Enterovirus A71 2A-S125A acts as an attenuated vaccine candidate, indicating a universal approach in developing enterovirus vaccines.

Enteroviruses are important human pathogens with diverse serotypes, posing a major challenge to develop vaccines for individual serotypes, the success of polio vaccines in controlling and eradicating polio, along with the recent emergence and high prevalence of enterovirus-caused infectious diseases, highlights the importance of enterovirus vaccine development. Given our previous report on enteroviruses weakened by the 2 A S/T125A mutation, we assessed the potential of the EV-A71 2A-125A mutant as a vaccine candidate to address this challenge. We found that the 2A-125A mutant caused transient mild symptoms, low viral loads, and no significant pathological changes mild pathological changes in hSCARB2-KI mice, producing long-lasting cross-neutralizing antibodies against two EV-A71 wild strains. Pre-exposure to the 2A-125A mutant substantially protected against the EV-A71 Isehara wild-type strain, causing minor pathologies, significantly reducing muscle and lung inflammation, and preventing neurological damage, with reduced viral loads in vivo. Pre-exposure also distinctly suppressed the expression of pro-inflammatory cytokines, correlating to the severity of clinical symptoms. Collectively, the EV-A71 2A-125A mutant was attenuated and could generate a robust and protective immune response, suggesting its potential as a vaccine candidate and global solution for specific enterovirus vaccine development.

Phosphorylation of enteroviral 2Apro at Ser/Thr125 benefits its proteolytic activity and viral pathogenesis.

Enteroviral 2A proteinase (2Apro ), a well-established and important viral functional protein, plays a key role in shutting down cellular cap-dependent translation, mainly via its proteolytic activity, and creating optimal conditions for Enterovirus survival. Accumulated data show that viruses take advantage of various signaling cascades for their life cycle; studies performed by us and others have demonstrated that the extracellular signal-regulated kinase (ERK) pathway is essential for enterovirus A71 (EV-A71) and other viruses replication. We recently showed that ERK1/2 is required for the proteolytic activity of viral 2Apro ; however, the mechanism underlying the regulation of 2Apro remains unknown. Here, we demonstrated that the 125th residue Ser125 of EV-A71 2Apro or Thr125 of coxsackievirus B3 2Apro , which is highly conserved in the Enterovirus, was phosphorylated by ERK1/2. Importantly, 2Apro with phosphor-Ser/Thr125 had much stronger proteolytic activity toward eukaryotic initiation factor 4GI and rendered the virus more efficient for multiplication and pathogenesis in hSCARB2 knock-in mice than that in nonphospho-Ser/Thr125A (S/T125A) mutants. Notably, phosphorylation-mimic mutations caused deleterious changes in 2Apro catalytic function (S/T125D/E) and in viral propagation (S125D). Crystal structure simulation analysis showed that Ser125 phosphorylation in EV-A71 2Apro enabled catalytic Cys to adopt an optimal conformation in the catalytic triad His-Asp-Cys, which enhances 2Apro proteolysis. Therefore, we are the first to report Ser/Thr125 phosphorylation of 2Apro increases enteroviral adaptation to the host to ensure enteroviral multiplication, causing pathogenicity. Additionally, weakened viruses containing a S/T125A mutation could be a general strategy to develop attenuated Enterovirus vaccines.

High throughput detection of antibiotic residues in milk by time-resolved fluorescence immunochromatography based on QR code.

Herein, we have successfully established a novel, rapid, and simple lateral-flow immunoassay based on time-resolved fluorescence and biotin-streptavidin to detect the residues of various antibiotics in milk. The fluorescence signal and sensitivity of immunochromatography were enhanced through biotinylated antibody coupled with streptavidin europium microspheres. Moreover, due to the use of a QR Code and fluorescent reader, quantitative detection and real-time data uploading can be achieved. Under the optimal conditions, the various antibiotic residues were detected in the milk samples. The results showed that the limits of detection of tylosin, lincomycin and doxycycline were 0.10, 0.06, and 0.27 ng/mL, respectively. The recoveries of the spiked milk samples were 88.9%~127%, with coefficients of variation less than 11%, and the test strip can be stored at room temperature for 12 months. This study shows that the proposed time-resolved fluorescence immunoassay is sensitive, rapid and reliable, and has the potential to be used for detection of veterinary antibiotic residues in food safety fields.