The Viral Load of Epstein-Barr Virus in Blood of Children after Hematopoietic Stem Cell Transplantation

Objective To detect the Epstein-Barr virus(EBV)viral load of children after hematopoietic stem cell transplantation(HSCT)using chip digital PCR(cdPCR).Methods The sensitivity of cdPCR was determined using EBV plasmids and the EBV B95-8 strain.The specificity of EBV cdPCR was evaluated using the EBV B95-8 strain and other herpesviruses(herpes simplex virus 1,herpes simplex virus 2,varicella zoster virus,human cytomegalovirus,human herpesvirus 6,and human herpesvirus 7).From May 2019 to September 2020,64 serum samples of children following HSCT were collected.EBV infection and the viral load of serum samples were detected by cdPCR.The epidemiological characteristics of EBV infections were analyzed in HSCT patients.Results The limit of detection of EBV cdPCR was 110 copies/mL,and the limit of detection of EBV quantitative PCR was 327 copies/mL for the pUC57-BALF5 plasmid.The result of EBV cdPCR was up to 121 copies/mL in the EBV B95-8 strain,and both were more sensitive than that of quantitative PCR.Using cdPCR,the incidence of EBV infection was 18.75%in 64 children after HSCT.The minimum EBV viral load was 140 copies/mL,and the maximum viral load was 3,209 copies/mL using cdPCR.The average hospital stay of children with EBV infection(184±91 days)was longer than that of children without EBV infection(125±79 days),P=0.026.Conclusion EBV cdPCR had good sensitivity and specificity.The incidence of EBV infection was 18.75%in 64 children after HSCT from May 2019 to September 2020.EBV cdPCR could therefore be a novel method to detect EBV viral load in children after HSCT.

Coxsackievirus B3 Infection Triggers Autophagy through 3 Pathways of Endoplasmic Reticulum Stress

Objective Autophagy is a highly conserved intracellular degradation pathway. Many picornaviruses induce autophagy to benefit viral replication, but an understanding of how autophagy occurs remains incomplete. In this study, we explored whether coxsackievirus B3(CVB3) infection induced autophagy through endoplasmic reticulum(ER) stress. Methods In CVB3-infected HeLa cells, the specific molecules of ER stress and autophagy were detected using Western blotting, reverse transcription polymerase chain reaction(RT-PCR), and confocal microscopy. Then PKR-like ER protein kinase(PERK) inhibitor, inositol-requiring protein-1(IRE1) inhibitor, or activating transcription factor-6(ATF6) inhibitor worked on CVB3-infected cells, their effect on autophagy was assessed by Western blotting for detecting microtubule-associated protein light chain 3(LC3). Results CVB3 infection induced ER stress, and ER stress sensors PERK/eIF2α, IRE1/XBP1, and ATF6 were activated. CVB3 infection increased the accumulation of green fluorescent protein(GFP)-LC3 punctuation and induced the conversion from LC3-Ⅰ to phosphatidylethanolamine-conjugated LC3-1(LC3-Ⅱ). CVB3 infection still decreased the expression of mammalian target of rapamycin(mTOR) and p-mTOR. Inhibition of PERK, IRE1, or ATF6 significantly decreased the ratio of LC3-Ⅱ to LC3-Ⅰ in CVB3-infected HeLa cells. Conclusion CVB3 infection induced autophagy through ER stress in HeL a cells, and PERK, IRE1, and ATF6 a pathways participated in the regulation of autophagy. Our data suggested that ER stress may inhibit mTOR signaling pathway to induce autophagy during CVB3 infection.

Exploration of IRES Elements within the ORF of the Coxsackievirus B3 Genome

Objective This study aimed to identify internal ribosome entry sites(IRESs)in the open reading frame(ORF)of the Coxsackievirus B3(CVB3)genome.Methods The sequences of P1,P2,or P3 of the CVB3 genome or the truncated sequences from each antithymocyte globulin(ATG)to the end of the P1,P2,or P3 gene were inserted into the pEGFP-N1vector.After transfection,possible IRES-dependent green fluorescent protein(GFP)-fused proteins were detected by anti-GFP western blotting.The sequences of possible IRESs were inserted into specific Fluc/Rluc bicistronic vectors,in which the potential IRESs were determined according to the Fluc/Rluc activity ratio.Expression of Fluc and Rluc mRNA of the bicistronic vector was detected by RT-qPCR.Results After transfection of full length or truncated sequences of the P1,P2,or P3 plasmids,six GFPfused protein bands in P1,six bands in P2 and nine bands in P3 were detected through western blotting.Two IRESs in VP2(1461–1646 nt)and VP1(2784–2983 nt)of P1;one IRES in 2C(4119–4564 nt)of P2;and two IRESs in 3C(5634–5834 nt)and 3D(6870–7087 nt)of P3 were identified according to Fluc/Rluc activity ratio.The cryptic promoter was also excluded by RT-qPCR.Conclusion Five IRESs are present in the CVB3 coding region.