Viral respiratory diseases: vaccines and antivirals.

Acute respiratory diseases, most of which are generally attributed to viruses, account for about 6% of all deaths and for about 60% of the deaths associated with all respiratory disease. The huge cost attributable to viral respiratory infections as a result of absenteeism and the disruption of business and the burden of medical care makes control of these diseases an important objective. The viruses that infect the respiratory tract fall taxonomically into five viral families. Although immunoprophylaxis would appear to be the logical approach, the development of suitable vaccines has been confronted with numerous obstacles, including antigenic drift and shift in the influenzaviruses, the large number of antigenically distinct immunotypes among rhinoviruses, the occurrence after immunization of rare cases of a severe form of the disease following subsequent natural infection with respiratory syncytial virus, and the risk of oncogenicity of adenoviruses for man. Considerable expenditure on the development of new antiviral drugs has so far resulted in only three compounds that are at present officially approved and licensed for use in the USA. Efforts to improve the tools available for control should continue and imaginative and inventive approaches are called for. However, creativity and ingenuity must operate within the constraints imposed by economic, political, ethical, and legal considerations.

Simian virus nomenclature, 1980.

Approximately 75 simian viruses, counterparts of other animal viruses, are recognized. Nomenclature of these isolates, in general, consists of an SV (simian virus) or SA (simian agent) numerical series with no attempt to group them according to virus families. The biologic characteristics of these viruses indicate they may be classified into recognized families and groups. A simple sequential numerical designation is recommended as a nomenclature within virus families and groups. Finalization of nomenclature would follow approval by the Study Groups of the International Committee on Taxonomy of Viruses.

Immunoperoxidase staining for detection of Colorado tick fever virus, and a study of congenital infection in the mouse.

Indirect immunoperoxidase (IP) staining was evaluated for sensitivity and specificity in detecting Colorado tick fever (CTF) virus antigen in infected cell cultures and infected mouse tissues, and then was applied to a study of congenital CTF infection in mice. The sensitivity of IP staining was comparable to that of immunofluorescence staining in detecting CTF antigen in infected cell cultures. Endogenous peroxidase activity of mouse tissues caused nonspecific reactivity in the IP system, but this could be abolished by treatment with sodium azide and hydrogen peroxide without destroying CTF antigen. Offspring of mice infected with CTF virus during the 2nd week of pregnancy showed a highly significant increase in the incidence of stillbirths and neonatal deaths as compared with offspring of uninfected controls. CTF antigen or virus was demonstrable in only a low proportion (7%) of embryos, ill newborns or stillborns examined, but a high proportion of mice examined at a time when maternal antibody would be lost (6 and 12 weeks) showed CTF antibody, indicating a higher incidence of infection. IP staining showed potential for use in studies of viral pathogenesis in the mouse model.

Possible pitfalls in the study of IgG receptors produced by herpesvirus-infected cells. Brief report.

The presence of specific antibodies to human herpesviruses in certain purified human IgG preparations from commercial sources may produce misleading results when such preparations are used in studied on the IgG receptors produced by virus-infected cells.

Comparative sensitivity of various cell culture systems for isolation of viruses from wastewater and fecal samples.

In efforts to define the most sensitive cell culture systems for recovery of viruses from wastewaters, 181 samples were inoculated in parallel into tube cultures of various cell types and were plaqued in bottle and petri dish cultures of three types of monkey kidney cells. Polioviruses were recovered most frequently in the RD line of human rhabdomyosarcoma cells, group A coxsackieviruses in RD and human fetal diploid kidney (HFDK) cells, group B coxsackieviruses in the BGM line of African green monkey kidney cells, echoviruses in RD and primary rhesus monkey kidney (RhMK) cells, and reoviruses in RhMK cells. BGM cells were unsatisfactory for recovery of viruses other than polioviruses and group B coxsackieviruses, and a line of fetal rhesus monkey kidney (MFK) was not a satisfactory substitute for primary RhMK. With RhMK cells, comparable numbers of virus isolations were made in tube cultures and in plaque assays conducted in bottle cultures, but with BGM and MFK cells, fewer isolations were made by plaquing than by inoculation of tube cultures. In comparative plaque assays on fecal samples under three different overlays in bottle and plate cultures of RhMK, BGM, and MFK cells, it was found that plaquing in the most sensitive system, RhMK, was less efficient for virus recovery than was inoculation of tube cultures of RhMK or HFDK cells. Overall, plaque assays performed in petri dishes in a CO(2) incubator yielded fewer virus isolates than did parallel plaque assays performed in closed bottle cultures. Other limitations of plaque assays for recovery of human enteric viruses are discussed.

Photochemical inactivation of DNA and RNA viruses by psoralen derivatives.

Western equine encephalitis virus, and RNA virus, and herpes simplex virus type I, a DNA virus, were efficiently inactivated in less than I min by exposure to long-wave ultraviolet light (320 to 380 nm) in the presence of several psoralen derivatives. The psoralen photochemical reaction was chosen for study due to its known specificity for nucleic acids. Neither the light nor any of the drugs alone caused appreciable inactivation. The inactivation kinetics and dependence on light intensity and on different derivatives of psoralen were studied. The high solubility of a new aminomethyl psoralen derivative was found to be advantageous in the photochemical inactivation of the RNA virus, but was not in the case of the more easily inactivated DNA virus. Within its limited solubility range trimethylpsoralen was superior to its aminomethyl derivative on a molar basis for the inactivation of both types of viruses under most of the conditions studied.

Analysis of antibody assay methods and classes of viral antibodies in serodiagnosis of cytomegalovirus infection.

Forty-nine serum pairs with antibody to cytomegalovirus (CMV) were evaluated for rises in antibody titer (greater than or equal to fourfold) by indirect hemagglutination (IHA) and complement fixation (CF), using a freeze-thaw antigen (FT) and a glycine extract antigen (GE). In this sample CF-FT detected more rises in antibody titer than did CF-GE. IHA detected the least number. The apparent reason for stationary antibody titers with CF-GE and IHA was the presence of high antibody titers in the first serum specimen. Separation of immunoglobulin classes of 20 serum pairs by sucrose gradient centrifugation indicated that these antibodies with IHA were of the immunoglobulin M (IgM) class and those with CF-GE were of the IgG class. By separation of immunoglobulin classes, rises in IgG CMV antibody titers were seen with IHA, rises not observed in the whole serum because of high IgM antibody titers in the first serum specimen. Absence of rises in antibody titers with CF-FT was due in part to too early sampling of the second serum specimen (less than 21 days) and in part to an apparent inability of some individuals to respond with antibody reactive with FT antigen. CF-GE and CF-FT antibodies of the IgM class were detected in some sera, usually in specimens collected more than 10 days after the onset of symptoms. Although reactive with CMV antigen, the specificity of these IgM antibodies in relation to rheumatoid factor requires clarification.

Role of rheumatoid factor in complement fixation and indirect hemagglutination tests for immunoglobulin M antibody to cytomegalovirus.

Absorption of immunoglobulin M (IgM)-rheumatoid factor (RF) from serum samples by reaction with insolubilized gamma globulin reduced the complement-fixing (CF) antibody titer to cytomegalovirus (CMV) antigen to less than 1:2 in the IgM fraction of some, but not all, sera. Thus, IgM-CF activity in some sera appeared to be due to specific IgM anti-CMV antibody and in other sera to complexes of IgM-RF with antiviral IgG antibody. Prozones were present in the CF tests on IgM fractions. Increasing the concentration of antigen from 2 to 4 U reduced the prozone titer by one or two double dilutions. This observation suggested that a competition for antigen may be operating at low dilutions of IgM antibody fractions. Removal of RF had little or no effect on the reaction of the IgM fraction of sera with CMV by the indirect hemagglutination test.

Comparison of immunofluorescence and immunoperoxidase staining for identification of rubella virus isolates.

To explore possible advantages which immunoperoxidase (IP) staining might have over immunofluorescence (IF) staining for identifying rubella virus isolates, direct comparative studies were done on the same coded clinical materials using the same rubella immune rabbit serum as the primary antiserum in both systems. The rubella immune rabbit serum and conjugated anti-rabbit immune globulins could be used more dilute in the IP system than in the IF system. Both IP and IF staining detected rubella antigen in all specimens which were positive by interference. IP staining also detected low levels of rubella antigen in a few additional specimens which had originally been positive for rubella virus, but which on retesting were negative by interference and IF staining. With second-cell-culture-passage material, IP and IF staining showed comparable specificity, and the few specimens which reacted nonspecifically generally did so in both systems. Cell cultures inoculated directly with urine specimens showed greater nonspecificity by IP than by IF, but this activity could be abolished by pretreatment with sodium azide and peroxide; other methods tried for inactivating endogenous peroxidase activity destroyed rubella antigen as well. The intensity of staining for positive specimens was comparable in the two systems. However, more antigen was demonstrable in both systems when BHK-21 cells were inoculated as a cell suspension and then permitted to grow into monolayers than when the same specimens were inoculated into preformed monolayers. IP staining was considered to be a highly satisfactory alternative to IF staining for identification of rubella virus isolates.

The development of Colorado tick fever virus within cells of the haemopoietic system.

Electron microscopic examination of haemopoietic liver tissue from mice infected in utero or when newborn showed inclusions of Colorado tick fever virus within erythroblasts, reticulocytes and erythrocytes. Inclusions were also seen within erythroblastoid cells undergoing mitosis. Other evidence of virus replication within erythropoietic cells was the presence of intracytoplasmic and intranuclear fibres, which have been shown to be associated with Colorado tick fever virus replication. The findings reported here support the hypothesis that virus replication within infected erythropoietic cells occurs concurrently with differentiation of the infected cell, resulting in the presence of virions within erythrocytes.

Anti-complement immunofluorescence test for antibodies to human cytomegalovirus.

An anti-complement immunofluorescence (ACIF) test that detects human cytomegalovirus (CMV) antigen in the nuclei of infected cells was used for assay of CMV antibodies in human sera. Various factors influencing the sensitivity and specificity of the ACIF test system were investigated, and results were applied to the development of a procedure which could be completed in a relatively short length of time and gave reproducible results. Results obtained in the ACIF test were compared with those obtained in complement fixation, indirect hemagglutination, and neutralization tests, and the ACIF test was shown to be suitable for detection of significant antibody titer rises and stationary levels of CMV antibody. Heterotypic antibody responses were not seen with sera from other human herpesvirus infections. The nonspecific cytoplasmic staining that occurs in indirect immunofluorescence tests for CMV did not occur in the ACIF system, and sera that were anti-complementary in complement fixation tests could be examined satisfactorily by ACIF. Thus, the test is a valuable supplemental or back-up procedure for the serodiagnosis of CMV infection.

Improved glycine-extracted complement-fixing antigen for human cytomegalovirus.

High-titered, sensitive, and stable complement-fixing antigens for human cytomegalovirus were consistently produced from human diploid fibroblast cells infected at a high multiplicity of virus, harvested after 7 days of incubation, and sonically treated immediately in 0.1 M glycine buffer, pH 9.5.

Distinction between envelope antigens of murine xenotropic and ecotropic type C viruses by immunoelectron microscopy.

The indirect ferritin-labelled antibody technique was used to determine the reactivity of an antiserum prepared against the NZB xenotropic virus with three murine xenotropic viruses, a feline xenotropic virus and a murine ecotropic virus. The envelope antigens of the xenotropic type C viruses isolated from the NZB, NIH Swiss and C57L mice were tagged with ferritin. The feline RD114 virus was not. Gross murine leukaemia virus was tagged, but only at high serum concentrations. The cross-reactivity titre of Gross virus to anti-NZB serum was removed by a serum dilution which was still reactive to xenotropic viruses. This difference in reactivity titres between a xenotropic and an ecotropic virus was sufficient to distinguish one from the other in doubly infected cultures. Specific tagging of membrances of cells infected by xenotropic virus was also observed.

Complications and immunological studies of measles virus infection in antithymocyte-treated hamsters.

A study was done to investigate possible late central nervous system (CNS) complications of latent measles infection in hamsters. Signs of CNS disease, consisting of myoclonus and paralysis, occurred in some weanling hamsters inoculated intraperitoneally at 3 or 9 days of age with antithymocyte serum (ATS) and measles virus, but no late complications in adult life attributable to measles virus were seen. A single series of ATS injections plus an injection of measles virus resulted in sustained antibody formation postulated due to establishment of a latent viral infection, whereas similar treatment with normal rabbit serum plus virus caused no or minimal antibody response. The majority of hamsters receiving ATS as newborns and ATS plus virus as weanlings also did not produce antibody. This differential effect of ATS may be due to combined elimination of suppressor and helper cells in the latter ATS-treated group and of suppressor cells only in the former ATS-treated group. Cellular immunity could not be evaluated since lymphocytes from inoculated and uninoculated hamsters released equal and variable amounts of 51Cr for both infected and noninfected labeled, allogeneic hamster target cells.

Complement-fixing reactivity of Varicella-Zoster virus subunit antigens with sera from homotypic infections and heterotypic Herpes simplex virus infections.

Various subunit antigens of varicella-zoster (V-Z) virus were examined for complement-fixing (CF) activity with sera from homotypic infections and from herpes simplex virus (HSV) infections in which a CF antibody titer rise was demonstrated with crude V-Z antigen. The subunit antigens included nucleocapsids, envelopes, a soluble antigen produced from infected culture fluids by sucrose density gradient centrifugation, a soluble antigen produced by reducing the volume of clarified infected culture fluids, a soluble antigen derived from infected cell lysates, a "viral" antigen consisting largely of enveloped particles with a few nucleocapsids, and a cell membrane-associated antigen. None was more suitable than crude V-Z antigen for serodifferentiation of V-Z virus and HSV infections. The envelope antigen, cell membrane antigen, and the soluble antigen prepared by density gradient centrifugation showed little reactivity with sera from varicella and HSV infections, but gave high antibody titers with sera from zoster infections, suggesting that a secondary V-Z virus infection is required to produce an antibody response to these subunit antigens. Patients with varicella and zoster infections and the selected patients with HSV infections all showed significant CF antibody responses to the other V-Z subunit antigens.

Classic slow virus diseases.

Two human disease, kuru and Creutzfeldt-Jakob disease, and two animal diseases, scrapie and mink encephalopathy, comprise the group designated the subacute spongiform encephalopathies. Studies on these four classic conditions have generated a new philosophy, new concepts, and new technology that provide a basis for the study of chronic diseases and latent infections of man and animals. These aspects are discussed more broadly and in variable detail in the references listed on the following page.