Host nutritional status affects alphavirus virulence, transmission, and evolution

Abstract

Malnourishment, specifically overweight/obesity and undernourishment, affects more than 2.5 billion people worldwide, with the number affected ever-increasing. Concurrently, emerging viral diseases, particularly those that are mosquito-borne, have spread dramatically in the past several decades, culminating in outbreaks of several viruses worldwide. Both forms of malnourishment are known to lead to an aberrant immune response, which can worsen disease outcomes and reduce vaccination efficacy for viral pathogens such as influenza and measles. Given the increasing rates of malnutrition and spread of arthropod-borne viruses (arboviruses), there is an urgent need to understand the role of host nutrition on the infection, virulence, and transmission of these viruses. To address this gap in knowledge, we infected lean, obese, and undernourished mice with arthritogenic arboviruses from the genus Alphavirus and assessed morbidity, virus replication, transmission, and evolution. Obesity and undernourishment did not consistently influence virus replication in the blood of infected animals except for reductions in virus in obese mice late in infection. However, morbidity was increased in obese mice under all conditions. Using Mayaro virus (MAYV) as a model arthritogenic alphavirus, we determined that both obese and undernourished mice transmit virus less efficiently to mosquitoes than control (lean) mice. In addition, viral genetic diversity and replicative fitness were reduced in virus isolated from obese compared to lean controls. Taken together, nutrition appears to alter the course of alphavirus infection and should be considered as a critical environmental factor during outbreaks.

Fig 1. Weights of mice following feeding on different diets.

Groups of C57BL/6N mice (7 mice per group) were fed a 45% fat (Obese), 5% protein (LP), or a control (Lean) diet for 8–10 weeks. A. Weights were measured weekly or bi-weekly following the initiation of feeding. Weights are plotted as the percent change compared to initial weight at the onset of feeding. B. Leptin was measured following 8–10 weeks of feeding using an ELISA. Statistical comparisons were made using a one-way ANOVA with Dunnett’s correction compared to the control group. The level of significance is represented as follows—**** p<0.0001. For A, the error bars represent standard error. For B, the whiskers represent the minimum and maximum points, the box limits represent the 25^th and 75^th percentile, and the horizontal line represents the median. Each graph represents data obtained from at least two independent experiments. The results from one representative experiment are presented.

Fig 2. Mice with differing nutritional status have altered morbidity following alphavirus infection.

Groups of C57BL/6N mice (A-D) or Balb/c (E-F) were fed either a 45% fat (Obese), 5% protein (LP), or control (Lean) diet for 8–10 weeks. Mice were then infected with 10⁴ PFU of either Mayaro virus (MAYV, 7 mice per group) or chikungunya virus (CHIKV, 14 mice per group) or 10³ PFU of Ross River virus (RRV, 5 mice per group) in the hind left footpad and monitored for weight change (A, C, and E), footpad swelling (B, D), and, for RRV, mortality (F). For A-E, statistical analyses were performed by two-way ANOVA with Dunnett’s correction compared to the control group. For F, statistical analysis was performed by the Mantel-Cox test. The error bars represent standard error. The level of significance is represented as follows—* p<0.05, ** p<0.01, *** p<0.001, ****p<0.0001. Besides E-F, each graph represents data obtained from at least two independent experiments. The results from one representative experiment are presented.

Fig 3. Nutrition alters viral replication kinetics.

Groups of C57BL/6N (A-C, 7 mice per group) or Balb/c mice (D, 5 mice per group) were fed either a 45% fat (Obese), 5% protein (LP), or control (Lean) diet for 8–10 weeks. Mice were then infected with either Mayaro virus (MAYV, A-B), chikungunya virus (CHIKV, C), or Ross River virus (RRV, D). At different times post-infection, the virus was quantified either by plaque assay (A, D) or qRT-PCR (B, C). GE = genome equivalents. Statistical analyses were performed by two-way ANOVA with Dunnett’s correction compared to the control group. The level of significance is represented as follows—* p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. The whiskers represent the minimum and maximum points, the box limits represent the 25^th and 75^th percentile, and the horizontal line represents the median. Except for D, each graph represents data obtained from at least two independent experiments. The results from one representative experiment are presented.

Fig 4. Local viral replication is increased in obese mice.

Groups of albino C57BL/6N mice (5 mice per group) were fed either a 45% fat (Obese, B), 5% protein (LP, C) or control diet (Lean, D) for 8–10 weeks. Mice were then infected with Mayaro virus (MAYV) expressing nano-luciferase (nLuc) to track replication. At different time points post-infection, mice were anesthetized, injected intraperitoneally with 20 μg/mouse of the furimazine substrate and imaged using a Perkins Elmer IVIS Spectrum machine. Mice were imaged for 15 seconds within 5 minutes of substrate administration, and photon flux (photons/second) was quantified in the hind left footpad using Living Image software (A). The whiskers represent the minimum and maximum points, the box limits represent the 25^th and 75^th percentile, and the horizontal line represents the median. The data are presented as flux or photons/second (p/s) present within the left footpad. The flux is used to quantify the amount of light emitted from the footpad. Representative images of mice fed different diets, infected with MAYV-nLuc, and then imaged six days following infection (B-D). An uninfected control mouse is presented as the leftmost mouse in each image. Statistical analyses were performed by two-way ANOVA with Dunnett’s correction compared to the control group. The level of significance is represented as follows—** p<0.01.

Fig 5. Viral replication in obese mice results in decreased genetic diversity.

Groups of C57BL/6N mice (7 mice per group) were fed either a 45% fat (Obese), 5% protein (LP), or control diet (Lean) for 8–10 weeks. Mice were then infected with Mayaro virus (MAYV) and blood was collected two days later. RNA was isolated from the serum and next-generation sequencing libraries were prepared and then sequenced on an Illumina NextSeq 500. The FASTQ files were trimmed for adapter and low-quality sequences using BBDuk. The trimmed reads were then analyzed using the ViVAN pipeline with a coverage cutoff of >100x and a p-value filter of 0.05. From the output of ViVAN, we compared both total and non-synonymous Shannon entropy (A-B), Region variation (C-D), and Region heterogeneity (E-F) as markers of genetic diversity. The whiskers represent the minimum and maximum points, the box limits represent the 25^th and 75^th percentile, and the horizontal line represents the median. Statistical analyses were performed by one-way ANOVA with Dunnett’s correction compared to the control group. The level of significance is represented as follows—* p<0.05, ** p<0.01.

Fig 6. Virus isolated from obese mice reduced early in vivo fitness.

Relative fitness of Mayaro virus (MAYV) isolated from mice fed different diets was tested by measuring an indirect measurement, genome to PFU ratio (GE:PFU), and an in vivo direct competition assay. Groups of C57BL/6N mice (5 mice per group) were fed either a 45% fat (Obese), 5% protein (LP), or control diet (Lean) for 8–10 weeks. Mice were then infected with Mayaro virus (MAYV) and bled at different time points post-infection. GE:PFU ratios were calculated by dividing the number of RNA genomes present in the serum of mice by the number of PFUs in the same sample (A). Statistical comparisons were performed using a two-way ANOVA with Dunnett’s correction compared to the control group. For direct in vivo competition assays, serum containing MAYV collected on two days post-infection of individual mice fed different diets were mixed at a 1:1 ratio with a genetically marked reference virus and then this mixture was used to infect new, lean C57BL/6N mice. For each mouse tested, one new mouse was infected (n = 7). Following infection of mice with the mixtures, serum samples were collected for two consecutive days, the mice were euthanized, and footpads were collected. Fitness was measured using a multiplex qRT-PCR developed in-house to discriminate between the wild-type MAYV sequence and the marked reference virus. Fitness is shown for each group in the footpad (B) and serum for one (C) and two-days (D) post-infection. The whiskers represent the minimum and maximum points, the box limits represent the 25^th and 75^th percentile, and the horizontal line represents the median. Statistical analyses were performed by one-way ANOVA with Dunnett’s correction compared to the control group. The level of significance is represented as follows—* p<0.05, ** p<0.01.

Fig 7. Virus from obese mice decreased infectivity to mosquitoes and lower transmission rates.

Groups of C57BL/6N mice (7 mice per group) were fed either a 45% fat (Obese), 5% protein (LP), or control (Lean) diet for 8–10 weeks. Mice were then infected with Mayaro virus (MAYV), and two days later were individually used to feed groups of Aedes aegypti mosquitoes. Serum was also collected to measure viremia two days post-infection by plaque assay (A). Mosquitoes were dissected ten days post-feeding on the viremic mice, at which point bodies (for infection, B and D) and saliva (for transmission potential, C and E-F) were collected for individual mosquitoes. The infection and transmission rates were calculated for the mice individually within a group (B-C) and for all individual mice combined within a group (D-E) based on the presence or absence of infectious virus tested by plaque assay. The number of RNA copies were calculated by qRT-PCR (F). The whiskers represent the minimum and maximum points, the box limits represent the 25^th and 75^th percentile, and the horizontal line represents the median. Statistical analyses for A-C and F were performed by one-way ANOVA with Dunnett’s correction compared to the control group. For comparing rates in D and E, a two-tailed Fisher’s exact test was used. The level of significance is represented as follows—* p<0.05, ** p<0.01.

Fig 8. Obese mice have reduced chronic RNA levels.

Groups of C57BL/6N mice (7 mice per group) were fed either a 45% fat (Obese), 5% protein (LP), or control (Lean) diet for 8–10 weeks. Mice were then infected with Mayaro virus (MAYV) and after 65 days post-infection footpads were collected. Viral RNA was quantified using qRT-PCR. The whiskers represent the minimum and maximum points, the box limits represent the 25^th and 75^th percentile, and the horizontal line represents the median. Statistical analyses were performed by one-way ANOVA with Dunnett’s correction compared to the control group. The level of significance is represented as follows—* p<0.05.