Background EV71 is one of the important pathogens of Hand-foot-and-mouth disease (HFMD), which causes serious neurological symptoms. Several studies have speculated that there will be interaction between 5 ' UTR and 3D protein. However, whether 5 ' UTR interacts with the 3D protein in regulating virus replication has not been clarified.Methods Four 5'UTR mutation sites (nt88C/T, nt90-102-3C, nt157G/A and nt574T/A) and two 3D protein mutation sites (S37N and R142K) were mutated or co-mutated using virulent strains as templates. The replication of these mutant viruses and their effect on autophagy were determined.Results 5'UTR single-point mutant strains, except for EGFP-EV71(nt90-102-3C), triggered replication attenuation. The replication ability of them was weaker than that of the parent strain the virulent strain SDLY107 which is the fatal strain that can cause severe neurological complications. While the replication level of the co-mutant strains showed different characteristics. 5 co-mutant strains with interaction were screened: EGFP-EV71(S37N-nt88C/T), EGFP-EV71(S37N-nt574T/A), EGFP-EV71(R142K-nt574T/A), EGFP-EV71(R142K-nt88C/T), and EGFP-EV71(R142K-nt157G/A). The results showed that the high replicative strains significantly promoted the accumulation of autophagosomes in host cells and hindered the degradation of autolysosomes. The low replicative strains had a low ability to regulate the autophagy of host cells. In addition, the high replicative strains also significantly inhibited the phosphorylation of AKT and mTOR.Conclusions EV71 5'UTR interacted with the 3D protein during virus replication. The co-mutation of S37N and nt88C/T, S37N and nt574T/ A, R142K and nt574T/A induced incomplete autophagy of host cells and promoted virus replication by inhibiting the autophagy pathway AKT-mTOR. The co-mutation of R142K and nt88C/T, and R142K and nt157G/A significantly reduced the inhibitory effect of EV71 on the AKT-mTOR pathway and reduced the replication ability of the virus.

Objective:To explore the relationship between severe fever with thrombocytopenia syndrome virus (SFTSV) Gc and its N-glycosylation site and viral infectivity, a recombinant pseudovirus containing SFTSV Gc glycosylation site mutant was constructed.Methods:The eukaryotic expression vectors pcDNA3.1(+ )-GC, PCDNA3.1(+ )-GC(N291Q), PCDNA3.1(+ )-GC(N352Q) and PCDNA3.1(+ )-GC (N374Q) were constructed by site-directed mutagenesis and homologous recombination. After their successful expression in 293T cells, we infected VSVΔG-Fluc*G pseudovirus, constructed four recombinant pseudoviruses and tested their effects on the cell force of infection.Results:Double digestion identification and sequence determination confirmed the successful construction of eukaryotic expression vectors pcDNA3.1 (+ )-Gc, pcDNA3.1 (+ )-Gc(N291Q), pcDNA3.1 (+ )-Gc(N352Q) and pcDNA3.1 (+ )-Gc(N374Q). Indirect immunofluorescence and Western Blotting result indicated the successful expression of all the four recombinant plasmids. SFTSV Gc recombinant pseudoviruses are specific for infecting Vero cells. Pseudovirus infection capacity was decreased significantly after the glycosylation site mutation, and the mutant strain with the glycosylation site at position 352 had the lowest level of infectivity ( P<0.001, P=0.001). Conclusions:The glycosylation site of SFTSV Gc may be associated with the infectious effect of the viral infection, and the amino acid mutation at position 352 has the greatest effect on the viral infectivity.

Objective:To rescue enterovirus group A type 71 (EV-A71) VP1 protein mutant strain (Val147→Ala) and explore the effect of this locus on viral virulence.Methods:The SDLY107 constructed plasmid pMD19-T-EGFP-107 was used as template, the recombinant plasmid pMD19-T-EGFP-107 (VP1-1) was constructed by site-directed mutation and reverse genetics. The recombinant plasmid was transfected into BSR-T7/5 cells, and the transfection products were subcultured in RD cells for three times. The mutant strain SDLY-EGFP-107(VP1-1) was successfully saved. The viral replication was detected by qRT-PCR, and the cell damage caused by the virus was detected by cell proliferation (CCK-8) and lactate dehydrogenase (LDH) tests.Results:The target fragment was amplified by overlapping fusion PCR, and the size was about 3.4 kb. The recombinant plasmid was identified by double enzyme digestion and the mutation was verified by sequencing. Obvious fluorescence was observed 24 hours after transfection of BSR-T7/5 cells with recombinant plasmid and 24 hours after inoculation into RD cells. The replication ability of mutant strain SDLY107 (VP1-1) in RD cells was weaker than that of virulent strain SDLY107 ( t=9.58, P<0.001) by qRT-PCR. CCK-8 and LDH tests showed that the cytotoxicity of mutant strain SDLY-EGFP-107(VP1-1) in RD cells ( t=106.60, P<0.001; t=39.88, P<0.001), SH-SY5Y cells ( t=18.72, P<0.001; t=19.09, P<0.001) were significantly weaker than that of virulent strain SDLY107. Conclusions:The mutant strain SDLY-EGFP-107(VP1-1) was successfully saved, which confirmed that the mutation of the 147th amino acid site of VP1 protein could reduce the replication and cell damage of the virus, providing a basis for further study of the role of VP1 protein in the pathogenesis of EV-A71.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging hemorrhagic fever with high mortality. Severe cases progressed rapidly, with deaths occurring within 2 weeks. Therefore, constructing a model to predict disease progression among hospitalized patients plays an important role in clinical practice. The development cohort included 121 patients with SFTS, 25 with severe SFTS, and 96 with mild SFTS. Two of the 64 variables were independent risk factors, including neurological symptoms (odds ratio [OR], 12.915; 95% confidence interval [CI], 3.342-49.916; P < 0.001) and aspartate aminotransferase/alanine aminotransferase levels (OR, 1.891; 95% CI, 1.272-2.813; P = 0.002). The model's area under the curve (AUC) was 0.882 (95% CI: 0.808-0.956). The mean AUC value obtained from the internal validation was 0.883 (95% CI: 0.809-0.957). The AUC in the external validation cohort was 0.873 (95% CI: 0.775-0.972). This model can be used to identify severely ill patients as early as possible with high predictive value, stability, and repeatability. This model can help clinicians with their treatment plans.