Poor outcome of adenovirus infections in adult hematopoietic stem cell transplant patients with sustained adenovirus viremia.

The outcome of adenovirus (ADV) infections in adult hematopoietic stem cell transplant (HSCT) patients remains poorly characterized. We studied 14 adults and 3 children, who had undergone HSCT and had developed ADV viremia. Peak ADV DNA levels were significantly higher in patients with ADV diseases than in those without (P=0.03). All children survived the ADV infections. Among the 14 adult HSCT patients, 11 were treated with cidofovir, 2 with ribavirin, and 1 did not receive antiviral treatment. Six of the 13 (46%) treated patients developed ADV diseases and 3 of them (23%) died of ADV infections. Sustained viremia (≥3 positive polymerase chain reaction assays during follow-up) was detected in all patients who finally died of ADV infections. However, 2 adults having had transient ADV viremia either survived or died of diseases other than ADV infections. Our study indicates that the outcome of adult HSCT patients with sustained ADV viremia may be poor, even for those who have received anti-ADV treatment.

Mesenchymal stem cells are susceptible to human herpesviruses, but viral DNA cannot be detected in the healthy seropositive individual.

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.

Mumps virus infection of the developing mouse brain--appearance of structural virus proteins demonstrated with monoclonal antibodies.

Newborn mice and hamsters were inoculated intracerebrally with mumps virus strains of high and low neurovirulence, Kilham and RW, respectively and with an egg-adapted patient isolate. The presence of viral antigen in brain tissue was analyzed with the immunofluorescence technique employing monoclonal antibodies against nucleoprotein (NP), polymerase (P), matrix (M), hemagglutinin-neuraminidase (HN) and fusion (F) mumps virus components. As expected, hamsters developed a fatal encephalitis eight to nine days after infection with the Kilham strain and synthesis of all five structural viral antigens was identified. In contrast, mice infected with any of the virus strains did not develop signs of disease, but in brain material collected on days nine and 12 after infection viral antigen was present in many neurons. However, only NP and P antigens were demonstrable and no infectious virus was present. The antibody response in mice developed later than in hamsters. Neurons in the mouse brain may exert a host cell restriction on the virus maturation, and mice offer a suitable host for the establishment of defective, persistent mumps virus infections.

A novel approach to the study of glycolipid receptors for viruses. Binding of Sendai virus to thin-layer chromatograms.

A method for the binding of virus to a silica gel thin-layer chromatogram is presented. After development the chromatogram is overlayed with the 125I-labelled virus and the bound virus is autoradiographed. Alternatively, the unlabelled virus may be detected after exposure to monoclonal antibody and labelled anti-antibody. The Sendai virus strain used did not bind to brain gangliosides earlier proposed to be receptors, but bound to human erythrocyte gangliosides. This finding may be explained by the existence of Sendai virus variants with different receptor specificities.

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.

Sendai virus M protein is found in two distinct isoforms defined by monoclonal antibodies.

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.

Inclusion body myositis and paramyxoviruses.

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.

Respiratory syncytial virus infection in hospitalized patients and healthy children in El Salvador.

Nasopharyngeal specimens from 42 children less than one-year old hospitalized with bronchiolitis or pneumonia in El Salvador were analyzed for the presence of subgroup-specific respiratory syncytial virus (RSV) antigens by the indirect immunofluorescence technique. The antigen RSV-A was demonstrated in 28 children, RSV-B in three, and in one child subgroup, specificity could not be determined. The male:female ratio in the RSV-infected children was 1.9:1. The most severe disease, requiring intensive care, was observed in two infants with RSV-B infection. Determination of serum IgG, IgA, and IgM antibodies in acute and convalescent sera showed that none of the tests alone had sufficient sensitivity for routine diagnostic purposes, although, in combination, they provided a correct diagnosis in 86% of the RSV-infected children. A seroprevalence study of IgG, IgA, and IgM antibodies in 206 healthy children showed that a primary RSV infection is usually acquired during the first year of life in El Salvador. These results also indicated that reinfections with RSV frequently occur during the first 3-4 years of life and suggest that the occurrence of serum RSV IgA antibodies may be a marker of reinfection.

Intertypic differentiation and detection of intratypic variants among canine and phocid morbillivirus isolates by kinetic neutralization using a novel immunoplaque assay.

Intertypic antigenic differences and the intratypic variability of the closely related canine (CVD) and phocid distemper viruses (PDV) were examined using a molecular (monoclonal antibodies specific for the H- and F-glycoproteins) and a functional (kinetic neutralization, KN) approach. KN studies were carried out using a novel immunoplaque technique which combined conventional plaque assay and antigen-specific enzyme-immunostaining techniques. Morbillivirus isolates of canine and phocid origin clearly formed two separate groups. Minor antigenic differences were also evident within each cluster. A distemper isolate of mustelid origin was distinguishable from both CDV- and PDV-like prototype viruses by kinetic neutralization.

Restricted virus protein translation in canine distemper virus inclusion body polioencephalitis.

In this study, inclusion body polioencephalitis, an uncommon form of canine distemper virus (CDV)-induced encephalitis, was investigated for viral protein and mRNA expression by immunohistochemistry (IH) and in situ hybridization and, in addition, infiltrating cells were characterized by IH. Lesions were predominantly found in the grey matter of the brain stem and the immune response, dominated by T cells, was associated with a strong MHC II upregulation. Abundant expression of all viral protein mRNAs and reduced or lacking protein translation, especially of the matrix protein were the most important findings, indicating that restricted virus infection in the grey matter might represent a mechanism for viral persistence in distemper polioencephalitis.

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.

Studies on manifestations of canine distemper virus infection in an urban dog population.

An upsurge of canine distemper was recognized at the beginning of 1991 in the urban dog population of the Copenhagen area. The outbreak had the characteristics of a virulent morbillivirus introduction in a partly immune population, where the disease primarily was manifested in young individuals. Testing of single serum samples for the presence of canine distemper virus (CDV) IgM antibodies using an IgM ELISA confirmed current and recent CDV infections in an urban dog population, where the use of attenuated CDV vaccines was widespread. In 49 out of 66 sera from clinical cases suspected of canine distemper we detected CDV IgM antibodies, as compared to the detection of viral antigen by indirect immunofluorescence in 27 of 65 specimens of conjunctival cells. The antigenic make-up of isolates from acute and subacute clinical cases was investigated with a panel of 51 monoclonal antibodies directed against CDV and the related phocine distemper virus. The isolates exhibited an homogeneous reaction pattern and shared overall antigenic characteristics of the CDV prototype. The majority of cases were diagnosed among unvaccinated dogs and individuals with unknown or obscure vaccination record. However, severe clinical cases were also diagnosed in vaccinated individuals.

Round table on morbilliviruses in marine mammals.

Since 1988 morbilliviruses have been increasingly recognized and held responsible for mass mortality amongst harbour seals (Phoca vitulina) and other seal species. Virus isolations and characterization proved that morbilliviruses from seals in Northwest Europe were genetically distinct from other known members of this group including canine distemper virus (CDV), rinderpest virus, peste des petits ruminants virus and measles virus. An epidemic in Baikal seals in 1987 was apparently caused by a morbillivirus closely related to CDV so that two morbilliviruses have now been identified in two geographically distant seal populations, with only the group of isolates from Northwest Europe forming a new member of the genus morbillivirus: phocid distemper virus (PDV). Because of distemper-like disease, the Baikal seal morbillivirus was tentatively named PDV-2 in spite of its possible identity with CDV. The appearance of morbilliviruses in the Mediterranean Sea causing high mortality amongst dolphins should further increase the research activities on protection strategies for endangered species of marine mammals.

Spot synthesis of overlapping peptides on paper membrane supports enables the identification of linear monoclonal antibody binding determinants on morbillivirus phosphoproteins.

In order to map antigenic domains on the P-protein of morbillivirus, a series of overlapping peptides, representing the P-protein sequences of phocid distemper virus strain 2558/Han88 and canine distemper virus strain Onderstepoort, were synthesized on a paper support by the spot-technique. The reactivity of six monoclonal antibodies with the peptides was tested in an enzyme immunoassay and compared to their reactivity in Western blots and in an ELISA using detergent extracts from virus-infected cells. Three linear determinants could be localized on the P-protein. Two antibody-binding sites were delineated within the C-terminal (between amino acids 307-322 and 382-400, respectively), and a third one was located on the N-terminal part (amino acids 13-31) of the protein. Fine mapping of this binding site revealed that this was a part of an antigenic domain. In Western blots, the monoclonal antibodies reacting with this domain also reacted with a second protein which was possibly the V-protein.

Antigenic heterogeneity amongst the field isolates of Newcastle Disease Virus (NDV) in relation to the vaccine strain. Part II: studies on viruses isolated from domestic birds in Israel.

Forty three Newcastle disease virus (NDV) strains isolated before and during 1997 in Israel from domestic birds were studied by means of the three panels of monoclonal antibodies prepared against all the viral envelope proteins in order to reveal the possible antigenic differences between them and the VH strain used in Israel for poultry vaccination. Three isolates were found to have significant antigenic differences in the hemagglutinin-neuraminidase (HN) and fusion (F) glycoproteins as compared to the vaccine strain. As to the matrix protein, almost all the viruses isolated during the year 1997 were found to have considerable differences from the vaccine strain in two of four antigenic sites.

Antigenic epitope characterization of matrix protein of Newcastle disease virus using monoclonal antibody approach: contrasting variability amongst NDV strains.

A panel of 15 monoclonal antibodies (MABs) against matrix (M) protein of Newcastle disease virus (NDV) was obtained and the specificity towards the M protein was proven by radioimmunoprecipitation assay and antigen capture enzyme-linked immunosorbent assay (ELISA). Further studies were directed to antigenic epitope mapping of the M protein by means of this panel. The epitope characterization was performed by competitive antibody-binding assay by means of labelling each MAB with biotin [3]. At least three clear non-overlapping and two partially overlapping groups were determined, each including four, one, eight, one, and one MAB, respectively. All the above MABs appeared to be induced by structural epitopes formed in conditions of tertiary structure of the native M antigen. Twelve reference and 51 recently isolated local NDV strains have been studied by means of this MAB panel, several lineages having been revealed. The high stability of some epitopes and different variability of the others was demonstrated. No correlation between the above lineages and some other properties of the studied NDV strains (host specificity, date and place of isolation) has been found.

Comparative characteristics of the Israeli Newcastle disease virus field strains by means of a wide panel of monoclonal antibodies against hemagglutinin-neuraminidase glycoprotein.

Fourteen mouse monoclonal antibodies (MAB) were tested for their ability to react with 15 reference and 52 local Newcastle disease virus (NDV) strains isolated in Israel during the last decade from feral birds. All the field isolates had no antigenic difference when examined by classic serological tests. However, MAB-mediated analysis revealed wide antigenic heterogeneity amongst the studied viruses. By the pattern of the MAB reactivity, all the isolates could be distributed into 13 groups.

Variability of antigenic epitopes of the fusion protein of Newcastle disease virus.

Using a panel of 10 monoclonal antibodies (Mab) against fusion (F) protein of Newcastle disease virus (NDV), strain Australia-Victoria, three non-overlapping antigenic sites (F1, F2 and F3) and one site partially overlapping with the sites F1 and F2 (F1.2) have been identified. The sites F2 and F3 are clusters that each include four antigenic epitopes. The antigenic stability of the above epitopes was estimated by comparison of the binding capacity of the corresponding Mabs towards 63 NDV strains isolated in different years and places from various avian species. The results demonstrated high variability of the site F1.2 and of all the four epitopes of the site F2. At the same time, the only epitope of the site F1 can be defined as highly conservative: the corresponding Mab gave positive binding with 60 from 63 NDV strains, one from the four epitopes pertaining to site F3 was the most conservative--the corresponding Mab reacted with all the 63 strains used in the studies, while the other three Mabs showed rather low stability--the corresponding Mabs reacted with 34-39 NDV strains. Thus, as opposed to the published data asserting antigenic stability of the F protein contrary to the high variability of the haemagglutinin-neuraminidase (HN) protein, our results have revealed a number of variable epitopes on the F protein. This demonstrates an evolutionary changeability of the F protein, which is of importance from the theoretical (viral antigenic evolution) as well as practical point of view.

Antigenic heterogeneity among the field isolates of Newcastle disease virus (NDV) in relation to the vaccine strain: 1. Studies on viruses isolated from wild birds in Israel.

In order to reveal the viruses strongly differing from the VH NDV strain used in Israel for poultry vaccination, 54 NDV strains isolated during the last 15 years in Israel from feral birds were studied by means of the panels of 39 monoclonal antibodies. Six isolates were found to have considerable antigenic differences in envelope proteins as compared to the vaccine strain. In four cases, the differences were related mostly to the hemagglutinin-neuraminidase glycoprotein, in one case to the fusion glycoprotein, and in one case to the matrix protein.

Antigenic characterization of the nucleocapsid protein of Newcastle disease virus by means of a new panel of monoclonal antibodies.

Nine monoclonal antibodies (MAB) against nucleocapsid protein (NP) of Newcastle disease virus (NDV) have been prepared and characterized. All the MABs were classified into three groups by means of the competitive binding assay. At least three antigenic sites were delineated on the NP. The 1st site includes two closely located epitopes; the 2nd site includes two related and two distinct epitopes; the 3rd site includes two closely related and one distinct epitopes.