Pathogenesis and transmission of kilham rat virus infection in rats

The Kilham rat virus (RV) was found to produce mortality in newborn rats after intracerebral, intravenous, or subcutaneous administration of virus. Both infectious virus and viral hemagglutinins were detected in a variety of tissues and in the blood and urine of experimentally infected rats. Contact control rats housed with infected littermates did not develop disease but did produce antibody to RV. Horizontal virus transmission was also evidenced by the seroconversion of antibody-negative mothers whose litters were infected with RV. The level of maternal antibody was found to be the determining factor in the susceptibility or refractiveness of newborn rats to RV infection. If the mother had no detectable hemagglutination-inhibiting (HI) antibody titer (less than 10) or a low antibody titer (10 or 20), her offspring were highly susceptible to RV. However, the litters of rats with HI titers of 40 or greater were afforded protection when challenged with RV; the higher the maternal antibody level the more solid was the protection conferred. Vertical transmission of RV was also demonstrated. Litters born of mothers infected with RV several days before delivery died within 7 to 9 days of a disease identical to that seen in infected newborns and virus was recovered from a variety of tissues. Results of mother-litter exchange experiments also indicated vertical transmission (rather than transmission through milk) occurs, since litters of infected mothers developed the disease when nursed by normal mothers, whereas litters of normal mothers remained normal although they were nursed by infected mothers.     

IgA Deficiency and Influenza Infection

A prospective study of influenza infection was carried out on 90 blood donors deficient for serum IgA as tested with double immunodiffusion. Half of them lacked IgA even by radioimmunoassay (RIA). A correlation existed between serum haemagglutination-inhibiting (HI) antibody and resistance to infection, suggesting that the serum HI antibody was an important determinant of protection. The rate of infection as evidenced by a fourfold or greater rise in HI titre, was about the same in the RIA-negative and RIA-positive donors and only slightly higher than the corresponding rate in pregnant women and in a ship's crew.     

Immunity to influenza in Ferrets

Summary Ferrets inoculated with 300 CCA of inactivated influenza A2/Hong Kong virus vaccine did not produce serum HI antibody, and were completely susceptible to subsequent infection with live A2/Hong Kong virus. Immunization of ferrets with A2/Hong Kong vaccine in Al(OH)₃ induced low levels of serum HI antibody; these animals showed a slightly reduced febrile reaction and reduced titres of virus were recovered from nasal washings following challenge virus infection. Ferrets immunized with inactivated A2/Hong Kong vaccine in Freund's incomplete adjuvant produced relatively high titres of serum HI antibody, but did not produce local antibody detectable in nasal washings. After challenge infection, these animals showed a modified febrile reaction, lower titres of virus were recovered from nasal washings and nasal symptoms were reduced. These results, together with results of similar studies, indicated that the degree of immunity to challenge virus infection was related to the titre of serum HI antibody. However, none of the methods used to induce serum HI antibody gave as solid an immunity as found following live virus infection, although immunization could induce levels of serum HI antibody comparable to that found following virus infection.     

Single radial hemolysis test for rubella immunity and recent infection.

The single radial hemolysis (SRH) test was compared with the hemagglutination inhibition (HI) test for establishing rubella immune status and diagnosing recent infection. Correlation between mean SRH diameters and HI titers greater than or equal to 1:8 was high (R = 0.99). It is suggested that a level of greater than or equal to 5 IU represents low-level antibody and that greater than or equal to 15 IU is a conservative threshold for designation of immunity. Of 343 sera tested, only 1 false-positive was found by SRH with the 5 IU cutoff level. The SRH test could detect serum antibody levels as low as 2.5 IU, whereas 15 IU was generally the limit of resolution of the HI test. Data from sucrose density gradient fractionation of serum demonstrated that neither rubella-specific immunoglobulin M (IgM) nor early postinfection HI-reactive IgG was detected by SRH. However, diagnostic changes in antibody titer measured by SRH were in general greater than those measured by HI. The SRH test showed diagnostic titer changes in some sera containing specific IgM for which no such changes were detected by the HI test. A 2.5-mm difference in hemolytic zone diameters (a fourfold rise in international units) between acute- and convalescent-phase serum pairs was chosen as being of diagnostic significance. This difference was less than the minimum observed difference of 2.9 mm from 59 serum pairs showing diagnostic changes by HI and far exceeded (greater than 3.6 standard deviations) the within-test and individual variability seen for 95 pregnant women from whom samples were obtained during each trimester.     

A contribution of cellular immunity to protection against influenza in man

The degree of lymphocyte transformations and leukocyte migration inhibition (LMI) in the presence of inactivated A/Scotland/74 (H3N2) influenza virus vaccine was measured in blood samples collected from 56 medical student volunteers. At the same time the volunteers were skin tested, using the same vaccine. Using the antigenically similar WRL 105 (H3N2), recombinant influenza virus, the level of haemagglutination-inhibiting (HI) antibodies in serum, and neutralizing antibodies in nasal washings collected from the volunteers, were also determined. Each volunteer was then inoculated with live, attenuated WRL 105 influenza virus vaccine and infections demonstrated by virus isolations and serology.Correlations between the ability to infect the volunteers and the various parameters of humoral and cellular immunity were then determined. The results showed a good correlation between the level of serum HI antibody and infection. Thus 16 of 20 volunteers with serum HI antibody titres of 110, but only 6 of 20 volunteers with antibody levels of 130, showed evidence of infection. No direct correlation was observed between any of the other parameters measured and infection by WRL 105 virus. However, when the LMI and serum HI antibody levels were considered together, a contribution of cellular immunity, as measured by the LMI test, could be found. Of 19 volunteers with low serum HI antibody and low LMI levels, 16 were infected, whereas of 13 volunteers with low HI antibody, but with high LMI levels, only 6 showed evidence of infection with WRL 105 influenza virus.     

Influenza infection in ferrets: role of serum antibody in protection and recovery.

The passive administration of ferret antiserum to Ao (H0N1) influenza virus failed to protect the recipient ferrets from subsequent infection with homologous virus. This susceptibility to infection was observed even when the passively acquired serum hemagglutination inhibition (HI) titer was similar to peak convalescent titers. It is therefore concluded that serum antibody alone is probably not a major factor in the prevention of influenza infection. This does not rule out a possible role for serum antibody in prevention of illness. Subsequent to infection, ferrets that had received passive antisera failed to develop high levels of serum HI antibody. In fact, many had no detectable serum antibody (less than 1:8). These animals shed virus for periods of time quite similar to those of infected control animals, which did develope serum antibody. From these data it was concluded that detectable serum HI antibody does not play a significant role in the recovery of ferrets from influenza infection. Interferon was present in high concentrations in the secretions a few days prior to cessation of virus shedding, but it is not clear whether this was the cause of the recovery or merely a concomitant event. Twenty-one days after initial infection two-thirds of the ferrets that had received passive antibody and all control animals were immune to reinfection with the homologous influenza virus. Since the former group had little or no detectable serum HI antibody but most members were immune, there must be some other host mechanism to account for the immunity.     

Immunity to influenza virus infection induced by heterologous,inactivated vaccines

The ability of inactivated influenza A vaccines to induce serum HI antibody and immunity to challenge infection was studied in hamsters and in volunteers. Groups of hamsters were immunized with 200 IU of influenza virus A/Scotland/74, A/Port Chalmers/73, A/England/72, or A/Hong Kong/68. The serum HI antibody response of animals to, and immunity to challenge infection was directly related to the known relationship between the vaccine and test viruses. Thus, hamsters given A/Hong Kong/68 or A/England/72 vaccine produced serum HI antibody and immunity to A/Hong Kong virus infection, and animals given A/Scotland/74, A/Port Chalmers/73, and A/England/72 produced antibody and immunity to A/Scotland infection.In a volunteer study, groups of students were immunized with 400 IU of the same vaccines as used above. The ability to infect these volunteers with WRL 105 virus given 4 weeks later was directly related to the vaccine-induced serum HI antibody to the challenge virus. The highest titers of serum HI antibody to A/Scotland virus were found in volunteers inoculated with homologous vaccine, lower titers were found in volunteers given A/Port Chalmers or A/England/ 72 vaccine and the lowest levels were seen in volunteers given A/Hong Kong/68 vaccine: the largest number of infections by the challenge virus was seen in volunteers given A/Hong Kong/68 vaccine, less were observed in volunteers given A/England/72 vaccine, and least were found in groups given A/Port Chalmers or A/Scotland/74 vaccine. Compared with the incidence of infection in volunteers given B/Hong Kong/73 vaccine, all groups given heterologous influenza A vaccines showed some immunity to challenge infection.     

Direct enzyme immunoassay for detection of specific IgG antibody to rubella virus by use of labelled antigen

A new solid phase enzyme immunoassay (EIA) for detection of rubella-specific immunoglobulin G (IgG) antibody was developed. The test uses polystyrene microtiter strips coated with rabbit anti-human IgG immunoglobulins as the solid phase and an enzyme-labelled semipurified rubella antigen as indicator. The direct EIA was compared with hemagglutination inhibition (HI), single radial hemolysis (SRH), radioimmunoassay (RIA) and time-resolved fluoroimmunoassay (TR-FIA) using 52 serum specimens from patients with remote rubella infection. The overall agreement of direct EIA with HI was 96.1%, with SRH and RIA 98.1% and with TR-FIA 100%. The linear regression coefficient varied from 0.77 to 0.91, the best being obtained with direct EIA and SRH. The direct EIA was also suitable for diagnosis of acute infections, as a significant increase in antibody levels was detected in all paired specimens tested from patients with acute rubella infection. The sensitivity and were comparable to those of the assays employed. An advantage of the present assay is that the same method and same labelled antigen can be used to test for different classes of antibody using simply a solid phase with capture antibodies of different chain specificity.     

Modulation of lethal and persistent rat parvovirus infection by antibody

Summary Two day-old athymic (rnu/rnu) and euthymic (rnu/+) rat pups nursing immune or non-immune dams were inoculated oronasally with the Yale strain of rat virus (RV-Y). All athymic and euthymic pups (57/57) from immune dams remained clinically normal, whereas 51 of 66 athymic and euthymic pups from non-immune dams died within 30 days. Infectious RV was detected by explant culture in 12 of 15 surviving pups of both genotypes from non-immune dams 30 days after inoculation, but in none of the 57 surviving pups from immune dams. RV-Y DNA was detected by Southern blotting in kidneys of surviving athymic pups from non-immune dams but was not detected in pups from immune dams. Euthymic pups from immune dams appeared not to produce endogenous antibody to RV after virus challenge, whereas euthymic pups from non-immune dams produced high-titered RV immune serum. Pups of both genotypes given immune serum prior to or with RV were fully protected from disease and persistent infection, whereas pups given immune serum 24 hours after RV were partially protected. These studies show that RV antibody offers significant protection against lethal and persistent RV infection.     

Comparison of the hemagglutination inhibition procedure and an enzyme-linked immunosorbent assay for detection of specific antibodies to pneumonia virus of mice in experimentally infected laboratory rats.

An enzyme-linked immunosorbent assay (ELISA) and the hemagglutination inhibition (HI) test were used to evaluate the response of laboratory rats to experimental infection with pneumonia virus of mice. The ELISA procedure was more sensitive than the HI test and detected very low levels of antibodies early in the course of infection. At 5 days postinfection, ELISA detected antibody increases in five of five animals, whereas only two of five increases were detected by the HI test. At 9 days postinfection, the HI test failed to detect one titer increase measured by ELISA. Later during the course of infection, increases were detected by both tests. The ELISA procedure was, in general, more sensitive for detecting low levels of antibody than was the HI test, but was equal in sensitivity when high titers of antibody were measured.     

Single-serum diagnosis of rubella by combined use of the hemagglutination inhibition and passive hemagglutination tests.

We explored the feasibility of using the passive hemagglutination (PHA) test in combination with the hemagglutination inhibition (HI) test for the single-serum diagnosis of rubella. We found by sedimentation analysis of the serum that early 7S antibody produced within 1 month after the onset of the rash had high HI but much lower PHA titers, whereas the PHA titers of antibody produced later were slightly higher than the HI titers. (19S antibody in the early serum had some PHA activity.) This disparity between the HI and PHA activities of the early 7S antibody could be used for the routine diagnosis of rubella. A comparison of the HI titers of the test serum with the PHA titers of mercaptoethanol-treated serum constitutes a simple method for determining whether the serum sample was taken shortly or remotely after the infection.     

Immunity following intranasal administration of an inactivated,freeze-dried A/England/42/72 vaccine

A group of 23 student volunteers were each inoculated intranasally with 400 IU of inactivated, freeze-dried A/England/42/72 vaccine. Only one volunteer showed a four-fold rise in serum HI antibody following immunization, and the mean increase in serum HI antibody (gmt) for all volunteers did not increase two-fold. Thirteen of the volunteers developed detectable levels of nasal wash neutralizing antibody after immunization; local antibody was most commonly found in volunteers who produced a detectable but less than four-fold fise in serum antibody titre, and who produced nasal washings with relatively high concentrations of protein and secretory IgA. Four weeks after immunization, the vaccinees and a matched group of control subjects were inoculated with attenuated A/England/42/72 (MRC-7) virus. Evidence of infection was found in 14 of 23 (61 per cent) of control subjects and in seven of 23 (30 per cent) of immunized volunteers. This result showed a significant protection (P = 0.04) against challenge virus infection for volunteers given intranasal vaccine.     

Passive hemagglutination test for measles immunity and serodiagnosis.

A passive hemagglutination (PHA) test for measles was evaluated in comparison with hemagglutination inhibition (HI) and neutralization (NT) tests. The PHA test determines exclusively the level of antibody directed to the hemagglutinin protein of measles virus. The ratio of PHA to HI titer was 1 to 32 (geometric mean, 6.5) for the first 5 weeks of infection but declined to near unity thereafter. It gradually increased again to 4 to 32 (geometric mean, 11.7) over several years. The initial high PHA titer relative to the HI titer was most likely due to the presence of the immunoglobulin M antibody known to be efficient in agglutination, because 2-mercaptoethanol (2ME) treatment of sera reduced the PHA titer to a level similar to that of the HI titer. The PHA titer in sera obtained after the convalescent phase was insensitive to 2ME, and the relative increase in the PHA over the HI titer was presumably a result of increased antibody avidity. In some individuals, the HI titer fell to below detectable levels several years after either natural infection or vaccination, but the PHA as well as the NT titer remained positive. The PHA titer was therefore a more reliable and more sensitive indicator of immune status against measles than the HI titer. The decrease in PHA titer by 2ME treatment provided evidence of a current or very recent infection. PHA was found to be useful both for assessing immunity status and for serodiagnosis.     

Clinical and serologic effects of live attenuated serum inhibitor-resistant influenza B vaccine in seronegative adults.

The clinical effects, nasal and serum antibody responses, and virus excretion of a live attenuated serum inhibitor-resistant influenza B virus vaccine, R75, was evaluated in 43 seronegative healthy adults by a random double-blind study. Symptom responses were minimal and were not significantly different between vaccine and placebo groups. No fevers, abnormalities in physical examination or laboratory testing developed during 4 weeks of observation. Among vaccinees, 10 (48%) developed serum hemagglutination-inhibition (HI) antibodies, 16 (76%) developed serum neutralization (N) antibodies and 4 (19%) developed nasal N antibodies. The GMT responses from study day 0 to day 28 were 4.0 to 10.4 for serum HI, 1.8 to 9.8 for serum N, and 1.0 to 1.4 for nasal N. There were no significant titer changes in the placebo group. No virus excretion was detected. Although there are some questions concerning the relationship of antibody levels to protection, the low antibody responses in this study are an indication that R75 is not sufficiently immunogenic.     

Simple hemagglutination inhibition test for the diagnosis of toxoplasmosis.

A simple hemagglutination inhibition (HI) test for the serological diagnosis of toxoplasmosis has been developed and evaluated. A total of 84 human and 120 mouse serum samples were tested by the newly developed HI test and compared with an immunoglobulin G-indirect fluorescent antibody test. Statistical analysis of serum titers obtained by using the HI test and the immunoglobulin G-indirect fluorescent antibody test showed a correlation coefficient of 0.89. The diagnostic efficacy of HI when compared with the immunoglobulin G-indirect fluorescent antibody diagnostic test results was 96.43% for human sera and 100% for mouse sera. The unique hemagglutination antigen, derived from Toxoplasma gondii (Rh strain) exotoxin, spontaneously binds with mouse or rat erythrocytes, causing the hemagglutination reaction. In this study, 2, 4, or 8 hemagglutinating units of T. gondii exotoxin was used with Swiss/Webster mouse erythrocytes as an indicator for the HI assay. The results indicate that 8 hemagglutinating units is optimal because this concentration has the least unexplained variability. T. gondii exotoxin was stable for at least 18 months at -70 degrees C. The Toxoplasma HI test we report in this paper is shown to be a fast, easy, highly specific, and sensitive test for the diagnosis of toxoplasmosis.     

Use of immunoenzyme analysis employing a staphylococcal protein A conjugate with peroxidase in the serodiagnosis of influenza

A test-system was developed on the basis of solid-phase enzyme-immunoassay using protein A/peroxidase conjugate for the determination of antibody levels to influenza virus in sera of humans who had experienced a natural infection or received a live influenza vaccine. The accurate observation of the test conditions was demonstrated to give the results well correlating with those of the HI test. The use of isolated hemagglutinin as the antigen considerably increased the specificity of the enzyme-immunoassay and in a number of cases detected a 4-fold or higher rise of antibody titres to hemagglutinin in paired sera of the vaccinees where the HI test showed no rise in antibody titres.     

Enzyme immunoassay for detection of antibodies against eastern equine encephalomyelitis virus in sentinel chickens

We developed an enzyme immunoassay (EIA) for the detection of immunoglobulin M (IgM) and IgG subclass antibodies directed against eastern equine encephalomyelitis (EEE) virus in chickens. The assays were compared with the serum plaque reduction neutralization test (PRNT) and the hemagglutination inhibition (HI) test for ability to detect antibodies against EEE virus in laboratory-infected birds. No cross-reactivity was detected in serum from chickens inoculated with St. Louis encephalitis or Highlands J virus. The interval after infection when EEE virus-specific antibodies were first detected by IgM and IgG EIAs was found to be similar to that determined by the PRNT and HI tests: 2 to 4 days. The IgG EIA, PRNT, and HI test detected antibodies to EEE virus for at least 27 to 30 days after inoculation. In contrast, serum from five of seven chickens did not contain detectable IgM 30 days after infection. Similarly, in all three naturally infected sentinel chickens from Maryland, IgM class antibody was undetectable 1 to 5 weeks after IgM was initially detected. EIAs provide simple and rapid alternatives to traditional tests for monitoring EEE virus infections in sentinel chicken flocks. Moreover, the IgM EIA provides a means to separate recently infected chickens from those infected greater than or equal to 1 month earlier.     

Relevance of detection of immunoglobulin M antibody response in birds used for arbovirus surveillance.

Young chickens were inoculated with 5,000 PFU of eastern equine encephalitis (EEE) virus and bled at intervals thereafter for determinations of hemagglutination-inhibiting (HI), neutralizing (N), immunoglobulin M (IgM), and IgG antibodies. HI, N, and IgM antibodies were first detected 4 days after infection, and IgG was detected 7 days after infection. All four antibodies persisted through day 90 after infection. HI, N, and IgM antibody titers remained elevated and were not cross-reactive with the related alphavirus western equine encephalitis (WEE) virus. IgG antibody titers also remained high, but heterologous reactivity to WEE virus increased with time after infection. Serum samples from sentinel chickens and wild birds infected in nature with EEE, WEE, or St. Louis encephalitis virus and submitted to this laboratory from state and local health departments were tested for IgM antibody by using anti-chicken IgM for capture and for IgG antibodies to the EEE and WEE viruses. There was essentially complete correlation between HI, N, and either IgM (indicating recent infections) or IgG (indicating more remote infections) antibody. We conclude that the IgM antibody capture enzyme immunoassay can be used as a specific and sensitive assay to replace the routinely used HI test for detecting antibody in sentinel chickens and in young, wild birds used for arbovirus surveillance. The test is rapid and relatively inexpensive and can be performed in essentially all adequately supplied laboratories.     

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.     

Characterization of rubella virus-specific antibody responses by using a new synthetic peptide-based enzyme-linked immunosorbent assay.

Rubella virus (RV)-specific immunoglobulin G antibodies were studied by enzyme-linked immunosorbent assay (ELISA) techniques in sera from RV (RA 27/3)-vaccinated individuals, patients experiencing natural RV infection, congenital rubella syndrome patients, and individuals failing to respond to repeated RV immunization. Results obtained by using whole-RV ELISAs (detergent-solubilized M33 strain or intact Gilchrist strain) and hemagglutination inhibition (HAI) and neutralization (NT) assays were compared with results obtained with the same sera by using ELISAs employing a synthetic peptide, BCH-178, representing a putative neutralization domain on the RV E1 protein. Murine RV E1-specific monoclonal antibodies with HAI and NT activities exhibited strong reactivity in ELISAs with BCH-178 peptide. In sera from RA 27/3-vaccinated individuals collected at 0 (prevaccine), 1, 2, 3, 4, 5, 6, 12, and 24 to 52 weeks postvaccine, the development of E1-peptide-reactive antibodies closely paralleled increases in RV-specific antibodies measured by whole-RV ELISAs and HAI and NT assays. Similarly, sequential serum samples obtained from patients during acute and convalescent phases of natural RV infection showed a coordinate increase in RV-specific antibodies as measured by whole-RV and peptide ELISAs. Conversely, congenital rubella syndrome patient sera, although exhibiting high levels of antibody in whole-RV ELISAs, had little or no antibody directed to the neutralization domain peptide. Sera from patients failing to respond to repeated RV immunization contained very low levels of RV-specific antibody in all ELISAs. Our results that the sequence represented by BCH-178 peptide may be a previously unidentified neutralization epitope for human antibodies on the RV E1 protein and may prove useful in determining effective RV immunity.