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.

Mechanism of blood-brain barrier disruption by an Escherichia coli from lambs with severe diarrhea and meningoencephalitis.

Escherichia coli (E. coli) is a common conditional pathogen that is associated with a variety of infections in humans and animals. Although there are increasing reports regarding the infection of E. coli to domestic animals and poultry, the infection of E. coli in lambs is relatively less reported, especially on meningoencephalitis. Here, we reported the isolation of an E. coli strain designated as NMGCF-19 from lambs characterized with severe diarrhea and neurological disorder, and demonstrated that NMGCF-19 as the causative agent has the ability to disrupt the blood-brain barrier (BBB) to cause the meningoencephalitis using a mouse model. Investigation on the mechanism regarding the NMGCF-19-related meningoencephalitis revealed a significant decreased expression of ZO-1 and occludin in mouse brain tissue in comparison with the control mice. Moreover, infection of NMGCF-19 increased the expression of proinflammatory cytokines TNF-α, IL-6, IL-1ß and IL-18, up-regulated HMGB1 level, and activated TLR2/TLR4/MyD88 and NLRP3 inflammasome pathways. These findings indicated that NMGCF-19 likely invades the brain tissue by disrupting the tight junction (TJ) architecture and causes the meningoencephalitis via increasing inflammatory response and activating TLR2/TLR4/MyD88 and NLRP3 inflammasome pathways.

Discovery of a virus of the species Enterovirus F in goats.

Here, we report two novel enteroviruses, designated as SD-S67 and SD-S68, isolated from a goat farm. Their complete genome sequences were determined and found to be 7455 and 7465 nucleotides in length, respectively. Molecular characterization revealed that SD-S67 is closely related to bovine enterovirus strain 261 and that SD-S68 to caprine enterovirus strain CEV-JL14. Phylogenetic analysis showed that SD-S67 clustered with members of the species Enterovirus F, and that SD-S68 clustered with enteroviruses of goats and sheep. Recombination analysis showed that SD-S67 is likely to have undergone several recombination events in the process of its evolution. To the best of our knowledge, this is the first report of an enterovirus F isolate from a goat and of a coinfection with enteroviruses of different species in the same goat herd.

Genetic diversity and recombination of bovine enterovirus strains in China.

Bovine enterovirus (BEV) consisting of enterovirus species E (EV-E) and F (EV-F) is the causative agent associated with respiratory and gastrointestinal diseases in cattle. Here, we reported the characterization, genetic diversity, and recombination of novel BEV strains isolated from the major cattle-raising regions in China during 2012-2018. Twenty-seven BEV strains were successfully isolated and characterized. Molecular characterization demonstrated that the majority of these novel BEV strains (24/27) were EV-E, while only few strains (3/27) were EV-F. Sequence analysis revealed the diversity of the circulating BEV strains such as species and subtypes where different species or subtype coinfections were detected in the same regions and even in the same cattle herds. For the EV-E, two novel subtypes, designated as EV-E6 and EV-E7, were revealed in addition to the currently reported EV-E1-EV-E5. Comparative genomic analysis revealed the intraspecies and interspecies genetic exchanges among BEV isolates. The representative strain HeN-B62 was probably from AN12 (EV-F7) and PS-87-Belfast (EV-F3) strains. The interspecies recombination between EV-E and EV-F was also discovered, where the EV-F7-AN12 might be from EV-E5 and EV-F1, and EV-E5-MexKSU/5 may be recombined from EV-F7 and EV-E1. The aforementioned results revealed the genetic diversity and recombination of novel BEV strains and unveiled the different BEV species or subtype infections in the same cattle herd, which will broaden the understanding of enterovirus genetic diversity, recombination, pathogenesis, and prevention of disease outbreaks. IMPORTANCE: Bovine enterovirus (BEV) infection is an emerging disease in China that is characterized by digestive, respiratory, and reproductive disorders. In this study, we first reported two novel EV-E subtypes detected in cattle herds in China, unveiled the coinfection of two enterovirus species (EV-E/EV-F) and different subtypes (EV-E2/EV-E7, EV-E1/EV-E7, and EV-E3/EV-E6) in the same cattle herds, and revealed the enterovirus genetic exchange in intraspecies and interspecies recombination. These results provide an important update of enterovirus prevalence and epidemiological aspects and contribute to a better understanding of enterovirus genetic diversity, evolution, and pathogenesis.

A Neonatal Murine Model for Caprine Enterovirus Infection and the Viral Tissue Tropism.

As the first caprine enterovirus identified from goat herds characterized by severe diarrhea with a high morbidity and mortality rate, the underlying pathogenesis and tissue tropism for CEV-JL14 remains largely unknown. Here, we reported the establishment of a neonatal murine model for caprine enterovirus and the unveiling of the tissue tropism and underlying pathogenesis for CEV-JL14 enterovirus. Susceptible murine strains, the infective dose, the infective routes, viral loads, and tissue tropism for CEV-JL14 infection were determined. The findings showed that ICR mice were susceptible to CEV-JL14 infection via all infection routes. Tissue viral load analysis showed that CEV-JL14 was detected in almost all tissues including the heart, liver, spleen, lung, kidney, intestine, brain, and muscle, with significantly higher viral loads in the heart, liver, lung, kidney, and intestine. These results revealed the pattern of viral load and tropism for CEV-JL14 and provided a model system for elucidating the pathogenesis of CEV-JL14 viruses.

Molecular Analysis of Caprine Enterovirus Circulating in China during 2016-2021: Evolutionary Significance.

Here, we report the characterization of 13 novel caprine/ovine enterovirus strains isolated from different regions in China during 2016-2021. Immunoperoxidase monolayer assay showed that these viral strains shared strong cross-reaction with the previously reported caprine enterovirus CEV-JL14. Alignment analysis of the complete nucleotide sequences revealed 79.2%-87.8% and 75.0%-76.7% sequence identity of these novel caprine enterovirus strains to CEV-JL14 and TB4-OEV, respectively. Phylogenetic analyses clustered these novel strains to EV-G based on the amino acid sequences of P1 and 2C+3CD. Moreover, phylogenetic analysis of these caprine enterovirus strains identified three new EV-G types using VP1 sequences. These results demonstrate the genetic variations and the evolution of caprine enterovirus.

Unveiling of the epidemiological patterns for caprine/ovine enterovirus infection in China.

Caprine/ovine enterovirus (CEV/OEV) infection is an emerging disease and remains largely unknown for its infection distribution, epidemic pattern, and the underlying contribution factors. Here, we report the investigation on CEV/OEV infection pattern and the underlying contribution factors by employing a sandwich ELISA kit for detection of CEV/OEV antigen. Epidemiological investigation revealed a wide range of infection rates of CEV/OEV from 19.80%-39.00% on goat/sheep farms in the major goat/sheep-raising provinces as such Henan, Shandong, Ningxia, Jilin, Inner Mongolia autonomous region, and Xinjiang autonomous region in China. Epidemic patterns and infection rates for CEV/OEV were affected by the breeds, raising mode, regions, and seasons. CEV/OEV infection rates were varied in different regions in China and significantly higher in the diarrheal herds (40.30%) than these in non-diarrheal herds (13.83%). Moreover, infection rate was higher in sheep (24.59%) than that in goats (9.84%), even dramatic difference among different breeds of goat or sheep. Out of different breeds of goat, Boer (20.13%) had the highest infection rate, followed by local breed (5.62%) and Saanen (2.61%). Among these breeds of sheep, higher infection rates were detected in local breed sheep (42.86%) and small-tailed Han sheep (35.91%) than these of Hu sheep (13.41%) and Dorper sheep (16.34%). Furthermore, raising modes were showed to contribute to the infection rate, where higher rates were detected among goats/sheep in captivity (27.10%) than these in free-range (12.27%) and semi-free range (19.24%). Additionally, CEV/OEV infection rate had obvious seasonality, while they increased from year 2015 to 2019. In summary, we investigated the CEV/OEV infection among the goat/sheep herds from different regions in China, revealed the epidemic pattern and the contribution factors to the infection, which provided the epidemiological data for future prevention and control of this emerging infection.

Unveiling of Evolution Pattern for HY12 Enterovirus Quasispecies and Pathogenicity Alteration.

Enterovirus, like the majority of RNA viruses, evolves to survive the changeable environments by a variety of strategies. Here, we showed that HY12 virus evolved to alter its characteristics and pathogenicity by employing a non-synonymous mutation. Analyses of 5'UTR, VP1 and VP2 gene sequences revealed the existence of HY12 virus in an array of mutants defined as quasispecies. The determination of diversity and complexity showed that the mutation rate and complexity of HY12 virus quasispecies increased, while the proportion of HY12 VP1 and VP2 consensus (master) sequences decreased with increasing passages. Synonymous mutation and non-synonymous mutation analysis displayed a positive selection for HY12 quasispecies evolution. A comparison of HY12 virus in different passages demonstrated that HY12 virus altered its characteristic, phenotype, and pathogenicity via non-synonymous mutation. These findings revealed the evolution pattern for HY12 virus, and the alteration of HY12 virus characteristics and pathogenicity by mutation.

Disruption of blood-brain barrier by an Escherichia coli isolated from canine septicemia and meningoencephalitis.

Escherichia coli (E. coli) is one of the common pathogenic bacteria in veterinary clinical infection. As an opportunistic microorganism, E. coli normally does not cause diseases. However, it causes infections under certain circumstance to domesticated animal and poultry, resulting in severe diarrhea, septicemia, and respiratory infections. Although there are increasing reports regarding the infections of E. coli to domestic animals and poultry, the infection of E. coli in dogs is relatively less reported, especially on septicemia and meningoencephalitis. Here, we reported the isolation and identification of an E. coli isolate named CEC-GZL17 from dogs characterized by septicemia and sudden death, and found that CEC-GZL17 is able to cause meningoencephalitis. Exploration on the potential mechanism underlying meningoencephalitis demonstrated that CEC-GZL17 infection significantly increases TNF-α expression and inhibits ZO-1 and occludin expressions in brain tissue, indicating that the E coli likely use the mechanism to penetrate the blood-brain barrier via disrupting tight junction architecture, thus leading to the invasion to brain tissue.