Draft genome sequences of antibiotic-resistant Serratia and Enterobacter species isolated from imported fresh produce in Georgia, USA.

Imported foods play an essential role in food security and in fulfilling consumer demand. However, these foods can also carry antibiotic-resistant bacteria, which might be introduced into the country of importation. Here, we report the draft genomes of antibiotic-resistant bacteria that were isolated from imported fresh produce in Georgia, USA.

Human Norovirus Surrogate Is Highly Stable in Berry Smoothies and under In Vitro Simulated Digestion.

Human noroviruses are major causes of foodborne outbreaks linked to berries. The overall goal of this study was to investigate the persistence of a human norovirus surrogate, Tulane virus (TV), in berry smoothies and under simulated digestion through the gastrointestinal track. Two types of smoothies were prepared from blueberries and strawberries. Tulane virus was spiked into each smoothie and incubated either at 37 or 4 °C for 2, 60, and 120 min. Furthermore, the virus-spiked smoothies were subjected to sequential oral (2 min), gastric (10 and 60 min), and intestinal (15 and 120 min) digestion according to the standardized INFOGEST model. Quantification of infectious TV was carried out using the TCID50 assay. At 4 °C, in both berry smoothies, TV infectivity did not show significant changes throughout the 120 min period. At 37 °C, TV infectivity showed significant reduction (~0.5 log TCID50/mL) only in blueberry smoothies starting at 60 min. During the oral, gastric, and intestinal digestion phases, the mean log reduction in TV infectivity in blueberry did not exceed ~0.5 log, while infectious TV in strawberry smoothies under all phases was stable. Given the notable stability of infectious viruses in berry smoothies and the gastrointestinal tract, prevention of norovirus contamination of berries is paramount to reduce virus outbreaks linked to berries.

Replication of Human Norovirus in Human Intestinal Enteroids Is Affected by Fecal Sample Processing.

Human intestinal enteroids (HIEs) culture is an emerging model for assessing the infectivity of human noroviruses (HuNoVs). The model is based on detecting an increase in HuNoV RNA post-infection of HIEs. HuNoV fecal samples used for HIE infection are traditionally processed by serial filtration. Recently, processing HuNoV fecal samples by serial centrifugation was shown to retain vesicles containing HuNoV. The objective of this study was to investigate whether serially centrifuged fecal samples, RNA extraction kit (QIAamp versus MagMaX) and HIE age (newer versus older) affect HuNoV RNA fold increase in HIE. HuNoV GII.1, GII.4 and GII.6 fecal samples were prepared by serial centrifugation and filtration and the viral RNA in HIE was quantified at 1 and 72 h post-infection (hpi) following RNA extraction and RT-qPCR. The serially filtered GII.1, GII.4 and GII.6 showed successful replication in HIE, resulting in mean log increases of 2.2, 2 and 1.2, respectively, at 72 vs. 1 hpi. In contrast, only serially centrifuged GII.1 showed consistently successful replication. However, using newer HIE passages and the MagMAX kit resulted in mean log fold increases for serially centrifuged GII.1, GII.4 and GII.6 (1.6, 2.3 and 1.8 log, respectively) that were similar to serially filtered samples. Therefore, HuNoV fecal sample processing and HIE age can affect virus replication in the HIE model.

Screening Commercial Tea for Rapid Inactivation of Infectious SARS-CoV-2 in Saliva.

SARS-CoV-2 infects the oral mucosa and is shed in salivary fluids. Traditionally, tea has been used by various cultures to treat respiratory ailments. The objective of this study was to identify commercially available teas that can rapidly inactivate infectious SARS-CoV-2 in saliva. Initially, tea (n = 24) was prepared as 40 mg/mL infusions and incubated with SARS-CoV-2 resuspended in water, for 5 min at 37 °C. Then, five teas that showed >3 log reduction in virus infectivity were further investigated at 40 and 10 mg/mL infusions for 60 and 10 s contact time with SARS-CoV-2 resuspended in saliva. Tea polyphenols were measured using the Folin-Ciocalteu assay. SARS-CoV-2 infectivity was quantified on Vero-E6 cell line using TCID50 assay. At 10 mg/mL infusion, black tea showed the highest reduction (3 log, i.e., 99.9%) of infectious SARS-CoV-2 within 10 s. Green, mint medley, eucalyptus-mint, and raspberry zinger teas showed similar inactivation of SARS-CoV-2 (1.5-2 log, i.e., 96-99% reduction). At 40 mg/mL infusions, all five teas showed >3 log reduction in virus infectivity within 10 s. Tea polyphenol but not pH was significantly correlated to virus reduction. Time-of-addition assay revealed that the five teas displayed preventive effects (0.5-1 log, i.e., 68-90% reduction) against SARS-CoV-2 infection of Vero-E6 cells as well as during post-virus infection (1.2-1.9 log, i.e., 94-98%). However, the highest inhibitory effect was observed when the teas were added at the time of virus infection (2-3 log, i.e., 99-99.9%). Our results provide insights into a rapid at-home intervention (tea drinking or gargling) to reduce infectious SARS-CoV-2 load in the oral cavity which might also mitigate infection of the oral mucosa.

In vitro digestion of SARS-CoV-2 contaminated berries reveals high inactivation of infectious virus during gastrointestinal passage.

IMPORTANCE: During the pandemic, news outlets occasionally reported on the detection of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) RNA on various foods, raising concerns over contaminated foods initiating infections. Coronavirus disease 2019 (COVID-19) patients often experience gastrointestinal symptoms and shed SARS-CoV-2 RNA in their feces. In addition, active virus replication in the gastrointestinal tract was shown; however, infectious viruses were rarely detected in feces. We previously showed that SARS-CoV-2 remained infectious on frozen berries for at least a month. Here, in vitro digestion models showed that SARS-CoV-2 on berries exhibits minimal inactivation at the oral phase and the virus may escape gastric inactivation early during feeding. However, high intestinal inactivation of the virus on berries suggested that SARS-CoV-2 was less likely to initiate infection in the small intestine. In contrast, the oral cavity is a potential site where infection might be initiated, providing more input for the gastrointestinal tract. High intestinal inactivation might explain the difficulty of detecting infectious SARS-CoV-2 in feces but not of virus RNA.

The Effect of Water Hardness and pH on the Efficacy of Peracetic Acid and Sodium Hypochlorite against SARS-CoV-2 on Food-Contact Surfaces.

Sodium hypochlorite (NaOCl) and peracetic acid (PAA) are commonly used disinfectants with a maximum recommended concentration of 200 ppm for food-contact surfaces. The objectives of this study were to assess the effect of pH and water hardness on NaOCl and PAA efficacy against SARS-CoV-2 on stainless steel (SS). The two disinfectants were prepared at 200 ppm in water of hardness 150 or 300 ppm with the final pH adjusted to 5, 6, 7, or 8. Disinfectants were applied to virus-contaminated SS for one minute at room temperature following the ASTM E2197 standard assay. SARS-CoV-2 infectivity was quantified using TCID50 assay on Vero-E6 cells. In general, increasingly hard water decreased the efficacy of NaOCl while increasing the efficacy of PAA. Hard water at 300 ppm significantly increased virus log reduction with PAA at pH 8 by ~1.5 log. The maximum virus log reductions were observed at pH 5 for both NaOCl (~1.2 log) and PAA (~2 log) at 150 and 300 ppm hard water, respectively. In conclusion, PAA performed significantly better than NaOCl with harder water. However, both disinfectants at 200 ppm and one minute were not effective (≤3 log) against SARS-CoV-2 on contaminated food-contact surfaces, which may facilitate the role of these surfaces in virus transmission.

First report of the mobile colistin resistance gene, mcr-1.26, in multidrug-resistant Escherichia coli isolated from retail chicken meat.

OBJECTIVES: We isolated a highly colistin-resistant Escherichia coli, strain 58, from fresh chicken wings in Lebanon. Here, we performed in-depth phenotypic and genomic analyses to identify the resistome of the isolate, focusing on the determinants that encoded colistin resistance. METHODS: The minimum inhibitory concentration (MIC) of colistin and resistance to other antibiotics were determined using the broth microdilution method and the Kirby-Bauer disk diffusion assay, respectively. Whole-genome sequencing (WGS) and different software available at the Center of Genomic Epidemiology were used to predict the resistome, the sequence type (ST), and the presence of virulence genes and plasmid replicon types. RESULTS: Susceptibility testing revealed that E. coli 58 exhibited multidrug resistance, including against colistin (MIC = 32 µg/mL). Whole-genome sequencing analyses showed that E. coli 58 carried 26 antimicrobial resistance genes associated with resistance to polymyxins (mcr-1.26), ß-lactams (blaTEM-1b and blaCMY-2), fosfomycin (fosA4), aminoglycosides (aac(3)-IId, aadA2b, aadA5, partial aadA1, aph(3'')-Ia, aph(3')-Ia, and aph(6)-Id), tetracyclines (tetA and tetM), quinolones (qnrS1), sulphonamides (sul2 and sul3), trimethoprim (dfrA14, dfrA17, and dfrA5), phenicols (floR and cmlA1), macrolides (mphA), lincosamides (lnu(F)), quaternary ammonium compounds (partial qacL and qacE), and peroxides (sitABCD). mcr-1.26 was located on an IncX4 plasmid and induced colistin resistance in otherwise naïve E. coli and Salmonella Enteritidis. Escherichia coli 58 was predicted to be a human pathogen and belonged to ST3107. CONCLUSION: To our knowledge, this is the first report of mcr-1.26 in poultry meat worldwide. We previously reported mcr-1.26 in an MDR E. coli (ST2207) isolated from a pigeon in Lebanon, which suggests that it might be spreading in different animal hosts and genetic backgrounds.

Draft Genome Sequences of Antibiotic-Resistant Serratia sp. Strains Isolated from Raw Sewage and Sediment Sludge in Georgia, USA.

Sewage has been established as a prime matrix for monitoring the emergence and dissemination of etiologic agents and antibiotic resistance determinants in a population. Here, we report the draft genomes and the acquired resistance genes of 11 antibiotic-resistant Serratia sp. isolates that were detected in samples from wastewater treatment plants.

Draft Genome Sequences of Multidrug-Resistant Escherichia coli Isolated from River Water.

The spread of antibiotic resistance poses a critical challenge worldwide. Contaminated environments can become reservoirs, spreading antibiotic-resistant bacteria and genetic determinants of resistance to humans directly or indirectly. Here, we report the draft genome sequence, the resistome, virulence genes, and sequence types of seven multidrug-resistant Escherichia coli strains isolated from river water.

SARS-CoV-2 remains infectious for at least a month on artificially-contaminated frozen berries.

The potential transmission of SARS-CoV-2 via food has been controversial since the beginning of the COVID-19 pandemic. To investigate these concerns, reliable detection methods and data on virus die-off rates in various foods are needed. Here, an FDA-standard method for the detection of enteric viruses' RNA from soft fruits was modified for the recovery of infectious SARS-CoV-2. Then, the survival of SARS-CoV-2 on berries was investigated as well as the effectiveness of washing virus-contaminated berries with water. The modified method did not significantly reduced log infectivity titers of recovered viruses, but berries did. The detection limit of the method for infectious SARS-CoV-2 was ∼2.97 log TCID50/g of berries. On SARS-CoV-2-inoculated berries that were stored at 4 °C for 7 days, significant reductions in SARS-CoV-2 infectivity were observed over time. In contrast, on frozen berries, infectious SARS-CoV-2 was recovered for 28 days without significant reductions. Washing SARS-CoV-2-inoculated berries with water removed >90% of infectious viruses within 10 min; however, infectious viruses were detected in wash water. Therefore, on fresh berries infectious viruses are markedly inactivated over time and can be largely removed by washing with water. However, the prolonged survival of SARS-CoV-2 on frozen berries suggests that the virus can potentially spread through frozen fruits.

Human Norovirus Histo-Blood Group Antigen (HBGA) Binding Sites Mediate the Virus Specific Interactions with Lettuce Carbohydrates.

Lettuce is often implicated in human norovirus (HuNoV) foodborne outbreaks. We identified H-like histo-blood group antigens (HBGAs) on lettuce leaves as specific binding moieties for virus-like particles (VLPs) of HuNoV GII.4/HS194/2009 strain. The objective of this study was to determine whether HuNoV-lettuce binding is mediated through the virus HBGA binding sites (HBS). Toward this objective, VLPs of historical HuNoV GII.4 strains (1987, 1997, 2002, 2004 and 2006) with known natural mutations in their HBS, two newly generated VLP mutants of GII.4/HS194/2009 (D374A and G443A) and a VLP mutant (W375A) of GI.1/Norwalk/1968 along with its wild type VLPs, which displays distinct HBS, were investigated for their binding to lettuce. ELISA revealed that historical GII.4 strains binding to lettuce was dependent on their HBGAs profiles. The VLP mutants D374A and G443A lost binding to HBGAs and displayed no to minimal binding to lettuce, respectively. The VLPs of GI.1/Norwalk/1968 strain bound to lettuce through an H-like HBGA and the binding was inhibited by fucosidase digestion. Mutant W375A which was previously shown not to bind to HBGAs, displayed significantly reduced binding to lettuce. We conclude that the binding of HuNoV GII.4 and GI.1 strains to lettuce is mediated through the virus HBS.

Tissue Distribution and Visualization of Internalized Human Norovirus in Leafy Greens.

Lettuce has been implicated in human norovirus (HuNoV) outbreaks. The virus is stable on the leaf surface for at least 2 weeks; however, the dynamics of virus internalization have not been fully investigated. The purpose of this study was to assess the internalization and distribution of HuNoV and two surrogate viruses, porcine sapovirus (SaV) and Tulane virus (TV), in lettuce and spinach. Viral inoculations through the roots of seedlings and the petiole of leaves from mature plants were performed, and the viruses were tracked on days 1 and 6 post-root inoculation and at 16 h and 72 h post-petiole inoculation. Confocal microscopy was used to visualize root-internalized HuNoV. In both lettuce and spinach, (i) HuNoV was internalized into the roots and leaves at similar RNA titers, whereas surrogate viruses were more restricted to the roots, (ii) all three viruses were stable inside the roots and leaves for at least 6 days, and (iii) HuNoV disseminated similarly inside the central veins and leaf lamina, whereas surrogate viruses were more restricted to the central veins. Infectious TV, but not SaV, was detectable in all tissues, suggesting that TV has greater stability than SaV. HuNoV was visualized inside the roots' vascular bundle and the leaf mesophyll of both plants. In conclusion, using surrogate viruses may underestimate the level of HuNoV internalization into edible leaves. The internalization of HuNoV through roots and cut leaves and the dissemination into various spinach and lettuce tissues raise concerns of internal contamination through irrigation and/or wash water.IMPORTANCE Human noroviruses are the leading cause of foodborne outbreaks, with lettuce being implicated in the majority of outbreaks. The virus causes acute gastroenteritis in all age groups, with more severe symptoms in children, the elderly, and immunocompromised patients, contributing to over 200,000 deaths worldwide annually. The majority of deaths due to HuNoV occur in the developing world, where limited sanitation exists along with poor wastewater treatment facilities, resulting in the contamination of water resources that are often used for irrigation. Our study confirms the ability of lettuce and spinach to internalize HuNoV from contaminated water through the roots into the edible leaves. Since these leafy greens are consumed with minimal processing that targets only surface pathogens, the internalized HuNoV presents an added risk to consumers. Thus, preventive measures should be in place to limit the contamination of irrigation water. In addition, better processing technologies are needed to inactivate internalized viral pathogens.

Recognition of Histo-Blood Group Antigen-Like Carbohydrates in Lettuce by Human GII.4 Norovirus.

UNLABELLED: Human norovirus (HuNoV) genogroup II genotype 4 (GII.4) strains account for about 80% of the gastroenteritis outbreaks in the United States. Contaminated food is a major transmission vehicle for this virus. In humans, pigs, and oysters, histo-blood group antigens (HBGAs) act as attachment factors for HuNoVs. In lettuce, although the virus-like particles (VLPs) of a GII.4 HuNoV were found to bind to cell wall carbohydrates, the exact binding site has not been investigated. Here, we show the presence of HBGA-like carbohydrates in the cell wall of lettuce. The digestion of lettuce leaves with cell wall-degrading enzymes exposed more binding sites and significantly increased the level of binding of GII.4 HuNoV VLPs. Competition assays showed that both the HBGA monoclonal antibody, recognizing the H type, and plant lectins, recognizing α-l-fucose in the H type, effectively inhibited VLP binding to lettuce tissues. Lettuce cell wall components were isolated and their NoV VLP binding characteristics were tested by enzyme-linked immunosorbent assays. The binding was inhibited by pretreatment of the lettuce cell wall materials with α-1,2-fucosidase. Collectively, our results indicate that H-type HBGA-like carbohydrates exist in lettuce tissues and that GII.4 HuNoV VLPs can bind the exposed fucose moiety, possibly in the hemicellulose component of the cell wall. IMPORTANCE: Salad crops and fruits are increasingly recognized as vehicles for human norovirus (HuNoV) transmission. A recent study showed that HuNoVs specifically bind to the carbohydrates of the lettuce cell wall. Histo-blood group antigens (HBGAs) are carbohydrates and are known as the attachment factors for HuNoV infection in humans. In this study, we show the presence of HBGA-like carbohydrates in lettuce, to which HuNoVs specifically bind. These results suggest that specifically bound HuNoVs cannot be removed by simple washing, which may allow viral transmission to consumers. Our findings provide new information needed for developing potential inhibitors to block binding and prevent contamination.

Abiotic Stress and Phyllosphere Bacteria Influence the Survival of Human Norovirus and Its Surrogates on Preharvest Leafy Greens.

Foodborne outbreaks of human noroviruses (HuNoVs) are frequently associated with leafy greens. Because there is no effective method to eliminate HuNoV from postharvest leafy greens, understanding virus survival under preharvest conditions is crucial. The objective of this study was to evaluate the survival of HuNoV and its surrogate viruses, murine norovirus (MNV), porcine sapovirus (SaV), and Tulane virus (TV), on preharvest lettuce and spinach that were subjected to abiotic stress (physical damage, heat, or flood). We also examined the bacteria culturable from the phyllosphere in response to abiotic stress and in relation to viral persistence. Mature plants were subjected to stressors 2 days prior to inoculation of the viruses on leaves. We quantified the viral RNA, determined the infectivity of the surrogates, and performed bacterial counts on postinoculation days (PIDs) 0, 1, 7, and 14. For both plant types, time exerted significant effects on HuNoV, MNV, SaV, and TV RNA titers, with greater effects being seen for the surrogates. Infectious surrogate viruses were undetectable on PID 14. Only physical damage on PID 14 significantly enhanced HuNoV RNA persistence on lettuce, while the three stressors differentially enhanced the persistence of MNV and TV RNA. Bacterial counts were significantly affected by time and plant type but not by the stressors. However, bacterial counts correlated significantly with HuNoV RNA titers on spinach and with the presence of surrogate viruses on both plant types under various conditions. In conclusion, abiotic stressors and phyllosphere bacterial density may differentially influence the survival of HuNoV and its surrogates on lettuce and spinach, emphasizing the need for the use of preventive measures at the preharvest stage.

Determination of the infectious titer and virulence of an original US porcine epidemic diarrhea virus PC22A strain.

The infectious dose of a virus pool of original US PEDV strain PC22A was determined in 4-day-old, cesarean-derived, colostrum-deprived (CDCD) piglets. The median pig diarrhea dose (PDD50) of the virus pool was determined as 7.35 log10 PDD50/mL, similar to the cell culture infectious titer, 7.75 log10 plaque-forming units (PFU)/mL. 100 PDD50 caused watery diarrhea in all conventional suckling piglets (n = 12) derived from a PEDV-naive sow, whereas 1000 and 10 000 PDD50 did not cause diarrhea in piglets derived from two PEDV-field exposed-recovered sows. This information is important for future PEDV challenge studies and validation of PEDV vaccines.

Postharvest Survival of Porcine Sapovirus, a Human Norovirus Surrogate, on Phytopathogen-Infected Leafy Greens.

Leafy greens are increasingly being recognized as an important vehicle for human noroviruses (HuNoV), which cause recurring gastroenteritis outbreaks. Leafy greens often become infected by phytopathogens in the field, which may cause symptoms on the edible parts. Whether plant pathogen infections enhance the survival of HuNoV on leafy greens is unknown. Lettuce and spinach plants were infected with a bacterium, Xanthomonas campestris pv. vitians strain 701a, and with Cucumber mosaic virus strain Fny, respectively. The survival rate of porcine sapovirus (SaV), a HuNoV surrogate, on infected and noninfected postharvest leaves was then assessed. In addition, acibenzolar-S-methyl, a commercial chemical elicitor of plant systemic defense, was used to assess whether stimulating the plant host defense affects the postharvest survival of SaV. Leaves harvested from control and treated plants were inoculated with SaV and incubated for 7 days at 4°C. The infectivity (tissue culture infectious dose affecting 50% of the culture [TCID50]/ml) and RNA (genomic equivalent/ml) titers of SaV were assayed using immunohistochemistry staining and SaV-specific TaqMan real-time reverse transcription PCR. Our results showed that cucumber mosaic virus Fny induced mild, nonnecrotic symptoms on spinach leaves and had no effect on SaV survival. In contrast, X. campestris pv. vitians 701a induced small localized necrotic lesions and significantly enhanced SaV survival on lettuce leaves. Treatment with acibenzolar-S-methyl was effective in reducing X. campestris pv. vitians 701a-induced lesions on infected lettuce plants but had no direct effect on SaV survival when used on healthy lettuce plants. These findings indicate that phytopathogen-induced necrotic lesions may enhance the postharvest survival of HuNoV on lettuce leaves. Therefore, preventive measures aiming to maintain healthy plants and minimize preharvest biological damage are expected to improve the safety of leafy greens.

Feline Calicivirus, Murine Norovirus, Porcine Sapovirus, and Tulane Virus Survival on Postharvest Lettuce.

Human norovirus (HuNoV) is the leading cause of foodborne illnesses, with an increasing number of outbreaks associated with leafy greens. Because HuNoV cannot be routinely cultured, culturable feline calicivirus (FCV), murine norovirus (MNV), porcine sapovirus (SaV), and Tulane virus (TV) have been used as surrogates. These viruses are generated in different cell lines as infected cell lysates, which may differentially affect their stability. Our objective was to uniformly compare the survival of these viruses on postharvest lettuce while evaluating the effects of cell lysates on their survival. Viruses were semipurified from cell lysates by ultrafiltration or ultracentrifugation followed by resuspension in sterile water. Virus survival was examined before and after semipurification: in suspension at room temperature (RT) until day 28 and on lettuce leaves stored at RT for 3 days or at 4°C for 7 and 14 days. In suspension, both methods significantly enhanced the survival of all viruses. On lettuce, the survival of MNV in cell lysates was similar to that in water, under all storage conditions. In contrast, the survival of FCV, SaV, and TV was differentially enhanced, under different storage conditions, by removing cell lysates. Following semipurification, viruses showed similar persistence to each other on lettuce stored under all conditions, with the exception of ultracentrifugation-purified FCV, which showed a higher inactivation rate than MNV at 4°C for 14 days. In conclusion, the presence of cell lysates in viral suspensions underestimated the survivability of these surrogate viruses, while viral semipurification revealed similar survivabilities on postharvest lettuce leaves.

Antigenic relationships among porcine epidemic diarrhea virus and transmissible gastroenteritis virus strains.

UNLABELLED: Porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus (TGEV) are economically important swine enteropathogenic coronaviruses. These two viruses belong to two distinct species of the Alphacoronavirus genus within Coronaviridae and induce similar clinical signs and pathological lesions in newborn piglets, but they are presumed to be antigenically distinct. In the present study, two-way antigenic cross-reactivity examinations between the prototype PEDV CV777 strain, three distinct U.S. PEDV strains (the original highly virulent PC22A, S indel Iowa106, and S 197del PC177), and two representative U.S. TGEV strains (Miller and Purdue) were conducted by cell culture immunofluorescent (CCIF) and viral neutralization (VN) assays. None of the pig TGEV antisera neutralized PEDV and vice versa. One-way cross-reactions were observed by CCIF between TGEV Miller hyperimmune pig antisera and all PEDV strains. Enzyme-linked immunosorbent assays, immunoblotting using monoclonal antibodies and Escherichia coli-expressed recombinant PEDV and TGEV nucleocapsid (N) proteins, and sequence analysis suggested at least one epitope on the N-terminal region of PEDV/TGEV N protein that contributed to this cross-reactivity. Biologically, PEDV strain CV777 induced greater cell fusion in Vero cells than did U.S. PEDV strains. Consistent with the reported genetic differences, the results of CCIF and VN assays also revealed higher antigenic variation between PEDV CV777 and U.S. strains. IMPORTANCE: Evidence of antigenic cross-reactivity between porcine enteric coronaviruses, PEDV and TGEV, in CCIF assays supports the idea that these two species are evolutionarily related, but they are distinct species defined by VN assays. Identification of PEDV- or TGEV-specific antigenic regions allows the development of more specific immunoassays for each virus. Antigenic and biologic variations between the prototype and current PEDV strains could explain, at least partially, the recurrence of PEDV epidemics. Information on the conserved antigenicity among PEDV strains is important for the development of PEDV vaccines to protect swine from current highly virulent PEDV infections.

Cell culture isolation and sequence analysis of genetically diverse US porcine epidemic diarrhea virus strains including a novel strain with a large deletion in the spike gene.

The highly contagious and deadly porcine epidemic diarrhea virus (PEDV) first appeared in the US in April 2013. Since then the virus has spread rapidly nationwide and to Canada and Mexico causing high mortality among nursing piglets and significant economic losses. Currently there are no efficacious preventive measures or therapeutic tools to control PEDV in the US. The isolation of PEDV in cell culture is the first step toward the development of an attenuated vaccine, to study the biology of PEDV and to develop in vitro PEDV immunoassays, inactivation assays and screen for PEDV antivirals. In this study, nine of 88 US PEDV strains were isolated successfully on Vero cells with supplemental trypsin and subjected to genomic sequence analysis. They differed genetically mainly in the N-terminal S protein region as follows: (1) strains (n=7) similar to the highly virulent US PEDV strains; (2) one similar to the reportedly US S INDEL PEDV strain; and (3) one novel strain most closely related to highly virulent US PEDV strains, but with a large (197aa) deletion in the S protein. Representative strains of these three genetic groups were passaged serially and grew to titers of ∼5-6log10 plaque forming units/mL. To our knowledge, this is the first report of the isolation in cell culture of an S INDEL PEDV strain and a PEDV strain with a large (197aa) deletion in the S protein. We also designed primer sets to detect these genetically diverse US PEDV strains.