Absolute Humidity Influences the Seasonal Persistence and Infectivity of Human Norovirus

Abstract

Norovirus (NoV) is one of the main causative agents of acute gastroenteritis worldwide. In temperate climates, outbreaks peak during the winter season. The mechanism by which climatic factors influence the occurrence of NoV outbreaks is unknown. We hypothesized that humidity is linked to NoV seasonality. Human NoV is not cultivatable, so we used cultivatable murine norovirus (MNV) as a surrogate to study its persistence when exposed to various levels of relative humidity (RH) from low (10% RH) to saturated (100% RH) conditions at 9 and 25°C. In addition, we conducted similar experiments with virus-like particles (VLPs) from the predominant GII-4 norovirus and studied changes in binding patterns to A, B, and O group carbohydrates that might reflect capsid alterations. The responses of MNV and VLP to humidity were somewhat similar, with 10 and 100% RH exhibiting a strong conserving effect for both models, whereas 50% RH was detrimental for MNV infectivity and VLP binding capacity. The data analysis suggested that absolute humidity (AH) rather than RH is the critical factor for keeping NoV infectious, with an AH below 0.007 kg water/kg air being favorable to NoV survival. Retrospective surveys of the meteorological data in Paris for the last 14 years showed that AH average values have almost always been below 0.007 kg water/kg air during the winter (i.e., 0.0046 ± 0.0014 kg water/kg air), and this finding supports the fact that low AH provides an ideal condition for NoV persistence and transmission during cold months.

FIG 1

Effect of the RH level on MNV survival during the evaporation step. Droplets (20 μl) were spotted onto glass coupons and directly placed in various RH environments for 1 h for drying. For each experimental condition, N represents the mean titer by plaque assay of three independent experiments (n = 3). N0 represents the mean titer of the MNV inoculum before the drying process. The N/N0 ratio is given as a percentage and is indicated on the ordinate for this figure and the following figures. Error bars represent the standard deviations of at least three repeated experiments.

FIG 2

Effect of RH on MNV survival during the equilibrium phase. All of the coupons were dried for 1 h at room temperature in a chamber with 10% RH before being placed in RHs ranging from 10 to 100% (ordinate). Incubation times at room temperature are given in hours and are indicated on the right side of the graph. For each experimental condition, N represents the mean titer by plaque assay of three independent experiments (n = 3). N0 represents the mean titer of the MNV inoculum, which had been drier for 1 h at 10% RH prior to resuspension. Error bars represent the standard deviations of at least three individual experiments. Each drying condition is color coded, and the coding system is indicated on the right side of the graph.

FIG 3

Persistence of the MNV genome under various RH levels. Bars—1 h (dark gray bar) and 20 h (light gray bar)—are defined on the right side of the graph. The ratio between the number of MNV genome copies (N), which was determined by real-time RT-PCR, and the N0 (the number of recovered genome copies after 1 h of drying at 10% RH) is indicated as a percentage (ordinate). Error bars represent the standard deviations of three repeated experiments (n = 3).

FIG 4

Saliva binding assays of the GII.4 Osaka variant (Cairo4 strain). VLPs had been dried for 1 week under different conditions at room temperature and then resuspended in PBS at 500 ng/μl. Twofold serial dilutions of resuspended VLPs were tested on 1,000-fold-diluted saliva samples from individuals that were typed for secretor (A to C) and nonsecretor (D) phenotypes. Saliva samples from O (A), A (B), B (C), and nonsecretor (D) individuals were characterized for the presence of the ABO and Lewis antigens, as documented previously (61). For the positive control, purified VLPs were diluted in PBS and stored at −80°C for 1 week prior to being used for the binding assay. The binding experiments were performed in duplicate for each sample, and the mean values are given on the graph (optical density at 450 nm [OD₄₅₀], ordinate). Each RH is color coded, and the coding system is indicated on the right side of the graph with the incubation period indicated in parentheses.

FIG 5

(A) SDS-PAGE of the VLP from the saliva binding assay. One aliquot of 1 μg each of the VLPs that had been resuspended in buffer after 1 week of drying under different RHs was resolved under denaturing conditions by SDS-PAGE using NuPAGE bis-Tris gel. VLPs were diluted in TGEB buffer and directly resolved by SDS-PAGE (lane 2) or stored at −40°C (lane 3) or 25°C (lane 4). For the drying experiments, VLP were dried under 10% RH at 25°C for 1 h (lane 5) and 1 week (lane 6). For 35 to 100% RH, VLP were first dried for 1 h at 10% RH and then immediately placed in RHs that are indicated above the gel (lanes 7 through 10). Resolved proteins were then transferred onto a nitrocellulose membrane prior to immunoblotting. VP1 protein was labeled with in-house specific MAbs which were detected with anti-mouse alkaline phosphatase-labeled antibody. Alkaline phosphatase activity from the mouse-specific alkaline phosphatase-labeled secondary antibody was detected with NBT-BCIP solution. The complete (VP1) and truncated (VP1*) capsid proteins for GII.4 VLPs, indicated by arrows, have been described (38). Lanes corresponding to RH levels and controls are indicated above the gel. The molecular mass (in kilodaltons) (lane 1) is indicated on the left side of the gel. (B) Electron micrographs of VLPs after storage at 25°C under various RH levels. VLPs were dried for 1 h at 10% RH before being placed at the various RH levels. Subpanels: A and B, control, C and D, drying at 10% RH for 1 h; E and F, 1 week at 10% RH; G and H, 1 week at 35% RH; I and J, 1 week at 55% RH; K and L, 1 week at 85% RH; M and N, 1 week at 100% RH. Preparations were observed at ×80,000 and ×150,000 magnification. Scale bars correspond to 100 nm, except for panel J (2 μm).

FIG 6

Distribution of daily temperature and AH in Montsouris Park, Paris, France, between 2000 and 2013. Temperature (expressed in °C) and AH (expressed in kg water/kg air) are indicated on the ordinate and abscissa, respectively. Minimal and maximal daily temperatures and corresponding AH are indicated by dots on the graph. Each dot corresponds to 1 day. Th RH is indicated by a curve on the graph and is plotted against temperature and AH. A vertical line is drawn across the graph for AH corresponding to 0.007 kg water/kg air. The percentage of days for the 14-year survey for which AH was below 0.007 kg water/kg air is indicated on the graph for each season: spring, blue dots; summer, green dots; autumn, brown dots; and winter, red dots.