GRP78 recognizes EV-F 3D protein and activates NF-κB to repress virus replication by interacting with CHUK/IKBKB.

Enteroviruses are the causative agents associated with several human and animal diseases, posing a significant threat to human and animal health. As one of the host immune defense strategies, innate immunity plays a crucial role in defending against invading pathogens, where the host utilizes a variety of mechanisms to inhibit or eliminate the pathogen. Here, we report a new strategy for the host to repress enterovirus replication by the 78 kDa glucose-regulated protein (GRP78), also known as heat shock protein family A member 5 (HSPA5). The GRP78 recognizes the EV-encoded RNA-dependent RNA polymerases (RdRPs) 3D protein and interacts with the nuclear factor kappa B kinase complex (CHUK) and subunit beta gene (IKBKB) to facilitate the phosphorylation and nuclear translocation of NF-κB, which induces the production of inflammatory factors and leads to a broad inhibition of enterovirus replication. These findings demonstrate a new role of GRP78 in regulating host innate immunity in response to viral infection and provide new insights into the mechanism underlying enterovirus replication and NF-κB activation.IMPORTANCEGRP78 is known as a molecular chaperone for protein folding and plays a critical role in maintaining protein folding and participating in cell proliferation, cell survival, apoptosis, and metabolism. However, the functions of GRP78 to participate in enterovirus genome replication and innate immune responses are rarely documented. In this study, we explored the functions of the EV-3D-interacting protein GRP78 and found that GRP78 inhibits enterovirus replication by activating NF-κB through binding to EV-F 3D and interacting with the NF-κB signaling molecules CHUK/IKBKB. This is the first report that GRP78 interacts with CHUK/IKBKB to activate the NF-κB signaling pathway, which leads to the expression of the proinflammatory cytokines and inhibition of enterovirus replication. These results demonstrate a unique mechanism of virus replication regulation by GRP78 and provide insights into the prevention and treatment of viral infections.

Proliferation characteristics of coxsackievirus A10 in mice and immune protection ability of experimental inactivated vaccine.

Coxsackievirus A10 (CVA10) is the main pathogen of hand, foot, and mouth disease in China. However, there are no CVA10-specific drugs and vaccines, and the pathogenesis and effects of this virus in the body are unknown. We investigated the effect of a clinically isolated CVA10 virus strain (CVA10-25) to investigate its effect in suckling mice through different infection routes. We observed the dynamic distribution and proliferation of the virus in mouse tissues by infecting suckling mice with different doses of the virus and mice of different ages with the same dose of the virus. We also analysed the pathological characteristics after infection. A formaldehyde-inactivated experimental vaccine was prepared to immunise 5-week-old BALB/c female mice three times, and newborn suckling mice were tested for the presence of maternally transmitted antibodies. The viral load in each organ after intracerebral administration was higher than that after intraperitoneal administration; the peroral administration route did not cause disease in mice. Mouse paralysis and death after infection were related to age. The skeletal muscles, heart, and lung showed histopathological changes after infection. We established a 2-day-old BALB/c suckling mouse model that could be infected intracranially to study the pathogenesis and pathology of CVA10. Maternally transmitted antibodies protected the mice against the virus. This study provides a reference for CVA10-related pathogenesis and vaccine research.

SAMHD1 Inhibits Multiple Enteroviruses by Interfering with the Interaction between VP1 and VP2 Proteins.

Sterile alpha motif and histidine-aspartic acid domain-containing protein 1 (SAMHD1) possesses multiple biological activities such as virus restriction, innate immunity regulation, and autoimmunity. Our previous study demonstrated that SAMHD1 potently inhibits the replication of enterovirus 71 (EV71). In this study, we observed that SAMHD1 also restricts multiple enteroviruses (EVs), including coxsackievirus A16 (CA16) and enterovirus D68 (EVD68), but not coxsackievirus A6 (CA6). Mechanistically, SAMHD1 competitively interacted with the same domain in VP1 that binds to VP2 of EV71 and EVD68, thereby interfering with the interaction between VP1 and VP2 , and therefore viral assembly. Moreover, we showed that the SAMHD1 T592A mutant maintained the EV71 inhibitory effect by attenuating the interaction between VP1 and VP2, whereas the T592D mutant failed to. We also demonstrated that SAMHD1 could not inhibit CA6 because a different binding site is required for the SAMHD1 and VP1 interaction. Our findings reveal the mechanism of SAMHD1 inhibition of multiple EVs, and this could potentially be important for developing drugs against a broad range of EVs. IMPORTANCE Enterovirus causes a wide variety of diseases, such as hand, foot, and mouth disease (HFMD), which is a severe public problem threatening children under 5 years. Therefore, identifying essential genes which restrict EV infection and exploring the underlying mechanisms are necessary to develop an effective strategy to inhibit EV infection. In this study, we report that host restrictive factor SAMHD1 has broad-spectrum antiviral activity against EV71, CA16, and EVD68 independent of its well-known deoxynucleoside triphosphate triphosphohydrolase (dNTPase) or RNase activity. Mechanistically, SAMHD1 restricts EVs by competitively interacting with the same domain in VP1 that binds to VP2 of EVs, thereby interfering with the interaction between VP1 and VP2, and therefore viral assembly. In contrast, we also demonstrated that SAMHD1 could not inhibit CA6 because a different binding site is required for the SAMHD1 and CA6 VP1 interaction. Our study reveals a novel mechanism for the SAMHD1 anti-EV replication activity.

Human Enterovirus Diversity by Next-Generation Sequencing Analysis in Urban Sewage Samples From Buenos Aires Metropolitan Area, Argentina: A Retrospective Study.

Human Enteroviruses (hEVs) are responsible for a wide variety of human diseases. During hEVs infection, virions are excreted in human feces and the fecal-oral route is the primary pathway for person-to-person transmission. Sewage surveillance could help in monitoring hEVs circulation and describing their diversity in a specific population. In this study, sewage samples collected in Buenos Aires Metropolitan Area (Argentina) were retrospectively studied through an amplicon-deep sequencing approach and phylogenetic analyses to characterize hEVs spread. We identified 17 different hEVs types belonging to A, B, and C species. To the best of our knowledge, this is the first report in Buenos Aires for 7 identified hEV-C types. Phylogenetic analyses suggest several introductions of coxsackievirus B4, echovirus 1, and echovirus 9 in the country, along with the national spread reached by some variants. Besides, well-supported monophyletic groups of Argentine, Uruguayan, and Brazilian strains unveiled regional circulation patterns for some variants. These results extend our knowledge about hEVs circulation in Buenos Aires and might exhort authorities to implement more active sewage surveillance in the region.

Viral Interference as a Factor of False-Negative in the Infectious Adenovirus Detection Using Integrated Cell Culture-PCR with a BGM Cell Line.

This study investigated the influence of viral interference on the detection of enteric viruses using the integrated cell culture (ICC)-PCR with a BGM cell line. It was possible to detect 102 plaque-forming units (PFU)/flask of enterovirus 71 (EV71) in spite of the presence of 104 PFU/flask of adenovirus 40 (AdV40). Meanwhile, 104 PFU/flask of AdV40 was not detected in the presence of 102 PFU/flask of EV71. This inhibition of AdV40 detection using ICC-PCR was attributable to the growth of EV71, because the addition of a growth inhibitor of EV71 (rupintrivir) neutralized the detection inhibition of AdV40. The growth inhibition of AdV40 under co-infection with EV71 is probably caused by the immune responses of EV71-infected cells. AdV is frequently used as a fecal contamination indicator of environmental water, but this study demonstrated that false-negative detection of infectious AdV using ICC-PCR could be caused by the co-existence of infectious EV in a water sample. The addition of rupintrivir could prevent false-negative detection of AdV using ICC-PCR. This study, therefore, emphasizes the importance of confirming the presence of multiple enteric viruses in a sample derived from environmental water prior to the application of ICC-PCR because the viral interference phenomenon may lead to the false-negative detection of target viruses.

Detection of Human Adenovirus, Rotavirus, and Enterovirus in Tap Water and Their Association with the Overall Quality of Water in Karachi, Pakistan.

Drinking water supplies in the developing world often serve as a biosphere for various organisms. Viral gastroenteritis is a neglected area of research in Pakistan, there are no data for the prevalence of enteric viruses in drinking water of the largest city of Karachi. The present study aimed to provide a survey of the existence of enteric viruses: human adenovirus (HAdV), human enteroviruses (hEV), and genotype A rotavirus (GARV) in tap water. Using a simple PCR approach, we detected 20%, 43%, and 23% of HAdV, hEV, and GARV in tap water samples, respectively. We have also shown an overall quality deficit of tap water at the pumping station and consumer tap. We have found no sample free from bacterial contaminations. The ranges for a total number of the heterotrophic plate count and coliform were found 8.7 × 102-4.5 × 106 CFU/mL and 210 to uncountable coliforms/100 mL, respectively. Moreover, we assessed the efficiency of small-scale water treatment methods for the removal of viruses.

Reduction of Pathogenic and Indicator Viruses at a Drinking Water Treatment Plant in Southern Louisiana, USA.

Monthly sampling was conducted at a drinking water treatment plant (DWTP) in Southern Louisiana, USA from March 2017 to February 2018 to determine the prevalence and reduction efficiency of pathogenic and indicator viruses. Water samples were collected from the DWTP at three different treatment stages (raw, secondary-treated, and chlorinated drinking water) and subjected to quantification of seven pathogenic viruses and three indicator viruses [pepper mild mottle virus (PMMoV), tobacco mosaic virus (TMV), and crAssphage] based on quantitative polymerase chain reaction. Among the seven pathogenic viruses tested, only Aichi virus 1 (AiV-1) (7/12, 58%) and noroviruses of genogroup II (NoVs-GII) (2/12, 17%) were detected in the raw water samples. CrAssphage had the highest positive ratio at 78% (28/36), and its concentrations were significantly higher than those of the other indicator viruses for all three water types (P < 0.05). The reduction ratios of AiV-1 (0.7 ± 0.5 log10; n = 7) during the whole treatment process were the lowest among the tested viruses, followed by crAssphage (1.1 ± 1.9 log10; n = 9), TMV (1.3 ± 0.9 log10; n = 8), PMMoV (1.7 ± 0.8 log10; n = 12), and NoVs-GII (3.1 ± 0.1 log10; n = 2). Considering the high abundance and relatively low reduction, crAssphage was judged to be an appropriate process indicator during drinking water treatment. To the best of our knowledge, this is the first study to assess the reduction of crAssphage and TMV during drinking water treatment.

Reduction of Human Enteric and Indicator Viruses at a Wastewater Treatment Plant in Southern Louisiana, USA.

This study assessed wastewater quality through the quantification of four human enteric viruses and the applicability of pepper mild mottle virus (PMMoV) and tobacco mosaic virus (TMV) as indicators of viral reduction during wastewater treatment. Thirty-three samples were collected from three steps of a wastewater treatment plant in Southern Louisiana, USA for a year between March 2017 and February 2018. Noroviruses of genogroup I were the most prevalent human enteric viruses in influent samples. The concentrations of PMMoV in influent samples (5.9 ± 0.7 log10 copies/L) and biologically treated effluent samples (5.9 ± 0.5 log10 copies/L) were significantly higher than those of TMV (P < 0.05), and the reduction ratio of PMMoV (1.0 ± 0.8 log10) was found comparable to those of TMV and Aichi virus 1. Because of the high prevalence, high correlations with human enteric viruses, and lower reduction ratios, PMMoV was deemed an appropriate indicator of human enteric viral reduction during wastewater treatment process.

Applicability of crAssphage, pepper mild mottle virus, and tobacco mosaic virus as indicators of reduction of enteric viruses during wastewater treatment.

This study was conducted to evaluate the applicability of crAssphage, pepper mild mottle virus (PMMoV), and tobacco mosaic virus (TMV) as indicators of the reduction of human enteric viruses during wastewater treatment. Thirty-nine samples were collected from three steps at a wastewater treatment plant (raw sewage, secondary-treated sewage, and final effluent) monthly for a 13-month period. In addition to the three indicator viruses, eight human enteric viruses [human adenoviruses, JC and BK polyomaviruses, Aichi virus 1 (AiV-1), enteroviruses, and noroviruses of genogroups I, II, and IV] were tested by quantitative PCR. Indicator viruses were consistently detected in the tested samples, except for a few final effluents for crAssphage and TMV. The mean concentrations of crAssphage were significantly higher than those of most tested viruses. The concentrations of crAssphage in raw sewage were positively correlated with the concentrations of all tested human enteric viruses (p <0.05), suggesting the applicability of crAssphage as a suitable indicator to estimate the concentrations of human enteric viruses in raw sewage. The reduction ratios of AiV-1 (1.8 ± 0.7 log10) were the lowest among the tested viruses, followed by TMV (2.0 ± 0.3 log10) and PMMoV (2.0 ± 0.4 log10). Our findings suggested that the use of not only AiV-1 and PMMoV but also TMV as indicators of reductions in viral levels can be applicable during wastewater treatment.