Virus shedding kinetics and unconventional virulence tradeoffs.

Tradeoff theory, which postulates that virulence provides both transmission costs and benefits for pathogens, has become widely adopted by the scientific community. Although theoretical literature exploring virulence-tradeoffs is vast, empirical studies validating various assumptions still remain sparse. In particular, truncation of transmission duration as a cost of virulence has been difficult to quantify with robust controlled in vivo studies. We sought to fill this knowledge gap by investigating how transmission rate and duration were associated with virulence for infectious hematopoietic necrosis virus (IHNV) in rainbow trout (Oncorhynchus mykiss). Using host mortality to quantify virulence and viral shedding to quantify transmission, we found that IHNV did not conform to classical tradeoff theory. More virulent genotypes of the virus were found to have longer transmission durations due to lower recovery rates of infected hosts, but the relationship was not saturating as assumed by tradeoff theory. Furthermore, the impact of host mortality on limiting transmission duration was minimal and greatly outweighed by recovery. Transmission rate differences between high and low virulence genotypes were also small and inconsistent. Ultimately, more virulent genotypes were found to have the overall fitness advantage, and there was no apparent constraint on the evolution of increased virulence for IHNV. However, using a mathematical model parameterized with experimental data, it was found that host culling resurrected the virulence tradeoff and provided low virulence genotypes with the advantage. Human-induced or natural culling, as well as host population fragmentation, may be some of the mechanisms by which virulence diversity is maintained in nature. This work highlights the importance of considering non-classical virulence tradeoffs.

Impact of Vaccination and Pathogen Exposure Dosage on Shedding Kinetics of Infectious Hematopoietic Necrosis Virus (IHNV) in Rainbow Trout.

Vaccine efficacy in preventing clinical disease has been well characterized. However, vaccine impacts on transmission under diverse field conditions, such as variable pathogen exposure dosages, are not fully understood. We evaluated the impacts of vaccination on disease-induced host mortality and shedding of infectious hematopoietic necrosis virus (IHNV) in Rainbow Trout Oncorhynchus mykiss. Fish, in up to three different genetic lines, were exposed to different dosages of IHNV to simulate field variability. Mortality and viral shedding of each individual fish were quantified over the course of infection. As the exposure dosage increased, mortality, number of fish shedding virus, daily virus quantity shed, and total amount of virus shed also increased. Vaccination significantly reduced mortality but had a much smaller impact on shedding, such that vaccinated fish still shed significant amounts of virus, particularly at higher viral exposure dosages. These studies demonstrate that the consideration of pathogen exposure dosage and transmission are critical for robust inference of vaccine efficacy.

Method for serial passage of infectious hematopoietic necrosis virus (IHNV) in rainbow trout.

Transmission is a fundamental component of pathogen fitness. A better understanding of pathogen transmission can greatly improve disease management. In particular, controlled studies of multiple rounds of natural transmission (i.e. serial passage) can provide powerful epidemiological and evolutionary inferences. However, such studies are possible in only a few systems because of the challenges in successfully initiating and maintaining transmission in the laboratory. Here we developed an efficient and reproducible cohabitation method for conducting controlled experiments investigating the effects of serial passage on infectious hematopoietic necrosis virus (IHNV) in rainbow trout. This method was used to investigate the transmission efficiency and kinetics of viral shedding of IHNV over 3 serial passages. Transmission efficiency decreased from 100 to 62.5% over the passage steps and was associated with a decrease in virus shedding into water. A shift in the peak of viral shedding was also observed, from Day 2 post immersion for passage 0 to at least 24 h later for all subsequent passages. Finally, the characterization of viruses after 1 round of transmission and propagation on cells showed no change in glycoprotein (G gene) sequences or viral virulence compared to the ancestral virus stock. The methods developed provide valuable tools for reproducible population-level studies of IHNV epidemiology and evolution.

The role of virulence in in vivo superinfection fitness of the vertebrate RNA virus infectious hematopoietic necrosis virus.

We have developed a novel in vivo superinfection fitness assay to examine superinfection dynamics and the role of virulence in superinfection fitness. This assay involves controlled, sequential infections of a natural vertebrate host, Oncorhynchus mykiss (rainbow trout), with variants of a coevolved viral pathogen, infectious hematopoietic necrosis virus (IHNV). Intervals between infections ranged from 12 h to 7 days, and both frequency of superinfection and viral replication levels were examined. Using virus genotype pairs of equal and unequal virulence, we observed that superinfection generally occurred with decreasing frequency as the interval between exposures to each genotype increased. For both the equal-virulence and unequal-virulence genotype pairs, the frequency of superinfection in most cases was the same regardless of which genotype was used in the primary exposure. The ability to replicate in the context of superinfection also did not differ between the genotypes of equal or unequal virulence tested here. For both genotype pairs, the mean viral load of the secondary virus was significantly reduced in superinfection while primary virus replication was unaffected. Our results demonstrate, for the two genotype pairs examined, that superinfection restriction does occur for IHNV and that higher virulence did not correlate with a significant difference in superinfection fitness. To our knowledge, this is the first assay to examine the role of virulence of an RNA virus in determining superinfection fitness dynamics within a natural vertebrate host.

Microplastics exacerbate virus-mediated mortality in fish.

Microplastics are a persistent and increasing environmental hazard. They have been reported to interact with a variety of biotic and abiotic environmental stressors, but the ramifications of such interactions are largely unknown. We investigated virus-induced mortalities in a commercially important salmonid following exposure to microplastics, plastic microfibers, and natural (non-plastic) microparticles. Microplastics or microparticles alone were not lethal. Mortality increased significantly when fish were co-exposed to virus and microplastics, particularly microfibers, compared to virus alone. This presents the unique finding that microplastics (not natural microparticulate matter) may have a significant impact on population health when presented with another stressor. Further, we found that mortality correlated with host viral load, mild gill inflammation, immune responses, and transmission potential. We hypothesize that microplastics can compromise host tissues, allowing pathogens to bypass defenses. Further research regarding this mechanism and the interplay between microplastics and infectious disease are paramount, considering microplastics increasing environmental burden.

In vivo fitness associated with high virulence in a vertebrate virus is a complex trait regulated by host entry, replication, and shedding.

The relationship between pathogen fitness and virulence is typically examined by quantifying only one or two pathogen fitness traits. More specifically, it is regularly assumed that within-host replication, as a precursor to transmission, is the driving force behind virulence. In reality, many traits contribute to pathogen fitness, and each trait could drive the evolution of virulence in different ways. Here, we independently quantified four viral infection cycle traits, namely, host entry, within-host replication, within-host coinfection fitness, and shedding, in vivo, in the vertebrate virus Infectious hematopoietic necrosis virus (IHNV). We examined how each of these stages of the viral infection cycle contributes to the fitness of IHNV genotypes that differ in virulence in rainbow trout. This enabled us to determine how infection cycle fitness traits are independently associated with virulence. We found that viral fitness was independently regulated by each of the traits examined, with the largest impact on fitness being provided by within-host replication. Furthermore, the more virulent of the two genotypes of IHNV we used had advantages in all of the traits quantified. Our results are thus congruent with the assumption that virulence and within-host replication are correlated but suggest that infection cycle fitness is complex and that replication is not the only trait associated with virulence.

Aquaculture Reuse Water, Genetic Line, and Vaccination Affect Rainbow Trout (Oncorhynchus mykiss) Disease Susceptibility and Infection Dynamics.

Infectious hematopoietic necrosis virus (IHNV) and Flavobacterium psychrophilum are major pathogens of farmed rainbow trout. Improved control strategies are desired but the influence of on-farm environmental factors that lead to disease outbreaks remain poorly understood. Water reuse is an important environmental factor affecting disease. Prior studies have established a replicated outdoor-tank system capable of varying the exposure to reuse water by controlling water flow from commercial trout production raceways. The goal of this research was to evaluate the effect of constant or pulsed reuse water exposure on survival, pathogen prevalence, and pathogen load. Herein, we compared two commercial lines of rainbow trout, Clear Springs Food (CSF) and Troutex (Tx) that were either vaccinated against IHNV with a DNA vaccine or sham vaccinated. Over a 27-day experimental period in constant reuse water, all fish from both lines and treatments, died while mortality in control fish in spring water was <1%. Water reuse exposure, genetic line, vaccination, and the interaction between genetic line and water exposure affected survival (P<0.05). Compared to all other water sources, fish exposed to constant reuse water had 46- to 710-fold greater risk of death (P<0.0001). Tx fish had a 2.7-fold greater risk of death compared to CSF fish in constant reuse water (P ≤ 0.001), while risk of death did not differ in spring water (P=0.98). Sham-vaccinated fish had 2.1-fold greater risk of death compared to vaccinated fish (P=0.02). Both IHNV prevalence and load were lower in vaccinated fish compared to sham-vaccinated fish, and unexpectedly, F. psychrophilum load associated with fin/gill tissues from live-sampled fish was lower in vaccinated fish compared to sham-vaccinated fish. As a result, up to forty-five percent of unvaccinated fish were naturally co-infected with F. psychrophilum and IHNV and the coinfected fish exhibited the highest IHNV loads. Under laboratory challenge conditions, co-infection with F. psychrophilum and IHNV overwhelmed IHNV vaccine-induced protection. In summary, we demonstrate that exposure to reuse water or multi-pathogen challenge can initiate complex disease dynamics that can overwhelm both vaccination and host genetic resistance.

Competitive release and facilitation of drug-resistant parasites after therapeutic chemotherapy in a rodent malaria model.

Malaria infections frequently consist of mixtures of drug-resistant and drug-sensitive parasites. If crowding occurs, where clonal population densities are suppressed by the presence of coinfecting clones, removal of susceptible clones by drug treatment could allow resistant clones to expand into the newly vacated niche space within a host. Theoretical models show that, if such competitive release occurs, it can be a potent contributor to the strength of selection, greatly accelerating the rate at which resistance spreads in a population. A variety of correlational field data suggest that competitive release could occur in human malaria populations, but direct evidence cannot be ethically obtained from human infections. Here we show competitive release after pyrimethamine curative chemotherapy of acute infections of the rodent malaria Plasmodium chabaudi in laboratory mice. The expansion of resistant parasite numbers after treatment resulted in enhanced transmission-stage densities. After the elimination or near-elimination of sensitive parasites, the number of resistant parasites increased beyond that achieved when a competitor had never been present. Thus, a substantial competitive release occurred, markedly elevating the fitness advantages of drug resistance above those arising from survival alone. This finding may explain the rapid spread of drug resistance and the subsequently brief useful lifespans of some antimalarial drugs. In a second experiment, where subcurative chemotherapy was administered, the resistant clone was only partly released from competitive suppression and experienced a restriction in the size of its expansion after treatment. This finding raises the prospect of harnessing in-host ecology to slow the spread of drug resistance.

Limited available evidence supports theoretical predictions of reduced vaccine efficacy at higher exposure dose.

Understanding the causes of vaccine failure is important for predicting disease dynamics in vaccinated populations and planning disease interventions. Pathogen exposure dose and heterogeneity in host susceptibility have both been implicated as important factors that may reduce overall vaccine efficacy and cause vaccine failure. Here, we explore the effect of pathogen dose and heterogeneity in host susceptibility in reducing efficacy of vaccines. Using simulation-based methods, we find that increases in pathogen exposure dose decrease vaccine efficacy, but this effect is modified by heterogeneity in host susceptibility. In populations where the mode of vaccine action is highly polarized, vaccine efficacy decreases more slowly with exposure dose than in populations with less variable protection. We compared these theoretical results to empirical estimates from a systematic literature review of vaccines tested over multiple exposure doses. We found that few studies (nine of 5,389) tested vaccine protection against infection over multiple pathogen challenge doses, with seven studies demonstrating a decrease in vaccine efficacy with increasing exposure dose. Our research demonstrates that pathogen dose has potential to be an important determinant of vaccine failure, although the limited empirical data highlight a need for additional studies to test theoretical predictions on the plausibility of reduced host susceptibility and high pathogen dose as mechanisms responsible for reduced vaccine efficacy in high transmission settings.

In vivo virus growth competition assays demonstrate equal fitness of fish rhabdovirus strains that co-circulate in aquaculture.

A novel virus growth competition assay for determining relative fitness of RNA virus variants in vivo has been developed using the fish rhabdovirus, Infectious hematopoietic necrosis virus (IHNV), in juvenile rainbow trout (Oncorhynchus mykiss). We have conducted assays with IHNV isolates designated B, C, and D, representing the three most common genetic subtypes that co-circulate in Idaho trout farm aquaculture. In each assay, groups of 30 fish were immersed in a 1:1 mixture of two genotypes of IHNV, and then held in individual beakers for a 72h period of in vivo competitive virus replication. Progeny virus populations in each fish were analyzed for the presence and proportion of each viral genotype. In two independent assays of the B:C isolate pair, and two assays of the B:D pair, all fish were co-infected and there was a high level of fish-to-fish variation in the ratio of the two competing genotypes. However, in each assay the average ratio in the 30-fish group was not significantly different from the input ratio of 1:1, indicating equal or nearly equal viral fitness on a host population basis, under the conditions tested.

Transmission stage investment of malaria parasites in response to in-host competition.

Conspecific competition occurs in a multitude of organisms, particularly in parasites, where several clones are commonly sharing limited resources inside their host. In theory, increased or decreased transmission investment might maximize parasite fitness in the face of competition, but, to our knowledge, this has not been tested experimentally. We developed and used a clone-specific, stage-specific, quantitative PCR protocol to quantify Plasmodium chabaudi replication and transmission stage densities in mixed-clone infections. We co-infected mice from two strains with an avirulent and virulent parasite clone and found competitive suppression of in-host (blood-stage) parasite densities and generally corresponding reductions in transmission stage production, with the virulent clone obtaining overall competitive superiority. In response to competitive suppression, there was little evidence of any alteration in transmission stage investment, apart from a small reduction by one of the two clones in one of the two host strains. This alteration did not result in a competitive advantage, although it might have reduced the disadvantage. This study supports much of the current literature, which predicts that conspecific in-host competition will result in a competitive advantage and positive selection for virulent clones and thus the evolution of higher virulence.

Replication and shedding kinetics of infectious hematopoietic necrosis virus in juvenile rainbow trout.

Viral replication and shedding are key components of transmission and fitness, the kinetics of which are heavily dependent on virus, host, and environmental factors. To date, no studies have quantified the shedding kinetics of infectious hematopoietic necrosis virus (IHNV) in rainbow trout (Oncorhynchus mykiss), or how they are associated with replication, making it difficult to ascertain the transmission dynamics of this pathogen of high agricultural and conservation importance. Here, the replication and shedding kinetics of two M genogroup IHNV genotypes were examined in their naturally co-evolved rainbow trout host. Within host virus replication began rapidly, approaching maximum values by day 3 post-infection, after which viral load was maintained or gradually dropped through day 7. Host innate immune response measured as stimulation of Mx-1 gene expression generally followed within host viral loads. Shedding also began very quickly and peaked within 2days, defining a generally uniform early peak period of shedding from 1 to 4days after exposure to virus. This was followed by a post-peak period where shedding declined, such that the majority of fish were no longer shedding by day 12 post-infection. Despite similar kinetics, the average shedding rate over the course of infection was significantly lower in mixed compared to single genotype infections, suggesting a competition effect, however, this did not significantly impact the total amount of virus shed. The data also indicated that the duration of shedding, rather than peak amount of virus shed, was correlated with fish mortality. Generally, the majority of virus produced during infection appeared to be shed into the environment rather than maintained in the host, although there was more retention of within host virus during the post-peak period. Viral virulence was correlated with shedding, such that the more virulent of the two genotypes shed more total virus. This fundamental understanding of IHNV shedding kinetics and variation at the individual fish level could assist with management decisions about how to respond to disease outbreaks when they occur.

Transmission routes maintaining a viral pathogen of steelhead trout within a complex multi-host assemblage.

This is the first comprehensive region wide, spatially explicit epidemiologic analysis of surveillance data of the aquatic viral pathogen infectious hematopoietic necrosis virus (IHNV) infecting native salmonid fish. The pathogen has been documented in the freshwater ecosystem of the Pacific Northwest of North America since the 1950s, and the current report describes the disease ecology of IHNV during 2000-2012. Prevalence of IHNV infection in monitored salmonid host cohorts ranged from 8% to 30%, with the highest levels observed in juvenile steelhead trout. The spatial distribution of all IHNV-infected cohorts was concentrated in two sub-regions of the study area, where historic burden of the viral disease has been high. During the study period, prevalence levels fluctuated with a temporal peak in 2002. Virologic and genetic surveillance data were analyzed for evidence of three separate but not mutually exclusive transmission routes hypothesized to be maintaining IHNV in the freshwater ecosystem. Transmission between year classes of juvenile fish at individual sites (route 1) was supported at varying levels of certainty in 10%-55% of candidate cases, transmission between neighboring juvenile cohorts (route 2) was supported in 31%-78% of candidate cases, and transmission from adult fish returning to the same site as an infected juvenile cohort was supported in 26%-74% of candidate cases. The results of this study indicate that multiple specific transmission routes are acting to maintain IHNV in juvenile fish, providing concrete evidence that can be used to improve resource management. Furthermore, these results demonstrate that more sophisticated analysis of available spatio-temporal and genetic data is likely to yield greater insight in future studies.

Vaccine Effects on Heterogeneity in Susceptibility and Implications for Population Health Management.

Heterogeneity in host susceptibility is a key determinant of infectious disease dynamics but is rarely accounted for in assessment of disease control measures. Understanding how susceptibility is distributed in populations, and how control measures change this distribution, is integral to predicting the course of epidemics with and without interventions. Using multiple experimental and modeling approaches, we show that rainbow trout have relatively homogeneous susceptibility to infection with infectious hematopoietic necrosis virus and that vaccination increases heterogeneity in susceptibility in a nearly all-or-nothing fashion. In a simple transmission model with an R0 of 2, the highly heterogeneous vaccine protection would cause a 35 percentage-point reduction in outbreak size over an intervention inducing homogenous protection at the same mean level. More broadly, these findings provide validation of methodology that can help to reduce biases in predictions of vaccine impact in natural settings and provide insight into how vaccination shapes population susceptibility.IMPORTANCE Differences among individuals influence transmission and spread of infectious diseases as well as the effectiveness of control measures. Control measures, such as vaccines, may provide leaky protection, protecting all hosts to an identical degree, or all-or-nothing protection, protecting some hosts completely while leaving others completely unprotected. This distinction can have a dramatic influence on disease dynamics, yet this distribution of protection is frequently unaccounted for in epidemiological models and estimates of vaccine efficacy. Here, we apply new methodology to experimentally examine host heterogeneity in susceptibility and mode of vaccine action as distinct components influencing disease outcome. Through multiple experiments and new modeling approaches, we show that the distribution of vaccine effects can be robustly estimated. These results offer new experimental and inferential methodology that can improve predictions of vaccine effectiveness and have broad applicability to human, wildlife, and ecosystem health.

Characterization of infectious dose and lethal dose of two strains of infectious hematopoietic necrosis virus (IHNV).

The ability to infect a host is a key trait of a virus, and differences in infectivity could put one virus at an evolutionary advantage over another. In this study we have quantified the infectivity of two strains of infectious hematopoietic necrosis virus (IHNV) that are known to differ in fitness and virulence. By exposing juvenile rainbow trout (Oncorhynchus mykiss) hosts to a wide range of virus doses, we were able to calculate the infectious dose in terms of ID50 values for the two genotypes. Lethal dose experiments were also conducted to confirm the virulence difference between the two virus genotypes, using a range of virus doses and holding fish either in isolation or in batch so as to calculate LD50 values. We found that infectivity is positively correlated with virulence, with the more virulent genotype having higher infectivity. Additionally, infectivity increases more steeply over a short range of doses compared to virulence, which has a shallower increase. We also examined the data using models of virion interaction and found no evidence to suggest that virions have either an antagonistic or a synergistic effect on each other, supporting the independent action hypothesis in the process of IHNV infection of rainbow trout.

Potential drivers of virulence evolution in aquaculture.

Infectious diseases are economically detrimental to aquaculture, and with continued expansion and intensification of aquaculture, the importance of managing infectious diseases will likely increase in the future. Here, we use evolution of virulence theory, along with examples, to identify aquaculture practices that might lead to the evolution of increased pathogen virulence. We identify eight practices common in aquaculture that theory predicts may favor evolution toward higher pathogen virulence. Four are related to intensive aquaculture operations, and four others are related specifically to infectious disease control. Our intention is to make aquaculture managers aware of these risks, such that with increased vigilance, they might be able to detect and prevent the emergence and spread of increasingly troublesome pathogen strains in the future.

PCR detection of lizard malaria parasites: prevalence of Plasmodium infections with low-level parasitemia differs by site and season.

Plasmodium-specific polymerase chain reaction (PCR) primers allowed detection of infections with very low-level parasitemia for 3 species of malaria parasites infecting Anolis lizards at 2 Caribbean sites, Puerto Rico and Saba, Netherlands Antilles. A verification study, using a single-tube nested PCR to eliminate contamination, showed that infections as low as 1 parasite per millions of erythrocytes could be detected by amplifying a 673 bp fragment of the cytochrome b gene. Very low-level parasitemia infections, subpatent under the microscope, were common in A. sabanus on Saba sites, with no significant seasonal difference (31% of infections appearing uninfected by microscopic examination in summer were found infected by PCR, 38% in winter). At the Puerto Rico site, the subpatent infections were also common in A. gundlachi, but were more prevalent in winter (53%) than in summer (17%). A similar high frequency of subpatent infections is known from studies on human and bird malaria, but a previous PCR-based study on a temperate lizard malaria system found few such low-level infections. Differences in the prevalence of subpatent infections by site and season suggest transmission biology may select for distinct life history strategies by the parasite.

Manipulation of the vertebrate host's testosterone does not affect gametocyte sex ratio of a malaria parasite.

Gametocyte sex ratio of the malaria parasite Plasmodium mexicanum is variable in its host, the western fence lizard (Sceloporus occidentalis), both among infections and within infections over time. We sought to determine the effect of host physiological quality on the gametocyte sex ratio in experimentally induced infections of P. mexicanum. Adult male lizards were assigned to 4 treatment groups: castrated, castrated + testosterone implant, sham implant, and unmanipulated control. No significant difference in gametocyte sex ratio was found among the 4 treatment groups. Two other analyses were performed. A surgery stress analysis compared infection sex ratio of castrated, castrated + testosterone implant, and sham implant groups with the unmanipulated control group. A testosterone alteration analysis compared infection sex ratio of the castrated and castrated + testosterone implant groups with the sham implant and unmanipulated control groups. Again, no significant difference was observed for these 2 comparisons. Thus, physiological changes expected for experimentally induced variation in host testosterone and the stress of surgery were not associated with any change in the gametocyte sex ratio. Also, theex-periment suggests testosterone is not a cue for shaping the sex ratio of gametocytes in P. mexicanum. These results are related to the evolutionary theory of sex ratios as applied to malaria parasites.

Viral fitness does not correlate with three genotype displacement events involving infectious hematopoietic necrosis virus.

Viral genotype displacement events are characterized by the replacement of a previously dominant virus genotype by a novel genotype of the same virus species in a given geographic region. We examine here the fitness of three pairs of infectious hematopoietic necrosis virus (IHNV) genotypes involved in three major genotype displacement events in Washington state over the last 30 years to determine whether increased virus fitness correlates with displacement. Fitness was assessed using in vivo assays to measure viral replication in single infection, simultaneous co-infection, and sequential superinfection in the natural host, steelhead trout. In addition, virion stability of each genotype was measured in freshwater and seawater environments at various temperatures. By these methods, we found no correlation between increased viral fitness and displacement in the field. These results suggest that other pressures likely exist in the field with important consequences for IHNV evolution.