Persistent infection of cells in culture by measles virus. II. Effect of measles antibody on persistently infected HeLa sublines and recovery of a HeLa clonal line persistently infected with incomplete virus

Rustigian, Robert (Tufts University School of Medicine, Boston, Mass.). Persistent infection of cells in culture by measles virus. II. Effect of measles antibody on persistently infected HeLa clonal line persistently infected with incomplete virus. J. Bacteriol. 92:1805-1811. 1966.-The effect of viral antibody on persistent infection of HeLa cells by the Edmonston strain of measles virus was investigated by culturing cells from three persistently infected clones in medium supplemented with human immune globulin. The three infected HeLa clones were isolated from a persistently infected parent line. Two sublines which were grown in the presence of measles antibody developed a nonyielder state, wherein there is no detectable virus infectious for normal HeLa cultures. There is, however, continued synthesis of intracellular viral antigen and formation of viral intracytoplasmic inclusion bodies. The development of a nonyielder state was associated with a marked decrease in the degree of hemadsorption in cultures of both sublines. Further studies of the viral properties of non-yielder HeLa cell populations were made with a clone obtained from one of these sublines by plating under antibody. Persistent infection in this line was characterized by synthesis of incomplete virus even when the cells were cultured thereafter in anti-body-free medium. This was evidenced by (i) failure to recover infectious virus from the clonal population despite continued formation of intracellular viral antigen and viral intracytoplasmic inclusion bodies in a majority of the cells, (ii) the presence of only a few cells with surface viral antigen(s) including hemagglutinin, and (iii) the relatively weak antibody response to viral envelope antigen(s) after injection of cells into guinea pigs.     

Mechanisms for adaptation of simian immunodeficiency virus to replication in alveolar macrophages

In contrast to the simian immunodeficiency virus SIVmac239, which replicates poorly in rhesus monkey alveolar macrophages, a variant with nine amino acid changes in envelope (SIVmac239/316E) replicates efficiently and to high titer in these same cells. We examined levels of viral DNA, RNA, antigen, and infectious virus to identify the nature of the block to SIVmac239 replication in these cells. Low levels of viral antigen (0.1 to 1.0 ng of p27 per ml) and infectious virus (100 to 1,000 infectious units per ml) were produced in the supernatant 1 to 4 days after SIVmac239 infection, but these levels did not increase subsequently. SIVmac239 DNA was synthesized in these macrophage cultures during the initial 24 h after infection, but the levels did not increase subsequently. Quantitation of the numbers of infectious cells in cultures over time and the results of experiments in which cells were reexposed to SIVmac239 after the initial exposure indicated that only a small proportion of cells were susceptible to SIVmac239 infection in these alveolar macrophage cultures and that the vast majority (>95%) of cells were refractory to SIVmac239 infection. In contrast to the results with SIVmac239, the levels of viral antigen, infectious virus, and viral DNA increased exponentially 2 to 7 days after infection by SIVmac239/316E, reaching levels greater than 100 ng of p27 per ml and 100,000 infectious units per ml. Since SIVmac239/316E has previously been described as a virus capable of infecting cells in a relatively CD4-independent fashion, we examined the levels of CD4 expression on the surface of fresh and cultured alveolar macrophages from rhesus monkeys. The levels of CD4 expression were extremely low, below the limit of detection by flow cytometry, on greater than 99% of the macrophages. CCR5(+) cells were profoundly depleted only from alveolar macrophage cultures infected with SIVmac239/316E. High concentrations of an antibody to CD4 delayed but did not block replication of SIVmac239/316E. The results suggest that the adaptation of SIVmac316 to efficient replication in alveolar macrophages results from its ability to infect these cells in a CD4-independent fashion or in a CD4-dependent fashion even at extremely low levels of surface CD4 expression. Since resident macrophages in brains and lungs of humans also express little or no CD4, our findings predict the presence of human immunodeficiency virus type 1 that is relatively CD4 independent in the lung and brain compartments of infected people.     

Kinetic study of the replication of a cell-culture-adapted hepatitis A virus

The kinetics of replication of hepatitis A virus (LCDC-01) was studied in foetal rhesus monkey kidney cells (FRhK-4). Cells infected at a multiplicity of infection (MOl) of 2.0 showed no viral antigen production until 12 h post-infection using radioimmuno assay (RIA); however, at 48 h post-infection a logarithmic increase in antigen concentration began, which peaked by day 7. Similar patterns were observed with cultures infected with lower MOI (0.20 and 0.02) but events were delayed by about 24 h. In contrast, detection of antigen by fluorescence antibody methods occured at only 72 h after inoculation, with either 2.0 or 0.02 MOI, and peaked by day 9. The production of infectious virus did not begin until 24 h post-infection as measured by RIA and gradually peaked by day 6. Viral RNA was first detected 24 h post-infection by hybridization assay. The amount of viral RNA in the infected cells increased significantly between days 4 to 7. Restriction in the synthesis of RNA or infectious virus was not observed.     

Studies on transmission of hepatitis A virus to squirrel monkeys

Non-human primates have been playing an essential role in the study of hepatitis A virus (HAV) biology, pathogenesis and for testing candidate HAV vaccines. This study was to determine the suitability of squirrel monkeys (Saimiri sciureus) as animal model for HAV infection. Animals were inoculated, either intragastrically or intravenously, with a Brazilian HAV isolate (HAF-203). Alanine aminotransferase (ALT) and anti-HAV antibodies (IgM and total) were monitored. Feces were daily collected for HAV antigen and HAV RNA detection. Samples of liver tissue were obtained by biopsy before inoculation at peak ALT levels and/or when anti-HAV antibodies developed, and at necropsy for morphological examination. Monkeys inoculated by the intravenous route rapidly developed significant elevations of serum ALT, anti-HAV antibodies, and liver histologic changes, while the only evidence of HAV infection in intragastrically inoculated animals was the seroconversion. Moreover, squirrel monkeys excreted very low levels of HAV detectable in only few fecal samples after amplification by RT-PCR, different from humans and other non-human primate species that eliminate large quantities of virus during the late incubation period. The unusual onset of hepatitis A in experimentally infected squirrel monkeys represent an important obstacle for its use as animal model for the study of this viral infection. However, they can represent a valuable tool for the obtention of hyperimmune sera for HAV, in the view of the very high titer of anti-HAV developed (10⁵) 24 days after a single intravenous inoculation.     

Squirrel monkeys support replication of BK virus more efficiently than simian virus 40: an animal model for human BK virus infection

We performed experiments to test the suitability of squirrel monkeys (Saimiri sciureus) as an experimental model for BK virus (BKV) and simian virus 40 (SV40) infection. Four squirrel monkeys received intravenous inoculation with BKV Gardner strain, and six squirrel monkeys received intravenous inoculation with SV40 777 strain. Eight of 10 monkeys received immunosuppression therapy, namely, cyclophosphamide subcutaneously either before or both before and after viral inoculation. The presence of viral infection was assessed by quantitative real-time PCR amplification of viral DNA from blood, urine, and 10 tissues. We found that squirrel monkeys were susceptible to infection with BKV, with high viral copy number detected in blood and viral genome detected in all tissues examined. BKV genome was detected in urine from only one monkey, while three monkeys manifested focal interstitial nephritis. BKV T antigen was expressed in renal peritubular capillary endothelial cells. By contrast, SV40 was detected at very low copy numbers in only a few tissues and was not detected in blood. We conclude that the squirrel monkey is a suitable animal for studies of experimental BKV infection and may facilitate studies of viral entry, pathogenesis, and therapy.     

Primate retroviruses: immunological cross-reactivity between major structural proteins of new and old world primate virus isolates.

The major 35,000-molecular-weight internal antigen (p35) of the squirrel monkey retrovirus (SMRV) was isolated and partially characterized. Immunological analysis of SMRV p35 led to the demonstration of antigenic determinants common to SMRV and the Mason-Pfizer monkey virus (MPMV). A broadly reactive competition immunoassay was developed utilizing antiserum to MPMV to precipitate 125I-labeled SMRV p35. Although the major structural proteins of MPMV and SMRV competed with equal efficiency in this assay, type B and type C oncornavirus proteins lacked detectable reactivity. Antibodies reactive with the major structural proteins of both MPMV and SMRV were observed in sera of several normal rhesus monkeys with known prior exposure to MPMV-infected animals. These findings demonstrate the ability of sera from naturally immunized primates to recognize broadly reactive interspecies antigenic determinants shared by the major structural proteins of type D oncornaviruses, and they suggest possible horizontal transmission of MPMV among rhesus monkeys. Although sera from a number of squirrel monkeys contained antibody to SMRV p35, the possibility that this latter reactivity was due to endogenous virus activation rather than horizontal transmission cannot be ruled out.     

Encephalitis induced by a canine distemper virus in squirrel monkeys

Intracerebral inoculation with canine distemper virus (CDV) caused acute neurological signs of viral encephalitis in squirrel monkeys. Electroencephalogram revealed an abnormal sharp wave and seizure discharges resembling those of epilepsy. There was parenchymal inflammation, perivascular cuffs, neuronal degeneration, and glial reactions. Virus antigen was detected immunohistologically in the neurons and ependymal cells. Thus, CDV infection in squirrel monkeys provides an animal model for viral encephalitis and epilepsy.     

Persistent infection with herpes simplex virus type 1 in an Ia antigen-positive murine macrophage cell line

The interaction of herpes simplex virus type 1 (HSV-1) with murine macrophage cell lines was examined. The cell lines appeared to be moderately permissive for HSV-1 replication, though the yield of the virus was limited compared with that in Vero cells. Furthermore, the murine macrophage cell line SL-1, bearing Ia antigen, was persistently infected with HSV-1 for over one year, and was designated SL-1/KOS. Persistent infection could not be established in an Ia antigen-negative macrophage cell line, SL-4. In the SL-1/KOS culture, there was a small number of infected cells as revealed by infectious center assay. Treatment with monoclonal antibody against HSV-1 cured the persistent infection. Therefore maintenance of the persistent infection is considered to be due to a carrier culture consisting of a minority of infected cells and a majority of uninfected cells. In the SL-1/KOS cultures a low level of interferon (IFN) was found. When a large amount of exogenous recombinant murine IFN-beta (10(5)-10(6) international units/ml) was added to the culture, virus production diminished to undetectable levels. These results suggest that IFN plays an important role in the maintenance of persistent infection. In long-term persistently infected cultures, syncytium formation appeared and the virus from such cultures had a different DNA structure from that of the virus originally used for infection as revealed by restriction endonuclease analysis.     

Rapid detection of SVC virus antigen in infected cell cultures and clinically diseased carp by the enzyme-linked immunosorbent assay (ELISA)

An ELISA was developed using a rabbit antiserum with high levels of binding antibody against SVC virus (SVCV). Modifications were made to the standard assay which increased the sensitivity and resulted in a rapid ELISA (rELISA) which could détéct SVCV antigen in cell cultures and in extracts from infected carp in approximately 1 h. When other cell-grown fish rhabdoviruses were tested, pike fry rhabdovirus (PFRV) antigen cross-reacted in the rELISA but only at a low level. Virus antigen was détécted by the rELISA in clinically and sub-clinically diseased carp during an SVC epizootic but the sensitivity of détéction of subclinical levels of virus was not as great as that of isolation of infectious virus in cell culture. However, antigen was détécted in some fish extracts which failed to yield infectious virus. Fresh extracts of organs from healthy carp were found to give false positive reactions in the rELISA. Kidney and liver extracts from these fish were found to contain a heat labile, non-specific binding factor and further modification of the rapid assay was undertaken which successfully reduced the effect of this factor.     

Hepatitis A and B: serologic survey of human and nonhuman primate sera

Sera of humans and seven species of nonhuman primates were tested by radioimmunoassay and enzyme immunoassay for the presence of hepatitis A antibody, hepatitis B surface antigen and antibody to hepatitis B surface antigen. The outcome of testing a total of 276 serum or plasma specimens was as follows: with the exception of squirrel monkeys (0%) and cotton-top marmosets (0%), a considerable percentage of all other species tested had detectable antibodies to hepatitis A virus: humans 45.9%, chimpanzees 36.6%, baboons 38.2%, vervets 57.9%, cebus monkeys 40.0% and common marmosets 50.0%. Only one human and two chimpanzees were carriers of hepatitis B surface antigen. Antibodies to hepatitis B surface antigen were detected in human (11.3%), chimpanzees (29.9%), baboons (36.2%) and squirrel monkeys (5%). Chimpanzees showed an increasing prevalence of antibodies to hepatitis A virus and hepatitis B surface antigen with age.     

Complement-Fixing Antigen from BHK-21 Cell Cultures Infected with Lymphocytic Choriomeningitis Virus

Infection of BHK-21 cells with lymphocytic choriomeningitis (LCM) virus resulted in the production of significant titers of complement-fixing (CF) antigen. The antigen was spontaneously released from the cells, but the highest titer of 1:16 was recovered by disruption of the infected cells by freeze-thawing in tryptose phosphate broth. The antigen could be partially separated from infectious virus by centrifugation. Furthermore, it was possible to detect LCM virus infection of cell cultures by the production of the CF antigen, but this method proved less sensitive than titration by intracerebral inoculation of mice. The CF antigen from cell cultures was at least as sensitive and specific as the reference antigen prepared from infected guinea pig spleen.     

Growth of measles virus in various human lymphoid cell lines.

Neurovirulent TYCSA strain and attenuated Schwarz strain of measles virus and Halle strain of subacute sclerosing panencephalitis (SSPE) virus replicated in cultures of human lymphoid cell lines of the T-cell type, MOLT-3, MOLT-4 and CCRF-CEM. TYCSA and Halle strains grew rapidly, but Schwarz strain grew slowly in these cell lines. Furthermore, these three strains established persistent infection in CCRF-CEM cells but not in the other cell lines. In these persistently infected cultures an almost entire population of cells were shown to be infected and infectious virus was produced constantly for over 100 days. Cells persistently infected with Schwarz strain contained nucleocapsid structures in both the nucleus and cytoplasm and produced low titered infectious virus, whereas nucleocapsid structures were observed only in the cytoplasm of cells persistently infected with either TYCSA or Halle strain and the titers of infectious virus produced from these cells were high.     

Restricted distribution of potato leafroll virus antigen in resistant potato genotypes and its effect on transmission of the virus by aphids

Enzyme-linked immunosorbent assay was used to measure the concentration of potato leafroll virus (PLRV) antigen in different parts of field-grown secondarily infected plants of three potato genotypes known to differ in resistance to infection. The antigen concentration in leaves of cv. Maris Piper (susceptible) was 10–30 times greater than that in cv. Pentland Crown or G 7445(1), a breeder's line (both resistant). Differences between genotypes in antigen concentration were smaller in petioles and tubers (5–10-fold) and in above-ground stems (about 4-fold), and were least in below-ground stems, stolons and roots (about 2-fold). PLRV antigen, detected by fluorescent antibody staining of tissue sections, was confined to phloem companion cells. In Pentland Crown, the decrease in PLRV antigen concentration in leaf mid-veins and petioles, relative to that in Maris Piper, was proportional to the decrease in number of PLRV-containing companion cells; this decrease was greater in the external phloem than in the internal phloem. The spread of PLRV infection within the phloem system seems to be impaired in the resistant genotypes. Green peach aphids (Myzuspersicae) acquired < 2800 pg PLRV/aphid when fed for 4 days on infected field-grown Maris Piper plants and < 58% of such aphids transmitted the virus to Physalis floridana test plants. In contrast, aphids fed on infected Pentland Crown plants acquired <120 pg PLRV/aphid and <3% transmitted the virus to P. floridana. The ease with which M. persicae acquired and transmitted PLRV from field-grown Maris Piper plants decreased greatly after the end of June without a proportionate drop in PLRV concentration. Spread of PLRV in potato crops should be substantially decreased by growing cultivars in which the virus multiplies to only a limited extent.     

Hematologic and immunologic responses in common marmosets (Callithrix jacchus) infected with Plasmodium knowlesi and Epstein-Barr virus

The hematologic and immunologic responses to infection with either the Epstein-Barr virus alone or infection with Epstein-Barr virus and Plasmodium knowlesi were studied using common marmosets (Callithrix jacchus). The assays performed included complete blood cell counts, determinations of natural killer cell activity, and determinations of antibody titers to Epstein-Barr virus early antigen, virus capsid antigen and the nuclear antigen. While no animal showed signs of lymphoproliferative disease, it was found that animals infected with Epstein-Barr virus became positive for early antigen, virus capsid antigen and nuclear antigen at low levels. No difference in antibody titers between Epstein-Barr virus infected animals and co-infected animals was observed. An increase also was found in the number of leukocytes in all groups, and an increase in natural killer cells following infection with Epstein-Barr virus. Some depression in natural killer cells was observed in the co-infected animals when compared to Epstein-Barr virus infected animals.     

Rabies virus antinucleoprotein antibody protects against rabies virus challenge in vivo and inhibits rabies virus replication in vitro.

We previously reported that A/WySnJ mice vaccinated via a tail scratch with a recombinant raccoon poxvirus (RCN) expressing the rabies virus internal structural nucleoprotein (N) (RCN-N) were protected against a street rabies virus (D. L. Lodmell, J. W. Sumner, J.J. Esposito, W.J. Bellini, and L. C. Ewalt, J. Virol. 65:3400-3405, 1991). To improve our understanding of the mechanism(s) of this protection, we investigated whether sera of A/WySnJ mice that had been vaccinated with RCN-N but not challenged with street rabies virus had anti-rabies virus activity. In vivo studies illustrated that mice inoculated in the footpad with preincubated mixtures of anti-N sera and virus were protected. In addition, anti-N sera inoculated into the site of virus challenge protected mice. The antiviral activity of anti-N sera was also demonstrated in vitro. Infectious virus was not detected in cultures 24 h following infection with virus that had been preincubated with anti-N sera. At later time points, infectious virus was detected, but inhibition of viral production was consistently > or = 99% compared with control cultures. The protective and antiviral inhibitory activity of the anti-N sera was identified as anti-N antibody by several methods. First, absorption of anti-N sera with goat anti-mouse immunoglobulin serum, but not normal goat serum, removed the activity. Second, radioimmuno-precipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of sucrose density gradient-fractionated anti-N sera showed that antiviral activity was present only in the fraction containing anti-N antibody. Finally, absorption of anti-N sera with insect cells infected with a baculovirus expressing the N protein removed the protective activity. These data indicate that anti-N antibody is a component of the resistance to rabies virus infections.     

Human immunodeficiency virus type 1 neutralization measured by flow cytometric quantitation of single-round infection of primary human T cells

There is currently intensive research on the design of novel human immunodeficiency virus type 1 (HIV-1) vaccine immunogens that can elicit potent neutralizing antibodies. A prerequisite for comparing and optimizing these strategies is the ability to precisely measure neutralizing antibody responses. To this end, we sought to develop an assay that directly quantifies single-round HIV-1 infection of peripheral blood mononuclear cells (PBMC). Initial experiments demonstrated that essentially all productively infected PBMC could be identified by flow cytometric detection of intracellular p24 antigen (p24-Ag). After infection of PBMC with HIV-1, p24(+) lymphocytes could be distinguished beginning 1 day postinfection, and the majority of CD8(-) T cells were p24-Ag positive by 3 to 4 days postinfection. To directly quantify first-round infection, we included a protease inhibitor in PBMC cultures. The resulting 2-day assay was highly sensitive and specific for the detection of HIV-1-infected PBMC. Serial dilutions of virus stocks demonstrated that the number of target cells infected was directly related to the amount of infectious virus input into the assay. In neutralization assays, the flow cytometric enumeration of first-round infection of PBMC provided quantitative data on the number of target cells infected and on the inactivation of infectious virus due to reaction with antibody. We also used this single-round assay to compare the percentage of cells expressing p24-Ag to the number of copies of HIV-1 gag per 100 PBMC. The precision and reproducibility of this assay will facilitate the measurement of HIV-1 neutralization, particularly incrementally improved neutralizing antibody responses generated by new candidate vaccines.