Niche theory‐based modeling of assembly processes of viral communities in bats

Understanding the assembly processes of symbiont communities, including viromes and microbiomes, is important for improving predictions on symbionts’ biogeography and disease ecology. Here, we use phylogenetic, functional, and geographic filters to predict the similarity between symbiont communities, using as a test case the assembly process in viral communities of Mexican bats. We construct generalized linear models to predict viral community similarity, as measured by the Jaccard index, as a function of differences in host phylogeny, host functionality, and spatial co‐occurrence, evaluating the models using the Akaike information criterion. Two model classes are constructed: a “known” model, where virus–host relationships are based only on data reported in Mexico, and a “potential” model, where viral reports of all the Americas are used, but then applied only to bat species that are distributed in Mexico. Although the “known” model shows only weak dependence on any of the filters, the “potential” model highlights the importance of all three filter types—phylogeny, functional traits, and co‐occurrence—in the assemblage of viral communities. The differences between the “known” and “potential” models highlight the utility of modeling at different “scales” so as to compare and contrast known information at one scale to another one, where, for example, virus information associated with bats is much scarcer.     

Transcription of Simian Virus 40 II. Hybridization of RNA Extracted from Different Lines of Transformed Cells to the Separated Strands of Simian Virus 40 DNA

The amount of simian virus 40 (SV40) DNA present in various SV40-transformed mouse cell lines and “revertants” isolated from them was determined. The number of viral DNA copies in the different cell lines ranged from 1.35 to 8.75 copies per diploid quantity of mouse cell DNA and from 2.2 to 14 copies per cell. The revertants had the same number of viral DNA copies per diploid quantity of mouse cell DNA as their parental cell lines. (However, they showed an increased number of viral DNA copies per cell due to their increased amount of DNA.) By using separated strands of SV40 DNA, the extent of each DNA strand transcribed into stable RNA species was determined for the transformed and “revertant” cell lines. From 30 to 80% of the “early” strand and from 0 to 20% of the “late” strand was present as stable RNA species in the cell lines tested. There was no alteration in the pattern of the stable viral RNA species present in three concanavalin A-selected revertants, whereas in a fluorodeoxyuridine-selected revertant there appeared to be less viral-specific RNA present in the cells.     

The viral era: New biotechnologies give humans an unprecedented control over Nature and require appropriate safeguards

New biotechnologies such as gene drives and engineered viruses herald a viral era that would give humans exceptional power over any organism at the level of the genotype. Subject Categories: Synthetic Biology & Biotechnology, S&S: Economics & Business, Ecology

We are entering a new phase in our relationship with nature: after mechanization, automation and digitalization, a new era of autonomous technical objects is dawning. The most advanced of these technologies are characterized by viral behaviour. The COVID‐19 pandemic has again aptly demonstrated the power of viral systems: not only because of the SARS‐CoV‐2 virus'' ability to jump into and rapidly spread among the human population while wreaking havoc with human societies, but also because some of the vaccines developed against the virus are themselves based on viruses. Both developments give us some ideas of the possible impact of new biotechnologies that aim to create artefacts with viral behaviour in order to shape and control our natural environment. In this essay, the focus is on the use of genetically engineered organisms and the genetic manipulation of wild species. This change has a more direct relationship to our natural environment than autonomous software artefacts such as computer apps or digital viruses that “live” in their artificial “ecosystems” of information‐processing devices. The development of artificial biological systems will therefore require new methods for monitoring and intervention given their potential to autonomously spread within natural ecosystems.     

Studies on Persistent Infections of Tissue Culture VI. Reversible Changes in Newcastle Disease Virus Populations as a Result of Passage in L Cells or Chick Embryos

Populations of the Victoria strain of Newcastle disease virus (NDV), reisolated from persistently infected L-cell cultures and passed twice in the embryonated hen's egg (NDV_L-E-2), were found to differ strikingly from the original, chick embryo-adapted virus (NDV_o). After exposure of L cells to NDV_o at high multiplicities of infection, all cells became abortively infected; they produced only small aggregates of viral antigen and few, if any, infectious virus particles, but they yielded large amounts of interferon. No cytopathic effects (CPE) were noted, and the cultures survived readily as viral carriers. In contrast, NDV_L-E-2 yielded under similar conditions large quantities of viral antigen and infectious virus particles, but no detectable interferon, and the cultures were rapidly destroyed. This change in “virulence” was at least partially reversible by further serial passages of NDV_L-E-2 in chick embryos, as was evident from a consecutive decrease in CPE with a concomitant increasingly rapid recovery of the L-cell cultures, gradually diminishing yields of infectious viral progeny, and the returning of a capacity to induce interferon synthesis. Thus, NDV_L-E-16 resembled NDV_o in many aspects, except for a less striking reduction in its ability to replicate in L cells. Although a selection of viral variants under the given sets of conditions has not been entirely excluded, the establishment of “avirulence” appears to be largely explained by a gradual accumulation of noninfectious, interferon-inducing components in the course of serial passages in the embryonated hen's egg, and the acquisition of “virulence” by a loss of these components. The evidence is as follows. (i) By a step-wise decrease in the dose of virus and restriction of the analyses to the first infectious cycle, a multiplicity of infection was ultimately reached for all “avirulent” populations at which infected cells produced normal yields of infectious viral progeny; i.e., the interferon-inducing components were diluted to noneffective levels. The lowest multiplicity which resulted in a measurable reduction in infectious virus replication was also the last one to induce detectable interferon synthesis. (ii) All viral clones derived from “avirulent” populations behaved like NDV_L-E-2 rather than like the parent viral suspensions, except that some of them elicited small amounts of interferon in L cells. The interferon-inducing components were reduced or lost in the cloning procedures. The nature of the interferon-inducing components has not been established. These components, which were neutralized by rabbit sera against “virulent” NDV_L-E-2 populations, may represent largely inactive or incomplete virus particles; however, the infectious virus-hemagglutinin ratios of “avirulent” populations were mostly of an order similar to those of “virulent” populations. The interferon-inducing components aborted the infectious process in cells simultaneously invaded by infectious virus particles. The implications of these findings are discussed.     

Sequential Formation of Vaccinia Virus Proteins and Viral Deoxyribonucleic Acid Replication

In vaccinia virus-infected cell cultures, cellular protein synthesis was inhibited 50% at 2 hr postinfection (PI) and 80 to 90% by 4 hr PI. Input virus was responsible for this inhibition. Five early proteins, coded for by the viral genome, could be detected at 2 to 3 hr PI. Normally, their synthesis did not continue beyond 6 hr PI, at which time synthesis of a different set of proteins began. When DNA replication was blocked, synthesis of these early proteins continued until 9 to 12 hr PI. The bulk of the proteins which were incorporated into mature virus were synthesized at 8 hr PI and thereafter. The time of their formation was close to the time at which virus maturation occurred. However, 15% of the protein found in mature virus was synthesized early in the infectious cycle. The quantity of “early viral protein” which was not incorporated into mature virus was almost as large as the quantity of viral protein which did appear in mature virus. The “early” and “late” proteins could be shown to have separate and distinct immunological properties. The role of this large quantity of “early” protein is discussed.     

Coxsackievirus B Exits the Host Cell in Shed Microvesicles Displaying Autophagosomal Markers

Coxsackievirus B3 (CVB3), a member of the picornavirus family and enterovirus genus, causes viral myocarditis, aseptic meningitis, and pancreatitis in humans. We genetically engineered a unique molecular marker, “fluorescent timer” protein, within our infectious CVB3 clone and isolated a high-titer recombinant viral stock (Timer-CVB3) following transfection in HeLa cells. “Fluorescent timer” protein undergoes slow conversion of fluorescence from green to red over time, and Timer-CVB3 can be utilized to track virus infection and dissemination in real time. Upon infection with Timer-CVB3, HeLa cells, neural progenitor and stem cells (NPSCs), and C2C12 myoblast cells slowly changed fluorescence from green to red over 72 hours as determined by fluorescence microscopy or flow cytometric analysis. The conversion of “fluorescent timer” protein in HeLa cells infected with Timer-CVB3 could be interrupted by fixation, suggesting that the fluorophore was stabilized by formaldehyde cross-linking reactions. Induction of a type I interferon response or ribavirin treatment reduced the progression of cell-to-cell virus spread in HeLa cells or NPSCs infected with Timer-CVB3. Time lapse photography of partially differentiated NPSCs infected with Timer-CVB3 revealed substantial intracellular membrane remodeling and the assembly of discrete virus replication organelles which changed fluorescence color in an asynchronous fashion within the cell. “Fluorescent timer” protein colocalized closely with viral 3A protein within virus replication organelles. Intriguingly, infection of partially differentiated NPSCs or C2C12 myoblast cells induced the release of abundant extracellular microvesicles (EMVs) containing matured “fluorescent timer” protein and infectious virus representing a novel route of virus dissemination. CVB3 virions were readily observed within purified EMVs by transmission electron microscopy, and infectious virus was identified within low-density isopycnic iodixanol gradient fractions consistent with membrane association. The preferential detection of the lipidated form of LC3 protein (LC3 II) in released EMVs harboring infectious virus suggests that the autophagy pathway plays a crucial role in microvesicle shedding and virus release, similar to a process previously described as autophagosome-mediated exit without lysis (AWOL) observed during poliovirus replication. Through the use of this novel recombinant virus which provides more dynamic information from static fluorescent images, we hope to gain a better understanding of CVB3 tropism, intracellular membrane reorganization, and virus-associated microvesicle dissemination within the host.     

BeeDoctor,a Versatile MLPA-Based Diagnostic Tool for Screening Bee Viruses

The long-term decline of managed honeybee hives in the world has drawn significant attention to the scientific community and bee-keeping industry. A high pathogen load is believed to play a crucial role in this phenomenon, with the bee viruses being key players. Most of the currently characterized honeybee viruses (around twenty) are positive stranded RNA viruses. Techniques based on RNA signatures are widely used to determine the viral load in honeybee colonies. High throughput screening for viral loads necessitates the development of a multiplex polymerase chain reaction approach in which different viruses can be targeted simultaneously. A new multiparameter assay, called “BeeDoctor”, was developed based on multiplex-ligation probe dependent amplification (MLPA) technology. This assay detects 10 honeybee viruses in one reaction. “BeeDoctor” is also able to screen selectively for either the positive strand of the targeted RNA bee viruses or the negative strand, which is indicative for active viral replication. Due to its sensitivity and specificity, the MLPA assay is a useful tool for rapid diagnosis, pathogen characterization, and epidemiology of viruses in honeybee populations. “BeeDoctor” was used for screening 363 samples from apiaries located throughout Flanders; the northern half of Belgium. Using the “BeeDoctor”, virus infections were detected in almost eighty percent of the colonies, with deformed wing virus by far the most frequently detected virus and multiple virus infections were found in 26 percent of the colonies.     

Initiation of Vaccinia Virus Infection in Actinomycin D-pretreated Cells

The early steps in vaccinia virus infection were studied in HeLa cells which had been treated with actinomycin D (1 μg/ml) and then incubated for several hours in fresh medium prior to infection. Initiation of infection occurred in such cells even though the synthesis of cellular ribonucleic acid and deoxyribonucleic acid (DNA) was severely depressed. Thymidine kinase was synthesized in amounts that exceeded those found in untreated, infected cells. The breakdown of viral “cores” to liberate viral DNA and the synthesis of viral specific DNA-polymerase also occurred but were somewhat delayed. A deoxyribonuclease resembling an exonuclease was made by the infected, pretreated cells. The time course for these events suggested that the genetic code for synthesis of thymidine kinase can be expressed before “cores” are broken down, but the DNA-polymerase can be synthesized only after liberation of the viral DNA. The amount of viral specific DNA-polymerase which was made after infection was proportional to the total number of virus synthesizing sites even beyond the point where all the cells were infected with one infectious particle. A similar relationship was observed for the amount of thymidine kinase formed and for the rate of viral DNA synthesis from ³H-thymidine.     

Avian Tumor Virus Proteins and RNA in Uninfected Chicken Embryo Cells

The content of proteins P19 and P15 (mol wt 19,000 and 15,000, respectively) of avian leukovirus in various types of uninfected chicken embryos has been determined by radioimmunoassay. All chicken embryos examined, including embryos which have thus far been classified as group specific (gs) antigen negative by complement fixation tests, contained these viral proteins as well as P27 as previously reported. The embryos known as “gs antigen-positive” type contained about five times as much of these viral proteins as did the “gs antigen-negative” type. The ratio of the three viral proteins was similar for all types of embryos, suggesting that the genes for these proteins are coordinately controlled. In contrast to the relatively high levels of viral internal proteins in gs antigen-negative cells, the amounts of virus-specific RNA detectable by molecular hybridization were extremely low. The levels of helper activity, which presumably reflect the level of viral envelope glycoprotein, were also generally low or undetectable in these cells. Thus, the expression of the gene for envelope glycoprotein does not appear to be controlled coordinately with the genes for viral internal proteins.     

Composition and Function of T Cell Subpopulations Are Slow to Change Despite Effective Antiretroviral Treatment of HIV Disease

The ability to reconstitute a normal immune system with antiretroviral therapy in the setting of HIV infection remains uncertain. This study aimed to characterize quantitative and qualitative aspects of various T cell subpopulations that do not improve despite effective ART. CD4∶CD8 ratio was evaluated in HIV-infected subjects with viral loads >10,000 copies/µl (“non-controllers”, n = 42), those with undetectable viral loads on ART (“ART-suppressed”, n = 53), and HIV-uninfected subjects (n = 22). In addition, T cell phenotype and function were examined in 25 non-controllers, 18 ART-suppressed, and 7 HIV-uninfected subjects. CD4∶CD8 ratio in non-controllers, ART-suppressed, and HIV-uninfected subjects was 0.25, 0.48, and 1.95 respectively (P<0.0001 for all comparisons). The increased ratio in ART-suppressed compared to non-controllers was driven by an increase of CD4+ T cells, with no change in the expanded CD8+ T cell population. Expansion of differentiated (CD28−CD27−CD45RA+/−CCR7−) T cell subpopulations persisted despite ART and minimal changes were noted in naïve T cell frequencies over time. Increased number of CD8+CD28− T cells and increased CD8+ CMV-specific T cell responses were associated with a decreased CD4∶CD8 ratio. Measures of T cell function demonstrated persistence of high frequencies of CD8+ T cells producing IFN–γ. Lastly, though all CD8+ subpopulations demonstrated significantly lower Ki67 expression in ART-suppressed subjects, CD4+ T cell subpopulations did not consistently show this decrease, thus demonstrating different proliferative responses in the setting of T cell depletion. In summary, this study demonstrated that CD4∶CD8 ratios remained significantly decreased and naïve T cell numbers were slow to increase despite long-term viral suppression on ART. In addition, there is a evidence of differential regulation of the CD4+ and CD8+ T cell subpopulations, suggesting independent homeostatic regulation of the two compartments.     

COVID‐19 and the gain of function debates: Improving biosafety measures requires a more precise definition of which experiments would raise safety concerns

The COVID‐19 pandemic has rekindled debates about gain‐of‐function experiments. This is an opportunity to clearly define safety risks and appropriate countermeasures. Subject Categories: Economics, Law & Politics, Microbiology, Virology & Host Pathogen Interaction, Science Policy & Publishing

The so‐called “gain of function” research has been recently debated in the context of viral research on coronaviruses and whether it is too risky to undertake such experiments. However, the meaning of “gain of function” or “GOF” in a science policy context has changed over time. The term was originally coined to describe two controversial research projects on H5N1 avian influenza virus and was later applied to specific experiments on coronavirus. Subsequent policies and discussions have attempted to define GOF in different ways, but no single definition has been widely accepted by the community. The fuzzy and imprecise nature of the term has led to misunderstandings and has hampered discussions on how to properly assess the benefit of such experiments and biosafety measures.

The fuzzy and imprecise nature of the term GOF has led to misunderstandings and has hampered discussions on how to properly assess the benefit of such experiments and biosafety measures

     

MANDIBULAR HERPES ZOSTER—With Report on the Use of Cortisone in a Case with Geniculate Ganglion Symptoms

Herpes zoster, an acute specific viral infection, occurs more commonly than is generally supposed. It should be differentiated from other diseases involving the ear and skin; it must be considered as a possible etiologic agent in some palsies of the facial, glossopharyngeal or vagal nerves.The type of cephalic herpes zoster should be carefully differentiated; cases involving the “geniculate zone” may be other than “Ramsay Hunt''s syndrome.” This syndrome is now defined as a herpes zoster eruption of the external ear at the “geniculate zone” with involvement of the seventh or seventh and eighth nerves.The “topognostic” method is the best for determining the level at which the facial nerve has been affected.It is questioned whether there is a single outstanding therapeutic agent for this disease. Cortisone had no apparent therapeutic effect in a case reported herein.     

Membrane Uncoating of Intact Enveloped Viruses

Experiments in the 1960s showed that Sendai virus, a paramyxovirus, fused its membrane with the host plasma membrane. After membrane fusion, the virus spontaneously “uncoated” with diffusion of the viral membrane proteins into the host plasma membrane and a merging of the host and viral membranes. This led to deposit of the viral ribonucleoprotein (RNP) and interior proteins in the cell cytoplasm. Later work showed that the common procedure then used to grow Sendai virus produced damaged, pleomorphic virions. Virions, which were grown under conditions that were not damaging, made a connecting structure between virus and cell at the region where the fusion occurred. The virus did not release its membrane proteins into the host membrane. The viral RNP was seen in the connecting structure in some cases. Uncoating of intact Sendai virus proceeds differently from uncoating described by the current standard model developed long ago with damaged virus. A model of intact paramyxovirus uncoating is presented and compared to what is known about the uncoating of other viruses.Enveloped virus entry at the plasma membrane includes binding of the virion to one or more receptors, changes in the virion components, membrane fusion, and membrane uncoating. The term “membrane uncoating” is being used to describe the separation of internal virion components from the viral membrane so the internal components can enter the cell. The term “uncoating” is sometimes used to mean the release of the viral genome from the capsid or other structures that have also entered the cell, but in this review, the term “membrane uncoating” will be used to represent only the separation of the virion internal contents and the viral envelope.Much of the original model of membrane fusion and uncoating was generally accepted as a result of a 1968 paper by Morgan and Howe (41). That paper provided strong evidence that Sendai virus (a paramyxovirus) entered a cell by fusion of the viral membrane with the cell plasma membrane. After membrane fusion, the virion rapidly lost its structure as the viral membrane merged with the host membrane and its components became part of the host membrane. The viral ribonucleoprotein (RNP) and internal proteins were released into the cytoplasm. This model of membrane uncoating is still generally accepted. For instance, in a 2007 virology text (24), this model was presented and illustrated with a figure from the Morgan and Howe paper. (The same figure is shown here as Fig. 2B.)Later, it was shown that Sendai viruses, which had been grown in fertilized chicken eggs, had different properties depending whether they had been harvested after growth for roughly 1 day (“early harvest”) or for several days (“late harvest”). The early-harvest viruses appear to be intact, but the late-harvest viruses have a different morphology and appear to be damaged (20, 26).This review summarizes data showing that intact early-harvest Sendai viruses uncoat quite differently from the way damaged late-harvest Sendai viruses uncoat. A model of intact paramyxovirus membrane uncoating is presented. The membrane uncoating of some other enveloped viruses that enter at the plasma membrane is compared to that described by this model.     

Fundamental Identifiability Limits in Molecular Epidemiology

Viral phylogenies provide crucial information on the spread of infectious diseases, and many studies fit mathematical models to phylogenetic data to estimate epidemiological parameters such as the effective reproduction ratio (R_e) over time. Such phylodynamic inferences often complement or even substitute for conventional surveillance data, particularly when sampling is poor or delayed. It remains generally unknown, however, how robust phylodynamic epidemiological inferences are, especially when there is uncertainty regarding pathogen prevalence and sampling intensity. Here, we use recently developed mathematical techniques to fully characterize the information that can possibly be extracted from serially collected viral phylogenetic data, in the context of the commonly used birth-death-sampling model. We show that for any candidate epidemiological scenario, there exists a myriad of alternative, markedly different, and yet plausible “congruent” scenarios that cannot be distinguished using phylogenetic data alone, no matter how large the data set. In the absence of strong constraints or rate priors across the entire study period, neither maximum-likelihood fitting nor Bayesian inference can reliably reconstruct the true epidemiological dynamics from phylogenetic data alone; rather, estimators can only converge to the “congruence class” of the true dynamics. We propose concrete and feasible strategies for making more robust epidemiological inferences from viral phylogenetic data.     

A human coronavirus evolves antigenically to escape antibody immunity

There is intense interest in antibody immunity to coronaviruses. However, it is unknown if coronaviruses evolve to escape such immunity, and if so, how rapidly. Here we address this question by characterizing the historical evolution of human coronavirus 229E. We identify human sera from the 1980s and 1990s that have neutralizing titers against contemporaneous 229E that are comparable to the anti-SARS-CoV-2 titers induced by SARS-CoV-2 infection or vaccination. We test these sera against 229E strains isolated after sera collection, and find that neutralizing titers are lower against these “future” viruses. In some cases, sera that neutralize contemporaneous 229E viral strains with titers >1:100 do not detectably neutralize strains isolated 8–17 years later. The decreased neutralization of “future” viruses is due to antigenic evolution of the viral spike, especially in the receptor-binding domain. If these results extrapolate to other coronaviruses, then it may be advisable to periodically update SARS-CoV-2 vaccines.     

Analysis of Minimal Functions of Simian Virus 40 II. Enhancement of Oncogenic Transformation in Vitro by UV Irradiation

After light UV irradiation (5,000 to 10,000 ergs/mm²) “complete” and “defective” simian virus 40 (SV40) showed an enhancement of oncogenic transformation capacity in Syrian hamster kidney cells in vitro up to 180 and 270% of the controls, respectively. Simultaneously with the enhancement of transformation, an increase in T-antigen induction was observed in CV-1 cells infected with light UV-irradiated SV40; infectivity, however, was correspondingly reduced by 1 log₁₀. After strong UV irradiation (10,000 to 80,000 ergs/mm²) of “complete” and “defective” SV40, transformation capacity in vitro proved to be the most resistant viral function. It was only slightly reduced in comparison with a 4 to 5 log₁₀ reduction of infectivity. T-antigen induction of SV40 was also equally resistant to strong UV irradiation. We found no evidence of “multiplicity reactivation” involved in the high resistance of transformation capacity of SV40 after UV irradiation. Syrian hamster kidney cells transformed in vitro by UV-irradiated SV40 contained the SV40-specific T-antigen and showed the same morphology and growth characteristics as cells transformed by non-irradiated “complete” or “defective” SV40. They induced malignant tumors after subcutaneous inoculation into Syrian hamsters.     

Dissecting the Dynamics of HIV-1 Protein Sequence Diversity

The rapid mutation of human immunodeficiency virus-type 1 (HIV-1) and the limited characterization of the composition and incidence of the variant population are major obstacles to the development of an effective HIV-1 vaccine. This issue was addressed by a comprehensive analysis of over 58,000 clade B HIV-1 protein sequences reported over at least 26 years. The sequences were aligned and the 2,874 overlapping nonamer amino acid positions of the viral proteome, each a possible core binding domain for human leukocyte antigen molecules and T-cell receptors, were quantitatively analyzed for four patterns of sequence motifs: (1) “index”, the most prevalent sequence; (2) “major” variant, the most common variant sequence; (3) “minor” variants, multiple different sequences, each with an incidence less than that of the major variant; and (4) “unique” variants, each observed only once in the alignment. The collective incidence of the major, minor, and unique variants at each nonamer position represented the total variant population for the position. Positions with more than 50% total variants contained correspondingly reduced incidences of index and major variant sequences and increased minor and unique variants. Highly diverse positions, with 80 to 98% variant nonamer sequences, were present in each protein, including 5% of Gag, and 27% of Env and Nef, each. The multitude of different variant nonamer sequences (i.e. nonatypes; up to 68%) at the highly diverse positions, represented by the major, multiple minor, and multiple unique variants likely supported variants function both in immune escape and as altered peptide ligands with deleterious T-cell responses. The patterns of mutational change were consistent with the sequences of individual HXB2 and C1P viruses and can be considered applicable to all HIV-1 viruses. This characterization of HIV-1 protein mutation provides a foundation for the design of peptide-based vaccines and therapeutics.     

An Inexpensive,Accurate, and Precise Wet-Mount Method for Enumerating Aquatic Viruses

Viruses affect biogeochemical cycling, microbial mortality, gene flow, and metabolic functions in diverse environments through infection and lysis of microorganisms. Fundamental to quantitatively investigating these roles is the determination of viral abundance in both field and laboratory samples. One current, widely used method to accomplish this with aquatic samples is the “filter mount” method, in which samples are filtered onto costly 0.02-μm-pore-size ceramic filters for enumeration of viruses by epifluorescence microscopy. Here we describe a cost-effective (ca. 500-fold-lower materials cost) alternative virus enumeration method in which fluorescently stained samples are wet mounted directly onto slides, after optional chemical flocculation of viruses in samples with viral concentrations of <5 × 10⁷ viruses ml⁻¹. The concentration of viruses in the sample is then determined from the ratio of viruses to a known concentration of added microsphere beads via epifluorescence microscopy. Virus concentrations obtained by using this wet-mount method, with and without chemical flocculation, were significantly correlated with, and had precision equivalent to, those obtained by the filter mount method across concentrations ranging from 2.17 × 10⁶ to 1.37 × 10⁸ viruses ml⁻¹ when tested by using cultivated viral isolates and natural samples from marine and freshwater environments. In summary, the wet-mount method is significantly less expensive than the filter mount method and is appropriate for rapid, precise, and accurate enumeration of aquatic viruses over a wide range of viral concentrations (≥1 × 10⁶ viruses ml⁻¹) encountered in field and laboratory samples.