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Inducement of respiratory mucosal immunogenicity for nasal vaccine due to amine- and amide-inactivation of parainfluenza virus type 1, Sendai.

The protective effects in mice by nasal vaccination of amine- and amide-inactivated Sendai viruses were investigated by a contact exposure experiment, immunofluorescent examination of the entire respiratory tract, and checking the serum HI antibody development. Of 10 monoamines, ethanolamine and 2-methoxyethylamine vaccines induced complete protection, and methylamine, ethylamine, n-propylamine, n-butylamine, 2-ethoxyethylamine, diethylamine and triethylamine vaccines brought about almost complete protection or lower respiratory infection. The methoxyamine-treated mouse conferred the least protection. Of 5 diamines, 1,3-diaminopropane vaccine inhibited completely the infection, but hydrazine, ethylenediamine, putrescine, and cadaverine vaccines produced regional infection. Two polyamines, spermine and spermidine, did not inactivate the virus. Of 4 amides, only semicarbazide vaccine conferred complete mucosal defense, while acetamide, propionamide, and isonicotinic acid hydrazide vaccines lead to regional infection. Serum HI titers developed by vaccination were low on the whole, following their slight rise, fall or maintenance postexposure. In effect, the 4 vaccines inactivated by a best-suited interstrand cross-link between phosphate groups in helix of viral RNA brought about the strongest protection, and showed the necessity of a definite length of molecules for inactivants.

Administration, Intranasal↗

Protection of cotton rats by immunization with the human parainfluenza virus type 3 fusion (F) glycoprotein expressed on the surface of insect cells infected with a recombinant baculovirus.

The antigenicity, immunogenicity and efficacy of the human PIV3 fusion (F) glycoprotein expressed in insect cells by a baculovirus vector were studied. The results indicate that the PIV3 F glycoprotein expressed by a recombinant baculovirus is antigenically authentic as determined using a panel of PIV3 F specific monoclonal antibodies. Only a low level of antibody was stimulated by immunization of animals with infected cells, but the antibody appeared to be of high quality. Immunized animals were also moderately protected against PIV3 challenge. These results indicate that the baculovirus expression system is a reasonable source of authentic PIV3 F protein for use in a subunit vaccine.

Animals↗

Precise mapping of the replication and transcription promoters of human parainfluenza virus type 3.

The terminal RNA regions of the genomic and antigenomic RNAs of the paramyxoviruses and rhabdoviruses are known to contain sequences essential for directing RNA replication and transcription. The 3' terminus (leader region) of the negative-sense, genomic RNA of the rhabdoviruses and paramyxoviruses is known as the leader (Le) promoter and directs synthesis of positive-sense replication and transcription products. The 3' terminus of the antigenome is termed the trailer complementary (TrC) promoter and directs the synthesis of genomic RNA. By creating mutations in the corresponding regions of an HPIV3 minireplicon in which the viral protein coding sequences were replaced by the luciferase gene, we were able to precisely define the elements of the leader promoter involved in directing positive-strand replication of HPIV3. Nucleotides 1 through 12 (from the terminus) formed a domain critical for replication. The region from nucleotides 13 through 55 was important but not crucial for replication, while G residues at positions 79, 85, and 91 comprised another domain critical for replication. It was also shown that the TrC promoter is similar, though not identical, to the Le promoter. Nucleotides 1 through 12 of the TrC promoter were critical for synthesis of genomic RNA, though specific positions behaved differently from the corresponding positions of the Le promoter. While many of these mutations could not be analyzed for transcription because they completely abrogated genomic RNA synthesis (the template for transcription), we were surprised to find that no mutations in the leader promoter which decreased replication had any significant effect on transcription. However, mutations in the intergenic sequence and gene start signal following the leader and preceding the luciferase message severely decreased transcription, but not replication.

Base Sequence↗

The infection of mouse preimplantation embryos to Sendai virus (parainfluenza I).

To provide information on the susceptibility of mouse embryos to Sendai virus, it was investigated if viral replication occurs in the preimplantation embryo at different stages of development, with or without the zona pellucida (ZP). Mice were induced to superovulate, and embryos were collected on Days 2, 3 and 4 after mating. The ZP was removed by digestion with 0.5% pronase. Embryos were exposed to Sendai virus, washed, and allowed to develop in fresh culture medium. The presence of viral antigen in the embryonic cells was examined by the fluorescent antibody test (FAT). Specific immunofluorescence was demonstrated in the ZP-free morula and ZP-intact blastocyst. However, viral antigen was not detected in the ZP-intact two-cell, four-cell, eight-cell or morula stage embryos. Infected embryos developed normally to expanded blastocysts. These findings show that mouse embryonic cells are permissive hosts to Sendai virus replication and that the ZP played the role of a barrier against the virus.

Journal Article↗

Type-specific detection of parainfluenza viruses by enzyme-immunoassay and radioimmunoassay in nasopharyngeal specimens of patients with acute respiratory disease.

A four-year solid phase enzyme-immunoassay (EIA) and radioimmunoassay (RIA) techniques were applied for the type-specific detection of parainfluenza type 1, 2 and 3 virus antigens in sonicated nasopharyngeal specimens of patients with acute respiratory disease. Guinea-pig antiviral immunoglobulins as the secondary antibodies, and horseradish peroxidase-labelled swine anti-rabbit immunoglobulins (EIA), or 125I-labelled sheep anti-rabbit IgG (RIA) as the indicator antibodies. A total of 174 nasopharyngeal specimens collected by mucus extractor were tested, and the results were compared with those obtained by a routinely used immunofluorescence (IF) technique. The same number of positive specimens were achieved by the EIA and the RIA and 3/4, 4/4, and 19/20 immunofluorescence (IF)-positive nasopharyngeal specimens were positive by the parainfluenza type 1, 2 and 3 immunoassays respectively. In addition, four parainfluenza type 1 and three parainfluenza type 3 virus-positive specimens were found by the immunoassays out of 146 parainfluenza IF-negative specimens. The type-specificities of the parainfluenza immunoassays were confirmed by showing that no cross-reactions occurred when purified immunizing antigens and the EIA- and RIA-positive clinical specimens were cross-tested. The results indicate that parainfluenza type-specific antigens can be detected directly in nasopharyngeal specimens by the immunoassays and the preliminary findings with a small number of positive specimens suggest that these assays have a diagnostic potential which is similar or slightly better than the IF techniques.

Acute Disease↗

A second receptor binding site on human parainfluenza virus type 3 hemagglutinin-neuraminidase contributes to activation of the fusion mechanism.

The hemagglutinin-neuraminidase (HN) protein of paramyxoviruses carries out three discrete activities that each affect the ability of HN to promote viral fusion and entry: receptor binding, receptor cleaving (neuraminidase), and triggering of the fusion protein. The interrelationship between the receptor binding and fusion-triggering functions of HN has not been clear. For human parainfluenza type 3 (HPIV3), one bifunctional site on HN can carry out both receptor binding and neuraminidase activities, and this site's receptor binding can be inhibited by the small receptor analog zanamivir. We now report experimental evidence, complemented by computational data, for a second receptor binding site near the HPIV3 HN dimer interface. This second binding site can mediate receptor binding even in the presence of zanamivir, and it differs from the second receptor binding site of the paramyxovirus Newcastle disease virus in its function and its relationship to the primary binding site. This second binding site of HPIV3 HN is involved in triggering F. We suggest that the two receptor binding sites on HPIV3 HN each contribute in distinct ways to virus-cell interaction; one is the multifunctional site that contains both binding and neuraminidase activities, and the other contains binding activity and also is involved in fusion promotion.

Alanine↗

Diagnosis of parainfluenza virus infection in children and older patients by detection of specific IgM antibody.

The significance of detecting specific antibody of the IgM class for the diagnosis of parainfluenza infections was examined. Paired sera from 763 children and adults admitted to the hospital for acute respiratory disease were tested for significant antibody titer rises in the hemagglutination inhibition (HI) test and for specific IgM antibody with the hemadsorption immunosorbent techniques (HIT). Sera were collected during two 6-month periods, January through June, 1982 and 1983. Evidence of parainfluenza infections was found in 122 patients (16%): 83 (25%) in 1982 and 39 (9.1%) in 1983. The HIT was superior to the HI test for detection of parainfluenza infection, in particular in infants and aged patients, 94 patients were positive only in the HIT test, 12 in the HI test, and 16 in both tests. In a control group of 120 persons (time- and age-matched to the patients of 1982) admitted for nonrespiratory illness, six (5%) showed parainfluenza IgM in their serum. Blocking experiments and retrospective clinical information indicated that the IgM antibody detected in these individuals is specific IgM acquired after a mild parainfluenza infection. Most (66%) patients showed IgM antibody titer rises or high titers (greater than 1,280) in both sera, and in 23%, a fall in IgM antibody titer was found. Detection of specific IgM antibody by HIT permitted early presumptive diagnosis in 71% of the patients with parainfluenza infection. IgM antibody persisted for 2-11 weeks. The HIT appears to be an important supplement for the diagnosis of parainfluenza infections.

Adolescent↗

Parainfluenza virus infection of cultured airway epithelial cells.

Studies of the cellular effects of respiratory viruses have generally used cultures of non-airway (particularly renal) epithelial cells. This requires the assumption that, despite the marked differences between renal epithelium and airway epithelium, the virus-host cell interactions in cultures of renal epithelium will be relevant to those in airway epithelium. To study viral infection of airway epithelial cells, we removed the epithelial cells from ferret tracheas using 0.1% pronase solution, and plated them at a density of 5 X 10(5) cells/cm2 in collagen-coated plastic tissue culture wells. Cultures grew to confluence after 5-7 days. Viral inocula, consisting of supernatants from parainfluenza type 1-infected rhesus monkey kidney cell monolayers, were added to the culture medium in a concentration 10(3) times that sufficient to produce infection in 50% of rhesus monkey kidney monolayers (TCID50). Cytopathic changes, consisting of cellular elongation and detachment, became apparent after 3-6 days, at which time the medium contained 5 X 10(8) TCID50/ml. The monolayer appeared to be uniformly infected as revealed by adsorption of guinea pig erythrocytes. Specific immunofluorescence revealed uniformly positive staining for parainfluenza type 1 antigens. The ability to infect pure cultures of airway epithelial cells with viruses will allow us to examine the effects of these viruses on epithelial cell function, and to study virus-host cell interactions in cell cultures derived from the natural host cell.

Animals↗

Immune responses and protection induced by DNA vaccines encoding bovine parainfluenza virus type 3 glycoproteins.

This study was designed to assess the parameters influencing the magnitude and type of immune responses generated to plasmids encoding the hemagglutinin/neuraminidase (HN) and fusion (F) proteins of bovine parainfluenzavirus type 3 (BPIV3). Mice immunized with plasmids expressing HN or F under control of the Rous sarcoma virus long terminal repeat promoter were primed, but they did not develop measurable immune responses. In contrast, strong humoral and cellular immune responses were induced with constructs containing the human cytomegalovirus immediate-early promoter and intron A. After immunization with both HN- and F-encoding plasmids, enhanced responses were observed. Analysis of in vitro protein synthesis confirmed that the presence of the intron is crucial for the expression of the BPIV3 HN gene. Plasmid encoding HN induced significantly higher serum antibody titers by intradermal injection than by intramuscular delivery, whereas antigen-specific T cell proliferation was stronger in intramuscularly injected mice. Both the isotype ratios and the cytokine profiles indicated a Th1-type response after intramuscular immunization and a mixed to Th2-type response in intradermally immunized mice. A plasmid encoding a truncated, secreted form of HN induced a Th2-type immune response, regardless of the route of delivery. In cotton rats, HN- and F-encoding plasmids conferred protection from BPIV3 challenge.

Animals↗

Evolution of in vitro persistence of two strains of canine parainfluenza virus. Brief report.

Lytic stock virus (CPI+) exhibited prominent CPE in Vero cell monolayers. The non-syncytial giant cell forming strain (CPI-) was isolated from brain tissues of a dog after experimental infection with syncytial giant cell-forming (CPI+) virus. In mixed infection experiments, CPI(-) virus interfered with the replication of CPI(+) virus in that simultaneously infected cells showed strand-forming CPE typical of CPI(-) and reduction of CPI(+) yield. The CPI(-) virus established immediate in vitro persistence, followed by a severe crisis at passage (p) 8 and several similar crises until p 31; no further crises occurred throughout the remaining observation period (p 80). The CPI(+) did not establish immediate in vitro viral persistence and experienced several lytic crises until p 29. No further crises were observed throughout the observation period (p 100).

Animals↗