Efficiency of viral entry determines the capacity of murine erythroleukemia cells to support persistent infections by mammalian reoviruses.

To determine mechanisms by which persistent viral infections are established and maintained, we initiated persistent infections of murine erythroleukemia (MEL) cells by using reovirus strains type 3 Abney and type 3 Dearing. Establishment of persistent reovirus infections of MEL cells was not associated with a significant cytopathic effect despite the presence of high titers of infectious virus in the cultures (>10(5) PFU/ml of culture lysate). Maintenance of persistently infected MEL-cell cultures was associated with coevolution of mutant viruses and cells. Mutant viruses produced greater yields than the parental wild-type (wt) strains in MEL cells cured of persistent infection and in cells treated with ammonium chloride, a weak base that blocks viral disassembly. Mutant cells supported growth of wt infectious subvirion particles, which are disassembly intermediates generated in vitro by treatment of virions with chymotrypsin, substantially better than growth of wt virions. These findings indicate that viral and cellular mutations selected during maintenance of persistently infected MEL-cell cultures affect acid-dependent proteolysis of virions during entry into cells. We also found that wt infectious subvirion particles produce greater yields than wt virions in wt MEL cells, which suggests that inefficient viral disassembly in MEL cells favors establishment of persistent infection. Therefore, steps in reovirus replication leading to viral disassembly appear to be critical determinants of the capacity of MEL cells to support both establishment and maintenance of persistent reovirus infections.     

Cells and viruses with mutations affecting viral entry are selected during persistent infections of L cells with mammalian reoviruses.

Previous studies demonstrated that both cellular and viral mutants are selected during maintenance of persistent infections established in murine L cells with high-passage stocks of mammalian reoviruses. In particular, when one culture was cured of persistent infection, the resulting cells were found to support the growth of viruses isolated from persistently infected cultures (termed PI viruses here) better than that of wild-type (wt) viruses (R. Ahmed, W. M. Canning, R. S. Kauffman, A. H. Sharpe, J. V. Hallum, and B. N. Fields, Cell 25:325-332, 1981). To address the nature of cellular and viral mutations selected during maintenance of persistent reovirus infections, we established independent, persistently infected cultures with L cells and high-passage stocks of wt reovirus. These cultures served as sources of new PI viruses and cured cells for study. We found that although wt viruses grew poorly in cured cells when infection was initiated with intact virions, they grew well in cured cells when infection was initiated with infectious subvirion particles generated from virions by in vitro treatment with chymotrypsin. This finding indicates that the block to growth of wt viruses in cured cells involves an early step that is unique to infection by virions, such as proteolytic processing in an endocytic compartment. We also found that PI viruses grew better than wt viruses in L cells treated with ammonium chloride, a weak base that inhibits the pH decrease in endosomes and lysosomes. Because ammonium chloride blocks an early step in infection by intact virions, probably the proteolytic processing of viral outer capsid proteins by acid-dependent cellular proteases in late endosomes or lysosomes, this finding indicates that PI viruses differ from wt viruses with respect to viral entry into cells. Therefore, these results indicate that both cells and viruses evolve mutations that affect one or more early steps in the viral growth cycle during maintenance of L-cell cultures persistently infected with reoviruses.     

Reovirus variants selected during persistent infections of L cells contain mutations in the viral S1 and S4 genes and are altered in viral disassembly.

Reoviruses isolated from persistently infected cultures (PI viruses) can grow in the presence of ammonium chloride, a weak base that blocks acid-dependent proteolysis of viral outer-capsid proteins during viral entry into cells. We used reassortant viruses isolated from crosses of wild-type (wt) reovirus strain, type 1 Lang, and three independent PI viruses, L/C, PI 2A1, and PI 3-1, to identify viral genes that segregate with the capacity of PI viruses to grow in cells treated with ammonium chloride. Growth of reassortant viruses in ammonium chloride-treated cells segregated with the S1 gene of L/C and the S4 gene of PI 2A1 and PI 3-1. The S1 gene encodes viral attachment protein sigma1, and the S4 gene encodes outer-capsid protein sigma3. To identify mutations in sigma3 selected during persistent reovirus infection, we determined the S4 gene nucleotide sequences of L/C, PI 2A1, PI 3-1, and four additional PI viruses. The deduced amino acid sequences of sigma3 protein of six of these PI viruses contained a tyrosine-to-histidine substitution at residue 354. To determine whether mutations selected during persistent infection alter cleavage of the viral outer capsid, the fate of viral structural proteins was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after treatment of virions of wt and PI viruses with chymotrypsin in vitro. Proteolysis of PI virus outer-capsid proteins sigma3 and mu1C occurred with faster kinetics than proteolysis of wt virus outer-capsid proteins. These results demonstrate that mutations in either the S1 or S4 gene alter acid-dependent disassembly of the reovirus outer capsid and suggest that increased efficiency of proteolysis of viral outer-capsid proteins is important for maintenance of persistent reovirus infections of cultured cells.     

Early stage of establishment of persistent Sendai virus infection: unstable dynamic phase and then selection of viruses which are tightly cell associated, temperature sensitive, and capable of establishing persistent infection

We obtained 157 cloned cell lines persistently infected with Sendai virus; these cell lines were generated independently of each other. Infectious viruses could be isolated from 123 of these cloned cell lines by inoculation of culture fluids or infected cells into embryonated eggs. The majority of the viruses carried by cells persistently infected with viruses showed high cytotoxicity and did not have the ability to establish persistent infection. The association of carried virus with cells became stronger and virus isolation correspondingly became more difficult as cells persistently infected with virus were subcultured. Viruses derived from virus-infected cells eventually acquired the ability to establish persistent infection, although the ways in which the viruses acquired this ability varied. The viruses also acquired temperature sensitivity as persistently infected cells were subcultured. First, the hemagglutinin-neuraminidase and M proteins acquired temperature sensitivity, and then the polymerase(s) did so. The M proteins were localized in the nuclei of cells infected with cloned viruses that had the ability to establish persistent infection. Cells infected with viruses capable of establishing persistent infection showed no or slight staining by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling. Specific amino acid substitutions accumulated in the M protein and the L protein as virus-infected cells were subcultured. This study shows that there is an unstable dynamic phase at an early stage of the establishment of persistent Sendai virus infection (steady state), and then viruses capable of establishing persistent infection are selected.     

Regulation of persistent infection with herpes simplex virus in vitro by hydrocortisone.

About 1% of Raji cells showed sensitivity to herpes simplex virus type 2 (HSV-2) infection when tested by infectious center assays or immunofluorescence tests, and the percentage did not change during cell passage. The addition of hydrocortisone to Raji cells persistently infected with HSV-2 caused a marked increase in virus production and in the number of HSV-producing cells. In the case of HSV-1, it was shown that the addition of hydrocortisone was required to maintain persistent infection. These observations suggest that control of replication of HSV-1 and HSV-2 in these cells is regulated by different mechanisms.     

Mutations in reovirus outer-capsid protein sigma3 selected during persistent infections of L cells confer resistance to protease inhibitor E64.

Mutations selected in reoviruses isolated from persistently infected cultures (PI viruses) affect viral entry into cells. Unlike wild-type (wt) viruses, PI viruses can grow in the presence of ammonium chloride, a weak base that blocks acid-dependent proteolysis of viral outer-capsid proteins in cellular endosomes during viral entry. In this study, we show that E64, an inhibitor of cysteine proteases such as those present in the endocytic compartment, blocks growth of wt reovirus by inhibiting viral disassembly. To determine whether PI viruses can grow in the presence of an inhibitor of endocytic proteases, we compared yields of wt and PI viruses in cells treated with E64. Prototype PI viruses L/C, PI 2A1, and PI 3-1 produced substantially greater yields than wt viruses type 1 Lang (T1L) and type 3 Dearing (T3D) in E64-treated cells. To identify viral genes that segregate with growth of PI viruses in the presence of E64, we tested reassortant viruses isolated from independent crosses of T1L and each of the prototype PI viruses for growth in cells treated with E64. Growth of reassortant viruses in the presence of E64 segregated exclusively with the S4 gene, which encodes viral outer-capsid protein sigma3. These results suggest that mutations in sigma3 protein selected during persistent infection alter its susceptibility to cleavage during viral disassembly. To determine the temporal relationship of acid-dependent and protease-dependent steps in reovirus disassembly, cells were infected with wt strain T1L or T3D, and medium containing either ammonium chloride or E64d, a membrane-permeable form of E64, was added at various times after adsorption. Susceptibility to inhibition by both ammonium chloride and E64 was abolished when either inhibitor was added at times greater than 60 min after adsorption. These findings indicate that acid-dependent and protease-dependent disassembly events occur with similar kinetics early in reovirus replication, which suggests that these events take place within the same compartment of the endocytic pathway.     

Persistent reovirus infections of L cells select mutations in viral attachment protein sigma1 that alter oligomer stability.

During maintenance of L-cell cultures persistently infected with reovirus, mutations are selected in viruses and cells. Cells cured of persistent infection support growth of viruses isolated from persistently infected cultures (PI viruses) significantly better than that of wild-type (wt) viruses. In a previous study, the capacity of PI virus strain L/C to grow better than wt strain type 1 Lang (T1L) in cured cells was mapped genetically to the S1 gene (R. S. Kauffman, R. Ahmed, and B. N. Fields, Virology 131:79-87, 1983), which encodes viral attachment protein sigma1. To investigate mechanisms by which mutations in S1 confer growth of PI viruses in cured cells, we determined the S1 gene nucleotide sequences of L/C virus and six additional PI viruses isolated from independent persistently infected L-cell cultures. The S1 sequences of these viruses contained from one to three mutations, and with the exception of PI 2A1 mutations in each S1 gene resulted in changes in the deduced amino acid sequence of sigma1 protein. Using electrophoresis conditions that favor migration of sigma1 oligomers, we found that sigma1 proteins of L/C, PI 1A1, PI 3-1, and PI 5-1 migrated as monomers, whereas sigma1 proteins of wt reovirus and PI 2A1 migrated as oligomers. These findings suggest that mutations in sigma1 protein affecting stability of sigma1 oligomers are important for the capacity of PI viruses to infect mutant cells selected during persistent infection. Since no mutation was found in the deduced amino acid sequence of PI 2A1 sigma1 protein, we used T1L X PI 2A1 reassortant viruses to identify viral genes associated with the capacity of this PI virus to grow better than wt in cured cells. The capacity of PI 2A1 to grow better than T1L in cured cells was mapped to the S4 gene, which encodes outer-capsid protein sigma3. This finding suggests that in some cases, mutations in sigma3 protein in the absence of sigma1 mutations confer growth of PI viruses in mutant cells. To confirm the importance of the S1 gene in PI virus growth in cured cells, we used T1L X PI 3-1 reassortant viruses to genetically map the capacity of this PI virus to grow better than wt in cured cells. In contrast to our results using PI 2A1, we found that growth of PI 3-1 in cured cells was determined by the sigma1-encoding S1 gene. Given that the sigma1 and sigma3 proteins play important roles in reovirus disassembly, findings made in this study suggest that stability of the viral outer capsid is an important determinant of the capacity of reoviruses to adapt to host cells during persistent infection.     

Persistent infection of cultured mammalian cells by Japanese encephalitis virus.

Persistent infections were established by serial undiluted passage of flavivirus Japanese encephalitis virus in a line of rabbit kidney cells (MA-111). The persistently infected cells resembled uninfected cells in most respects. Low levels of infectious virions were released from a small percentage of cells, and a larger and more variable percentage was shown to possess viral antigen by fluorescent-antibody staining. Released viruses were shown to interfere with replication of wild-type Japanese encephalitis virus. Persistently infected MA-111 cells could not be superinfected with homologous wild-type Japanese encephalitis virus but could be superinfected with two heterologous viruses. Transfer of cell culture medium from persistently infected MA-111 cells to a line of African green monkey kidney cells (Vero) resulted in similar persistent infections in the latter cells. Temperature sensitivity and host-cell interferon production were not involved in establishment or maintenance of persistence. Determination of ratios of physical particles to infectious particles revealed that many defective, noninfectious viruses were present, suggesting that defective interfering particles may be responsible for persistency.     

Biology of cloned cytotoxic T lymphocytes specific for lymphocytic choriomeningitis virus. VI. Migration and activity in vivo in acute and persistent infection

Cloned cytotoxic T lymphocytes (CTL) specific for lymphocytic choriomeningitis virus (LCMV) were adoptively transferred to syngeneic mice acutely or persistently (carrier mice) infected with LCMV. Although infectious virus was cleared from the spleens during acute LCMV infection begun 24 hr earlier and the spleens remained clear of virus for the 4 days of testing, there was no concomitant reduction of viral titers in lymph nodes. In contrast, adoptive transfer of cloned CTL into animals with persistent rather than acute LCMV infection resulted in deaths of syngeneic but not allogeneic recipients. LCMV-immune spleen cells taken 30 to 50 days after a primary immunization and activated by in vitro stimulation before transfer also caused death of syngeneic carrier mice. However, LCMV-immune spleen cell per se provoked no clinical manifestations when transferred but cleared infectious virus and viral nucleic acid sequences from syngeneic carrier mice. The migration of 51Cr-labeled, LCMV-specific, H-2-restricted cloned CTL was assessed in vivo. The circulation of these CTL clearly differed from that of spleen cells freshly isolated from uninfected mice and from non-LCMV-specific CTL clone. Further, the circulatory pattern of LCMV-specific, H-2-restricted, cloned CTL in carrier mice was markedly different than in uninfected animals; only 7% of the injected cells remained in the lungs of uninfected mice 8 hr after injection, whereas 30% had accumulated in the liver. However, 55% of the cells injected into carrier mice still remained in their lungs 8 to 16 hr later. Hence, LCMV-specific, H-2-restricted, cloned CTL have unique trafficking patterns in the presence of LCMV antigens and immune activities in vivo.     

Isolation of cold-sensitive mutants of measles virus from persistently infected murine neuroblastoma cells.

Clone NS20Y of the mouse neuroblastoma C1300 was infected with wild-type Edmonston measles virus, and, after a transition to a carrier culture, became persistently infected. Persistently infected clones were derived and characterized morphologically by the appearance of multinucleate giant cells and nucleocapsid matrices in cytoplasm and nucleus, but very few budding virus particles. Antimeasles antibodies markedly suppressed the expression of viral antigens and giant cells, and the effect was totally reversible. When the cells were cultured at 33 degrees C, the number of giant cells began to diminish and ultimately disappeared; in contrast, when cultured at 39 degrees C, the cultures invariably lysed. Yields at 33 degrees C were ca. 2 logs lower than those at 39 degrees C. Cells cultured at 33 degrees C produced relatively high levels of interferon, whereas those at 39 degrees C produced little or no interferon. When the persistently infected cultures were exposed to anti-interferon alpha/beta serum at a nonpermissive temperature, there was a marked increase in multinucleate cells, suggesting that maintenance of the persistence state and its regulation by temperature may be related to the production of interferon. Viral isolates from cells cultured at 39 degrees C were obtained, and 90% of viral clones were found to be cold sensitive. Complementation studies with different viral clones indicated that the cold-sensitive defect was probably associated with the same genetic function. Western blot analysis of the persistently infected cells indicated a significant diminution and expression of all measles-specific proteins at a nonpermissive temperature. Infection of NS20Y neuroblastoma cells with the cold-sensitive virus isolates resulted in the development of an immediate persistent infection, whereas infection of Vero or HeLa cells resulted in a characteristic lytic infection, suggesting that the cold-sensitive mutants may be selected or adapted for persistent infection in cells of neural origin.     

Role of the S4 gene in the establishment of persistent reovirus infection in L cells

We have studied the viral genes playing a role in persistence of reovirus in L cells. We established persistent infections by coinfecting L cells using wild-type reovirus type 2 and defective reovirus type 3. An analysis of the genomic double-stranded RNA pattern of the virus population selected during persistent infection revealed that the S4 gene was derived from the defective parent in three independently established lines, suggesting that the S4 gene plays an essential role in the establishment of persistent infection. The selection of recombinant viruses containing the S4 gene derived from the defective virus was specific for persistent infection, since it was not selected in lytic infections. Following the establishment of persistent infection, mutations in the S1 gene appeared in two of three cell lines. Thus mutations in the S4 gene play a critical role in the establishment of persistent infection, while mutations in the S1 gene play a role in the maintenance of the persistent infection.     

Coevolution of cells and viruses in a persistent infection of foot-and-mouth disease virus in cell culture.

Virus and cells evolve during serial passage of cloned BHK-21 cells persistently infected with foot-and-mouth disease virus (FMDV). These carrier cells, termed C1-BHK-Rc1 (J.C. de la Torre, M. Dávila, F. Sobrino, J. Ortín, and E. Domingo, Virology 145:24-35, 1985), become constitutively resistant to the parental FMDV C-S8c1. Curing of late-passage C1-BHK-Rc1 cells of FMDV by ribavirin treatment (J.C. de la Torre, B. Alarcón, E. Martínez-Salas, L. Carrasco, and E. Domingo, J. Virol. 61:233-235, 1987) did not restore sensitivity to FMDV C-S8c1. The resistance of C1-BHK-Rc1 cells to FMDV C-S8c1 was not due to an impairment of attachment, penetration, or uncoating of the particles but to some intracellular block that resulted in a 100-fold decrease in the amount of FMDV RNA in the infected cells. FMDV R59, the virus isolated from late-passage carrier cells, partly overcame the cellular block and was more cytolytic than FMDV C-S8c1 for BHK-21 cells. Sequencing of the VP1 gene from nine viral clones from C1-BHK-Rc1 cells showed genetic heterogeneity of 5 X 10(-4) substitutions per nucleotide. Mutations were sequentially fixed during persistence. In addition to resistance to FMDV C-S8c1, C1-BHK-Rc1 cells showed a characteristic round cell morphology, and compared with BHK-21 cells, they grew faster in liquid culture, were less subject to contact inhibition of growth, and had an increased ability to form colonies in semisolid agar. Reconstitution of a persistent infection was readily attained with late-passage C1-BHK-Rc1 cells and FMDV C-S8c1 or FMDV R59. The results suggest that coevolution of BHK-21 cells and FMDV contributes to the maintenance of persistence in cell culture.     

Defective interfering influenza viruses and host cells: establishment and maintenance of persistent influenza virus infection in MDBK and HeLa cells.

WSN (H0N1) influenza virus upon undiluted passages in different species of cells, namely, bovine kidney (MDBK), chicken embryo (CEF), and HeLa cells, produced a varying amount of defective interfering (DI) virus which correlated well with the ability of the species of cell to produce infectious virus. However, the nature of the influenza DI viral RNA produced from a single clonal stock was essentially identical in all three cells types, suggesting that these cells do not exert a great selective pressure in the amplification of specific DI viral RNAs either at early or late passages. DI viruses produced from one subtype (H0N1) could interfere with the replication of infectious viruses belonging to other subtypes (H1N1, H3N2). DI viral RNAs could also replicate with the helper function of other subtype viruses. The persistent infection of MDBK and HeLa cells could be initiated by coinfecting cells with both temperature-sensitive mutants (ts-) and DI influenza viruses. Persistently infected cultures cultures at early passages (up to passage 7) showed a cyclical pattern of cell lysis and virus production (crisis), whereas, at later passages (after passage 20), they produced little or no virus and were resistant to infection by homologous virus but not by heterologous virus. The majority of persistently infected cells, however, contained the complete viral genome since they expressed viral antigens and produced infectious centers. Selection of a slow-growing temperature-sensitive variant rather than the presence of DI virus or interferon appears to be critical in maintaining persistent influenza infection in these cells.     

Vaccinia virus infection attenuates innate immune responses and antigen presentation by epidermal dendritic cells

Langerhans cells (LCs) are antigen-presenting cells in the skin that play sentinel roles in host immune defense by secreting proinflammatory molecules and activating T cells. Here we studied the interaction of vaccinia virus with XS52 cells, a murine epidermis-derived dendritic cell line that serves as a surrogate model for LCs. We found that vaccinia virus productively infects XS52 cells, yet this infection displays an atypical response to anti-poxvirus agents. Whereas adenosine N1-oxide blocked virus production and viral protein synthesis during a synchronous infection, cytosine arabinoside had no effect at concentrations sufficient to prevent virus replication in BSC40 monkey kidney cells. Vaccinia virus infection of XS52 cells not only failed to elicit the production of various cytokines, including tumor necrosis factor alpha (TNF-alpha), interleukin-1beta (IL-1beta), IL-6, IL-10, IL-12 p40, alpha interferon (IFN-alpha), and IFN-gamma, it actively inhibited the production of proinflammatory cytokines TNF-alpha and IL-6 by XS52 cells in response to exogenous lipopolysaccharide (LPS) or poly(I:C). Infection with a vaccinia virus mutant lacking the E3L gene resulted in TNF-alpha secretion in the absence of applied stimuli. Infection of XS52 cells or BSC40 cells with the DeltaE3L virus, but not wild-type vaccinia virus, triggered proteolytic decay of IkappaBalpha. These results suggest a novel role for the E3L protein as an antagonist of the NF-kappaB signaling pathway. DeltaE3L-infected XS52 cells secreted higher levels of TNF-alpha and IL-6 in response to LPS and poly(I:C) than did cells infected with the wild-type virus. XS52 cells were productively infected by a vaccinia virus mutant lacking the K1L gene. DeltaK1L-infected cells secreted higher levels of TNF-alpha and IL-6 in response to LPS than wild-type virus-infected cells. Vaccinia virus infection of primary LCs harvested from mouse epidermis was nonpermissive, although a viral reporter protein was expressed in the infected LCs. Vaccinia virus infection of primary LCs strongly inhibited their capacity for antigen-specific activation of T cells. Our results highlight suppression of the skin immune response as a feature of orthopoxvirus infection.     

Initiation and maintenance of persistent infection by respiratory syncytial virus.

Propagation of cells infected with temperature-sensitive (ts) mutants of respiratory syncytial (RS) virus at nonpermissive temperature (39 degrees C) resulted in cytolytic, abortive, or persistent infection, depending on the mutant used to initiate infection. Five mutants from complementation group B produced cytolytic or abortive infections, whereas a single mutant (ts1) from group D and a noncomplbmenting mutant produced persistent infections. The persistently infected culture initiated by mutant ts1 (RS ts1/BS-C-1) has been maintained in serial culture for greater than 100 transfers, and infectious-center assays and immunofluorescent staining indicated that all cells harbored the RS virus genome. RS ts1/BS-C-1 cultures were resistant to superinfection by homologous and some heterologous viruses, and interferon-like activity against some heterologous viruses was present in the culture medium. Small amounts (0.002 to 0.2 PFU/cell) of infectious virus were present in the culture fluid, but autointerfering defective particles were not detected. This released virus formed small plaques and produced persistent infection of BS-C-1 cells at 37 degrees C. The RS ts1/BS-C-1 cells contained abundant RS virus antigen internally, but little at the surface, although the cells showed enhanced agglutinability by concanavalin A. Nucleocapsids and the 41,000-molecular-weight nucleoprotein were present in extracts of both nucleated and enucleated cells. No infectious RS virus was obtained by transfection of DNA from RS tsl/BS-C-1 cells to susceptible BS-C-1 or feline embryo cells under conditions allowing efficient transfection of a foamy virus proviral DNA. It was concluded that persistent infection was maintained in part by a non-ts variant of RS virus partially defective in maturation. The karyotype of the RS ts1/BS-C-1 culture differed from that of unifected cells.     

Effect of antiviral antibody on maintenance of long-term rubella virus persistent infection in Vero cells.

A Vero cell line with a long-term rubella virus persistent infection was maintained for 45 weeks in the presence of anti-rubella virus antibody of sufficient titer to completely neutralize the virus in the culture fluid to determine the effect of the presence of antibody on the maintenance of the persistent infection. Prior to antibody treatment, virus was continuously detected as plaque-forming units in the persistently infected culture fluid. Virus clones that were plaque purified from the persistently infected culture fluid were temperature sensitive and exhibited a reduced efficiency of replication and ability to induce cytopathic effects in Vero cells at the persistently infected culture temperature compared with the standard virus used to initiate the persistently infected culture. Defective interfering RNAs were the major intracellular virus-specific RNA species present in the persistently infected cells. Treatment with antibody failed to cure the persistently infected culture of virus, and the cells retained the ability to release virus after antibody treatment was discontinued. Interestingly, the presence of antibody led to the selection of a population of virus which was markedly less cytopathic for Vero cells than the virus population which was selected during persistent infection in the absence of antibody.     

Antigenic and genetic variation in cytopathic hepatitis A virus variants arising during persistent infection: evidence for genetic recombination.

Variants of hepatitis A virus (pHM175 virus) recovered from persistently infected green monkey kidney (BS-C-1) cells induced a cytopathic effect during serial passage in BS-C-1 or fetal rhesus kidney (FRhK-4) cells. Epitope-specific radioimmunofocus assays showed that this virus comprised two virion populations, one with altered antigenicity including neutralization resistance to monoclonal antibody K24F2, and the other with normal antigenic characteristics. Replication of the antigenic variant was favored over that of virus with the normal antigenic phenotype during persistent infection, while virus with the normal antigenic phenotype was selected during serial passage. Viruses of each type were clonally isolated; both were cytopathic in cell cultures and displayed a rapid replication phenotype when compared with the noncytopathic passage 16 (p16) HM175 virus which was used to establish the original persistent infection. The two cytopathic virus clones contained 31 and 34 nucleotide changes from the sequence of p16 HM175. Both shared a common 5' sequence (bases 30 to 1677), as well as sequence identity in the P2-P3 region (bases 3249 to 5303 and 6462 to 6781) and 3' terminus (bases 7272 to 7478). VP3, VP1, and 3Cpro contained different mutations in the two virus clones, with amino acid substitutions at residues 70 of VP3 and 197 and 276 of VP1 of the antigenic variant. These capsid mutations did not affect virion thermal stability. A comparison of the nearly complete genomic sequences of three clonally isolated cytopathic variants was suggestive of genetic recombination between these viruses during persistent infection and indicated that mutations in both 5' and 3' nontranslated regions and in the nonstructural proteins 2A, 2B, 2C, 3A, and 3Dpol may be related to the cytopathic phenotype.