Virus-specific responses of Heterosigma akashiwo to infection

We used flow cytometry to examine the process of cell death in the bloom-forming alga Heterosigma akashiwo during infection by a double-stranded DNA virus (OIs1) and a single-stranded RNA virus (H. akashiwo RNA virus [HaRNAV]). These viruses were isolated from the same geographic area and infect the same strain of H. akashiwo. By use of the live/dead stains fluorescein diacetate and SYTOX green as indicators of cellular physiology, cells infected with OIs1 showed signs of infection earlier than HaRNAV-infected cultures (6 to 17 h versus 23 to 29 h). Intracellular esterase activity was lost prior to increased membrane permeability during infection with OIs1, while the opposite was seen with HaRNAV-infected cultures. In addition, OIs1-infected cells accumulated in the cultures while HaRNAV-infected cells rapidly disintegrated. Progeny OIs1 viruses consisted of large and small morphotypes with estimated latent periods of 11 and 17 h, respectively, and about 1,100 and 16,000 viruses produced per cell, respectively. In contrast, HaRNAV produced about 21,000 viruses per cell and had a latent period of 29 h. This study reveals that the characteristics of viral infection in algae are virus dependent and therefore are variable among viruses infecting the same species. This is an important consideration for ecosystem modeling exercises; calculations based on in situ measurements of algal physiology must be sensitive to the diverse responses of algae to viral infection.     

CHARACTERIZATION OF HaRNAV,A SINGLE‐STRANDED RNA VIRUS CAUSING LYSIS OF HETEROSIGMA AKASHIWO (RAPHIDOPHYCEAE)1

HaRNAV, a novel virus that infects the toxic bloom‐forming alga Heterosigma akashiwo (Hada) Hada ex Hada et Chihara, was characterized based on morphology, pathology, nucleic acid type, structural proteins, and the range of host strains that it infects. HaRNAV is a 25‐nm single‐stranded RNA (ssRNA) virus with a genome size of approximately 9100 nucleotides. This is the first report of an ssRNA virus that causes lysis of a phytoplankton species. The virus particle is sensitive to chloroform and contains at least five structural proteins ranging in apparent size from 24 to 34 kDa. HaRNAV infection causes swelling of the endoplasmic reticulum and progeny virus particles assemble in the cytoplasm of the host, frequently in crystalline arrays. The infectivity of HaRNAV was tested against 15 strains of H. akashiwo isolated from Japanese waters, the Northeast Pacific, and the Northwest Atlantic. HaRNAV caused lysis of three strains from the Northeast Pacific and two strains from Japan but none from the Northwest Atlantic. The characterization of HaRNAV demonstrates that HaRNAV is a novel type of phytoplankton virus but has some similarities with plant viruses belonging to the Sequiviridae and to other known ssRNA viruses. Further genomic analysis, however, is necessary to determine any phylogenetic relationships. The discovery of HaRNAV emphasizes the diversity of H. akashiwo viral pathogens and, more importantly, algal–virus pathogens and the complexity of virus–host interactions in the environment.     

Viral Impacts on Total Abundance and Clonal Composition of the Harmful Bloom-Forming Phytoplankton Heterosigma akashiwo

Recent observations that viruses are very abundant and biologically active components in marine ecosystems suggest that they probably influence various biogeochemical and ecological processes. In this study, the population dynamics of the harmful bloom-forming phytoplankton Heterosigma akashiwo (Raphidophyceae) and the infectious H. akashiwo viruses (HaV) were monitored in Hiroshima Bay, Japan, from May to July 1998. Concurrently, a number of H. akashiwo and HaV clones were isolated, and their virus susceptibilities and host ranges were determined through laboratory cross-reactivity tests. A sudden decrease in cell density of H. akashiwo was accompanied by a drastic increase in the abundance of HaV, suggesting that viruses contributed greatly to the disintegration of the H. akashiwo bloom as mortality agents. Despite the large quantity of infectious HaV, however, a significant proportion of H. akashiwo cells survived after the bloom disintegration. The viral susceptibility of H. akashiwo isolates demonstrated that the majority of these surviving cells were resistant to most of the HaV clones, whereas resistant cells were a minor component during the bloom period. Moreover, these resistant cells were displaced by susceptible cells, presumably due to viral infection. These results demonstrated that the properties of dominant cells within the H. akashiwo population change during the period when a bloom is terminated by viral infection, suggesting that viruses also play an important role in determining the clonal composition and maintaining the clonal diversity of H. akashiwo populations. Therefore, our data indicate that viral infection influences the total abundance and the clonal composition of one host algal species, suggesting that viruses are an important component in quantitatively and qualitatively controlling phytoplankton populations in natural marine environments.     

Heterologous interference in Aedes albopictus cells infected with alphaviruses.

Maximum amounts of 42S and 26S single-stranded viral RNA and viral structural proteins were synthesized in Aedes albopictus cells at 24 h after Sindbis virus infection. Thereafter, viral RNA and protein syntheses were inhibited. By 3 days postinfection, only small quantities of 42S RNA and no detectable 26S RNA or structural proteins were synthesized in infected cells. Superinfection of A. albopictus cells 3 days after Sindbis virus infection with Sindbis, Semliki Forest, Una, or Chikungunya alphavirus did not lead to the synthesis of intracellular 26S viral RNA. In contrast, infection with snowshoe hare virus, a bunyavirus, induced the synthesis of snowshoe hare virus RNA in both A. Ablpictus cells 3 days after Sindbis virus infection and previously uninfected mosquito cells. These results suggested that at 3 days after infection with Sindbis virus, mosquito cells restricted the replication of both homologous and heterologous alphaviruses but remained susceptible to infection with a bunyavirus. In superinfection experiments the the alphaviruses were differentiated on the basis of plaque morphology and the electrophoretic mobility of their intracellular 26S viral RNA species. Thus, it was shown that within 1 h after infection with eigher Sindbis or Chikungunya virus, A. albopictus cells were resistant to superinfection with Sindbis, Chikungunya, Una, and Semliki Forest viruses. Infected cultures were resistant to superinfection with the homologous virus indefinitely, but maximum resistance to superinfection with heterologous alphaviruses lasted for approximately 8 days. After that time, infected cultures supported the replication of heterologous alphaviruses to the same extent as did persistently infected cultures established months previously. However, the titer of heterologous alphavirus produced after superinfection of persistently infected cultures was 10- to 50-fold less than that produced by an equal number of previously uninfected A. albopictus cells. Only a small proportion (8 to 10%) of the cells in a persistently infected culture was capable of supporting the replication of a heterologous alphavirus.     

Rapid expression of human immunodeficiency virus following activation of latently infected cells

The host cell activation state impacts the nature of human immunodeficiency virus infection. Activated cells facilitate productive infections; quiescent cells enable the virus to enter a latent state, the major obstacle to viral clearance. We wanted to understand how these differences affected viral gene expression. In quiescent cells activated prior to infection, viral RNA was seen 12 h postinfection; when cells were stimulated postinfection, viral RNA was not seen until 36 h postinfection. Up-regulation of viral RNA in latently infected cells occurred within 2 h poststimulation. This hierarchy also held true for viral protein production. These results may explain the rapid reemergence of viremia following termination of therapy.     

Bunyamwera Virus Replication in Cultured Aedes albopictus (Mosquito) Cells: Establishment of a Persistent Viral Infection

Bunyamwera virus replication was examined in Aedes albopictus (mosquito) cell cultures in which a persistent infection is established and in cytopathically infected BHK cells. During primary infection of A. albopictus cells, Bunyamwera virus reached relatively high titers (10⁷ PFU/ml), and autointerference was not observed. Three virus-specific RNAs (L, M, and S) and two virion proteins (N and G1) were detected in infected cells. Maximum rates of viral RNA synthesis and viral protein synthesis were extremely low, corresponding to <2% of the synthetic capacities of uninfected control cells. Viral protein synthesis was maximal at 12 h postinfection and was shut down to barely detectable levels at 24 h postinfection. Virus-specific RNA and nucleocapsid syntheses showed similar patterns of change, but later in infection. The proportions of cells able to release a single PFU at 3, 6, and 54 days postinfection were 100, 50, and 1.5%, respectively. Titers fell to 10³ to 10⁵ PFU/ml in carrier cultures. Persistently infected cultures were resistant to superinfection with homologous virus but not with heterologous virus. No changes in host cell protein synthesis or other cytopathic effects were observed at any stage of infection. Small-plaque variants of Bunyamwera virus appeared at approximately 7 days postinfection and increased gradually until they were 75 to 95% of the total infectious virus at 66 days postinfection. Temperature-sensitive mutants appeared between 23 and 49 days postinfection. No antiviral activity similar to that reported in A. albopictus cell cultures persistently infected with Sindbis virus (R. Riedel and D. T. Brown, J. Virol. 29: 51-60, 1979) was detected in culture fluids by 3 months after infection. Bunyamwera virus replicated more rapidly in BHK cells than in mosquito cells but reached lower titers. Autointerference occurred at multiplicities of infection of 10. Virus-specific RNA and protein syntheses were at least 20% of the levels in uninfected control cells. Host cell protein synthesis was completely shut down, and nucleocapsid protein accumulated until it was 4% of the total cell protein. We discuss these results in relation to possible mechanisms involved in determining the outcome of arbovirus infection of vertebrate and mosquito cells.     

Epstein-Barr virus RNA. VIII. Viral RNA in permissively infected B95-8 cells

More than 50 RNAs expressed by Epstein-Barr virus late in productive infection have been identified. B95-8-infected cells were induced to a relatively high level of permissive infection with the tumor promotor 12-O-tetradecanoylphorbol-13-acetate. Polyadenylated RNAs were extracted from the cell cytoplasm, separated by size on formaldehyde gels, transferred to nitrocellulose, and hybridized to labeled recombinant Epstein-Barr virus DNA fragments. Comparison of RNAs from induced cultures with RNAs from induced cultures also treated with phosphonoacetic acid to inhibit viral DNA synthesis identifies two RNA classes: a persistent early class of RNAs whose abundance is relatively resistant to viral DNA synthesis inhibition and a late class of RNAs whose abundance is relatively sensitive to viral DNA synthesis inhibition. The persistent early and late RNAs are not clustered but are intermixed and scattered through most of segments UL and US. The cytoplasmic polyadenylated RNAs expressed during latent infection were not detected in productively infected cells, indicating that different classes of viral RNA are associated with latent and productive infection. Non-polyadenylated small RNAs originally identified in cells latently infected with Epstein-Barr virus are expressed in greater abundance in productively infected cells and are part of the early RNA class.     

Growth Characteristics and Intraspecies Host Specificity of a Large Virus Infecting the Dinoflagellate Heterocapsa circularisquama

The growth characteristics and intraspecies host specificity of Heterocapsa circularisquama virus (HcV), a large icosahedral virus specifically infecting the bivalve-killing dinoflagellate H. circularisquama, were examined. Exponentially growing host cells were more sensitive to HcV than those in the stationary phase, and host cells were more susceptible to HcV infection in the culture when a higher percent of the culture was replaced with fresh medium each day, suggesting an intimate relationship between virus sensitivity and the physiological condition of the host cells. HcV was infective over a wide range of temperatures, 15 to 30°C, and the latent period and burst size were estimated at 40 to 56 h and 1,800 to 2,440 infective particles, respectively. Transmission electron microscopy revealed that capsid formation began within 16 h postinfection, and mature virus particles appeared within 24 h postinfection at 20°C. Compared to Heterosigma akashiwo virus, HcV was more widely infectious to H. circularisquama strains that had been independently isolated in the western part of Japan, and only 5.3% of the host-virus combinations (53 host and 10 viral strains) showed resistance to viral infection. The present results are helpful in understanding the ecology of algal host-virus systems in nature.     

Production of reovirus type-1 and type-3 from Vero cells grown on solid and macroporous microcarriers

Two strains of reovirus were propagated in Vero cells grown in stationary or microcarriers cultures. Vero cells grown as monolayers on T-flasks or in spinner cultures of Cytodex-1 or Cultispher-G microcarriers could be infected with reovirus serotype 1, strain Lang (T1L), and serotype 3, strain Dearing (T3D). A regime of intermittent low speed stirring at reduced culture volume was critical to ensure viral infection of cells in microcarrier cultures. The virus titre increased by 3 to 4 orders of magnitude over a culture period of 150 h. Titres of the T3D reovirus strain were higher (43%) compared to those of the T1L strain in all cultures. Titres were significantly higher in T-flask and Cytodex-1 microcarrier cultures compared to Cultispher-G cultures with respect to either reovirus type. The viral productivity in the microcarrier cultures was dependent upon the multiplicity of infection (MOI) and the cell/bead ratio at the point of infection. A combination of high MOI (5 pfu/cell) and high cell/bead loading (>400 for Cytodex-1 and >1,000 for Cultispher-G) resulted in a low virus productivity per cell. However, at low MOI (0.5 pfu/cell) the virus productivity per cell was significantly higher at high cell/bead loading in cultures of either microcarrier type. The maximum virus titre (8.5 x 10(9) pfu/mL) was obtained in Cytodex-1 cultures with a low MOI (0.5 pfu/cell) and a cell/bead loading of 1,000. The virus productivity per cell in these cultures was 4,000 pfu/cell. The lower viral yield in the Cultispher-G microcarrier cultures is attributed to a decreased accessibility of the entrapped cells to viral infection. The high viral productivity from the Vero cells in Cytodex-1 cultures suggests that this is a suitable system for the development of a vaccine production system for the Reoviridae viruses.     

Components of apoptotic pathways modulate HIV-1 latency in Jurkat cells

The ability of the human immunodeficiency virus type 1 (HIV-1) to establish latent infections serves as a major barrier for its cure. This process could occur when its host cells undergo apoptosis, but it is uncertain whether the components of the apoptotic pathways affect viral latency. Using the susceptible Jurkat cell line, we investigated the relationship of apoptosis-associated components with HIV-1 DNA levels using the sensitive real-time PCR assay. Here, we found that the expression of proapoptotic proteins, including Fas ligand (FasL), FADD, and p53, significantly decreased HIV-1 viral DNA in cells. In contrast, the expression of antiapoptotic molecules, such as FLIP, Bcl2, and XIAP, increased the levels of viral DNA. Furthermore, promoting cellular antiapoptotic state via the knockdown of Bax with siRNA and FADD with antisense mRNA or the treatment with the Caspase-3 inhibitor, Z-DEVD, also raised viral DNA. We also simultaneously measured viral RNA from supernatants of these cell cultures and found that HIV-1 latency is inversely proportional to viral replication. Furthermore, we demonstrated that HIV-1-infected cells that underwent the transient expression of FLIP- or XIAP-induced viral latency would then produce an increased level of viral RNA upon the reversal of these antiapoptotic effects via PMA treatment compared to LacZ control cells. Taken together, these data suggest that HIV-1 infection could be adapted to employ or even manipulate the cellular apoptotic pathway to its advantage: when the host cell remains in a pro-apoptotic state, HIV-1 favors active replication, while when the host cell prefers an anti-apoptotic state, the virus establishes viral latency and promotes latent reservoir seeding in a way which would enhance viral replication and cytopathogenesis when the cellular conditions shift to encourage the productive infection phase.     

A Novel Cre Recombinase Imaging System for Tracking Lymphotropic Virus Infection In Vivo

Background

Detection, isolation, and identification of individual virus infected cells during long term infection are critical to advance our understanding of mechanisms of pathogenesis for latent/persistent viruses. However, current approaches to study these viruses in vivo have been hampered by low sensitivity and effects of cell-type on expression of viral encoded reporter genes. We have designed a novel Cre recombinase (Cre)-based murine system to overcome these problems, and thereby enable tracking and isolation of individual in vivo infected cells.

Methodology/Principal findings

Murine gammaherpesvirus 68 (MHV-68) was used as a prototypic persistent model virus. A Cre expressing recombinant virus was constructed and characterised. The virus is attenuated both in lytic virus replication, producing ten-fold lower lung virus titres than wild type virus, and in the establishment of latency. However, despite this limitation, when the sEGFP7 mouse line containing a Cre-activated enhanced green fluorescent protein (EGFP) was infected with the Cre expressing virus, sites of latent and persistent virus infection could be identified within B cells and macrophages of the lymphoid system on the basis of EGFP expression. Importantly, the use of the sEGFP7 mouse line which expresses high levels of EGFP allowed individual virus positive cells to be purified by FACSorting. Virus gene expression could be detected in these cells. Low numbers of EGFP positive cells could also be detected in the bone marrow.

Conclusions/Significance

The use of this novel Cre-based virus/mouse system allowed identification of individual latently infected cells in vivo and may be useful for the study and long-term monitoring of other latent/persistent virus infections.     

Why does viral RNA sometimes persist after recovery from acute infections?

DNA viruses often persist in the body of their host, becoming latent and recurring many months or years later. By contrast, most RNA viruses cause acute infections that are cleared from the host as they lack the mechanisms to persist. However, it is becoming clear that viral RNA can persist after clinical recovery and elimination of detectable infectious virus. This persistence can either be asymptomatic or associated with late progressive disease or nonspecific lingering symptoms, such as may be the case following infection with Ebola or Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Why does viral RNA sometimes persist after recovery from an acute infection? Where does the RNA come from? And what are the consequences?

Most RNA viruses cause acute infections that are cleared from the host as they lack the mechanisms to persist; however, phenomena such as "long COVID" suggest that viral RNA can persist after clinical recovery and elimination of detectable infectious virus. This Unsolved Mystery article explores the meaning, origins and consequences of such persistent RNA.     

A NOVEL VIRUS (HaNIV) CAUSES LYSIS OF THE TOXIC BLOOM-FORMING ALGA HETEROSIGMA AKASHIWO (RAPHIDOPHYCEAE)

We describe a previously unknown virus that causes lysis of the toxic bloom-forming alga Heterosigma akashiwo (Hada) Hara et Chihara (Raphidophyceae). Heterosigma akashiwo nuclear inclusion virus (HaNIV) does not resemble other algal viruses described to date. HaNIV is small (ca. 30 nm diameter), is assembled in the nucleus, and forms crystalline arrays. We estimate that approximately 10⁵ HaNIV particles are released during lysis of a cell. During a time-course experiment, TEM revealed the first signs of HaNIV infection 24 h after viral addition, and by 74 h 98% of observed cells were visibly infected. The onset of cell lysis, as indicated by a decrease in the relative fluorescence of the cultures, was apparent by 42 h postinfection. The heterochromatin of infected cells is frequently found at the margin of the nucleoplasm, which is consistent with virus-mediated programmed cell death, or apoptosis. HaNIV is clearly different from other described viruses that infect algae, including other viral pathogens of H. akashiwo. These results indicate that viruses other than Phycodnaviridae are pathogens and cause mortality of microalgae in marine systems. It is likely that HaNIV plays an integral role in the population dynamics of H. akashiwo.     

RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells

Viral pathogens represent a significant public health threat; not only can viruses cause natural epidemics of human disease, but their potential use in bioterrorism is also a concern. A better understanding of the cellular factors that impact infection would facilitate the development of much-needed therapeutics. Recent advances in RNA interference (RNAi) technology coupled with complete genome sequencing of several organisms has led to the optimization of genome-wide, cell-based loss-of-function screens. Drosophila cells are particularly amenable to genome-scale screens because of the ease and efficiency of RNAi in this system ¹. Importantly, a wide variety of viruses can infect Drosophila cells, including a number of mammalian viruses of medical and agricultural importance ^2,3,4. Previous RNAi screens in Drosophila have identified host factors that are required for various steps in virus infection including entry, translation and RNA replication ⁵. Moreover, many of the cellular factors required for viral replication in Drosophila cell culture are also limiting in human cells infected with these viruses ^{4,6,7,8, 9}. Therefore, the identification of host factors co-opted during viral infection presents novel targets for antiviral therapeutics. Here we present a generalized protocol for a high-throughput RNAi screen to identify cellular factors involved in viral infection, using vaccinia virus as an example.     

Genome-wide siRNA Screening at Biosafety Level 4 Reveals a Crucial Role for Fibrillarin in Henipavirus Infection

Hendra and Nipah viruses (genus Henipavirus, family Paramyxoviridae) are highly pathogenic bat-borne viruses. The need for high biocontainment when studying henipaviruses has hindered the development of therapeutics and knowledge of the viral infection cycle. We have performed a genome-wide siRNA screen at biosafety level 4 that identified 585 human proteins required for henipavirus infection. The host protein with the largest impact was fibrillarin, a nucleolar methyltransferase that was also required by measles, mumps and respiratory syncytial viruses for infection. While not required for cell entry, henipavirus RNA and protein syntheses were greatly impaired in cells lacking fibrillarin, indicating a crucial role in the RNA replication phase of infection. During infection, the Hendra virus matrix protein co-localized with fibrillarin in cell nucleoli, and co-associated as a complex in pulldown studies, while its nuclear import was unaffected in fibrillarin-depleted cells. Mutagenesis studies showed that the methyltransferase activity of fibrillarin was required for henipavirus infection, suggesting that this enzyme could be targeted therapeutically to combat henipavirus infections.     

Human B cells on their route to latent infection--early but transient expression of lytic genes of Epstein-Barr virus

Epstein-Barr virus (EBV) is a human tumor virus and a paradigm of herpesviral latency. Mature naïve or memory B cells are EBV's preferred targets in vitro and in vivo. Upon infection of any B cell with EBV, the virus induces cellular proliferation to yield lymphoblastoid cell lines (LCLs) in vitro and establishes a latent infection in them. In these cells a ‘classical’ subset of latent viral genes is expressed that orchestrate and regulate cellular activation and proliferation, prevent apoptosis, and maintain viral latency. Surprisingly, little is known about the early events in primary human B cells infected with EBV. Recent analyses have revealed the initial but transient expression of additional viral genes that do not belong to the ‘classical’ latent subset. Some of these viral genes have been known to initiate the lytic, productive phase of EBV but virus synthesis does not take place early after infection. The early but transient expression of certain viral lytic genes is essential for or contributes to the initial survival and cell cycle entry of resting B cells to foster their proliferation and sustain a latent infection. This review summarizes the recent findings and discusses the presumed function(s) of viral genes expressed shortly but transiently after infection of B-lymphocytes with EBV.     

Defective interfering influenza viruses and host cells: establishment and maintenance of persistent influenza virus infection in MDBK and HeLa cells.

WSN (H0N1) influenza virus upon undiluted passages in different species of cells, namely, bovine kidney (MDBK), chicken embryo (CEF), and HeLa cells, produced a varying amount of defective interfering (DI) virus which correlated well with the ability of the species of cell to produce infectious virus. However, the nature of the influenza DI viral RNA produced from a single clonal stock was essentially identical in all three cells types, suggesting that these cells do not exert a great selective pressure in the amplification of specific DI viral RNAs either at early or late passages. DI viruses produced from one subtype (H0N1) could interfere with the replication of infectious viruses belonging to other subtypes (H1N1, H3N2). DI viral RNAs could also replicate with the helper function of other subtype viruses. The persistent infection of MDBK and HeLa cells could be initiated by coinfecting cells with both temperature-sensitive mutants (ts-) and DI influenza viruses. Persistently infected cultures cultures at early passages (up to passage 7) showed a cyclical pattern of cell lysis and virus production (crisis), whereas, at later passages (after passage 20), they produced little or no virus and were resistant to infection by homologous virus but not by heterologous virus. The majority of persistently infected cells, however, contained the complete viral genome since they expressed viral antigens and produced infectious centers. Selection of a slow-growing temperature-sensitive variant rather than the presence of DI virus or interferon appears to be critical in maintaining persistent influenza infection in these cells.     

Cell Tropism Predicts Long-term Nucleotide Substitution Rates of Mammalian RNA Viruses

The high rates of RNA virus evolution are generally attributed to replication with error-prone RNA-dependent RNA polymerases. However, these long-term nucleotide substitution rates span three orders of magnitude and do not correlate well with mutation rates or selection pressures. This substitution rate variation may be explained by differences in virus ecology or intrinsic genomic properties. We generated nucleotide substitution rate estimates for mammalian RNA viruses and compiled comparable published rates, yielding a dataset of 118 substitution rates of structural genes from 51 different species, as well as 40 rates of non-structural genes from 28 species. Through ANCOVA analyses, we evaluated the relationships between these rates and four ecological factors: target cell, transmission route, host range, infection duration; and three genomic properties: genome length, genome sense, genome segmentation. Of these seven factors, we found target cells to be the only significant predictors of viral substitution rates, with tropisms for epithelial cells or neurons (P<0.0001) as the most significant predictors. Further, one-tailed t-tests showed that viruses primarily infecting epithelial cells evolve significantly faster than neurotropic viruses (P<0.0001 and P<0.001 for the structural genes and non-structural genes, respectively). These results provide strong evidence that the fastest evolving mammalian RNA viruses infect cells with the highest turnover rates: the highly proliferative epithelial cells. Estimated viral generation times suggest that epithelial-infecting viruses replicate more quickly than viruses with different cell tropisms. Our results indicate that cell tropism is a key factor in viral evolvability.