A Multiplex Noninvasive Salivary Antibody Assay for SARS-CoV-2 Infection and Its Application in a Population-Based Survey by Mail.

Noninvasive salivary antibody immunoassays can enable low-cost epidemiological surveillance of infections. This study involved developing and validating a multiplex suspension immunoassay on the Luminex platform to measure immunoglobulin G (IgG) responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid and spike (S) proteins, and the spike protein's S1 and S2 subunits and receptor binding domain. Multiple versions of these recombinant proteins acquired from commercial and noncommercial sources were evaluated. Assay development and validation utilized saliva and serum samples from coronavirus disease 2019 (COVID-19) cases procured from commercial sources and negative controls from a prepandemic survey. Saliva was also collected in a demonstration survey by mail involving adult individuals in the United States who were diagnosed with SARS-CoV-2 infection 15 to 80 days prior to sample collection. The survey had an 83% valid sample return rate (192 samples from 38 states). Most COVID-19 cases (93%) reported mildly symptomatic or asymptomatic infections. The final salivary assay based on the best-performing spike and nucleocapsid proteins had a sensitivity of 87.1% (95% bootstrap confidence interval, 82.1 to 91.7%) and specificity of 98.5% (95.0 to 100%) using 227 and 285 saliva samples, respectively. The same assay had 95.9% (92.8 to 98.9%) sensitivity and 100% (98.4 to 100%) specificity in serum (174 and 285 serum samples, respectively). Salivary and serum antibody responses to spike and nucleocapsid proteins were strongly correlated in 22 paired samples (r = 0.88 and r = 0.80, respectively). Antibody responses peaked at approximately 50 days postonset; greater illness severity was associated with stronger responses. This study demonstrated that a salivary antibody assay can be used in large-scale population surveys by mail to better characterize public health impacts of COVID-19. IMPORTANCE Given the enormous impacts of the COVID-19 pandemic, developing tools for population surveillance of infection is of paramount importance. This article describes the development of a multiplex immunoassay on a Luminex platform to measure salivary immunoglobulin G responses to the spike protein, its two subunits and receptor binding domain, and the nucleocapsid protein of SARS-CoV-2. The assay validation utilized serum and saliva samples from prepandemic controls and recent COVID-19 cases. A survey by mail targeting recent COVID-19 cases across the United States also demonstrated the utility of safe, at-home self-collection of saliva. By incorporating multiple SARS-CoV-2 proteins, this assay may differentiate responses to natural SARS-CoV-2 infections from responses to most vaccines. Application of this noninvasive immunoassay in COVID-19 surveillance can help provide estimates of cumulative incidence rates of symptomatic and asymptomatic infections in various communities and subpopulations, temporal patterns of antibody responses, and risk factors for infection.

Peracetic Acid Sanitation on Arugula Microgreens Contaminated with Surface-Attached and Internalized Tulane Virus and Rotavirus.

Hydroponic production of vegetables is becoming more common, especially in regions with unfavorable climate for year-round crop production. However, if viruses are present in the hydroponics feed water, then there is a chance that infectious viruses will be internalized into the tissues of hydroponically grown vegetables. When this happens, surface sanitization of postharvest vegetables may not be effective because the sanitizer cannot disinfect the internalized viruses. In this study, we determined if the effectiveness of peracetic acid (PAA), a sanitizer used in the vegetable industry, is affected by the location of viruses (produce surface or interior tissue) in microgreen arugula. Either internally or externally contaminated hydroponically grown microgreen arugula was then treated with PAA at either 30 or 80 ppm for up to 3 min. The PAA disinfection efficacy was higher when the RV was on the arugula surface (approximately 5-log10 in PFU after 3 min of exposure), instead of the arugula interior (1.5-log10 in PFU after 3 min of exposure). However, PAA disinfection efficacy of TV was not dependent on the virus location in arugula. For both internalized TV and RV, the disinfection efficacy was less than 2-log10 in PFU using all the tested PAA concentrations and exposure times examined here. Thus, both the type and location of virus in fresh vegetables may influence the virus disinfection of postharvest vegetables. Therefore, the optimization of sanitation for postharvest fresh vegetables is needed to reduce foodborne viral infection risks.

The Basis of Peracetic Acid Inactivation Mechanisms for Rotavirus and Tulane Virus under Conditions Relevant for Vegetable Sanitation.

We determined the disinfection efficacy and inactivation mechanisms of peracetic acid (PAA)-based sanitizer using pH values relevant for vegetable sanitation against rotavirus (RV) and Tulane virus (TV; a human norovirus surrogate). TV was significantly more resistant to PAA disinfection than RV: for a 2-log10 reduction of virus titer, RV required 1 mg/liter PAA for 3.5 min of exposure, while TV required 10 mg/liter PAA for 30 min. The higher resistance of TV can be explained, in part, by significantly more aggregation of TV in PAA solutions. The PAA mechanisms of virus inactivation were explored by quantifying (i) viral genome integrity and replication using reverse transcription-quantitative PCR (RT-qPCR) and (ii) virus-host receptor interactions using a cell-free binding assay with porcine gastric mucin conjugated with magnetic beads (PGM-MBs). We observed that PAA induced damage to both RV and TV genomes and also decreased virus-receptor interactions, with the latter suggesting that PAA damages viral proteins important for binding its host cell receptors. Importantly, the levels of genome-versus-protein damage induced by PAA were different for each virus. PAA inactivation correlated with higher levels of RV genome damage than of RV-receptor interactions. For PAA-treated TV, the opposite trends were observed. Thus, PAA inactivates each of these viruses via different molecular mechanisms. The findings presented here potentially contribute to the design of a robust sanitation strategy for RV and TV using PAA to prevent foodborne disease.IMPORTANCE In this study, we examined the inactivation mechanisms of peracetic acid (PAA), a sanitizer commonly used for postharvest vegetable washing, for two enteric viruses: Tulane virus (TV) as a human norovirus surrogate and rotavirus (RV). PAA disinfection mechanisms for RV were mainly due to genome damage. In contrast, PAA disinfection in TV was due to damage of the proteins important for binding to its host receptor. We also observed that PAA triggered aggregation of TV to a much greater extent than RV. These studies demonstrate that different viruses are inactivated via different PAA mechanisms. This information is important for designing an optimal sanitation practice for postharvest vegetable washing to minimize foodborne viral diseases.

UV Inactivation of Rotavirus and Tulane Virus Targets Different Components of the Virions.

Enteric viruses are shed in fecal material by humans and other animals and are common contaminants in wastewater and surface water. Wastewater treatment plants often disinfect this effluent with low-pressure and medium-pressure UV lamps, which emit 254-nm and 220- to 280-nm irradiation, respectively. It is not known whether this treatment is efficacious against enteric viruses or how such treatments may inactivate these enteric viruses. This study examined UV disinfection for two enteric viruses: rotavirus (RV) (strain OSU with double-stranded RNA and a three-layer capsid) and Tulane virus (TV) (a cultivable surrogate for human norovirus with single-stranded RNA and a single-layer capsid). Viruses were treated with UV irradiation at 220 or 254 nm under conditions relevant to wastewater stabilization ponds, whose water is often used for irrigation. TV was susceptible to 220- or 254-nm UV at similar levels. It appears that UV irradiation inactivated TV by mutagenizing both its genome and capsid binding proteins. RV was more susceptible to UV at 220 nm than to UV at 254 nm. UV irradiation of RV at either 220 or 254 nm resulted in a virus that retained its ability to bind to its host cell receptor. After 220-nm treatment, the VP7 segment of the RV genome could not be amplified by PCR, suggesting that this treatment mutagenized the viral genome. However, this correlation was not observed when UV at 254 nm was used. Thus, RV and TV, with different genome and capsid contents, are targeted by UV irradiation in different ways.IMPORTANCE UV irradiation is becoming common for disinfection in water treatment plants, but little is known about the effectiveness of this treatment for enteric RNA viruses. Here, we observed that 220-nm UV irradiation was efficacious against rotavirus (RV) and Tulane virus (TV). UV irradiation at 254 nm inactivated TV to a greater extent than RV. Additional assays showed that UV irradiation compromised different portions of the RV and TV life cycles. UV irradiation decreased the binding of TV to its host receptor and mutagenized the TV genome. UV irradiation at 220 nm appeared to allow RV-host receptor interaction but halted RV genome replication. These findings provide knowledge about the disinfection of waterborne viruses, information that is important for the safe reuse or release of treated wastewater.

Roles of Vegetable Surface Properties and Sanitizer Type on Annual Disease Burden of Rotavirus Illness by Consumption of Rotavirus-Contaminated Fresh Vegetables: A Quantitative Microbial Risk Assessment.

Enteric viruses are often detected in water used for crop irrigation. One concern is foodborne viral disease via the consumption of fresh produce irrigated with virus-contaminated water. Although the food industry routinely uses chemical sanitizers to disinfect post-harvest fresh produce, it remains unknown how sanitizer and fresh produce properties affect the risk of viral illness through fresh produce consumption. A quantitative microbial risk assessment model was conducted to estimate (i) the health risks associated with consumption of rotavirus (RV)-contaminated fresh produce with different surface properties (endive and kale) and (ii) how risks changed when using peracetic acid (PAA) or a surfactant-based sanitizer. The modeling results showed that the annual disease burden depended on the combination of sanitizer and vegetable type when vegetables were irrigated with RV-contaminated water. Global sensitivity analyses revealed that the most influential factors in the disease burden were RV concentration in irrigation water and postharvest disinfection efficacy. A postharvest disinfection efficacy of higher than 99% (2-log10 ) was needed to decrease the disease burden below the World Health Organization (WHO) threshold, even in scenarios with low RV concentrations in irrigation water (i.e., river water). All scenarios tested here with at least 99.9% (3-log10 ) disinfection efficacy had a disease burden lower than the WHO threshold, except for the endive treated with PAA. The disinfection efficacy for the endive treated with PAA was only about 80%, leading to a disease burden 100 times higher than the WHO threshold. These findings should be considered and incorporated into future models for estimating foodborne viral illness risks.

Free Chlorine Disinfection Mechanisms of Rotaviruses and Human Norovirus Surrogate Tulane Virus Attached to Fresh Produce Surfaces.

To fill the knowledge gap on how effective free chlorine is against viral-contaminated produce, we inoculated the surfaces of outdoor- or greenhouse-grown kale and mustard with Rotavirus (RV) or a human norovirus surrogate (Tulane virus, TV) and then disinfected the leaves with free chlorine. Disinfection efficacies for RV strain OSU and Wa were approximately 1-log10 higher when attached to mustard than to kale. Similar disinfection efficacies were observed for TV attached to mustard or kale. When examining TV and RV OSU in suspension (not attached to leaf surfaces), TV was more resistant to free chlorine than RV OSU. Inactivation efficacies were higher for these viruses in suspension versus viruses attached to produce the surface. We also found that free chlorine damaged viral capsids, allowing free chlorine access to viral RNA to damage viral genomes. Exposure to free chlorine at 1.7 ppm over 1 min caused VP8* of RV OSU to lose its ability to bind to its host receptors. TV lost its ability to bind to its receptor only after exposure to free chlorine at 29 ppm over 1 min. Thus, to reduce foodborne viral infections, it is important to consider the differences in virus' reactivity and inactivation mechanisms with free chlorine.

Effect of Leaf Surface Chemical Properties on Efficacy of Sanitizer for Rotavirus Inactivation.

The use of sanitizers is essential for produce safety. However, little is known about how sanitizer efficacy varies with respect to the chemical surface properties of produce. To answer this question, the disinfection efficacies of an oxidant-based sanitizer and a new surfactant-based sanitizer for porcine rotavirus (PRV) strain OSU were examined. PRV was attached to the leaf surfaces of two kale cultivars with high epicuticular wax contents and one cultivar of endive with a low epicuticular wax content and then treated with each sanitizer. The efficacy of the oxidant-based sanitizer correlated with leaf wax content as evidenced by the 1-log10 PRV disinfection on endive surfaces (low wax content) and 3-log10 disinfection of the cultivars with higher wax contents. In contrast, the surfactant-based sanitizer showed similar PRV disinfection efficacies (up to 3 log10) that were independent of leaf wax content. A statistical difference was observed with the disinfection efficacies of the oxidant-based sanitizer for suspended and attached PRV, while the surfactant-based sanitizer showed similar PRV disinfection efficacies. Significant reductions in the entry and replication of PRV were observed after treatment with either disinfectant. Moreover, the oxidant-based-sanitizer-treated PRV showed sialic acid-specific binding to the host cells, whereas the surfactant-based sanitizer increased the nonspecific binding of PRV to the host cells. These findings suggest that the surface properties of fresh produce may affect the efficacy of virus disinfection, implying that food sanitizers should be carefully selected for the different surface characteristics of fresh produce. IMPORTANCE: Food sanitizer efficacies are affected by the surface properties of vegetables. This study evaluated the disinfection efficacies of two food sanitizers, an oxidant-based sanitizer and a surfactant-based sanitizer, on porcine rotavirus strain OSU adhering to the leaf epicuticular surfaces of high- and low-wax-content cultivars. The disinfection efficacy of the oxidant-based sanitizer was affected by the surface properties of the vegetables, while the surfactant-based sanitizer was effective for both high- and low-wax leafy vegetable cultivars. This study suggests that the surface properties of vegetables may be an important factor that interacts with disinfection with food sanitizers of rotaviruses adhering to fresh produce.

Histo-blood group antigen-like substances of human enteric bacteria as specific adsorbents for human noroviruses.

Histo-blood group antigens (HBGAs) have been suggested to be receptors or coreceptors for human noroviruses (HuNoVs) expressed on the intestinal epithelium. We isolated an enteric bacterium strain (SENG-6), closely related to Enterobacter cloacae, bearing HBGA-like substances from a fecal sample of a healthy individual by using a biopanning technique with anti-HBGA antibodies. The binding capacities of four genotypes of norovirus-like particles (NoVLPs) to Enterobacter sp. SENG-6 cells were confirmed by enzyme-linked immunosorbent assay (ELISA). Transmission electron microscopy demonstrated that NoVLPs bound mainly to extracellular polymeric substances (EPS) of Enterobacter sp. SENG-6, where the HBGA-like substances were localized. EPS that contained HBGA-like substances extracted from Enterobacter sp. SENG-6 was shown by enzyme-linked immunosorbent assay (ELISA) to be capable of binding to NoVLPs of a GI.1 wild-type strain (8fIIa) and a GII.6 strain that can recognize A antigen but not to an NoVLP GI.1 mutant strain (W375A) that loses the ability to bind to A antigen. Enzymatic cleavage of terminal N-acetyl-galactosamine residues in the bacterial EPS weakened bacterial EPS binding to the GI.1 wild-type strain (8fIIa). These results indicate that A-like substances in the bacterial EPS play a key role in binding to NoVLPs. Since the specific binding of HuNoVs to HBGA-positive enteric bacteria is likely to affect the transmission and infection processes of HuNoVs in their hosts and in the environment, further studies of human enteric bacteria and their binding capacity to HuNoVs will provide a new scientific platform for understanding interactions between two types of microbes that were previously regarded as biologically unrelated.