyxoviruses. Sendai virus caused a substantial neuronal lysis, while the RW strain of mumps virus caused a much less pronounced nerve cell loss. Early during infection, the subpopulation of IFN-gamma-like immunoreactive neurons was less susceptible to mumps virus. Virus antigen was rapidly lost from surviving IFN-gamma-like positive neurons infected with Sendai virus, while this remarkable self-curing effect occurred in both nerve cell populations at later time points after mumps virus infection. By quantitative enzyme-linked immunosorbent assay (ELISA) technique, increased levels of "neuronal IFN-gamma" were recorded at 10 hr and 30 hr after infection with Sendai and mumps virus, respectively. This study indicates a role for the neuronal IFN-gamma-like molecule in determining the outcome of a viral infection in sensory ganglia.

Paramyxovirus cell entry is controlled by the concerted action of two viral envelope glycoproteins, the fusion (F) and the receptor-binding (H) proteins, which together with a cell surface receptor mediate plasma membrane fusion activity. The paramyxovirus F protein belongs to class I viral fusion proteins which typically contain two heptad repeat regions (HR). Particular to paramyxovirus F proteins is a long intervening sequence (IS) located between both HR domains. To investigate the role of the IS domain in regulating fusogenicity, we mutated in the canine distemper virus (CDV) F protein IS domain a highly conserved leucine residue (L372) previously reported to cause a hyperfusogenic phenotype. Beside one F mutant, which elicited significant defects in processing, transport competence, and fusogenicity, all remaining mutants were characterized by enhanced fusion activity despite normal or slightly impaired processing and cell surface targeting. Using anti-CDV-F monoclonal antibodies, modified conformational F states were detected in F mutants compared to the parental protein. Despite these structural differences, coimmunoprecipitation assays did not reveal any drastic modulation in F/H avidity of interaction. However, we found that F mutants had significantly enhanced fusogenicity at low temperature only, suggesting that they folded into conformations requiring less energy to activate fusion. Together, these data provide strong biochemical and functional evidence that the conserved leucine 372 at the base of the HRA coiled-coil of F(wt) controls the stabilization of the prefusogenic state, restraining the conformational switch and thereby preventing extensive cell-cell fusion activity.

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.

These results demonstrated that cross-reactions between mumps virus, PIV-2, PIV-4, and SV5 involve both internal components and surface glycoproteins and that these viruses form a separate group within the paramyxovirus genus.

Inclusion body myositis (IBM) is a distinct type of muscle disease. The characteristic electron microscopic findings, intranuclear or intracytoplasmic inclusions composed of microtubular filaments, morphologically resemble paramyxovirus nucleocapsids. These findings and the reported immunoreactivity of the inclusions with mumps virus antibodies have suggested that inclusion body myositis is a chronic virus infection. We analyzed skeletal muscle specimens from three patients with characteristic light microscopic features and electron microscopically verified inclusions of IBM by immunocytochemistry using antibodies raised against members of the paramyxovirus group, and by in situ hybridization with a cRNA probe representing the mumps virus nucleocapsid gene. The specificity of the reactions was demonstrated with infected and uninfected cultured cells. No immunocytochemical staining or hybridization signal was observed in biopsy specimens from IBM patients. These findings speak against a paramyxovirus etiology of IBM.

An antigenic site (represented by 15 amino acids, residues 174 to 188, designated peptide 12) of the large glycoprotein G of respiratory syncytial virus was demonstrated to be subgroup specific in peptide enzyme-linked immunosorbent assay tests with murine monoclonal antibodies and human postinfection sera. The role of individual amino acids in this subgroup-specific site was determined by use of single-amino-acid-deletion sets of peptides. When monoclonal antibodies were reacted with the deletion sets, a broad amino acid dependence of 11 or 12 residues, Cys-176 (Ile-175 in subgroup B) to Cys-186, was found. Human postinfection sera exhibited a narrower reaction profile (for subgroup A, Cys-182 to Trp-183; for subgroup B, Cys-176 to Lys-183). Reduction of peptides on microtiter plates by treatment with dithiothreitol completely destroyed their antigenic activity in tests with monoclonal antibodies and human postinfection sera of subgroup B. A variant of peptide 12 containing all four cysteines of the G protein (represented by 16 amino acids, residues 172 to 187, designated peptide 12var) also was subgroup specific. We concluded that the activity of the antigenic site in tests with monoclonal antibodies for subgroups A and B appears to depend on intrapeptide disulfide bonds. Reactions with postinfection sera of subgroup B also may depend on a disulfide bond. In contrast, postinfection sera of subgroup A appeared to have the capacity to identify a subgroup-specific site in a linear form of the selected 15-amino-acid-long peptide. Treatment of peptides with dithiothreitol had no effect on their antigenic activity in tests with human postinfection sera of subgroup A. These findings have relevance for molecular engineering of peptide antigens for use in respiratory syncytial virus subgroup-specific site-directed serology.

The immunological relationships between distemper viruses, isolated from a seal and mink in Denmark and from a dog in Greenland, were investigated with 39 previously developed monoclonal antibodies (MAbs) directed against four major structural proteins of canine distemper virus (CDV). They were also investigated with 16 newly developed MAbs directed against the fusion (F) and large glycoprotein (named H in analogy with measles virus) of phocid distemper virus (PDV) isolated from a harbour seal (Phoca vitulina). These MAbs were reacted with the three different isolated viruses and with the LEC strain of measles virus, in ELISA and immunofluorescence tests. In addition, immunoprecipitation tests were carried out with some of the cross-reacting antibodies. All 55 MAbs reacted identically with distemper virus isolated from seals or mink. When the MAbs produced against CDV were tested, 37 of 39 antibodies reacted with a virus isolated from a sled dog diseased in an outbreak of distemper in Greenland prior to the epizootic among seals in the North Sea. Of the 39 antibodies, 25 reacted with PDV and distemper virus isolated from mink. Of these antibodies, only three of the nine antibodies directed against the H protein of CDV cross-reacted with PDV and distemper virus from mink. Eleven MAbs, reacting with six epitopes of the H protein of PDV, were produced. All 11 antibodies reacted with distemper virus from mink, two of the antibodies reacted with CDV and none reacted with measles virus. All five antibodies reacting with three different epitopes of the F protein of PDV reacted with distemper virus from mink and CDV. Of these five antibodies three, directed against two epitopes, reacted with measles virus. Of the two envelope proteins, the H protein shows pronounced immunological differences between PDV and CDV. In contrast, immunologically the F protein appears to be well conserved among morbilliviruses. It is concluded that the virus causing the epizootic in seals in the North Sea in 1988 may have infected mink on land, or, alternatively, the virus in the sea may have originated from virus-infected mink.

Direct immunofluorescent staining (DIF) of exfoliated cells and immunofluorescent staining of infected tissue culture cells (TCIF) are superior to conventional virus isolation (CVI) techniques for the diagnosis of respiratory virus infections. Out of 197 specimens from patients with respiratory syncytial virus (RSV), influenza or parainfluenza virus, measles, adenovirus or cytomegalovirus (CMV) infections, DIF recognised 84%, TCIF 82% and CVI only 42%. DIF and TCIF were particularly useful for the diagnosis of RSV and parainfluenza virus infections, and the optimal specimen for diagnosis was nasopharyngeal aspirate.

Mumps virus strains isolated during an epidemic in Lithuania in 1998 - 2000 were studied. Viruses of the neurovirulent C1 and non-neurovirulent C2 small hydrophobic (SH) genotype variant were sequenced for the haemagglutinin-neuraminidase (HN) and fusion (F) protein genes. Amino acid differences between C1 and C2 strains were found for both proteins. Two amino acid differences were of potential importance for the non-neurovirulent phenotype of the C2 virus. Four of 5 C2 strains exhibited the amino acid arginine instead of lysine at position 335 of the HN protein, and the amino acid phenylalanine was found instead of serine at amino acid position 195 of the F protein. Amino acid differences at these positions have previously been reported to associate with a change in neurovirulence and fusion activity. In addition, the HN gene of the neurovirulent Kilham strain of genotype A was sequenced. The deduced amino acid sequence showed different amino acids compared to both genotypes A and C on some positions. Notably, amino acid differences located in previously identified neutralizing epitopes were found at positions 266, 354 and 356 of the HN protein compared to other genotype A strains. The amino acid differences between Kilham virus strain and other genotype A strains and the similarity of the Kilham HN protein (7 positions) to neurovirulent genotype C strains on some amino acid positions may indicate a possible role for this protein in mumps virus neurovirulence.

Though mumps virus (MuV) is a monotypic virus, genetic variation between strains has been described. Viruses have been placed into genotypes designated A-L based on the nucleotide sequence of the small hydrophobic (SH) gene, which is the most variable gene in the mumps genome. Molecular characterisation of MuV is an important component of mumps surveillance because it can help identify the transmission pathways of the virus as well as distinguish between wild-type and vaccine strains. Here, we propose a standardized nomenclature and an analysis protocol for the genetic characterisation of mumps strains to facilitate expansion of molecular epidemiological studies. In addition to assigning standard reference strains for the recognized genotypes of MuV, a convention is proposed for naming for strains and criteria to designate a new genotype.

The epidemiology of respiratory syncytial virus (RSV) group A was followed by nucleotide sequencing of the variable parts of the glycoprotein (G) gene. The amino acid sequences of an aminoterminal (A-terminal, amino acids 90–132) and carboxyterminal (C-terminal, amino acids 262–298) portion of the G protein in 47 virus strains, collected in Stockholm, between 1965 and 2004, were determined. In phylogenetic analysis jointly with previously described genotypes (GA1 to GA7 and SAA1), 33 virus strains (isolated between 1991 and 2004) belonged to genotype GA5, seven to GA2, three to genotype GA1 (isolated before 1991), one to genotype GA4 (isolated in 1982) and three to genotype GA7 (isolated in 1993 and 2001). Genotype GA5 was predominant in four epidemics, between 2000/2001 and 2003/2004. Little or no variation with time of the C-terminal amino acid sequence of the G protein was found when the virus strains were compared within their own genotype. Identical and nearly identical nucleotide sequences were found between strains isolated more than 10 (GA5) and 25 (GA2) years apart. The A-terminal part of they G protein of genotype GA2 was highly conserved. In contrast, the A-terminal part of the G protein of genotype GA5 exhibited a pronounced variation in its amino acid sequence over time.

Six different genotypes of mumps virus, A, C, D, G, H and I, genotyped on the basis of the small hydrophobic protein gene sequence, were subjected to antigenic comparison. Monoclonal antibodies directed against the haemagglutinin–neuraminidase protein of the SBL-1 strain of genotype A were used in immunofluorescence tests with different mumps virus strains. In addition, the six virus genotypes were compared by cross-neutralization tests with human post-vaccination sera after vaccination with the Jeryl Lynn (JL) strain of mumps virus and with rabbit hyperimmune sera directed against the A or D genotypes of mumps virus. Genotypes C, D, G, H and I could not be antigenically separated. In contrast, three different virus strains of genotype A, SBL-1, JL and Kilham, were distinct and were found to represent three different serotypes within the A genotype of mumps virus. Vaccination of Swedish children with the JL strain of mumps virus resulted in clearly lower neutralization titres against the SBL-1 strain, which is endemic in Sweden, compared to the homologous vaccine titres.

The nucleotide sequences of a 300 bp segment carrying the small hydrophobic (SH) protein gene of a large number of virus strains, belonging to 10 different mumps virus genotypes denoted A-J, were compared. When virus strains belonging to the same genotype were compared intra-genotypically, a variation in the range 2-4% was recorded. The level of inter-genotypical variation was higher (8-18%). A consistent finding was the more pronounced variation found when the A genotype was compared to the B-J genotypes than when the B-J genotypes were compared among themselves. A similar relatively more pronounced genetic variation between the A and B-D genotypes has also been found previously with the hemagglutinin-neuraminidase and fusion protein genes. It is concluded that the establishment of a new genotype should be founded on a nucleotide variation of the SH gene of > or = 6% in relation to previously described genotypes.

Twenty-nine Danish virus isolates and 14 serum samples from patients with mumps were genotyped by nucleotide sequencing of the small hydrophobic (SH) protein gene and the deduced 57 amino acid sequences were aligned with sequences of mumps virus strains published previously. Four neurovirulent genotypes of the SH protein gene, genotypes C, D, H and a new genotype, designated J, were found. There was a dynamic fluctuation of the different genotypes over the two decade period of time. Genotype J was found from 1981 to 1988; genotypes C and H exhibited a similar distribution in time. Genotype D was found between 1979 and 1982, it then disappeared and reappeared again in 1996. From 1996 onwards, genotype D was found to be the predominant genotype, which is in contrast to the situation seen in the neighbouring country of Sweden, where, since 1985, only genotype A has been found.

In April 1999, a previously healthy 22-year-old woman was taken ill with fever and bilateral swelling of the parotid glands. A chronic course of disease extending from April to December was found with swelling of the parotid glands, fatigue, low grade fever, episodes of tachycardia and nightswetting. Mumps virus RNA of genotype A character based on the SH (small hydrophobic) protein gene classification was demonstrated in three serum samples collected during the course of clinical disease. Different criteria for reinfection were fulfilled including demonstration of IgG antibodies by ELISA in a preinfection serum sample. The preinfection serum sample of the patient was able to efficiently neutralize the infectivity of a heterologous genotype D strain but was unable to neutralize the homologous genotype A virus. The findings in the present study may offer an explanation of a mechanism behind previously observed vaccine failures and the occurrence of reinfection with heterologous mumps virus strains.

Twelve strains of mumps virus, belonging to the A, C and D genotypes of the small hydrophobic (SH) protein gene, were investigated by nucleotide sequencing of the fusion protein gene. The nucleotide sequences and deduced amino acid sequences were aligned and compared with previously reported sequences of the gene. In addition an antigenic comparison between the F protein of different strains of the A, C and D genotypes was performed with ten monoclonal antibodies directed against the F protein of genotype A. Phylogenetic analysis of the coding region of the F gene showed the expected clustering of the different genotypes, as previously determined from the SH protein gene. Comparison of the 538 long amino acid sequence of the protein showed that only a small number of amino acids differed between the viral strains. The A genotype differed from B, C and D whereas the latter showed fewer consistent amino acid differences between themselves. Nine of the ten monoclonal antibodies reacted with the C and D genotypes and one failed to react with these genotypes. It is concluded that the structure and antigenicity of the F protein is well conserved both intra- and intergenotypically over long periods of time.

Eighteen virus isolates and 22 serum samples collected between 1971 and 1997 from patients with mumps were genotyped by PCR with specific primer pairs for the A, C and D genotypes of the small hydrophobic (SH) protein gene. All serum samples were subjected to nucleotide sequence analysis of the gene, and the deduced 57-amino-acid sequences were aligned with previously published sequences from the USA, Canada, Portugal, the UK, France, Germany, Switzerland, Denmark, Sweden, Russia, China and Japan. The existence of six genotypes of the SH protein gene, named A to F, was confirmed. In the Stockholm area, co-circulation of genotypes A, C and D at different times was found. There was a striking difference in genotype between the virus isolates and the serum samples. The 18 virus isolates represented genotypes C and D, whereas the 22 serum samples contained genotype A. In most cases, the amino acid sequences of the 22 genotype A specimens were identical to the previously described SBL-1 strain of genotype A. Genotypes C and D were always associated with meningitis, and in some cases parotitis, whereas infection with genotype A most often resulted in parotitis and seldom in meningitis.

New series of escape mutants of human respiratory syncytial virus were prepared with monoclonal antibodies specific for the fusion (F) protein. Sequence changes selected in the escape mutants identified two new antigenic sites (V and VI) recognized by neutralizing antibodies and a group-specific site (I) in the F1 chain of the F molecule. The new epitopes, and previously identified antigenic sites, were incorporated into a refined prediction of secondary-structure motifs to generate a detailed antigenic map of the F glycoprotein.

The SBL-1 strain of mumps virus, grouping with genotype A on the basis of the small hydrophobic protein gene sequence, was grown in the presence of three different monoclonal antibodies. The monoclonal antibodies were directed against the haemagglutinin-neuraminidase (HN) protein and they inhibited haemagglutinating activity and infectivity of the virus. The HN genes of the nine neutralization-escape virus mutants were sequenced and the predicted amino acid sequences were compared with that of the parental virus. Amino acid substitutions were found at positions 269, 352 and 354, respectively, of the 582 amino acid long protein. The three monoclonal antibodies did not react with 35 virus strains isolated in Stockholm during the years 1970 to 1985. Thirteen and four of the strains were found to belong to the D and C genotypes, respectively. A type-specific neutralization antibody response was also found in sera of rabbits hyperimmunized with purified virions of genotype A and D. The genotype-specific difference in neutralizing activity in mice and rabbits was not corroborated by an overall difference in the amino acid sequence of the HN protein of the different genotypes. Further studies are needed to explore the efficacy of mumps virus vaccines for protection against homologous and heterologous genotypes of mumps virus.