Prime-boost vaccination with recombinant mumps virus and recombinant vesicular stomatitis virus vectors elicits an enhanced human immunodeficiency virus type 1 Gag-specific cellular immune response in rhesus macaques.

Intramuscular inoculation of rhesus macaques with one or more doses of recombinant vesicular stomatitis virus (rVSV) expressing human immunodeficiency virus type 1 (HIV-1) Gag (rVSVgag) typically elicits peak cellular immune responses of 500 to 1,000 gamma interferon (IFN-gamma) enzyme-linked immunospots (ELISPOTS)/10(6) peripheral blood lymphocytes (PBL). Here, we describe the generation of a novel recombinant mumps virus (rMuV) expressing HIV-1 Gag (rMuVgag) and measure the Gag-specific cellular immune responses detected in rhesus macaques following vaccination with a highly attenuated form of rVSV expressing HIV-1 Gag (rVSVN4CT1gag1) and rMuVgag in various prime-boost combinations. Notably, peak Gag-specific cellular immune responses of 3,000 to 3,500 ELISPOTS/10(6) PBL were detected in macaques that were primed with rMuVgag and boosted with rVSVN4CT1gag1. Lower peak cellular immune responses were detected in macaques that were primed with rVSVN4CT1gag1 and boosted with rMuVgag, although longer-term gag-specific responses appeared to remain higher in this group of macaques. These findings indicate that rMuVgag may significantly enhance Gag-specific cellular immune responses when administered with rVSVN4CT1gag1 in heterologous prime-boost regimens.

Rescue of mumps virus from cDNA.

A complete DNA copy of the genome of a Jeryl Lynn strain of mumps virus (15,384 nucleotides) was assembled from cDNA fragments such that an exact antigenome RNA could be generated following transcription by T7 RNA polymerase and cleavage by hepatitis delta virus ribozyme. The plasmid containing the genome sequence, together with support plasmids which express mumps virus NP, P, and L proteins under control of the T7 RNA polymerase promoter, were transfected into A549 cells previously infected with recombinant vaccinia virus (MVA-T7) that expressed T7 RNA polymerase. Rescue of infectious virus from the genome cDNA was demonstrated by amplification of mumps virus from transfected-cell cultures and by subsequent consensus sequencing of reverse transcription-PCR products generated from infected-cell RNA to verify the presence of specific nucleotide tags introduced into the genome cDNA clone. The only coding change (position 8502, A to G) in the cDNA clone relative to the consensus sequence of the Jeryl Lynn plaque isolate from which it was derived, resulting in a lysine-to-arginine substitution at amino acid 22 of the L protein, did not prevent rescue of mumps virus, even though an amino acid alignment for the L proteins of paramyxoviruses indicates that lysine is highly conserved at that position. This system may provide the basis of a safe and effective virus vector for the in vivo expression of immunologically and biologically active proteins, peptides, and RNAs.

Evolution of fitness in experimental populations of vesicular stomatitis virus.

The evolution of fitness in experimental clonal populations of vesicular stomatitis virus (VSV) has been compared under different genetic (fitness of initial clone) and demographic (population dynamics) regimes. In spite of the high genetic heterogeneity among replicates within experiments, there is a clear effect of population dynamics on the evolution of fitness. Those populations that went through strong periodic bottlenecks showed a decreased fitness in competition experiments with wild type. Conversely, mutant populations that were transferred under the dynamics of continuous population expansions increased their fitness when compared with the same wild type. The magnitude of the observed effect depended on the fitness of the original viral clone. Thus, high fitness clones showed a larger reduction in fitness than low fitness clones under dynamics with included periodic bottleneck. In contrast, the gain in fitness was larger the lower the initial fitness of the viral clone. The quantitative genetic analysis of the trait "fitness" in the resulting populations shows that genetic variation for the trait is positively correlated with the magnitude of the change in the same trait. The results are interpreted in terms of the operation of Muller's ratchet and genetic drift as opposed to the appearance of beneficial mutations.

Extreme fitness differences in mammalian and insect hosts after continuous replication of vesicular stomatitis virus in sandfly cells.

Continuous, persistent replication of a wild-type strain of vesicular stomatitis virus in cultured sandfly cells for 10 months profoundly decreased virus replicative fitness in mammalian cells and greatly increased fitness in sandfly cells. After persistent infection of sandfly cells, fitness was over 2,000,000-fold greater than that in mammalian cells, indicating extreme selective differences in the environmental conditions provided by insect and mammalian cells. The sandfly-adapted virus also showed extremely low fitness in mouse brain cells (comparable to that in mammalian cell cultures). It also showed an attenuated phenotype, requiring a nearly millionfold higher intracranial dose than that of its parent clone to kill mice. A single passage of this adapted virus in BHK-21 cells at 37 degrees C restored fitness to near neutrality and also restored mouse neurovirulence. These results clearly illustrate the enormous capacity of RNA viruses to adapt to changing selective environments.

RNA virus quasispecies: significance for viral disease and epidemiology.

The experimental evidence available for animal and plant RNA viruses, as well as other RNA genetic elements (viroids, satellites, retroelements, etc.), reinforces the view that many different types of genetic alterations may occur during RNA genome replication. This is fundamentally because of infidelity of genome replication and large population sizes. Homologous and heterologous recombination, as well as gene reassortments occur frequently during replication of retroviruses and most riboviruses, especially those that use enzymes with limited processivity. Following the generation of variant genomes, selection, which is dependent on environmental parameters in ways that are poorly understood, sorts out those genome fits enough to generate viable quasispecies. Chance events can also be destabilizing, as illustrated by recent results on fitness loss and other phenotypic changes accompanying bottleneck transmission. Variation, selection, and random sampling of genomes occur continuously and unavoidably during virus evolution. Evolution of RNA viruses is largely unpredictable because of the stochastic nature of mutation and recombination events, as well as the subtle effects of chance transmission events and host/environmental factors. Among environmental factors, alterations resulting from human intervention (deforestation, agricultural activities, global climatic changes, etc.) may alter dispersal patterns and provide new adaptive possibilities to viral quasispecies. Current understanding of RNA virus evolution suggests several strategies to control and diagnose viral diseases. The new generation of chemically defined vaccines and diagnostic reagents (monoclonal antibodies, peptide antigens, oligonucleotides for polymerase chain reaction amplification, etc.) may be adequate to prevent disease and detect some or even most of the circulating quasispecies of any given RNA pathogen. However, the dynamics of viral quasispecies mandate careful consideration of those reagents to be incorporated into diagnostic kits. Broadening diagnosis without jeopardizing specificity of detection will be challenging. There is a finite probability (impossible to quantify at present) that a defined vaccine may promote selection of escape mutants or a particular diagnostic kit may fail to detect a viral pathogen. Of particular concern are the potential long-term effects of weak selective pressures that may initially go unnoticed. Variant viruses resulting from evolutionary pressure imposed by vaccines or drugs may insidiously and gradually replace previous quasispecies. The great potential for variation and phenotypic diversity of some important RNA virus pathogens (human immunodeficiency virus, the hepatitis viruses, the newly recognized human hantaviruses, etc.) has become clear. Prevention and therapy should rely on multicomponent vaccines and antiviral agents to address the complexity of RNA quasispecies mutant spectra.(ABSTRACT TRUNCATED AT 400 WORDS)

Subclonal components of consensus fitness in an RNA virus clone.

Most RNA virus populations exhibit extremely high mutation frequencies which generate complex, genetically heterogeneous populations referred to as quasi-species. Previous work has shown that when a large spectrum of the quasi-species is transferred, natural selection operates, leading to elimination of noncompetitive (inferior) genomes and rapid gains in fitness. However, whenever the population is repeatedly reduced to a single virion, variable declines in fitness occur as predicted by the Muller's ratchet hypothesis. Here, we quantitated the fitness of 98 subclones isolated from an RNA virus clonal population. We found a normal distribution around a lower fitness, with the average subclone being less fit than the parental clonal population. This finding demonstrates the phenotypic diversity in RNA virus populations and shows that, as expected, a large fraction of mutations generated during virus replication is deleterious. This clarifies the operation of Muller's ratchet and illustrates why a large number of virions must be transferred for rapid fitness gains to occur. We also found that repeated genetic bottleneck passages can cause irregular stochastic declines in fitness, emphasizing again the phenotypic heterogeneity present in RNA virus populations. Finally, we found that following only 60 h of selection (15 passages in which virus yields were harvested after 4 h), RNA virus populations can undergo a 250% average increase in fitness, even on a host cell type to which they were already well adapted. This is a remarkable ability; in population biology, even a much lower fitness gain (e.g., 1 to 2%) can represent a highly significant reproductive advantage. We discuss the biological implications of these findings for the natural transmission and pathogenesis of RNA viruses.

The red queen reigns in the kingdom of RNA viruses.

Two clonal populations of vesicular stomatitis virus of approximately equal relative fitness were mixed together and allowed to compete during many transfers in vitro as large virus populations. Eventually, one or the other population suddenly excluded its competitor population, yet both the winners and losers exhibited absolute gains in fitness. Our results agree with the predictions of two major theories of classical population biology; the Competitive Exclusion Principle and the Red Queen's Hypothesis, where (in Lewis Carroll's words) "it takes all the running you can do to keep in the same place."

The correction of coagulometer effects on international normalized ratios: a multicentre evaluation.

One hundred and twenty-nine centres in the U.K. participated in a study to test the reliability of the three methods of correction for coagulometer effects on international normalized ratios (INR). Results from 37 centres which tested three warfarinized plasmas by the manual method were taken as the 'true' INR for the assessment of coagulometers. 63 centres (11 manual and 52 using coagulometers) determined their local International Sensitivity Index (ISI) in a calibration exercise. This was performed with a set of 20 lyophilized plasma calibrants with certified manual prothrombin times for the thromboplastin used in the study. The following methods of INR derivation were compared by assessing the percentage deviation from the three INR values established by 37 manual users: I. No coagulometer correction, i.e. (local PT/reference manual normal PT)manual ISI II. Coagulometer ratio correction, i.e. (local coagulometer PT/local coagulometer MNPT)manual ISI III. Local system ISI, i.e. (local coagulometer PT/local coagulometer MNPT)local system ISI IV. System ISI, i.e. (local coagulometer PT/local coagulometer MNPT)system ISI The local system ISI with the plasma calibrants (method III) gave the most reliable correction (mean deviation from 'true' INR 4.87%). The method which gave the least was with the coagulometer ratio correction, i.e. the manual ISI and local coagulometer MNPT (mean 11.25%). The system ISI tested with ACL coagulometers gave less correction than the local ISI calibration. The local system calibration with lyophilized plasmas also avoids some of the constraints on conventional thromboplastin calibrations.

A comparison of the nucleotide sequences of eastern and western equine encephalomyelitis viruses with those of other alphaviruses and related RNA viruses.

The complete nucleotide sequence of a 1982 Florida strain of eastern equine encephalomyelitis (EEE) virus, and partial sequence of the nonstructural protein genes of western equine encephalomyelitis (WEE) virus, were determined. The EEE virus genome was 11,678 nucleotides in length, excluding the cap nucleotide and poly(A) tail, and the nucleotide composition was 28% A, 24% G, 25% C, and 23% U. The organization of both EEE and WEE virus genomes was like that of other alphaviruses and included a termination codon between the nsP3 and nsP4 genes. Codon usage for 10 of 20 amino acids was nonrandom in the EEE genome, and dinucleotide CpG-containing codons were underutilized in both genomes. The slight CpG deficiency was similar to that seen in other alphaviruses and plant viruses in the alphavirus-like group, but less than that of poliovirus and yellow fever virus. This slight deficiency may reflect adaptation for replication in both CpG-deficient vertebrates, as well as insects which do not have CpG-deficient genomes. Phylogenetic analyses using nonstructural protein amino acid sequences indicated that alphaviruses evolved from a common ancestor which existed a few thousand years ago. An intercontinental introduction of an ancestral virus from the Old to New World, or vice versa, probably resulted in two main extant groups: one includes New World (EEE and Venezuelan equine encephalitis) viruses, while the other includes Old World (Sindbis, Middelburg, O'nyong-nyong, Ross River, and Semliki Forest) viruses. The position of WEE virus in the phylogenetic trees indicated that, in addition to its capsid gene (C. S. Hahn et al. (1988) Proc. Natl. Acad. Sci. USA 85, 5997-6001), WEE virus acquired its nonstructural genes from an EEE-like ancestor during recombination.

Many-trillionfold amplification of single RNA virus particles fails to overcome the Muller's ratchet effect.

We showed earlier that transfers of large populations of RNA viruses lead to fitness gains and that repeated genetic bottleneck transfers result in fitness losses due to Muller's ratchet. In the present study, we examined the effects of genetic bottleneck passages intervening between population passages, a process akin to some natural viral transmissions, using vesicular stomatitis virus as a model. Our findings show that the pronounced fitness increases that occur during two successive population passages cannot overcome the fitness decreases caused by a single intervening genetic bottleneck passage. The implications for natural transmissions of RNA viruses are discussed.

Genetic bottlenecks and population passages cause profound fitness differences in RNA viruses.

Repeated clone-to-clone (genetic bottleneck) passages of an RNA phage and vesicular stomatitis virus have been shown previously to result in loss of fitness due to Muller's ratchet. We now demonstrate that Muller's ratchet also operates when genetic bottleneck passages are carried out at 37 rather than 32 degrees C. Thus, these fitness losses do not depend on growth of temperature-sensitive (ts) mutants at lowered temperatures. We also demonstrate that during repeated genetic bottleneck passages, accumulation of deleterious mutations does occur in a stepwise (ratchet-like) manner as originally proposed by Muller. One selected clone which had undergone significant loss of fitness after only 20 genetic bottleneck passages was passaged again in clone-to-clone series. Additional large losses of fitness were observed in five of nine independent bottleneck series; the relative fitnesses of the other four series remained close to the starting fitness. In sharp contrast, when the same selected clone was transferred 20 more times as large populations (10(5) to 10(6) PFU transferred at each passage), significant increases in fitness were observed in all eight passage series. Finally, we selected several clones which had undergone extreme losses of fitness during 20 bottleneck passages. When these low-fitness clones were passaged many times as large virus populations, they always regained very high relative fitness. We conclude that transfer of large populations of RNA viruses regularly selects those genomes within the quasispecies population which have the highest relative fitness, whereas bottleneck transfers have a high probability of leading to loss of fitness by random isolation of genomes carrying debilitating mutations. Both phenomena arise from, and underscore, the extreme mutability and variability of RNA viruses.

Quantitation of relative fitness and great adaptability of clonal populations of RNA viruses.

We describe a sensitive, internally controlled method for comparing the genetic adaptability and relative fitness of virus populations in constant or changing host environments. Certain monoclonal antibody-resistant mutants of vesicular stomatitis virus can compete equally during serial passages in mixtures with the parental wild-type clone from which they were derived. These genetically marked "surrogate wild-type" neutral mutants, when mixed with wild-type virus, allow reliable measurement of changes in virus fitness and of virus adaptation to different host environments. Quantitative fitness vector plots demonstrate graphically that even clones of an RNA virus are composed of complex variant populations (quasispecies). Variants of greater fitness (competitive replication ability) were selected within very few passages of virus clones in new host cells or animals. Even clones which were well adapted to BHK21 cells gained further fitness during repeated passages in BHK21 cells.

The nucleotide sequence of the gene encoding the attachment protein H of canine distemper virus.

The sequence of the H gene and flanking sequences in the F and L genes of canine distemper virus (CDV) have been determined. The H gene of CDV (1946 nucleotides) contains one large open reading frame starting at position 21 and terminating at position 1835, encoding a protein of 604 amino acid residues. This protein contains three potential glycosylation sites in the extracellular domain and, like all other paramyxoviruses, a N-terminal membrane-spanning hydrophobic anchor domain. The deduced H protein sequence shows an identity of 36% with rinderpest virus (RPV) and measles virus (MV). The identities at the nucleotide level are higher (RPV 52% and MV 53%). The amino acid sequence shows conservation of all the structural determinants with the H proteins of MV and RPV. The data also show that CDV is evolutionarily equidistant to RPV and MV with respect to the H gene.

The nucleotide sequence of the gene encoding the F protein of canine distemper virus: a comparison of the deduced amino acid sequence with other paramyxoviruses.

The nucleotide sequence of the gene encoding the fusion protein of canine distemper virus was determined from cDNA clones derived from virus genome RNA and poly(A)+ RNA extracted from infected cells. The mRNA encoding the F protein is about 2300 nucleotides in length including the 3' poly(A) tail. There is a large open reading frame from nucleotides 86 to 2071 which begins at the first AUG codon in the F mRNA. This reading frame encodes a protein of 662 amino acid residues with a calculated mol. wt. of 73001. The first major hydrophobic domain in the amino acid sequence of the deduced protein (residues 104 to 130) may represent all or part of a signal sequence for cleavage of the N terminal part of the F2 protein. There are four potential N glycosylation sites in the F protein located within the F2 part of the molecule or the putative signal sequence, and one in the F1 portion. A second hydrophobic region corresponds to the proteolytic cleavage site which generates the F2 and F1 subunits. This stretches from residue 225 to 262 and the N terminal part of the F1 protein shows sequence conservation with the other paramyxoviruses. A third major hydrophobic domain near the C terminus of the F protein probably represents the membrane anchor for the F protein (residues 602 to 630). The F1 proteins of six paramyxoviruses are compared and shown to have substantial conservation of those residues important in the maintenance of tertiary structure of this protein.

Characterization of clones for the sixth (L) gene and a transcriptional map for morbilliviruses.

cDNA clones of the largest RNA transcript of the canine distemper and measles morbilliviruses were characterized. This presumably codes for the L protein of these viruses. mRNA 4 was identified as coding for the haemagglutinin protein of measles virus. From an analysis of readthrough transcripts representing tandem copies of two or three genes we established a transcriptional map and the gene order on the negative strand genome of the morbilliviruses to be 3'-N-P + C-M-F-H-L-5'. The data exclude the presence of small intervening genes between the six major genes of morbilliviruses and indicate the gene order to be similar to that of Sendai virus and different from that of simian virus 5.

cDNA cloning of the messenger RNAs of five genes of canine distemper virus.

Messenger RNAs from Vero cells infected with the Onderstepoort strain of canine distemper virus (CDV) were cloned into the PstI site of plasmid pAT153. Total polyadenylated RNA was used and resulting clones were screened with 32P-labelled cDNA probes from infected and mock-infected cells. The virus specificity of the clones was proven by Northern blot hybridization and by ability to select radioactive virus mRNAs labelled in vivo in the presence of actinomycin D. Clones from the N, P and M genes of CDV were identified by hybrid select translation; clones which presumably represent the H and F genes were also obtained. The clones allowed a designation of the major viral mRNA bands. Bicistronic mRNAs were identified, and their selection by various clones suggests a gene order of 3'-N-P-M-70K-65K-L-5' for this virus.