Abstract: Recombination is increasingly seen as an important means of shaping genetic diversity in RNA viruses. However, observed recombination frequencies vary widely among those viruses studied to date, with only sporadic occurrences reported in RNA viruses with negative-sense genomes. To determine the extent of homologous recombination in negative-sense RNA viruses, phylogenetic analyses of 79 gene sequence alignments from 35 negative-sense RNA viruses (a total of 2154 sequences) were carried out. Powerful evidence was found for recombination, in the form of incongruent phylogenetic trees between different gene regions, in only five sequences from Hantaan virus, Mumps virus and Newcastle disease virus. This is the first report of recombination in these viruses. More tentative evidence for recombination, where conflicting phylogenetic trees were observed (but were without strong bootstrap support) and/or where putative recombinant regions were very short, was found in three alignments from La Crosse virus and Puumala virus. Finally, patterns of sequence variation compatible with the action of recombination, but not definitive evidence for this process, were observed in a further ten viruses: Canine distemper virus, Crimean-Congo haemorrhagic fever virus, Influenza A virus, Influenza B virus, Influenza C virus, Lassa virus, Pirital virus, Rabies virus, Rift Valley Fever virus and Vesicular stomatitis virus. The possibility of recombination in these viruses should be investigated further. Overall, this study reveals that rates of homologous recombination in negative-sense RNA viruses are very much lower than those of mutation, with many viruses seemingly clonal on current data. Consequently, recombination rate is unlikely to be a trait that is set by natural selection to create advantageous or purge deleterious mutations.

Abstract: Completely revised and updated to reflect important advances in the field, "Principles of Virology, Second Edition" continues to fill the gap between simple introductory texts and very advanced reviews of major virus families, introducing upper-level undergraduates, graduate students, and medical students to all aspects of virology. The second edition retains all of the defining and much-praised features of the first edition, focusing on concepts and principles and presenting a comprehensive treatment from molecular biology to pathogenesis and infection control. Written in an engagingly readable style and generously illustrated with over 400 full-colour illustrations, this approachable volume offers detailed examples that illustrate common principles, specific strategies adopted by different viruses to ensure their reproduction, and the current state of virology research. The book is divided into chapters that focus on specific topics rather than individual viruses, and allows the student to visualize common themes that cut across virus families, emphasizing the shared features of different viruses. Drawing on the extensive teaching experience of each of its distinguished authors, "Principles of Virology" illustrates why and how animal viruses are studied and demonstrates, using well-studied systems, how the knowledge gained from such model viruses can be used to study viral systems about which our knowledge is still quite limited. A thorough introduction to principles of viral pathogenesis, a broad view of viral evolution, a discussion of how viruses were discovered, and how the discipline of virology came to be are also provided. A variety of special boxes highlight key experiments, background material, caveats, and much more. The text focuses on concepts and principles and covers not only aspects of molecular biology, but also pathogenesis, evolution, emergence, and control, and will also be a valuable resource for practicing physicians and scientists. New in the second edition: completely revised pathogenesis chapters; pathogenicity snapshots: an appendix highlighting teaching points for major viral diseases; expanded appendix on viral life cycles; new chapter on viral genomes and coding strategies; detailed glossary; expanded references after each chapter; and new textboxes.

Abstract: In the present study we report on evolution of calicivirus RNA from a patient with chronic diarrhea (i.e., lasting >2 years) and viral shedding. Partial sequencing of open reading frame 1 (ORF1) from 12 consecutive isolates revealed shedding of a genogroup II virus with relatively few nucleotide changes during a 1-year period. The entire capsid gene (ORF2) was also sequenced from the same isolates and found to contain 1,647 nucleotides encoding a protein of 548 amino acids with similarities to the Arg320 and Mx strains. Comparative sequence analysis of ORF2 revealed 32 amino acid changes during the year. It was notable that the vast majority of the cumulative amino acid changes (8 of 11) appeared within residues 279 to 405 located within the hypervariable domain (P2) of the capsid protein and hence were subject to immune pressure. An interesting and novel observation was that the accumulated amino acid changes in the P2 domain resulted in predicted structural changes, including disappearance of a helix structure, and thus a possible emergence of a new phenotype. FUT2 gene polymorphism characterization revealed that the patient is heterozygous at nucleotide 428 and thus Secretor+, a finding in accordance with the hypothesis of FUT2 gene polymorphism and calicivirus susceptibility. To our knowledge, this is the first report of RNA evolution of calicivirus in a single individual, and our data suggest an immunity-driven mechanism for viral evolution. We also report on chronic virus excretion, immunoglobulin treatment, and modification of clinical symptoms; our observations from these studies, together with the FUT2 gene characterization, may lead to a better understanding of calicivirus pathogenesis.

Abstract: Although the ultimate origins of RNA viruses are uncertain, it seems reasonable to assume that these infectious agents have a long evolutionary history, appearing with, or perhaps before, the first cellular life-forms (38). While the RNA viruses we see today may not date back quite this far, the evidence that some DNA viruses have evolved with their vertebrate hosts over many millions of years (24) makes an equally ancient history for RNA viruses a natural expectation. Yet a very different picture of RNA virus origins is painted if their gene sequences are compared; by using the best estimates for rates of evolutionary change (nucleotide substitution) and assuming an approximate molecular clock (21, 33), it can be inferred that the families of RNA viruses circulating today could only have appeared very recently, probably not more than about 50,000 years ago. Hence, if evolutionary rates are accurate and relatively constant, present-day RNA viruses may have originated more recently than our own species. Before discussing the solutions to this apparent paradox, it is important to determine exactly why the molecular clock estimates of RNA virus origins are so recent. The key to establishing a timescale of viral evolution lies in accurately determining the rate of nucleotide substitution. Most analyses undertaken to date suggest that the average rate of nucleotide substitution in RNA viruses is 10 3 substitutions per site per year, with an approximately fivefold range around this (21). The fact that broadly similar rates are found in RNA viruses with very different genome organizations and lifestyles implies that both the error rate associated with RNA polymerase, estimated to be about one mutation per genome replication (10), and the rate of viral replication are roughly constant. If the average substitution rate of 10 3 substitutions/site/year is accurate, then, on average, every nucleotide position will have fixed 1 substitution after 1,000 years of evolution (corresponding to an average divergence time between two lineages of only 500 years). This also corresponds to an evolutionary (corrected) distance (d) between two sequences of 1.0, the maximum that can be reliably estimated through sequence comparisons; larger distances will be inherently inaccurate because of uncounted multiple substitutions at single sites. Of course, reality is a little more complex because viral genomes are a patchwork of synonymous sites, where mutations do not change the encoded amino acid, and nonsynonymous sites, where mutations alter amino acids and which usually evolve more slowly. If we conservatively assume that the substitution

Abstract: The family Flaviviridae includes important human pathogens, such as dengue (DEN) virus, yellow fever (YF) virus and hepatitis C virus, many of which have emerged or re-emerged in recent years. Until recently, flavivirus evolution was thought to proceed in a clonal manner, with diversity generated mainly through the accumulation of mutational changes. However, this assumption has now been shown to be invalid, with homologous recombination demonstrated in all three genera of the Flaviviridae. Since recombination has important implications for the study of virus evolution, a survey of recombination in the viruses of the genus Flavivirus was carried out. Using envelope gene sequence data and a combination of graphical and phylogenetic analyses, hitherto unreported recombination in Japanese encephalitis virus and St Louis encephalitis virus was detected, as well as further recombinants in DEN virus. However, no evidence for recombination was found in West Nile or YF viruses, or in the tick-borne flavivirus group. It is proposed that the difference between the mosquito- and tick-borne viruses can be accounted for by their differing modes of transmission, whilst the variation among the mosquito-borne flaviviruses reflects both the ecology of the particular host and vector species and also bias in the sampling process.

Abstract: 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.

Abstract: Animals and plants evolved systems to permit non-cell-autonomous trafficking of RNA, whereas DNA plays a cell-autonomous role. In plants, plasmodesmata serve as the conduit for this phenomenon, and viruses have evolved to use this pathway for the spread of infectious nucleic acids. In this study, a plant DNA virus was used to explore the constraints imposed on the movement of DNA through this endogenous RNA trafficking pathway. The combined properties of the geminivirus-encoded movement protein and plasmodesmata were shown to impose a strict limitation on the size of the viral genome at the level of cell-to-cell movement. Size-increased viral genome components underwent homologous and nonhomologous recombination to overcome this strict limitation. Our results provide insights into the genetic mechanisms that underlie viral evolution and provide a likely explanation for why relatively few types of plant DNA viruses have evolved: they would have had to overcome the constraints imposed by an endogenous system operating to ensure that DNA acts in a cell-autonomous manner.

Abstract: Publisher Summary The picture beginning to form from genome analyses of viruses, unicellular organisms, and multicellular organisms is that viruses have shared functional modules with cells. A process of coevolution has probably involved exchanges of genetic information between cells and viruses for long evolutionary periods. From this point of view present-day viruses show flexibility in receptor usage and a capacity to alter through mutation their receptor recognition specificity. It is possible that for the complex DNA viruses, due to a likely limited tolerance to generalized high mutation rates, modifications in receptor specificity will be less frequent than for RNA viruses, albeit with similar biological consequences once they occur. It is found that different receptors, or allelic forms of one receptor, may be used with different efficiency and receptor affinities are probably modified by mutation and selection. Receptor abundance and its affinity for a virus may modulate not only the efficiency of infection, but also the capacity of the virus to diffuse toward other sites of the organism. The chapter concludes that receptors may be shared by different, unrelated viruses and that one virus may use several receptors and may expand its receptor specificity in ways that, at present, are largely unpredictable.

Abstract: A hallmark of the infectious cycle for many RNA viruses parasitizing multicellular hosts is the need to invade and successfully replicate in tissues that comprise a variety of cell types. Thus, multicellular hosts represent a heterogeneous environment to evolving viral populations. To understand viral adaptation to multicellular hosts, we took a double approach. First, we developed a mathematical model that served to make predictions concerning the dynamics of viral populations evolving in heterogeneous environments. Second, the predictions were tested by evolving vesicular stomatitis virus in vitro on a spatially structured environment formed by three different cell types. In the absence of gene flow, adaptation was tissue-specific, but fitness in all tissues decreased with migration rate. The performance in a given tissue was negatively correlated with its distance to the tissue hosting the population. This correlation decreased with migration rate.

Abstract: Despite extraordinary progress that has recently been made in biomedical sciences, viral infectious diseases still remain one of the most serious world health problems. Among the different types of viruses, those using RNA as their genetic material (RNA viruses and retroviruses) are especially dangerous. At present there is no medicine allowing an effective treatment of RNA-based virus infections. Many RNA viruses and retroviruses need only a few weeks to escape immune response or to produce drug-resistant mutants. This seems to be the obvious consequence of the unusual genetic variability of RNA-based viruses. An individual virus does not form a homogenous population but rather a set of similar but not identical variants. In consequence, RNA-based viruses can easily adapt to environmental changes, also those resulting from immune system response or therapy. The modifications identified within viral genes can be divided into two groups: point mutations and complex genome rearrangements. The former arises mainly during error-prone replication, whereas RNA recombination and generic reassortment are responsible for the latter. This article shortly describes major strategies used to control virus infections. Then, it presents the various mechanisms generating the genetic diversity of RNA-based viruses, which are most probably the main cause of clinical problems.

Abstract: RNA virus populations consist of complex and dynamic mutant distributions, rather than defined genomic sequences. This feature confers great adaptability on viruses and is partly responsible for current difficulties of viral disease prevention and control. Mutant distributions, also termed mutant swarms or mutant clouds, were first proposed in a theory of molecular evolution termed quasispecies theory. The theoretical formulation of quasispecies and its links to present day RNA viruses are discussed. The need to accommodate antiviral strategies to the dynamic nature of viral populations is emphasized. In particular, recent results on viral extinction associated with enhanced mutagenesis (virus entry into error catastrophe) are reviewed and presented as an example of how the understanding of viruses as quasispecies could lead to a potential practical application in medicine.

Abstract: Epistasis results when the fitness effects of a mutation change depending on the presence or absence of other mutations in the genome. The predictions of many influential evolutionary hypotheses are determined by the existence and form of epistasis. One rich source of data on the interactions among deleterious mutations that has gone untapped by evolutionary biologists is the literature on the design of live, attenuated vaccine viruses. Rational vaccine design depends upon the measurement of individual and combined effects of deleterious mutations. In the current study, we have reviewed data from 29 vaccine-oriented studies using 14 different RNA viruses. Our analyses indicate that (1) no consistent tendency towards a particular form of epistasis exists across RNA viruses and (2) significant interactions among groups of mutations within individual viruses occur but are not common. RNA viruses are significant pathogens of human disease, and are tractable model systems for evolutionary studies – we discuss the relevance of our findings in both contexts.

Abstract: The Bo17 gene of bovine herpesvirus 4 (BoHV-4) is the only viral gene known to date that encodes a homologue of the cellular core 2 β-1,6-N-acetylglucosaminyltransferase-mucin type (C2GnT-M). To investigate the origin and evolution of the Bo17 gene, we analyzed its distribution among BoHV-4 strains and determined the sequences of Bo17 from nine representative strains and of the C2GnT-M gene from six species of ruminants expected to encompass the group within which the gene acquisition occurred. Of 34 strains of BoHV-4, isolated from four different continents, all were found to contain the Bo17 gene. Phylogenetic analyses indicated that Bo17 was acquired from a recent ancestor of the African buffalo, implying that cattle subsequently acquired BoHV-4 by cross-species transmission. The rate of synonymous nucleotide substitution in Bo17 was estimated at 5 × 10−8 to 6 × 10−8 substitutions/site/year, consistent with previous estimates made under the assumption that herpesviruses have cospeciated with their hosts. The Bo17 gene acquisition was dated to around 1.5 million years ago. Bo17 sequences from BoHV-4 strains from African buffalo and from cattle formed two separate clades, estimated to have split about 700,000 years ago. Analysis of the ratio of nonsynonymous to synonymous nucleotide substitutions revealed a burst of amino acid replacements subsequent to the transfer of the cellular gene to the viral genome, followed by a return to a strong constraint on nonsynonymous changes during the divergence of contemporary BoHV-4 strains. The Bo17 gene represents the most recent of the known herpesvirus gene acquisitions and provides the best opportunity for learning more about this important process of viral evolution.

Abstract: The evolution of viral genomes has recently attracted considerable attention. We compare the sequences of two large viral genomes, EsV-1 and FirrV-1, belonging to the family of phaeoviruses which infect different species of marine brown algae. Although their genomes differ substantially in size, these viruses share similar morphologies and similar latent infection cycles. In fact, sequence comparisons show that the viruses have more than 60% of their genes in common. However, the order of genes is completely different in the two genomes, suggesting that extensive recombinational events in addition to several large deletions had occurred during the separate evolutionary routes from a common ancestor. We investigated genes encoding components of signal transduction pathways and genes encoding replicative functions in more detail. We found that the two genomes possess different, although overlapping, sets of genes in both classes, suggesting that different genes from each class were lost, perhaps randomly, after the separate evolution from an ancestral genome. Random loss would also account for the fact that more than one-third of the genes in one viral genome has no counterparts in the other genome. We speculate that the ancestral genome belonged to a cellular organism that had once invaded a primordial brown algal host.

Abstract: The rate of disease development in simian immunodeficiency virus (SIV) infection of macaques varies considerably among individual macaques. While the majority of macaques inoculated with pathogenic SIV develop AIDS within a period of 1 to 2 years, a minority exhibit a rapid disease course characterized by absence or transience of humoral and cellular immune responses and high levels of virus replication with widespread dissemination of SIV in macrophages and multinucleated giant cells. The goal of this study was to examine viral evolution in three SIVsmE543-3-inoculated rapid progressors to determine the contribution of viral evolution to the development of rapid disease and the effect of the absence of immune pressure upon viral evolution. PCR was used to amplify and clone the entire SIV genome from tissues collected at necropsy, and the course of viral evolution was assessed by env sequences cloned from sequential plasma samples of one rapid progressor (RP) macaque. The majority of sequence changes in RP macaques occurred in the envelope gene. Substitutions were observed in all three animals at specific conserved residues in envelope, including loss of a glycosylation site in V1/V2, a D-to-N/V substitution in a highly conserved GDPE motif, and a P-to-V/H/T substitution in the V3 loop analog. A cell-cell fusion assay revealed that representative env clones utilized CCR5 as a coreceptor, independent of CD4. The selection of specific substitutions in envelope in RP macaques suggests novel selection pressures on virus in such animals and suggests that viral variants that evolve in these animals may play a role in disease progression.

Abstract: This paper describes the study of hepatitis C virus (HCV) evolution in the largest cohort of HCV-infected blood donors (BDs)/blood recipients (BRs) reported to date (25 pairs). A molecular analysis of partial sequences in the E1 (envelope) and NS5-B (polymerase) genes was performed. Phylogenetic reconstruction showed that the evolution of dominant strains was qualitatively and quantitatively different in BDs and BRs. The evolutionary rate was significantly higher in BRs, in which, in addition, most substitutions observed were antonymous. These findings corroborate the hypothesis that a large part of virus evolution – which was evaluated to be equivalent to ∼20 years of chronic evolution – is acquired during the early phase of infection. These findings should be taken into account for the modelling of the long-term evolution of HCV and their possible contribution to improve our understanding of HCV natural history is discussed.

Abstract: This paper describes the study of hepatitis C virus (HCV) evolution in the largest cohort of HCVinfected blood donors (BDs)/blood recipients (BRs) reported to date (25 pairs). A molecular analysis of partial sequences in the E1 (envelope) and NS5-B (polymerase) genes was performed. Phylogenetic reconstruction showed that the evolution of dominant strains was qualitatively and quantitatively different in BDs and BRs. The evolutionary rate was significantly higher in BRs, in which, in addition, most substitutions observed were antonymous. These findings corroborate the hypothesis that a large part of virus evolution ‐ which was evaluated to be equivalent to ~20 years ofchronic evolution ‐ isacquired during theearly phaseofinfection. Thesefindings should betaken into account for the modelling of the long-term evolution of HCV and their possible contribution to improve our understanding of HCV natural history is discussed. During the second half of the twentieth century, several million persons have been contaminated by hepatitis C virus (HCV) following blood transfusion. Despite this extremely significant number of cases, virus evolution ‐ in particular during the early stages of infection ‐ remains poorly understood. The genome of HCV is replicated with the help of a viral RNA polymerase, which lacks the proofreading activity of DNA polymerase complexes and therefore confers on the virus a high rate of genome evolution. A number of studies have been dedicated to the analysis of HCV evolution over time, principally by determining and comparing virus sequences at two different times of the infection in humans (Ogata et al., 1991) or chimpanzees (Lu et al., 2001), or virus sequences from different individuals infected by the same virus strain (McAllister et al., 1998). In most of the cases, virus sequences could not be determined at the early stages of infection and therefore, data obtained are mostly reflecting the phenomena occurring during chronic infection. We describe here a molecular analysis based on the study of the largest cohort of HCV-infected blood donors (BDs)/blood recipients (BRs) reported to date (25 pairs). It reveals qualitative and quantitative differences in the apparent evolution of the dominant strains from BDs and their respective BRs. The significance and possible contribution of these findings to improve our understanding of HCV evolution are discussed.

Abstract: Viruses commonly cause gastrointestinal illnesses in dogs and cats that range in severity from mild diarrhoea to malignant neoplasia. Perpetual evolution of viruses is reflected in changing disease patterns, so that familiar viruses are sometimes discovered to cause new or unexpected diseases. For example, canine parvovirus (CPV) has regained the ability to infect felids and cause a panleucopenia-like illness. Feline panleucopenia virus (FPV) has been shown to cause fading in young kittens and has recently been implicated as a possible cause of feline idiopathic cardiomyopathy. Molecular scrutiny of viral diseases sometimes permits deeper understanding of pathogenesis and epizootiology. Feline gastrointestinal lymphomas have not, in the past, been strongly associated with retroviral infections, yet some of these tumours harbour retroviral proviruses. Feline leukaemia virus (FeLV) may play a role in lymphomagenesis, even in cats diagnosed as uninfected using conventional criteria. There is strong evidence that feline immunodeficiency virus (FIV) can also be oncogenic. The variant feline coronaviruses that cause invariably-fatal feline infectious peritonitis (FIP) arise by sporadic mutation of an ubiquitous and only mildly pathogenic feline enteric coronavirus (FECV); a finding that has substantial management implications for cat breeders and veterinarians. Conversely, canine enteric coronavirus (CECV) shows considerable genetic and antigenic diversity but causes only mild, self-limiting diarrhoea in puppies. Routine vaccination against this virus is not recommended. Although parvoviruses, coronaviruses and retroviruses are the most important known viral causes of canine and feline gastrointestinal disease, other viruses play a role. Feline and canine rotaviruses have combined with human rotaviruses to produce new, reassortant, zoonotic viruses. Some companion animal rotaviruses can infect humans directly. Undoubtedly, further viral causes of canine and feline gastrointestinal disease await discovery.

Abstract: More than 50 researchers and administrators from over a dozen countries attended a symposium on the emergence and control of zoonotic viral encephilitis. Held April 6–8, 2003, in a convivial setting at Les Pensieres, Veyrier du Lac, near Annecy in the French Alps, this meeting was one of a series on the emergence and control of infectious diseases, sponsored and organized by the Merieux Foundation. The general objectives were to review the biology of viral encephalitis, the virulence and genetic evolution of encephalitis viruses, and the factors involved in emergence of these diseases. Emergence or reemergence of viruses may be due to virus evolution, to the impact and influence of human populations on previously undisturbed ecosystems, or to better recognition. Clearly, we must understand the basic mechanisms by which these viruses emerge or reemerge and cause illnesses. Methods for detecting infections caused by neurotropic viruses and for detecting viruses or their genome sequences are available and improving. Methods for detecting antibodies also have improved. Examples of recently recognized viruses causing encephalitis in humans, livestock, or wildlife include Hendra and Nipah viruses (henipaviruses; family Paramyxoviridae, genus Henipavirus), both of which are neurotropic, and Australian bat lyssavirus (family Rhabdoviridae, genus Lyssavirus), also a neurotrope. All three viruses, and others related to them, have been shown to have fruit bats (Pteropus spp.) as their natural hosts. Progress is being made in understanding transcription regulation and cell fusion by henipaviruses. In addition, basic epidemiologic procedures and classical prevention strategies have been put into place to prevent infections caused by these viruses. Since 1988, a worldwide effort has been under way to eradicate the nonzoonotic but encephalitogenic poliomyelitis viruses. The number of cases has been reduced by 99%, and the natural occurrence of these viruses now is limited to seven countries. The system established to conduct surveillance and response may provide a model for use in tracking and controlling other viruses causing encephalitis. Long-term studies of ecologic parameters, seasonality, and changing virus and vector prevalence rates are being used to determine risk factors in various arbovirus infections, including Japanese encephalitis virus in Thailand, and are being applied for prevention and control. In Russia, where West Nile virus has long been recognized but has not caused any major diseases, recent detection of various virus genotypes suggests a melange of genotypes circulating in various areas and transported between areas by birds. Generation and maintenance of continuous genetic variation may lead to partial protection and escape mutants, which could provide a “pump” that generates more variants and “new” viruses. When these genotypes adapt to naive populations of birds, horses, and humans, in the presence of competent arthropod vectors, epidemics may arise and new opportunities for these viruses and virus variants may occur, perhaps including West Nile virus into the New World in 1999. Evidence presented suggests that little genomic variation in New World West Nile virus has occurred since its 1999 recognition there; this situation is likely to change. Continuing to make the classical epidemiologic observations that have characterized disease investigations in the previous half-century is important. However, to understand the overall effects of virus outbreaks, denominators are needed. Numerous presentations demonstrated that we are beginning to understand the molecular mechanisms leading to pathogenetic events. Further studies may provide information useful for the development of antiviral compounds and candidate vaccines. Attendees were provided with an overview of various transmission cycles of arboviruses, which are concomitantly diverse in regards to their hosts and vectors. Viral neuroinvasiveness appears to depend on the uniqueness of phylogenetically diverse hosts, their ages, genetic predispositions, immune status, virus origin, passage level, dose, and other factors—a complex situation to investigate and comprehend. Critical factors impacting neuroinvasiveness and neuronotropism must be coupled to cause encephalitis. Viral mutations may affect the ability of the virus to replicate in cells, altering viral virulence; however, specific genomic and polyprotein sequence changes may account for the high viremias and replication in neurons that are central to emergence. The extent of the roles of various proteins in virus infections, neuronal involvement, and apoptosis are being recognized. Now we are beginning to understand complex signaling mechanisms, antibody-producing cell types, cytokines, and the cellular responses and pathways leading to both disease and protection from disease. Considerable progress has been made in understanding the relationships between genetic and functional diversities, neuronal receptors, transport, and cellular protein-virus interactions. Such understanding is critical to further insights to neurotropism, pathogenesis, pathogenetic mechanisms, and immunogenicity. Phylogenetic trees were used to describe the evolution of encephalitic flaviviruses, geographic exclusion, virus persistence, and flaviviral recombination as a mechanism of flaviviral evolution. In addition, data were presented that illustrated the persistence of, and immune modulation by, alphaviruses, which, in concert, allow the virus to replicate while preventing the host from responding to its benefit. Other than the classical techniques of preventing infection, little was mentioned about disease control during this symposium. Control must be based on rapid recognition of early cases, subsequent immunization of persons or animals at risk, or immunization of persons or animals with the potential to be at risk, such as travelers, laboratory personnel, and attending clinicians. Attendees learned about diverse methods being used to develop vaccines. Representatives from the World Health Organization explained that organization’s plans for responding to disease emergence and for preventing zoonotic diseases from reaching human populations. New paradigms for field studies of zoonotic diseases are necessary. These approaches must include longitudinal and in-depth investigations of agent, host, habitat, and environment if we are to predict risk and respond in an appropriate manner. At this time, zoonotic disease control comprises prevention and public education and not much more. Progress is being made in rapid diagnosis, production of sophisticated vaccines, and understanding of the molecular mechanisms by which zoonotic viruses persist and cause disease.

Abstract: Macrophagetropic R5 human immunodeficiency virus type 1 (HIV-1) isolates often evolve into dualtropic R5X4 variants during disease progression. The structural basis for CCR5 coreceptor function has been studied in a limited number of prototype strains and suggests that R5 and R5X4 Envs interact differently with CCR5. However, differences between unrelated viruses may reflect strain-specific factors and do not necessarily represent changes resulting from R5 to R5X4 evolution of a virus in vivo. Here we addressed CCR5 domains involved in fusion for a large set of closely related yet functionally distinct variants within a primary isolate swarm, employing R5 and R5X4 Envs derived from the HIV-1 89.6PI quasispecies. R5 variants of 89.6PI could fuse using either N-terminal or extracellular loop CCR5 sequences in the context of CCR5/CXCR2 chimeras, similar to the unrelated R5 strain JRFL, but R5X4 variants of 89.6PI were highly dependent on the CCR5 N terminus. Similarly, R5 89.6PI variants and isolate JRFL tolerated N-terminal CCR5 deletions, but fusion by most R5X4 variants was markedly impaired. R5 89.6PI Envs also tolerated multiple extracellular domain substitutions, while R5X4 variants did not. In contrast to CCR5 use, fusion by R5X4 variants of 89.6PI was largely independent of the CXCR4 N-terminal region. Thus, R5 and R5X4 species from a single swarm differ in how they interact with CCR5. These results suggest that R5 Envs possess a highly plastic capacity to interact with multiple CCR5 regions and support the concept that viral evolution in vivo results from the emergence of R5X4 variants with the capacity to use the CXCR4 extracellular loops but demonstrate less-flexible interactions with CCR5 that are strongly dependent on the N-terminal region.