Modelling the evolution and spread of HIV immune escape mutants

During infection with human immunodeficiency virus (HIV), immune pressure from cytotoxic T-lymphocytes (CTLs) selects for viral mutants that confer escape from CTL recognition. These escape variants can be transmitted between individuals where, depending upon their cost to viral fitness and the CTL responses made by the recipient, they may revert. The rates of within-host evolution and their concordant impact upon the rate of spread of escape mutants at the population level are uncertain. Here we present a mathematical model of within-host evolution of escape mutants, transmission of these variants between hosts and subsequent reversion in new hosts. The model is an extension of the well-known SI model of disease transmission and includes three further parameters that describe host immunogenetic heterogeneity and rates of within host viral evolution. We use the model to explain why some escape mutants appear to have stable prevalence whilst others are spreading through the population. Further, we use it to compare diverse datasets on CTL escape, highlighting where different sources agree or disagree on within-host evolutionary rates. The several dozen CTL epitopes we survey from HIV-1 gag, RT and nef reveal a relatively sedate rate of evolution with average rates of escape measured in years and reversion in decades. For many epitopes in HIV, occasional rapid within-host evolution is not reflected in fast evolution at the population level.     

Optimal control of drug therapy: melding pharmacokinetics with viral dynamics

Pharmacokinetics were melded with a viral dynamical model to design an optimal drug administration regimen such that the basic reproductive number for the virus was minimized. One-compartmental models with two kinds of drug delivery routes, intravenous and extravascular with multiple dosages, and two drug elimination rates, first order and Michaelis-Menten rates, were considered. We defined explicitly the basic reproductive number for the viral dynamical model melded with pharmacokinetics. When the average plasma drug concentration was constant, intravenous administration of the drug with small dosages applied frequently minimized the basic reproductive number. For extravascular administration, the basic reproductive number initially decreases to a trough point and then increases as the drug dosage increases. When a therapeutic window is considered, numerical studies indicate that the wider the window, the smaller the basic reproductive number. Once the width of the therapeutic window is fixed, the basic reproductive number monotonously declines as the minimum therapeutic level increases. The findings suggest that the existence of drug dosage and drug administration interval that minimize the basic reproductive number could help design the optimal drug administration regimen.     

Estimating the impact of vaccination on acute simian-human immunodeficiency virus/simian immunodeficiency virus infections

The dynamics of HIV infection have been studied in humans and in a variety of animal models. The standard model of infection has been used to estimate the basic reproductive ratio of the virus, calculated from the growth rate of virus in acute infection. This method has not been useful in studying the effects of vaccination, since, for the vaccines developed so far, early growth rates of virus do not differ between control and vaccinated animals. Here, we use the standard model of viral dynamics to derive the reproductive ratio from the peak viral load and nadir of target cell numbers in acute infection. We apply this method to data from studies of vaccination in SHIV and SIV infection and demonstrate that vaccination can reduce the reproductive ratio by 2.3- and 2-fold, respectively. This method allows the comparison of vaccination efficacies among different viral strains and animal models in vivo.     

Estimating the basic reproductive number from viral sequence data

Epidemiological processes leave a fingerprint in the pattern of genetic structure of virus populations. Here, we provide a new method to infer epidemiological parameters directly from viral sequence data. The method is based on phylogenetic analysis using a birth-death model (BDM) rather than the commonly used coalescent as the model for the epidemiological transmission of the pathogen. Using the BDM has the advantage that transmission and death rates are estimated independently and therefore enables for the first time the estimation of the basic reproductive number of the pathogen using only sequence data, without further assumptions like the average duration of infection. We apply the method to genetic data of the HIV-1 epidemic in Switzerland.     

A large variation in the rates of synonymous substitution for RNA viruses and its relationship to a diversity of viral infection and transmission modes

RNA viruses successfully adapt to various environments by repeatedly producing new mutants, often through generating a number of nucleotide substitutions. To estimate the degree of variation in mutation rates of RNA viruses and to understand the source of such variation, we studied the synonymous substitution rate because synonymous substitution is exempt from functional constraints at the protein level, and its rate reflects the mutation rate to a great extent. We estimated the synonymous substitution rates for a total of 49 different species of RNA viruses, and we found that the rates had tremendous variation by 5 orders of magnitude (from 1.3 x 10(-7) to 6.2 x 10(-2) /synonymous site/year). Comparing the synonymous substitution rates with the replication frequencies and replication error rates for the RNA viruses, we found that the main source of the rate variation was differences in the replication frequency because the rates of replication error were roughly constant over different RNA viruses. Moreover, we examined a relationship between viral life strategies and synonymous substitution rates to understand which viral life strategies affect replication frequencies. The results show that the variation of synonymous substitution rates has been influenced most by either the difference in the infection modes or the differences in the transmission modes. In conclusion, the variation of mutation rates for RNA viruses is caused by different replication frequencies, which are affected strongly by the infection and transmission modes.     

Rapid complementation assays measuring replicative potential of human immunodeficiency virus type 1 envelope glycoprotein mutants.

Rapid assays which measure the ability of mutant human immunodeficiency virus type 1 envelope glycoproteins to mediate cell-free and/or cell-to-cell transmission of virus are described. By using these assays, envelope glycoprotein mutants with varying degrees of syncytium-forming ability were tested for ability to complement viral replication in trans. As expected, mutants that dramatically affect association of the gp120-gp41 envelope subunits, CD4 binding, or membrane fusion were unable to form syncytia or to support cell-free or cell-to-cell transmission. Surprisingly, some membrane fusion-defective mutants significantly attenuated in syncytium-forming ability were able to complement viral replication. Conversely, mutations in the carboxyl terminus of gp41 transmembrane glycoprotein, although not affecting syncytium-forming ability, significantly attenuated both forms of virus transmission. These results indicate that syncytium formation is not sufficient for cell-to-cell transmission of human immunodeficiency virus type 1. Furthermore, virus transmission appears to be less sensitive to inhibition of membrane fusion than is syncytium formation.     

栗疫病菌的营养体亲和性基因和dsRNAs对病毒传播的影响

研究了栗疫病菌[Cryphonectria parasitica(Murr.)Barr]的营养体亲和性基因及dsRNA病毒对菌株间病毒特征传播的影响。试验选用已知4个VC基因座位的15个Vc基因型菌株和3种dsRNA病毒,通过含病毒菌株与野生型菌株的配对培养,将病毒逐个转入不同Vc基因型菌株。将不同Vc基因型的含病毒菌株与具特定VC基因差异的野生型菌株配对培养,根据培养两周后野生型菌株培养性状的改变与否,统计菌株间的病毒传播率。结果表明,各个VC基因对菌株间病毒传播的影响不同;存在2个VC基因差异的菌株间病毒传播率低于相差1个VC基因的：病毒在相差1个VC基因的菌株间的传播多具单向性：不同类型的病毒在菌株间的传播率也有差异。     

Glycoprotein gp50-negative pseudorabies virus: a novel approach toward a nonspreading live herpesvirus vaccine.

Essential herpesvirus glycoproteins are involved in membrane fusion processes during infection, e.g., viral penetration and direct cell-to-cell transmission. We previously showed that the gD-homologous glycoprotein gp50 of pseudorabies virus (PrV) is essential for virus entry into target cells but proved to be dispensable for direct viral cell-to-cell spread in cell culture (I. Rauh and T. C. Mettenleiter, J. Virol. 65:5348-5456, 1991). For gp50-negative (gp50-) viruses, after phenotypic complementation necessary for primary infection, the only means of viral spread is by way of direct cell-to-cell transmission. In contrast, virus mutants lacking the essential gB-homologous glycoprotein gII after phenotypic complementation are only able to infect primary target cells and are blocked in further viral spread. To analyze how these in vitro phenotypes translate into virus replication in the animal, mice were infected intranasally with gp50- or gII- PrV mutants after prior phenotypic complementation by propagation on cell lines providing the essential glycoprotein in trans. Our results show that whereas the gII- mutants did not cause disease or any symptoms, gp50- mutants derived from two different PrV strains were fully virulent, with animals exhibiting severe symptoms ultimately leading to death. However, free infectious virus could not be recovered from either gp50- or gII- PrV-infected animals. We conclude that direct cell-to-cell transmission as the only means of viral spread of the gp50- mutants is sufficient for a full virulent phenotype in mice. After infection of pigs with phenotypically complemented gp50- PrV, only mild symptoms were observed, whereas the gII- mutant was totally avirulent. In both cases, shedding of infectious virus did not occur, in contrast to results with animals infected by gX- PrV that showed severe signs of disease and extensive virus shedding. After challenge infection with the highly virulent NIA-3 strain, the previously gII- PrV-infected animals exhibited severe symptoms, whereas the gp50- PrV-infected pigs showed a significant level of protection. In conclusion, vaccination with a PrV mutant lacking glycoprotein gp50, which is unable to spread between animals because of a lack of formation of free infectious virions, can confer on pigs protection against challenge infection. These results provide the basis for the development of new, nonspreading live herpesvirus vaccines based on gp50- PrV mutants.     

Construction of less neurovirulent polioviruses by introducing deletions into the 5'' noncoding sequence of the genome.

Viral attenuation may be due to lowered efficiency of certain steps essential for viral multiplication. For the construction of less neurovirulent strains of poliovirus in vitro, we introduced deletions into the 5' noncoding sequence (742 nucleotides long) of the genomes of the Mahoney and Sabin 1 strains of poliovirus type 1 by using infectious cDNA clones of the virus strains. Plaque sizes shown by deletion mutants were used as a marker for rate of viral proliferation. Deletion mutants of both the strains thus constructed lacked a genome region of nucleotide positions 564 to 726. The sizes of plaques displayed by these deletion mutants were smaller than those by the respective parental viruses, although a phenotype referring to reproductive capacity at different temperatures (rct) of viruses was not affected by introduction of the deletion. Monkey neurovirulence tests were performed on the deletion mutants. The results clearly indicated that the deletion mutants had much less neurovirulence than with the corresponding parent viruses. Production of infectious particles and virus-specific protein synthesis in cells infected with the deletion mutants started later than in those infected with the parental viruses. The rate at which cytopathic effect progressed was also slower in cells infected with the mutants. Phenotypic stability of the deletion mutant for small-plaque phenotype and temperature sensitivity was investigated after passaging the mutant at an elevated temperature of 37.5 degrees C. Our data strongly suggested that the less neurovirulent phenotype introduced by the deletion is very stable during passaging of the virus.     

The Genealogical Population Dynamics of HIV-1 in a Large Transmission Chain: Bridging within and among Host Evolutionary Rates

Transmission lies at the interface of human immunodeficiency virus type 1 (HIV-1) evolution within and among hosts and separates distinct selective pressures that impose differences in both the mode of diversification and the tempo of evolution. In the absence of comprehensive direct comparative analyses of the evolutionary processes at different biological scales, our understanding of how fast within-host HIV-1 evolutionary rates translate to lower rates at the between host level remains incomplete. Here, we address this by analyzing pol and env data from a large HIV-1 subtype C transmission chain for which both the timing and the direction is known for most transmission events. To this purpose, we develop a new transmission model in a Bayesian genealogical inference framework and demonstrate how to constrain the viral evolutionary history to be compatible with the transmission history while simultaneously inferring the within-host evolutionary and population dynamics. We show that accommodating a transmission bottleneck affords the best fit our data, but the sparse within-host HIV-1 sampling prevents accurate quantification of the concomitant loss in genetic diversity. We draw inference under the transmission model to estimate HIV-1 evolutionary rates among epidemiologically-related patients and demonstrate that they lie in between fast intra-host rates and lower rates among epidemiologically unrelated individuals infected with HIV subtype C. Using a new molecular clock approach, we quantify and find support for a lower evolutionary rate along branches that accommodate a transmission event or branches that represent the entire backbone of transmitted lineages in our transmission history. Finally, we recover the rate differences at the different biological scales for both synonymous and non-synonymous substitution rates, which is only compatible with the ‘store and retrieve’ hypothesis positing that viruses stored early in latently infected cells preferentially transmit or establish new infections upon reactivation.     

Prevention and control of emerging infections: a challenge for the 3rd millennium

In the last 30 years, several emerging infections due to novel viruses have been identified, from haemorrhagic fever viruses to HIV, from the SARS-Coronavirus to Avian influenza viruses. Ecological and genetic changes are important determinants of the emergence of new viral infections, driving to an increase of R0 (the basic reproductive number) through increasing the probability of transmission. The current H5N1 epidemic may be considered a prepandemic paradigm that needs thorough investigation.     

Effects of point mutations in the major capsid protein of beet western yellows virus on capsid formation,virus accumulation,and aphid transmission

Point mutations were introduced into the major capsid protein (P3) of cloned infectious cDNA of the polerovirus beet western yellows virus (BWYV) by manipulation of cloned infectious cDNA. Seven mutations targeted sites on the S domain predicted to lie on the capsid surface. An eighth mutation eliminated two arginine residues in the R domain, which is thought to extend into the capsid interior. The effects of the mutations on virus capsid formation, virus accumulation in protoplasts and plants, and aphid transmission were tested. All of the mutants replicated in protoplasts. The S-domain mutant W166R failed to protect viral RNA from RNase attack, suggesting that this particular mutation interfered with stable capsid formation. The R-domain mutant R7A/R8A protected approximately 90% of the viral RNA strand from RNase, suggesting that lower positive-charge density in the mutant capsid interior interfered with stable packaging of the complete strand into virions. Neither of these mutants systemically infected plants. The six remaining mutants properly packaged viral RNA and could invade Nicotiana clevelandii systemically following agroinfection. Mutant Q121E/N122D was poorly transmitted by aphids, implicating one or both targeted residues in virus-vector interactions. Successful transmission of mutant D172N was accompanied either by reversion to the wild type or by appearance of a second-site mutation, N137D. This finding indicates that D172 is also important for transmission but that the D172N transmission defect can be compensated for by a "reverse" substitution at another site. The results have been used to evaluate possible structural models for the BWYV capsid.     

Discussion: The Kermack-McKendrick epidemic threshold theorem

The paper reviews the work of Kermack and McKendrick on the development of simple mathematical models of the transmission dynamics of viral and bacterial infectious agents within population of hosts. The focus of attention is centred on the notion of a threshold density of susceptible hosts to trigger an epidemic and recent extensions of this idea as expressed in the definition of a basic or case reproductive rate of infection. The main body of the paper examines recent developments of the basic Kermack-McKendrick model with an emphasis on deterministic models that describe various types of heterogeneity in the processes that determine transmission between infected and susceptible persons. Particular attention is given to the role of behavioural heterogeneity within the framework of a contact or mixing matrix which defines “who acquires infection from whom”.     

Application of Arrhenius kinetic theory to viral eclipse: selection of bacteriophage phi X174 mutants.

Analysis of the bacteriophage phi X174 eclipse period in terms of Arrhenius kinetic theory suggests the following hypothesis: mutants should exist with two concomitant physiological characteristics as their phenotype. These are an eclipse rate lower than that of the wild type at permissive temperatures for plaque formation and an eclipse rate too low at lower temperatures to permit plaque development. Thus, enrichment of a mutagenized virus population for mutants that fail to eclipse during a short period at permissive temperatures should yield eclipse mutants with the cold-sensitive (cs; nonpermissive temperature, 25 degrees C), and not the temperature-sensitive (ts; nonpermissive temperature, 42 degrees C), plaque phenotype. In several trials, the frequency of the cs phenotype in the population increased from less than 0.2% to between 2 and 4% after the enrichment step, whereas the frequency of the ts phenotype remained unchanged (less than 0.2%). Moreover, 80% of these cs mutants have eclipse rates that are 3- to 40-fold lower than that of the wild type at both 37 degrees C and 25 degrees C. The successful application of the Arrhenius theory to phi X eclipse may provide insights into the molecular mechanism whereby the phi X174 genome is delivered into the host cell. Since the eclipse kinetics of other nonenveloped viruses are similar to those of phi X174, kinetic theory may be broadly applicable in the selection and characterization of viral eclipse mutants.     

Effect of Synaptic Transmission on Viral Fitness in HIV Infection

HIV can spread through its target cell population either via cell-free transmission, or by cell-to-cell transmission, presumably through virological synapses. Synaptic transmission entails the transfer of tens to hundreds of viruses per synapse, a fraction of which successfully integrate into the target cell genome. It is currently not understood how synaptic transmission affects viral fitness. Using a mathematical model, we investigate how different synaptic transmission strategies, defined by the number of viruses passed per synapse, influence the basic reproductive ratio of the virus, R₀, and virus load. In the most basic scenario, the model suggests that R₀ is maximized if a single virus particle is transferred per synapse. R₀ decreases and the infection eventually cannot be maintained for larger numbers of transferred viruses, because multiple infection of the same cell wastes viruses that could otherwise enter uninfected cells. To explain the relatively large number of HIV copies transferred per synapse, we consider additional biological assumptions under which an intermediate number of viruses transferred per synapse could maximize R₀. These include an increased burst size in multiply infected cells, the saturation of anti-viral factors upon infection of cells, and rate limiting steps during the process of synapse formation.     

Attenuation of Sindbis virus neurovirulence by using defined mutations in nontranslated regions of the genome RNA.

We examined a panel of Sindbis virus mutants containing defined mutations in the 5' nontranslated region of the genome RNA, in the 3' nontranslated region, or in both for their growth in cultured cells and virulence in newborn mice. In cultured cells, these viruses all had defects in RNA synthesis and displayed a wide range of growth rates. The growth properties of the mutants were often very different in mouse cells from those in chicken cells or in mosquito cells. We hypothesize that host factors, presumably proteins, interact with these nontranslated regions to promote viral replication and that the mammalian protein and the chicken or mosquito protein are sufficiently divergent that alterations in the viral RNA sequence can affect the interactions with these different host proteins in different ways. Some of the mutants were temperature sensitive for plaque formation, whereas one mutant was slightly cold sensitive in its growth in chicken cells. Upon inoculation into mice, viruses that grew well in cultured mouse cells retained their virulence, but mice that succumbed usually had extended survival times. One virulent mutant that grew slightly less well in cultured mouse cells than did the parental virus produced eight times as much virus in mouse brain following intracerebral inoculation, suggesting that changes in these regulatory regions may have tissue-specific as well as host-specific effects. Viruses that were severely crippled in their growth in mouse cells in culture were usually, but not always, attenuated in their virulence. In particular, temperature sensitivity was correlated with attenuation. The effect of two mutations was found to be cumulative, and double mutants that contained mutations in both the 5' and 3' nontranslated regions were more attenuated than was either single mutant. Three of four double mutants tested were severely crippled for virus production in cultured cells and were avirulent for mice, even when inoculated intracerebrally.     

Analysis of VSV mutant with attenuated cytopathogenicity: mutation in viral function, P, for inhibition of protein synthesis.

T1026, a ts mutant of VSV which is much less cytopathogenic than its parent, HR, and which can establish persistent infection under certain conditions, is a double mutant. In addition to its ts mutation in the virion RNA polymerase, T1026 has a second non-ts mutation in a viral function termed "P". This function is responsible for the inhibition of total protein synthesis in infected cells and acts chiefly at the level of translational initiation. In some cell systems, the inhibition of protein synthesis produced by P appears to be selective for cellular protein synthesis, whereas in other cell systems, both cellular and viral protein synthesis are inhibited. T1026 and its ts revertants are phenotypically P- -that is, cells infected with them show total protein synthesis rates equal to or greater than uninfected cells, while synthesizing viral proteins at the same or even greater rates than HR-infected cells. The P- mutation is correlated with failure to increase plaque size after 2-3 days of incubation. Since viral mutants obtained from persistently infected cultures in a variety of systems appear to be double mutants with a ts mutation in the virion RNA polymerase and a small plaque marker, we suggest that T1026 could represent a model for such mutants.     

Multiple viral genetic analyses detect low-level human immunodeficiency virus type 1 replication during effective highly active antiretroviral therapy

To evaluate human immunodeficiency virus type 1 (HIV-1) replication and selection of drug-resistant viruses during seemingly effective highly active antiretroviral therapy (HAART), multiple HIV-1 env and pol sequences were analyzed and viral DNA levels were quantified from nucleoside analog-experienced children prior to and during a median of 5.1 (range, 1.8 to 6.4) years of HAART. Viral replication was detected at different rates, with apparently increasing sensitivity: 1 of 10 by phylogenetic analysis; 2 of 10 by viral evolution with increasing genetic distances from the most recent common ancestor (MRCA) of infection; 3 of 10 by selection of drug-resistant mutants; and 6 of 10 by maintenance of genetic distances from the MRCA. When four- or five-drug antiretroviral regimens were given to these children, persistent plasma viral rebound did not occur despite the accumulation of highly drug-resistant genotypes. Among the four children without genetic evidence of viral replication, a statistically significant decrease in the genetic distance to the MRCA was detected in three, indicating the persistence of a greater number of early compared to recent viruses, and their HIV-1 DNA decreased by > or =0.9 log(10), resulting in lower absolute DNA levels (P = 0.007). This study demonstrates the variable rates of viral replication when HAART has suppressed plasma HIV-1 RNA for years to a median of <50 copies/ml and that combinations of four or five antiretroviral drugs suppress viral replication even after short-term virologic failure of three-drug HAART and despite ongoing accumulation of drug-resistant mutants. Furthermore, the decrease of cellular HIV-1 DNA to low absolute levels in those without genetic evidence of viral replication suggests that monitoring viral DNA during HAART may gauge low-level replication.