Plaque Purification Research Articles

Neuroattenuated bunyavirus variant: derivation, characterization, and revertant clones

A neuroattenuated variant bunyavirus, designated RFC/25B.5 (B.5), was selected by serial passage of a reassortant clone (RFC virus) of a California serogroup virus in BHK-21 cells, followed by plaque purification of that passaged stock. Based on its virulence index (ratio of PFU/50% lethal dose), clone B5 was over 40,000-fold less virulent than its unpassaged RFC parent after intracerebral (i.c.) inoculation into adult mice. Clone B.5 also exhibited markedly reduced neuroinvasiveness after subcutaneous injection into neonatal mice, although it retained its ability to replicate and kill suckling mice after i.c. injection. A murine neuroblastoma line (NA cells) can be used as an in vitro surrogate for the adult mouse brain, since clone B.5 replicated to at least 1,000-fold-lower titers in NA cells than did several neurovirulent California serogroup viruses. Clone B.5 replicated in BHK-21 cells at 37 degrees C to titers similar to those achieved by other California serogroup viruses but was temperature sensitive (ts) since its replication was markedly restricted at 38.9 degrees C. Ten ts revertant clones of B.5 virus were selected at 38.9 degrees C, and all of them lost their ts phenotype and regained the ability to replicate to high titer in NA cells and to kill adult mice after i.c. injection. Clone B.5 is the first described California serogroup virus which is truly attenuated after i.c. inoculation, and its availability will permit genetic analysis of bunyavirus neurovirulence.

Two amino acids, Phe 16 and Ala 776, on the polyprotein are most likely to be responsible for the diabetogenicity of encephalomyocarditis virus

The diabetogenic D variant of encephalomyocarditis virus (EMC-D) was previously shown to differ from the non-diabetogenic B variant (EMC-B) by 14 nucleotides out of 7829 bases. Similar approaches with a new nondiabetogenic variant, EMC-DV1, obtained by plaque purification of the EMC-D variant stock pool, enabled us to narrow down further the possible genomic area responsible for the diabetogenicity of EMC virus. EMC-DV1 does not induce interferon in vitro, differing from the highly interferon-inducing EMC-B. The complete nucleotide sequence of EMC-DV1 was determined by RNA-dependent DNA sequencing and cDNA sequencing. The genomic size and organization of EMC-DV1 are similar to those of EMC-D and EMC-B, with a long open reading frame encoding a polyprotein of 2292 amino acids. Comparative analyses of sequence information as well as biological activities of EMC-DV1 with EMC-D and EMC-B suggest that (i) the diabetogenicity is apparently distinct from the ability to induce interferon, which is probably due to the single U base insertion at position 765 in EMC-B, and (ii) the diabetogenicity of EMC virus is most probably controlled by one or both of two amino acids, Phe 16 (on the leader peptide) and Ala 776 (152nd amino acid on the VP1) on the polyprotein.

Synthesis of the membrane fusion and hemagglutinin proteins of measles virus, using a novel baculovirus vector containing the beta-galactosidase gene

An improved baculovirus expression vector was developed to expedite screening and facilitate oligonucleotide-directed mutagenesis. This vector contained twin promoters derived from the P10 and polyhedrin genes of Autographica californica nuclear polyhedrosis virus. The P10 promoter directed the synthesis of beta-galactosidase, whereas the polyhedrin promoter controlled the synthesis of foreign gene products. These two genes recombined with wild-type virus genome to yield recombinants which were polyhedrin negative, produced the foreign gene product, and formed blue plaques when beta-galactosidase indicator was present in the agarose overlay. An origin of replication derived from M13 or f1 bacteriophage was also included in the plasmid to permit the synthesis of single-stranded DNA. This template DNA was used to introduce or delete sequences through the process of site-specific mutagenesis. The measles virus virion possesses a membrane envelope which contains two glycoproteins: the hemagglutinin (H) and membrane fusion (F) proteins. The H polypeptide has receptor-binding and hemagglutinating activity, whereas the F protein mediates virus penetration of the host cell, formation of syncytia, and hemolysis of erythrocytes. Genes for these two glycoproteins were inserted into the NheI cloning site of the modified expression vector described above. The vector and purified wild-type viral DNA were introduced into Sf9 insect cells by calcium phosphate precipitation. A mixture of wild-type and recombinant virus was generated and used to infect Sf9 cells, which were subsequently overlaid with agarose. After 3 days, 0.1 to 1% of the plaques became blue in the presence of beta-galactosidase indicator. At least 70% of these blue viral colonies contained the foreign gene of interest as determined by dot blot analysis. Recombinant virus was separated from contaminating wild-type virus through several rounds of plaque purification. Insect cells were then infected with the purified recombinants, and synthesis of H and F proteins were verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by immunoblot detection and Coomassie blue staining. Glycosylation of the proteins appeared to be impaired somewhat, and the precursor to the F protein was not completely cleaved by the proteases present in insect host cells. On the other hand, both proteins appeared to be active in hemagglutination, hemolysis, and cell fusion assays. Levels of synthesis were in the order of 50 to 150 mg of protein per 10(8) cells.

Progressive Esophagitis from Acyclovir-Resistant Herpes Simplex

Clinically acquired acyclovir resistance in herpes simplex has usually been associated with a deficiency in viral thymidine kinase, which, in turn, has been linked with attenuated virulence in animal models. Diminished pathogenicity in thymidine kinase-deficient isolates has been partly responsible for controversies about the clinical significance of antiviral resistance. We report on a series of resistant virus isolates from a patient who had severe, progressive esophagitis. These isolates had various thymidine kinase activities, ranging from 2.8% to 130% when compared with the activity of the isolate obtained before treatment; the resistant isolate 615 retained enzyme activity as well as neurovirulence in an encephalitis model. Plaque purification showed a heterogeneous mixture containing at least one acyclovir-resistant, foscarnet-resistant plaque isolate (615.8) fully able to phosphorylate acyclovir. The 3.3-kbp BamHI fragment containing most of the DNA polymerase gene from isolate 615.8 was purified and used to successfully transfer both acyclovir and foscarnet resistance. Acquisition of in-vitro acyclovir resistance was associated with progression of clinical disease, as well as with maintenance of pathogenicity in an animal model and at least one mutation in viral DNA polymerase. Patients with herpes simplex infections that progress during acyclovir therapy should be observed for acquisition of resistance in the setting of antiviral chemotherapy; future studies should also consider the presence of heterogeneous virus populations in such patients.

Adenoviruses with nonidentical terminal sequences are viable.

Adenovirus genomes consist of linear DNA molecules containing inverted terminal repeat sequences (ITRs) of 100 to 200 base pairs. The importance of identical termini for viability of adenoviruses was investigated. The viral strains used in this study were wild-type adenovirus type 5 (Ad5) and a variant Ad2 strain with termini which were distinct from those of all other human adenoviruses sequenced to date. A hybrid virus (sub54), obtained by recombination between Ad2 and Ad5, derived the left 42 to 52% of its genome from Ad2 and the right 58 to 48% from Ad5. Southern blotting analysis with labeled oligodeoxynucleotides indicated that both Ad2 and Ad5 ITRs were present in sub54 viral DNA preparations, and successive plaque purifications of sub54 demonstrated that viruses with nonidentical terminal sequences were viable but were rapidly converted to viruses with identical ends. Cloning of the sub54 genome as a bacterial plasmid supported the observations made by analysis of sub54 virion DNA. A plasmid, pFG154, was isolated which contained the entire adenovirus genome with an Ad2 ITR at the left terminus covalently linked to an Ad5 ITR at the right terminus. Upon transfection of mammalian cells with pFG154, viral progeny were obtained which had all possible combinations of termini, thus confirming that molecules with nonidentical termini are viable. Pure populations of viruses with nonidentical termini could not be isolated, suggesting efficient repair of one end with the opposite terminus used as a template. A model for this process is proposed involving strand displacement replication and emphasizing the importance of panhandle formation (annealing of terminal sequences) as a replicative intermediate.

Effect of Serial Passage on Genetic Homogeneity of a Plaque Variant of Lymantria dispar Nuclear Polyhedrosis Virus (Hamden LDP-67)

Summary The wild-type Hamden gypsy moth baculovirus preparation (LDP-67) was subjected to plaque purification in IPLB-LD-652Y cells, resulting in three distinguishable classes of variants, based on the number of polyhedra produced per cell in vitro. Representatives were designated Ld-S (several polyhedra per cell), Ld-F (few polyhedra per cell) and Ld-V (variable numbers of polyhedra per cell). These representative phenotypic variants were compared to the wild-type by restriction enzyme analysis and by bioassay in Lymantria dispar larvae. With all enzymes tested the variants demonstrated restriction site polymorphism. The genome size of the three variants was estimated at 159 to 163 kb. In bioassay trials two of the variants, Ld-V and Ld-S, exhibited LD50 values approximately 3·9 times lower than the LD50 of wild-type virus. The Ld-F variant was not infective in feeding trials. The stability of Ld-S was tested by high multiplicity passage (HMP) in IPLB-LD-652Y cells. By the twentieth undiluted passage (P-20), the in vitro cytopathic effect of this variant was significantly altered. DNA isolated from the P-20 stock exhibited several differences in the restriction endonuclease profile relative to the early passage virus. Genomic alterations were more clearly visualized after plaque purification of the Ld-S (P-20) stock. Four of the five plaques chosen contained what appeared to be two distinct viral populations. One of these populations, which could be isolated by repeated plaque purification, had a restriction enzyme profile identical to the pre-HMP Ld-S variant. The second population, characterized by high M r BglII bands not present in the Ld-S profile, could never be plaque-purified of submolar amounts of the first population. The possibility that this population represents a defective interfering virus is discussed. The source of additional DNA in the P-20 variants was investigated by probing blots of these variants with labelled total cellular DNA. No specific hybridization to any of the fragments was obtained, suggesting that the P-20 variants arose by rearrangements involving solely viral DNA.

Heterogeneity within an HSV-1 Wild-Type Strain and Its Importance in Pathogenesis

A herpes simplex virus-type 1 low passage, clinical eye isolate, E-43 at P2, was compared with its variant progeny, SLi-43 at P8, in terms of ocular disease, cytopathic effects, and genomic variation. In New Zealand White (NZW) rabbits, E-43 produced mild epithelial defects and punctate lesions with full recovery by Day 10 postinfection (pi). SLi-43 caused dendritic lesions, progressing to geographic ulceration and death from herpes simplex virus encephalitis in 10 days postinfection. In RK, Hep-2, and Vero cells, E-43 displayed the syn+ phenotype (aggregation and cell rounding); SLi-43 showed the syn phenotype (syncytium formation). DNA digestion profiles of E-43, SLi-43, and isolates from the brains of infected animals showed that the genomic differences map within the terminal repeat of the unique long segment and the internal joint region, specifically in bands B, E, N, and S (Bam HI) and bands M and N (Hind III). Analysis of the DNA of virus recovered from the brain stem of SLi-43-infected, encephalitic rabbits demonstrated that an in vivo selection for neurotropic virions had taken place. Plaque purification of 20 clones from the original E-43 strain showed that one of 20 was the syn phenotype, indicating that the SLi-43 variant was present in the original E-43 isolate and did not develop de novo by rapid mutation. The parent-progeny relationship between E-43 and SLi-43 forms an ideal model in which to compare differences in pathogenicity at the genomic level, and underscores the importance of heterogeneity within a single herpes simplex virus-type 1 wild-type population in terms of variations in ocular disease.

Use of a Monoclonal Antibody Specific for Wild-type Yellow Fever Virus to Identify a Wild-type Antigenic Variant in 17D Vaccine Pools

Monoclonal antibodies (MAbs) against the Asibi wild-type strain of yellow fever (YF) virus were prepared and characterized. One of the MAbs (designated MAb 117) was shown, by cross-immunofluorescence tests with flaviviruses, to be specific for wildtype YF virus. This MAb was used in indirect immunofluorescence tests to identify wild-type antigenic variants in several different YF vaccine pools. Simultaneously, a vaccine-specific MAb prepared previously (MAb 864) was used to identify YF strain 17D vaccine type variants in the wild-type Asibi virus preparation. One variant, isolated by plaque purification from a 17D vaccine pool, possessed the wild-type epitope and was neurovirulent in infant mice whereas other variants, lacking the wild-type epitope but with vaccine-specific epitopes (identified by MAb 411), were avirulent in infant mice. Avirulent variants were able to infect mice and induce antibody. Virus-specific antigen was still detected in the brains of these mice 4 weeks after inoculation, suggesting that persistent infections were developing. These results to the potential risk of selection of wild-type variants in YF vaccine preparations. They also point to the potential risk of selection of wild-type variants in YF vaccine preparations and re-emphasize the need for modernization of techniques and more effective control measures to be taken during the production of YF vaccine.

Genotypic Transitions among Bluetongue Viral Isolates from Domestic Ruminants in Colorado during 1981–1984

Two predominant electropherotypes of bluetongue virus (BTV) serotype 11 isolates from cattle during a 1981-1984 field study in eastern Colorado were characterized. The genomes of strains isolated from the first 2 years of the study had 1 predominant electropherotype (CO81), with the exception of 1 isolate that differed only in the migration of segment 3. A second electropherotype (CO83), with differences in the migration of 4 segments, coexisted in the same region during 1983 and 1984 with strains having the CO81 RNA profile. The genomes of CO81 and CO83 were also distinguishable from those of the US prototype of BTV 11. Analysis of the polypeptides of representative strains of each electropherotype by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the proteins were very similar. The occurrence of the CO81 electropherotype was apparently the result of multiple viral infections since the positions of 7 segments had faint second bands and single-banded variants were isolated after serial plaque purifications. In addition, protein 7 of 1 of the CO81 isolates and protein 7 of the single-banded variant differed as shown by reverse phase-high performance liquid chromatography of 35S-methionine-labeled tryptic peptides.

Isolation of Rickettsia prowazekii with reduced sensitivity to gamma interferon

The growth of Rickettsia prowazekii Madrid E was monitored in mouse L929 cells subcultured for several weeks in the presence of gamma interferon (IFN-gamma) to determine whether the rickettsiae would be eliminated from or would persist in these cultures. R. prowazekii exhibited two distinct patterns in these IFN-gamma-treated cultures. In some cases, IFN-gamma-induced inhibition of rickettsial growth led to elimination of the rickettsiae from the L929 cell cultures; in other cases, the initial inhibition of rickettsial growth was followed by establishment of a persistent rickettsial infection in the IFN-gamma-treated L929 cells. During the first 3 days after infection, the growth rate of the L929 cells was significantly lower and higher percentages of the cells were killed in the IFN-gamma-treated, R. prowazekii-infected cultures than in the untreated, R. prowazekii-infected cultures or the mock-infected cultures, whether treated or untreated. This suppression of cell growth occurred in the infected, IFN-gamma-treated cultures that eventually exhibited the elimination pattern as well as the IFN-gamma-treated cultures that became persistently infected. It was not possible to predict the outcome of a particular infection from the early growth pattern of the culture. It was determined that the L929 cells in the persistently infected, IFN-gamma-treated cultures had not lost the ability to respond to IFN-gamma. These cells, after treatment with an antibiotic to eliminate the persistent rickettsiae, retained the ability to inhibit both the replication of vesicular stomatitis virus and the growth of R. prowazekii Madrid E after treatment with IFN-gamma. In contrast, rickettsiae isolated from two persistently infected, IFN-gamma-treated cultures were less sensitive than R. prowazekii Madrid E to the antirickettsial effects of IFN-gamma in standard L929 cells. The maintenance of the phenotype of these altered rickettsiae during plaque purification and passage in the absence of IFN-gamma suggests an alteration at the genetic level rather than phenotypic adaptation.

Genomic differences between the diabetogenic and nondiabetogenic variants of encephalomyocarditis virus

Plaque purification of the M variant of encephalomyocarditis (EMC-M) virus resulted in the isolation of two stable variants. One is a highly diabetogenic D variant (EMC-D) and the other is a nondiabetogenic B variant (EMC-B). The cDNA of EMC-D and EMC-B genomes were cloned and seven overlapping cDNA clones were selected to cover the entire genome except the 5′-end 310 bases which were determined by RNA-dependent DNA sequencing and enzymatic RNA sequencing. Each clone was restriction-mapped, subcloned, and sequenced. The genomes of EMC-D and EMC-B are composed of 7829 and 7825 bases, respectively. Both genomes contain a long open reading frame of 6876 nucleotides starting at position 830 on the consensus sequence, which encodes a polyprotein of 2292 amino acids. The sequences of EMC-D and EMC-B differ by two deletions, one insertion, and eight point mutations. The first deletion of 3 nucleotides is located in the 5′ poly(C) tract where EMC-B has 127 nucleotides compared with 130 nucleotides in EMC-D. The second deletion in EMC-B involves 2 nucleotides at the 3′-end polyadenylation site. A single base insertion of U occurs at the 5′ noncoding region of EMC-B. The eight point mutations are located in the polyprotein coding region. Two are silent and are each located in the structural gene 1B and in the nonstructural gene 2B. The remaining six mutations, one on the L gene and the other five on the 1D gene, introduce respective amino acid changes. It is concluded that the diabetogenic EMC-D viral genome (7829 bases) differs from the nondiabetogenic EMC-B viral genome (7825 bases) by 14 nucleotides out of 7829.

Biological function of the rotavirus protein VP4: Observations on porcine isolates from China

Rotaviruses isolated from pigs in China were grown in MA104 cells. One tissue-culture-adapted isolate consisted of two subpopulations (variants), the RNA profiles of which differed in the relative migration of RNA segment 4 only. The variants were separated by plaque purification and by recovery from limiting dilutions and remained genetically stable. The variant possessing the slower migrating RNA segment 4, called 4S, grew faster and formed large plaques after 4-6 days incubation, whereas the variant possessing the faster migrating RNA segment 4, called 4F, grew more slowly and formed only microscopic plaques after 10-14 days incubation. The protein product of the 4F RNA occurred in much lower concentration in infected cells than the product of the 4S RNA. The RNA segments 4 of the two variants were found to be closely related when tested by dot hybridization under stringent conditions. The 4S RNA is more resistant to denaturation with methyl mercuric hydroxide than is the 4F RNA. The relevance of these findings to the biological functions of rotaviruses is discussed.

Isolation and plaque purification of a syncytial forming component of myxoma virus

Using the endpoint dilution and plaque purification technique performed on rabbit kidney cells, two stable syncytial clones of myxoma virus have been isolated. No eosinophilic intracytoplasmic inclusion bodies nor intranuclear granulations could be detected in cells infected with these clones. Traditional plaques with the central area of detached cells surrounded by round cells also could not be recognized. However, when enough virus was added, one large complete syncytium developed in 24 h on the monolayer. Syncytial activity was associated with the entire particle. This was the first time that myxoma virus is reported to demonstrate such an intense syncytial activity.

Single amino acid change at the cleavage site of the fusion protein is responsible for both enhanced chymotrypsin sensitivity and trypsin resistance of a Sendai virus mutant, TR-5.

A trypsin-resistant mutant of Sendai virus, TR-5, which was obtained by passaging the wild-type (wt) virus in the presence of chymotrypsin followed by plaque purification, also had an enhanced susceptibility to activation by chymotrypsin. A trypsin-sensitive revertant, TSrev-58, derived from TR-5 had a decreased susceptibility to chymotrypsin and was susceptible to trypsin to the same degree as the wt virus. Based on the facts that TR-5 had an amino acid change (Arg----Ile) at residue 116 of the fusion (F) protein, which is the cleavage site for trypsin in the wt virus F protein, and that this change had reverted in TSrev-58, it was concluded that the above amino acid change was responsible for both the enhanced chymotrypsin sensitivity and the trypsin resistance of TR-5. In this paper, the cleavage site of the F protein of TR-5 by chymotrypsin was determined in comparison with those of the wt virus and TSrev-58. The cleavage sites were not different among these viruses, being located between residues 114 (Gln) and 115 (Ser), the two residues at the N-terminal side of the trypsin cleavage site. A possible reason for the increased susceptibility of TR-5 to chymotrypsin is discussed.

Generation of measles virus defective interfering particles and their presence in a preparation of attenuated live-virus vaccine.

By starting from a thrice-purified wild-type measles virus plaque, the generation of detectable subgenomic RNAs was achieved within a series of five serial infections of Vero cells. The evolution of these subgenomic RNAs was followed for seven serial passages and ended with the preparation of a highly interfering viral stock. On the other hand, the detection of discrete subgenomic RNAs was achieved during the first infection of Vero cells with at least one of three measles virus vaccine preparations tested. These subgenomic RNAs, which interfered very efficiently with the replication of the endogenous standard genomes upon vaccine infection but showed a moderate interfering activity with a standard virus stock derived by plaque purification from the vaccine preparation, resulted from the presence of defective interfering particles in the vaccine preparation. The relevance of this finding for the attenuation, stability, and potential capacity for persistent infection of such a vaccine is discussed.

RNA recombination of murine coronaviruses: recombination between fusion-positive mouse hepatitis virus A59 and fusion-negative mouse hepatitis virus 2.

It has previously been shown that the murine coronavirus mouse hepatitis virus (MHV) undergoes RNA recombination at a relatively high frequency in both tissue culture and infected animals. Thus far, all of the recombination sites had been localized at the 5' half of the RNA genome. We have now performed a cross between MHV-2, a fusion-negative murine coronavirus, and a temperature-sensitive mutant of the A59 strain of MHV, which is fusion positive at the permissive temperature. By selecting fusion-positive viruses at the nonpermissive temperature, we isolated several recombinants containing multiple crossovers in a single genome. Some of the recombinants became fusion negative during the plaque purification. The fusion ability of the recombinants parallels the presence or absence of the A59 genomic sequences encoding peplomers. Several of the recombinants have crossovers within 3' end genes which encode viral structural proteins, N and E1. These recombination sites were not specifically selected with the selection markers used. This finding, together with results of previous recombination studies, indicates that RNA recombination can occur almost anywhere from the 5' end to the 3' end along the entire genome. The data also show that the replacement of A59 genetic sequences at the 5' end of gene C, which encodes the peplomer protein, with the fusion-negative MHV-2 sequences do not affect the fusion ability of the recombinant viruses. Thus, the crucial determinant for the fusion-inducing capability appears to reside in the more carboxyl portion of the peplomer protein.

Site-specific oligonucleotide-directed mutagenesis using T4 DNA polymerase.

A simple and efficient mutagenesis procedure is described which uses both the 3'----5' exonuclease and 5'----3' polymerase activities of T4 DNA polymerase. Different types of mutation-deletion, insertion, and substitution-can be introduced into the DNA in a single reaction. The technique uses recombinant M13 single-stranded DNA and two complementary DNA oligonucleotides to target and control the extent of deletions catalyzed by T4 DNA polymerase. The second oligonucleotide not only directs ligation, but also serves as a template for insertion or substitution of nucleotides by T4 polymerase. Mutant phages in a genetically pure form can be obtained at high efficiency, allowing their characterization directly by nucleotide sequencing without prior enrichment, plaque purification, and screening. We tested the versatility of this method by manipulating five regions of cDNA encoding the structural proteins of eastern equine encephalitis virus.

Detection and purification of a recombinant human B lymphotropic virus (HHV-6) in the baculovirus expression system by limiting dilution and DNA dot-blot hybridization

A recombinant virus Ac373-HB was detected and purified from the transfection mixture of wild type and recombinant virus in the baculovirus expression system using a combination of limiting dilution and DNA dot-blot hybridization. This method allows for a quick and convenient way of detection and purification of recombinant virus without the need to use a plaque purification step. It is generally applicable to other expression systems besides the baculovirus system described here.

Egg-grown and tissue-culture-grown variants of influenza A (H3N2) virus with special attention to their use as antigens in seroepidemiology

A field strain of influenza A (H3N2) virus isolated in embryonated eggs during the 1984-5 influenza outbreak (A/Finland/13/85E) was compared in an antigenic analysis with virus from the same clinical specimen isolated in MDCK cell cultures (A/Finland/13/85M). The M-virus appeared to be more sensitive to haemagglutination-inhibiting antibodies against heterologous viruses than did the E-virus. The results of propagation and plaque purification experiments support the hypothesis that a single clinical specimen may consist of distinct antigenic variant subpopulations promoted selectively by the host during isolation procedures. Receptor-binding properties are discussed as a possible explanation for this selectivity. A set of 471 paired sera consisting of pre-epidemic and post-epidemic specimens taken from the same subjects in 1984-5 was studied for haemagglutination-inhibiting antibodies to six influenza A (H3N2) virus strains, including the E-virus and the M-virus from A/Finland/13/85. Of the antigens used, the M-virus detected significant antibody increases more frequently than did the E-virus (10.0 v. 5.9%). The superiority of the M-virus may rest primarily in its ability to pick out anamnestic antibody responses. Irrespective of this cross-reactivity, pre-epidemic antibody to the M-virus was fairly well associated with protection. In the set of sera (230 specimens) collected in summer 1985 to represent different age groups, the antibody status against the M-virus was significantly better than the status against the E-virus. The results suggest that, at least in some instances, antibody to MDCK-grown virus is a more accurate indicator of the immune status of a community than antibodies to egg-grown virus variants.

The creation of adenovirus genomes with viable, stable, internal redundancies centered about the E2b region

During the course of constructing new adenoviral strains by overlap recombination, we have discovered that internally redundant viable genomes can be created by end-to-end joining of the input DNA molecules. The cellular functions responsible for the end-joining activity frequently ligated the overhanging single strands of the complementary ends to form a novel restriction site at the junction. In 2 of the 17 cases analyzed in detail by restriction digestion, and some sequence determinations, the cellular functions had repaired the ends, presumably prior to end-joining. Four of the isolates had suffered deletions at the junction ranging in size from 13 to 532 bp. The isolate with the largest deletion also had an insertion of 14 by of unknown origin at the site of the deletion. All of the redundant isolates replicated as efficiently as isogenic unit length strains, and plaque dilution titrations obeyed one-hit kinetics, showing that the redundant genomes were nondefective. Nevertheless unit-length genomes were observed at a low level (some 5 to 10% of the total) in stocks of each isolate before and after plaque purification. They presumably arose by recombination between the redundant sequences either intra- or intermolecularly. Evidence from Southern blot analysis showed that molecules with three copies of the redundant sequences also arose and could be detected both in intracellular and in capsid viral DNA. These species would arise by unequal crossing-over between redundant genomes. The efficient replication of the redundant species demonstrates that the precise spatial relationships between splice donors and acceptors on either strand, in this region of the genome, do not have to be rigidly maintained. These data suggest that it may be possible to place other genetic information between the DNA polymerase and terminal protein precursor genes and have it expressed from the major late promoter in its normal location.