A novel porcine deltacoronavirus (PdCV) was first discovered in Ohio and Indiana in February 2014, rapidly spread to other states in the United States and Canada, and caused significant economic loss in the swine industry. The origin and virulence of this novel porcine coronavirus are not known. Here, we characterized U.S. PdCV isolates and determined their virulence in gnotobiotic and conventional piglets. Genome analyses revealed that U.S. PdCV isolates possess unique genetic characteristics and share a close relationship with Hong Kong and South Korean PdCV strains and coronaviruses (CoVs) of Asian leopard cats and Chinese ferret-badgers. The PdCV-positive intestinal content (Ohio CVM1) and the cell culture-adapted PdCV Michigan (MI) strain were orally inoculated into gnotobiotic and/or conventional piglets. Within 1 to 3 days postinfection, profuse watery diarrhea, vomiting, and dehydration were observed. Clinical signs were associated with epithelial necrosis in the gastric pits and small intestine, the latter resulting in severe villous atrophy. Mild interstitial pneumonia was identified in the lungs of PdCV-infected piglets. High levels of viral RNA (8 to 11 log RNA copies/g) were detected in intestinal tissues/luminal contents and feces of infected piglets, whereas moderate RNA levels (2 to 5 log RNA copies/g) were detected in blood, lung, liver, and kidney, indicating multisystemic dissemination of the virus. Polyclonal immune serum against PdCV but not immune serum against porcine epidemic diarrhea virus (PEDV) reacted with PdCV-infected small-intestinal epithelial cells, indicating that PdCV is antigenically distinct from PEDV. Collectively, we demonstrate for the first time that PdCV caused severe gastrointestinal diseases in swine.

e Porcine epidemic diarrhea coronavirus (PEDV) has significantly damaged America's pork industry. Here we investigate the receptor usage and cell entry of PEDV. PEDV recognizes protein receptor aminopeptidase N from pig and human and sugar coreceptor N-acetylneuraminic acid. Moreover, PEDV infects cells from pig, human, monkey, and bat. These results support the idea of bats as an evolutionary origin for PEDV, implicate PEDV as a potential threat to other species, and suggest antiviral strategies to control its spread. P orcine epidemic diarrhea coronavirus (PEDV) causes largescale outbreaks of diarrhea in pigs and an 80 to 100% fatality rate in suckling piglets (1-3). Since 2013, PEDV has swept throughout the United States, wiped out more than 10% of America's pig population in less than a year, and significantly damaged the U.S. pork industry (4-6). No vaccine or antiviral drug is currently available to keep the spread of PEDV in check. PEDV belongs to the ␣ genus of the coronavirus family (7,8), which also includes porcine transmissible gastroenteritis coronavirus (TGEV), bat coronavirus 512/2005 (BtCoV/512/2005), and human NL63 coronavirus (HCoV-NL63). Although both PEDV and TGEV infect pigs, PEDV is genetically more closely related to BtCoV/512/ 2005 than to TGEV, leading to the hypothesis that PEDV originated from bats (9).Receptor binding and cell entry are essential steps in viral infection cycles, critical determinants of viral host range and tropism, and important targets for antiviral interventions. An envelope-anchored spike protein mediates coronavirus entry into cells. The spike ectodomain consists of a receptor-binding subunit, S1, and a membrane fusion subunit, S2. S1 contains two domains, an N-terminal domain (S1-NTD) and a C-terminal domain (S1-CTD), both of which can potentially function as receptor-binding domains (RBDs) (Fig. 1A) (10, 11). The ability of coronavirus RBDs to recognize receptor orthologs from different species is one of the most important determinants of coronavirus host range and tropism (8,(12)(13)(14). HCoV-NL63 S1-CTD recognizes human angiotensin-converting enzyme 2 (ACE2), whereas TGEV S1-CTD recognizes porcine aminopeptidase N (APN), and its S1-NTD recognizes two sugar coreceptors, N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) (15-18). Usage of sugar coreceptors is linked to the enteric tropism of coronaviruses (18,19). It has been shown that PEDV uses porcine APN as its receptor (20). However, it is not known whether PEDV recognizes APN from other species or whether it uses sugar coreceptors. Addressing these questions will be critical for understanding the host range, tropism, and evolutionary origin of PEDV, for evaluating its potential risk to other species, particularly humans, and for developing effective vaccines and antiviral drugs to curb the spread of PEDV in pigs and to other species.To characterize the receptor usage of PEDV, here we identified the two S1 domains of PEDV based on the sequence similarity between PEDV and TGEV S1 subun...

Gamma irradiation is a nonthermal processing technology that has been used for the preservation of a variety of food products. This technology has been shown to effectively inactivate bacterial pathogens. Currently, the FDA has approved doses of up to 4.0 kGy to control food-borne pathogens in fresh iceberg lettuce and spinach. However, whether this dose range effectively inactivates food-borne viruses is less understood. We have performed a systematic study on the inactivation of a human norovirus surrogate (murine norovirus 1 [MNV-1]), human norovirus virus-like particles (VLPs), and vesicular stomatitis virus (VSV) by gamma irradiation. We demonstrated that MNV-1 and human norovirus VLPs were resistant to gamma irradiation. For MNV-1, only a 1.7-to 2.4-log virus reduction in fresh produce at the dose of 5.6 kGy was observed. However, VSV was more susceptible to gamma irradiation, and a 3.3-log virus reduction at a dose of 5.6 kGy in Dulbecco's modified Eagle medium (DMEM) was achieved. We further demonstrated that gamma irradiation disrupted virion structure and degraded viral proteins and genomic RNA, which resulted in virus inactivation. Using human norovirus VLPs as a model, we provide the first evidence that the capsid of human norovirus has stability similar to that of MNV-1 after exposure to gamma irradiation. Overall, our results suggest that viruses are much more resistant to irradiation than bacterial pathogens. Although gamma irradiation used to eliminate the virus contaminants in fresh produce by the FDA-approved irradiation dose limits seems impractical, this technology may be practical to inactivate viruses for other purposes, such as sterilization of medical equipment.

N6-methyladenosine (m6A) is the most prevalent internal modification of mRNAs in most eukaryotes. Here we show that RNAs of human respiratory syncytial virus (RSV) are modified by m6A within discreet regions and that these modifications enhance viral replication and pathogenesis. Knockdown of m6A methyltransferases decreases RSV replication and gene expression whereas knockdown of m6A demethylases has the opposite effect. The G gene transcript contains the most m6A modifications. Recombinant RSV variants expressing G transcripts that lack particular clusters of m6A display reduced replication in A549 cells, primary well differentiated human airway epithelial cultures, and respiratory tracts of cotton rats. One of the m6A-deficient variants is highly attenuated yet retains high immunogenicity in cotton rats. Collectively, our results demonstrate that viral m6A methylation upregulates RSV replication and pathogenesis and identify viral m6A methylation as a target for rational design of live attenuated vaccine candidates for RSV and perhaps other pneumoviruses.

ABSTRACTFresh produce is a major vehicle for the transmission of human norovirus (NoV) because it is easily contaminated during both pre- and postharvest stages. However, the ecology of human NoV in fresh produce is poorly understood. In this study, we determined whether human NoV and its surrogates can be internalized via roots and disseminated to edible portions of the plant. The roots of romaine lettuce growing in hydroponic feed water were inoculated with 1 × 106RNA copies/ml of a human NoV genogroup II genotype 4 (GII.4) strain or 1 × 106to 2 × 106PFU/ml of animal caliciviruses (Tulane virus [TV] and murine norovirus [MNV-1]), and plants were allowed to grow for 2 weeks. Leaves, shoots, and roots were homogenized, and viral titers and/or RNA copies were determined by plaque assay and/or real-time reverse transcription (RT)-PCR. For human NoV, high levels of viral-genome RNA (105to 106RNA copies/g) were detected in leaves, shoots, and roots at day 1 postinoculation and remained stable over the 14-day study period. For MNV-1 and TV, relatively low levels of infectious virus particles (101to 103PFU/g) were detected in leaves and shoots at days 1 and 2 postinoculation, but virus reached a peak titer (105to 106PFU/g) at day 3 or 7 postinoculation. In addition, human NoV had a rate of internalization comparable with that of TV as determined by real-time RT-PCR, whereas TV was more efficiently internalized than MNV-1 as determined by plaque assay. Taken together, these results demonstrated that human NoV and animal caliciviruses became internalized via roots and efficiently disseminated to the shoots and leaves of the lettuce.