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Introduction: Molecular epidemiology of viruses
seminars in VIROI..OGY, Vol 6, 1995: pp 357-358
Introduction: Molecular epidemiology of viruses Olen Kew and Neal Nathanson
The origins of epidemics have long been one of the deepest mysteries of medicine. The rise of m o d e r n epidemiology has paved the way for the control of infectious diseases through the understanding of the modes of transmission of the infecting microbial agents. 1 Learning the details of infectious spread is especially important to virology, because our main lines of defense are prevention of infection and maintenance of high levels of community or individual immunity. Integration of immunization with comprehensive epidemiologic surveillance has been the key to the control of many viral diseases. The application of molecular methods has added a new dimension to virologic surveillance. Many important, previously unanswerable, epidemiologic questions can now be definitively addressed using molecular tools. Links between cases and infections can be unambiguously established. Patterns of transmission can be resolved. Reservoir communities or host species can be clearly identified. The importance of virus variants to endemicity and epidemicity can be assessed. The impact of immunization programs can be indepe.ndently measured by monitoring the disappearance of specific virus lineages. Complex transmission patterns are occasionally found to underlie apparently simple epidemic curves, and cases that are temporally and geographically clustered are sometimes found to be associated with different lineages. As its name implies, molecular epidemiology is a hybrid discipline, combining the tools and concepts of classical epidemiology with those of microbiology, biochemistry, genetics and evolutionary biology. Genetic variability among infectious agents forms the the foundation of molecular epidemiology, for which the most powerful applications are in virology. This is the consequence of several basic properties of viruses. First, virus generation times are typically very short, such that virus populations can increase rapidly in a single host. If conditions favor efficient transmission of the virus to other hosts, a single f o u n d e r lineage From the Division of Viral and Rickettsial Diseases, Centers for Disease Control and prevention, Atlanta, GA, and *University of Pennslyvania Medical Centeg, Philadelphia, PA, USA ©1995 Academic Press Ltd
• 357
can expand explosively within a few days. Second, the polymerases catalyzing replication of RNA virus and retrovirus genomic templates have high error rates, primarily because they lack proofreading activities. Populations of RNA viruses and retroviruses are typically described as quasispecies, consisting of a broad collection of variants distributed about a consensus genomic sequence. Third, changing environmental conditions, such as the dynamics of immune responses in individual hosts or the availability of new host species, can drive virus populations towards rapid evolution. Fourth, the migration of human and animal hosts can facilitate the widespread dissemination of viral agents. Fifth, virus infections are often associated with cases of disease, prompting specimens for analysis to be taken at many different times and locations. Finally, viral genomes are sufficiently small to permit their detailed characterization by a variety of analytic methods, which differ in specificity and sensitivity. The resolving power of molecular epidemiologic methods is determined by three factors. The first, and most fundamental, factor is the overall rate of evolution of a particular virus in nature. The second is the capacity of the molecular tools to detect differences between viruses. T h e third factor is the location and size of the genomic interval selected for analysis. Selection of the most appropriate sample follows from the epidemiologic question u n d e r consideration. Small sequence samples may be sufficient for rapidly evolving viruses, provided that the sequences studied are representative of the total genome. Wider genomic surveys may be necessary when evolution rates are slow or when recombination or reassortment of gene segments are important sources of variability. Small samples may also be sufficient to detect changes associated with alterations in antigenicity, virulence, or resistance to antiviral agents. Virus evolution rates are controlled primarily by the frequency of passage through evolutionary bottlenecks rather than by the inherent error rates of viral polymerases. Some RNA viruses, whose genomes are highly mutable u n d e r laboratory conditions, evolve comparatively slowly in nature. A clear illustration o f
Introduction
DNA sequence analysis, frequently performed in combination with PCR, has made it the method of choice for most molecular epidemiologic investigations. Genomic comparisons have been complemented in many virus systems by phenotypic studies, especially those following antigenic evolution using panels of monoclonal antibodies or sera derived from the natural hosts. This issue represents a progress report on the lessons learned from molecular epidemiologic studies. Many different patterns of virus circulation have been revealed: multiple co-circulating lineages, temporally predominating lineages, geographic clustering of lineages, and the restriction of lineages to specific reservoir species. Several broad themes recur in these reviews. One is the impact of a changing global environment on human health. With the h u m a n population in closer contact than at any other time in history, viruses and other infectious agents can spread ever faster. Concurrently, increased contact with zoonofic reservoirs, coupled with the capacity of many viruses to cross species lines, raises the risks for infections of humans with virus variants selected in animal hosts. In some systems, recombination and reassortment play an important role in the generation of new variants. T h e r e is also the central theme of virus evolution in response to host defenses, with its implications for vaccine development and refinement. Finally, the impact of immunization on virus circulation is considered in the context of optimal immunization strategies and current progress towards the control of vaccine-preventable diseases. These brief reviews, opening with the classic influenza virus system and closing with the more recently characterized parvovirus system, together cover the major conceptual developments in the molecular'epidemiology of viruses.
this point is the contrast between the slow evolution of influenza A viruses in birds and the rapid evolution of the same viruses in humans. Other viruses, such as feline parvoviruses, whose DNA genomes are many orders of magnitude less mumble than those of RNA viruses, have evolved over short time periods to give rise to variants with important new phenotypes. Evolutionary bottlenecks may involve direct positive selection, such as when antigenic variants more resistant to host immune responses grow out from virus populations. Because virus populations in a single host can be very large, variants with increased replicafive fitness (under a specific set of conditions) may pre-exist in populations of viruses with relatively stable genomes. The variants with higher fitness can quickly predominate in the presence of intense new selective forces. The bottlenecks most frequently encountered by rapidly evolving RNA viruses appear not to involve functional selection. Instead, minority variants within the heterogeneous quasispecies are randomly sampled for passage through bottlenecks that arise when virus populations decline to very low numbers. This process of genetic drift is generally accompanied by the accumulation of 'neutral' mutations, a large proportion of which are substitutions to synonymous codons. The net effect of the bottlenecks is to produce new f o u n d e r lineages which can be distinguished from the preceding population using molecular techniques. Many different methods for sampling genomic variability have been used in molecular epidemiologic studies. Initial studies used nucleic acid hybridization, RNase T1 oligonucleofide fingerprinting of RNA genomes, restriction fragment length polymorphism (RFLP) analysis of DNA genomes, and high-resolution electrophoresis of genomic segments. More recently, various methods based u p o n the polyrnerase chain reaction (PCR) have been widely used. One PCR approach uses primers with consensus sequences to amplify diverse templates that are subsequently characterized by RFLP or sequencing. Another approach uses highly specific primers to selectively amplify particular lineages or variants in mixed virus populations. The increasing speed and efficiency of
References 1. Nathanson N (1990) Epidemiology,in Virology,2nd ed (Fields BN, Knipe DM, Chanock RM, Hirsch MS, MelnickJL, Monath TP, Roizman B, eds) pp 267-291. Raven Press, New York
358
Introduction: Molecular epidemiology of viruses Olen Kew and Neal Nathanson
The origins of epidemics have long been one of the deepest mysteries of medicine. The rise of m o d e r n epidemiology has paved the way for the control of infectious diseases through the understanding of the modes of transmission of the infecting microbial agents. 1 Learning the details of infectious spread is especially important to virology, because our main lines of defense are prevention of infection and maintenance of high levels of community or individual immunity. Integration of immunization with comprehensive epidemiologic surveillance has been the key to the control of many viral diseases. The application of molecular methods has added a new dimension to virologic surveillance. Many important, previously unanswerable, epidemiologic questions can now be definitively addressed using molecular tools. Links between cases and infections can be unambiguously established. Patterns of transmission can be resolved. Reservoir communities or host species can be clearly identified. The importance of virus variants to endemicity and epidemicity can be assessed. The impact of immunization programs can be indepe.ndently measured by monitoring the disappearance of specific virus lineages. Complex transmission patterns are occasionally found to underlie apparently simple epidemic curves, and cases that are temporally and geographically clustered are sometimes found to be associated with different lineages. As its name implies, molecular epidemiology is a hybrid discipline, combining the tools and concepts of classical epidemiology with those of microbiology, biochemistry, genetics and evolutionary biology. Genetic variability among infectious agents forms the the foundation of molecular epidemiology, for which the most powerful applications are in virology. This is the consequence of several basic properties of viruses. First, virus generation times are typically very short, such that virus populations can increase rapidly in a single host. If conditions favor efficient transmission of the virus to other hosts, a single f o u n d e r lineage From the Division of Viral and Rickettsial Diseases, Centers for Disease Control and prevention, Atlanta, GA, and *University of Pennslyvania Medical Centeg, Philadelphia, PA, USA ©1995 Academic Press Ltd
• 357
can expand explosively within a few days. Second, the polymerases catalyzing replication of RNA virus and retrovirus genomic templates have high error rates, primarily because they lack proofreading activities. Populations of RNA viruses and retroviruses are typically described as quasispecies, consisting of a broad collection of variants distributed about a consensus genomic sequence. Third, changing environmental conditions, such as the dynamics of immune responses in individual hosts or the availability of new host species, can drive virus populations towards rapid evolution. Fourth, the migration of human and animal hosts can facilitate the widespread dissemination of viral agents. Fifth, virus infections are often associated with cases of disease, prompting specimens for analysis to be taken at many different times and locations. Finally, viral genomes are sufficiently small to permit their detailed characterization by a variety of analytic methods, which differ in specificity and sensitivity. The resolving power of molecular epidemiologic methods is determined by three factors. The first, and most fundamental, factor is the overall rate of evolution of a particular virus in nature. The second is the capacity of the molecular tools to detect differences between viruses. T h e third factor is the location and size of the genomic interval selected for analysis. Selection of the most appropriate sample follows from the epidemiologic question u n d e r consideration. Small sequence samples may be sufficient for rapidly evolving viruses, provided that the sequences studied are representative of the total genome. Wider genomic surveys may be necessary when evolution rates are slow or when recombination or reassortment of gene segments are important sources of variability. Small samples may also be sufficient to detect changes associated with alterations in antigenicity, virulence, or resistance to antiviral agents. Virus evolution rates are controlled primarily by the frequency of passage through evolutionary bottlenecks rather than by the inherent error rates of viral polymerases. Some RNA viruses, whose genomes are highly mutable u n d e r laboratory conditions, evolve comparatively slowly in nature. A clear illustration o f
Introduction
DNA sequence analysis, frequently performed in combination with PCR, has made it the method of choice for most molecular epidemiologic investigations. Genomic comparisons have been complemented in many virus systems by phenotypic studies, especially those following antigenic evolution using panels of monoclonal antibodies or sera derived from the natural hosts. This issue represents a progress report on the lessons learned from molecular epidemiologic studies. Many different patterns of virus circulation have been revealed: multiple co-circulating lineages, temporally predominating lineages, geographic clustering of lineages, and the restriction of lineages to specific reservoir species. Several broad themes recur in these reviews. One is the impact of a changing global environment on human health. With the h u m a n population in closer contact than at any other time in history, viruses and other infectious agents can spread ever faster. Concurrently, increased contact with zoonofic reservoirs, coupled with the capacity of many viruses to cross species lines, raises the risks for infections of humans with virus variants selected in animal hosts. In some systems, recombination and reassortment play an important role in the generation of new variants. T h e r e is also the central theme of virus evolution in response to host defenses, with its implications for vaccine development and refinement. Finally, the impact of immunization on virus circulation is considered in the context of optimal immunization strategies and current progress towards the control of vaccine-preventable diseases. These brief reviews, opening with the classic influenza virus system and closing with the more recently characterized parvovirus system, together cover the major conceptual developments in the molecular'epidemiology of viruses.
this point is the contrast between the slow evolution of influenza A viruses in birds and the rapid evolution of the same viruses in humans. Other viruses, such as feline parvoviruses, whose DNA genomes are many orders of magnitude less mumble than those of RNA viruses, have evolved over short time periods to give rise to variants with important new phenotypes. Evolutionary bottlenecks may involve direct positive selection, such as when antigenic variants more resistant to host immune responses grow out from virus populations. Because virus populations in a single host can be very large, variants with increased replicafive fitness (under a specific set of conditions) may pre-exist in populations of viruses with relatively stable genomes. The variants with higher fitness can quickly predominate in the presence of intense new selective forces. The bottlenecks most frequently encountered by rapidly evolving RNA viruses appear not to involve functional selection. Instead, minority variants within the heterogeneous quasispecies are randomly sampled for passage through bottlenecks that arise when virus populations decline to very low numbers. This process of genetic drift is generally accompanied by the accumulation of 'neutral' mutations, a large proportion of which are substitutions to synonymous codons. The net effect of the bottlenecks is to produce new f o u n d e r lineages which can be distinguished from the preceding population using molecular techniques. Many different methods for sampling genomic variability have been used in molecular epidemiologic studies. Initial studies used nucleic acid hybridization, RNase T1 oligonucleofide fingerprinting of RNA genomes, restriction fragment length polymorphism (RFLP) analysis of DNA genomes, and high-resolution electrophoresis of genomic segments. More recently, various methods based u p o n the polyrnerase chain reaction (PCR) have been widely used. One PCR approach uses primers with consensus sequences to amplify diverse templates that are subsequently characterized by RFLP or sequencing. Another approach uses highly specific primers to selectively amplify particular lineages or variants in mixed virus populations. The increasing speed and efficiency of
References 1. Nathanson N (1990) Epidemiology,in Virology,2nd ed (Fields BN, Knipe DM, Chanock RM, Hirsch MS, MelnickJL, Monath TP, Roizman B, eds) pp 267-291. Raven Press, New York
358