The symptoms of children infected with SARS-CoV-2 are mainly asymptomatic, mild, moderate, and a few severe cases. To understand the immune response characteristics of children infected with SARS-COV-2 who do not develop severe cases, 82 children infected with the SARS-CoV-2 delta strain were recruited in this study. Our results showed that high levels of IgG, IgM, and neutralization antibodies appeared in children infected with SARS-CoV-2. SARS-CoV-2 induced upregulation of both pro-inflammatory factors including TNF-α and anti-inflammatory factors including IL-4 and IL-13 in the children, even IL-10. The expression of INF-α in infected children also showed a significant increase compared to healthy children. However, IL-6, one of the important inflammatory factors, did not show an increase in infected children. It is worth noting that a large number of chemokines reduced in the SARS-CoV-2-infected children. Subsequently, TCR Repertoire, TCRβ bias, and preferential usage were analyzed on data of TCR next-generation sequencing from 8 SARS-CoV-2-infected children and 8 healthy controls. We found a significant decrease in TCR clonal diversity and a significant increase in TCR clonal expansion in SARS-CoV-2-infected children compared to healthy children. The most frequent V and J genes in SARS-CoV-2 children were TRBV28 and TRBJ2-1. The most frequently VβJ gene pairing in SARS-CoV-2 infected children was TRBV20-1-TRBJ2-1. The strong antiviral antibody levels, low expression of key pro-inflammatory factors, significant elevation of anti-inflammatory factors, and downregulation of many chemokines jointly determine that SARS-CoV-2-infected children rarely develop severe cases. Overall, our findings shed a light on the immune response of non-severe children infected with SARS-CoV-2.

Viral myocarditis (VMC) is a common disease characterized by cardiac inflammation. AC-73, an inhibitor of CD147, disrupts the dimerization of CD147, which participates in the regulation of inflammation. To explore whether AC-73 could alleviate cardiac inflammation induced by CVB3, mice were injected intraperitoneally with AC-73 on the fourth day post-infection (dpi) and sacrificed on the seventh dpi. Pathological changes in the myocardium, T cell activation or differentiation, and expression of cytokines were analyzed using H&E staining, flow cytometry, fluorescence staining and multiplex immunoassay. The results showed that AC-73 alleviated cardiac pathological injury and downregulated the percentage of CD45+CD3+ T cells in the CVB3-infected mice. The administration of AC-73 reduced the percentage of activated CD4+ and CD8+ T cells (CD69+ and/or CD38+) in the spleen, while the percentage of CD4+ T cell subsets in the spleen was not changed in the CVB3-infected mice. In addition, the infiltration of activated T cells (CD69+) and macrophages (F4/80+) in the myocardium also decreased after the AC-73 treatment. The results also showed that AC-73 inhibited the release of many cytokines and chemokines in the plasma of the CVB3-infected mice. In conclusion, AC-73 mitigated CVB3-induced myocarditis by inhibiting the activation of T cells and the recruitment of immune cells to the heart. Thus, CD147 may be a therapeutic target for virus-induced cardiac inflammation.

Objective:A mouse model of pancreatitis induced by coxsackievirus B3 (CVB3) was established. The pathological change of pancreas and the infiltration of Th17/Treg cells were observed.Methods:The BALB/c mice were inoculated intraperitoneally with CVB3 to induce acute viral pancreatitis model. Then the pathological changes of pancreas were observed by HE staining; the viral RNA load and relative expression of cytokines (IFN-γ, IL-6 and IL-17) mRNA were detected by q-PCR; the proportion of infiltrated CD45 + CD3 + T cells, CD4 + and CD8 + T cells, Th17 and Treg cells in the pancreas was determined by flow cytometry. Results:Three days after CVB3 infection, the viral RNA load in pancreas was the highest (0.96±0.18) and gradually decreased with prolongation of infection. Compared with the 3 dpi group, the viral RNA load in pancreas was decreased (0.96±0.18 vs. 0.62±0.14) at 7 dpi, but there was no statistically significant difference. In addition, the infiltration of immune cell in pancreas increased significantly after 7dpi and the pathological score >2. The percent of infiltrated Th17 cells (1.05±0.21 vs. 22.13±5.79) and Treg cells (3.11±0.78 vs. 8.25±1.30) among CD4 + T cells significantly increased after infection (P<0.05), and the Th17/Treg also increased (P<0.01). Compared with the control group, the relative mRNA expression of IFN-γ (1.05±0.23 vs. 672.6±47.67), IL-6 (1.00±0.38 vs. 68.28±4.57), and IL-17 (1.01±0.11 vs. 54.15±7.94) in pancreas increased at 7 days after CVB3 infection ( P<0.01). Conclusions:The infiltration of Th17/Treg cells and the expression of related cytokines related cytokines IL-6 and IL-17 mRNA were upregulated in pancreas, which promoted the process of CVB3-induced pancreatitis.

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.

Objective:To analyze the epidemiological characteristics and genotypes of human rhinovirus (HRV) in patients with upper respiratory tract infection in Qingdao in the winter of 2020.Methods:Throat swab samples were collected from 101 patients with upper respiratory tract infection in Qingdao from November 2020 to January 2021. Quantitative PCR was used to detect 15 common respiratory viruses in the samples. HRV-positive samples were further analyzed with RT-PCR to amplify and sequence HRV VP4/VP2 gene. A phylogenetic tree was constructed based on the sequencing results and homology analysis was conducted.Results:Six common respiratory viruses were detected in the 101 patients. Thirty-four cases (34/101, 33.66%) were single pathogen infection and two cases were multiple infection (2/101, 1.98%). The positive rate of HRV was the highest (21.78%, 22/101). Twenty HRV VP4/VP2 sequences were successfully amplified. Phylogenetic analysis showed that there were 16 strains of HRV-A subtype and four strains of HRV-C subtype and 14 serotypes were involved.Conclusions:HRV was one of the leading viral pathogens causing upper respiratory tract infection in Qingdao in the winter of 2020 and the predominant subtype was HRV-A.

In the present study we investigated the changes in miRNA levels inhuman rhinovirus 16 (HRV16)-infected cells. A small RNA deep sequencing experiment was performed through next-generation sequencing. In total, 53 differentially expressed miRNAs were confirmed by RT-qPCR, including 37 known miRNAs and 16 novel miRNAs. Interaction networks between differentially expressed miRNAs and their targets were established by mirDIP and Navigator. The prediction results showed that QKI, NFAT5, BNC2, CELF2, LCOR, MBNL2, MTMR3, NFIB, PPARGC1A, RSBN1, TRPS1, WDR26, and ZNF148, which are associated with cellular differentiation and transcriptional regulation, were recognized by 12, 11, or 9 miRNAs. Many correlations were observed between transcriptional or post-transcriptional regulation of an miRNA and the expression levels of its target genes in HRV16-infected H1-HeLa cells.

As an alternative mechanism for cap-dependent (m7GpppN) translation, internal ribosome entry site (IRES)-dependent translation has been observed in the 5' untranslated regions (5' UTR) and coding regions of a number of viral and eukaryotic mRNAs. In this study, a series of 5' terminal truncated structural protein genes that were fused with GFP was used to screen for potential IRESs, and IRESs were identified using a bicistronic luciferase vector or GFP expression vector possessing a hairpin structure. Our results revealed that a putative IRES was located between nt 1982 and 2281 in the VP3 coding region of the human rhinovirus 16 (HRV16) genomes. We also demonstrated that effective IRES-initiated protein expression in vitro did not occur through splicing sites or cryptic promoters. We confirmed that thapsigargin (TG), an inducer of endoplasmic reticulum stress (ERS), facilitated increased IRES activity in a dose-dependent manner. Additionally, the secondary structure of the IRES was predicted online using the RNAfold web server.

Objectives: This study assessed the incidence and resistance of Mycoplasma pneumoniae (MP) in children in Qingdao, China, in 2019. Methods: We detected MP infection in 78 pharyngeal swabs from children with pneumonia by qPCR. The RepMP4 element in the P1 adhesin gene, domain V of the 23S rRNA gene, and the L4/L22 ribosomal pro-teins were amplified by nested PCR. Evolutionary analysis was conducted based on the P1 gene sequence. Resistance mutations in domain V of the 23S rRNA gene and L4/L22 ribosomal proteins were analysed. Results: The incidence of MP infection in children with pneumonia was 59.0% (46/78). The mean duration of MP infection was longer than that of non-MP infection. According to P1 gene sequencing of 21 samples, 12 (57.1%) were type 1 and 9 (42.9%) were type 2. Drug resistance mutations A2063G in domain V of 23S rRNA gene and T508C in L22 were identified from all sequenced MP. However, mutations at positions 2064 and 2617 were not found in this study. C162A mutation appeared in most type 2 samples. A430G mutation appeared in one type 1 sample and in several type 2 samples. T279C mutation in L22 was mostly found in type 2 samples. Conclusion: The incidence of MP infection was 59.0% in children with pneumonia in Qingdao in 2019. Type 1 MP infection was slightly more common than type 2, indicating that the genotype of MP is grad-ually shifting from type 1 to type 2. Macrolide resistance mutation A2063G could be detected in all sequenced MP. (c) 2021 The Author(s). Published by Elsevier Ltd on behalf of International Society for Antimicrobial Chemotherapy. This is an open access article under the CC BY-NC-ND license ( http://creativecommons.org/licenses/by-nc-nd/4.0/ )

Objective:To understand the prevalence of coxsackievirus A6 (CV-A6) in hospitalized patients with hand, foot and mouth disease in autumn and winter of 2019, and the genetic characteristics of VP1 region and the relation-ship between germline evolution.Methods:A total of 104 throat swabs from hospitalized patients with hand, foot and mouth disease in Qingdao in autumn and winter of 2019, and screen positive specimens were collected using enterovirus universal real-time PCR technology. Then the universal typing primers in the VP1 region were used to amplify the positive samples, the amplified segments were sequenced and BLAST comparison was performed. CV-A6 VP1 full-length primers were used to amplify the BLAST comparison of CV-A6 positive samples, sequence the ampli-fied products, analyze the homology of nucleotides and amino acids through DNAstar and MEGA software, and construct a phylogenetic tree.Results:Among 104 throat swab samples, 60 were positive for enterovirus, and the overall positive rate was 57.7%, of which the positive rate of CV-A6 was 26.9% (28/104), and the rate of coxsackievirus A16 (CV-A6) was 30.8% (32/104). Twenty-eight CV-A6 positive samples were subjected to full-length amplification, sequencing, comparison and homology analysis of the VP1 region, and 22 positive sequences were obtained. The nucleotide homology was 93.6% to 99.9%, and the amino acid homology was 98.0%-100%. Phylogenetic analysis showed that 22 CV-A6 strains belonged to the D3 subtype in the D genotype.Conclusions:The pathogens causing hospitalized hand, foot and mouth disease in Qingdao in autumn and winter of 2019 were CV-A6 and CV-A16. The prevalent strains of CV-A6 were mainly D3 subtypes.

Cap-dependent initiation of translation is a canonical mechanism adopted by eukaryotic cells. Internal ribosome entry site (IRES)-dependent translation is a mechanism distinct from 5′ cap-dependent translation. IRES elements are located mainly in the 5′-untranslated regions (UTRs) of viral and eukaryotic mRNAs. In addition, IRESs are found in the coding regions of some viral and eukaryotic genomes and initiate the translation of some functional truncated isoforms. Here, via IRES-initiated expression of proteins, bicistronic vectors and ribosome profiling of the human rhinovirus 16 (HRV16), we found that the coding region of the nonstructural proteins P2 and P3 contained 5 putative IRES elements. These 5 putative IRESs were located within nucleotides 4286-4585, 5002-5126, 6245-6394, 6619-6718 and 6629-6778 and initiated green fluorescent protein (GFP) expression in vitro . This alternative mechanism might be effective and economical for eliminating the time and raw material required to synthesize the full-length polyprotein.

Objective:To analyze the epidemiological characteristics and co-infections of pathogens in children with pneumonia in autumn and winter of 2019 in Qingdao.Methods:From August to November in 2019, 77 children with pneumonia in three hospitals in Qingdao were selected as the research subjects. Throat swabs were collected, nucleic acid was extracted, and 20 common respiratory pathogens were detected by single tube multiplex PCR.Results:Among the 77 cases, the incidence of pneumonia in boys (53.2%) was slightly higher than that in girls (46.7%). Children aged 1-2 years accounted for 10.3% of the total cases, children aged 3-6 years accounted for 61%, and children aged 7-13 years accounted for 20.7%. Twenty-nine cases (38.10%) had high white blood cells; 16 cases (20.77%) had high neutrophil count; 30 cases (38.96%) had high lymphocyte count; the pathogen detection rate was 77.92% of cases, among whom Mycoplasma pneumoniae (MP) was 59.74%, Bocavirus was 10.39%(8/77), adenovirus was 7.79% (6/77), rhinovirus was 3.89% (3/77), parainfluenza virus type 4 was 3.89% (3/77), Bordetella pertussis was 3.89% (3/77), parainfluenza virus type 2 was 2 2.59% (2/77), coronavirus nl63/hku1 was 2.59% (2/77), coronavirus OC43 was 2.59% (2/77), human metapneumovirus was 1.29% (1/77), Parainfluenza virus type 3 was 1.29%(1/77). The 24 cases of virus infection accounted for 31.16% (24/77). The co-infection with two pathogens accounted for 18.18%.Conclusions:Many kinds of pathogens were detected in children with pneumonia in autumn and winter of 2019, in Qingdao. The prevalence of Mycoplasma pneumoniae infection was the highest. Many common viral infections were found in the cases. A high proportion of co-infection was detected in these pneumonia cases.

Objective:To determine the viral load of human herpesvirus 6 A (HHV-6A), HHV-6B and chromosomal integrated HHV-6 (ciHHV-6) simultaneously through a triple chip digital PCR (tcdPCR) method for detection of HHV-6A/6B and ribonuclease P-30 (RPP30).Methods:According to optimal reaction conditions of real-time fluorescence quantitative PCR (RT-qPCR) method, the tcdPCR mehod of HHV-6A, HHV-6B and RPP30 was established. The sensitivity of tcdPCR was determined by virus cultures and the specificity of tcdPCR was detected with other herpesviruses. Subsequently, the tcdPCR of HHV-6A, HHV-6B and RPP30 was verified through 127 whole blood samples.Results:The consistency between RT-qPCR and tcdPCR for HHV-6 detection was good (R 2>0.97). And there was no cross-reaction with other herpesviruses. The 14 positive samples could be detected effectively by the tcdPCR of HHV-6A, HHV-6B and RPP30. The lowest detectable viral load of HHV-6A and HHV-6B was 50 copies/ml and 105 copies/ml, respectively. And the ratio of HHV-6/(RPP30/2) in 14 positive samples was less than 1. Conclusions:The tcdPCR has good sensitivity and specificity. And HHV-6 tcdPCR method can quantitatively detect the viral load of HHV-6 infection and the copy number of RPP30, and ciHHV-6 can be judged by ratio of HHV-6/(RPP30/2) in clinical samples.

Objective:To summarize the experience and effectiveness of biosafety management for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleic acid detection process in biosafety level 2 (BSL-2) laboratories.Methods:The samples were the human specimens received by the Department of Center for Virus Resource in the Viral Disease Control and Prevention Institute of China CDC from February 21, 2020 to November 5, 2020, including throat swabs, nasal swabs, sputum, urine and stool. In addition to routine biosafety management requirements, the biosafety management of personnel, positions, specimens, processes, and safety inspections during the testing process were actively conducted.Results:A total of 27 692 cases’ samples were tested for SARS-CoV-2 nucleic acid. there were (2.32±0.11) batches of samples were tested per day, (87.22±6.83) samples were tested on average per batch and (3.43±0.23) h were spent in the BSL-2 laboratories per day. There were no biosafety issue occurred during the test process.Conclusions:During the long-term SARS-CoV-2 nucleic acid detection process, the BSL-2 laboratory must actively strengthen biosafety management in many aspects to ensure the safe and orderly test of the work.

Objective:To construct the dual-luciferase reporter vector for identification of internal ribosome entry site (IRES).Methods:The hairpin structure was inserted between Renilla luciferase (R-Luc) and Firefly luciferase (F-Luc) genes based on psiCHECK-2 to form plasmid psiCHECK-IRES. IRES of Encephalomyocarditis virus (EMCV) was inserted between the hairpin structure and F-Luc genes of psiCHECK-IRES to form vector psiCHECK-IRES-EMCV. After psiCHECK-IRES-EMCV or psiCHECK-IRES was transfected into BHK-21 cells respectively, expressions of F-Luc and R-Luc were detected by RT-qPCR. Then Luciferase activity of transfected cells was detected with the dual-luciferase reporter assay system at 24 h post-transfection.Results:The hairpin structure was successfully inserted into psiCHECK-2 to form psiCHECK-IRES by sequencing. RT-qPCR result showed that there were the approximate expressing levels of mRNA between F-Luc and R-Luc. The result indicated that no aberrant monocistronic transcripts, which caused false positive F-Luc readings, were produced. Then IRES of EMCV was introduced into psiCHECK-IRES to form psiCHECK-IRES-EMCV. The F-Luc/R-Luc ratio in psiCHECK-IRES-EMCV-transfected cells was 53.35 times that of psiCHECK-IRES-transfected cells. The result confirmed that IRES of EMCV initiated effectively the translation of F-Luc.Conclusions:Dual-luciferase reporter vector psiCHECK-IRES was successfully constructed, which could be used to validate viruses and eukaryotic genes, the translation thereof was IRES-dependent.

Objective:To establish a real-time fluorescent quantitative PCR for the detection of torque teno virus types 7 (TTV7), 8 (TTV8) and 10 (TTV10) and analyze its performance in clinical sample detection.Methods:Specific primers were designed based on the gene sequences of TTV7, TTV8 and TTV10 in GenBank. Recombinant plasmids of pMD19-T-TTV7, pMD19-T-TTV8 and pMD19-T-TTV10 were constructed and used as positive standard control to establish a real-time fluorescent quantitative PCR based on FAM-Eclipse probe method. The specificity and sensitivity of the established method were evaluated. Moreover, it was validated in terms of clinical sample detection.Results:The standard curve equations of the real-time fluorescent quantitative PCR for detecting TTV7, TTV8 and TTV10 were y=-0.340 2 x+ 114.780 0 ( R2=0.998 8), y=-0.351 1 x+ 114.940 0 ( R2=0.995 3) and y=-0.348 9 x+ 115.020 0 ( R2=0.991 7), respectively, and there was no cross-reaction with other viruses. The detection sensitivity of the established method for TTV7, TTV8 and TTV10 were 108 copies/μl, 84 copies/μl and 98 copies/μl, and the positive detection rates in clinical pediatric serum samples were 10.9%, 2.1% and 4.3%, respectively. Conclusions:The established real-time fluorescent quantitative PCR for detection of TTV7, TTV8 and TTV10 was featured by strong specificity and high sensitivity, which could be used for rapid TTV detection in clinical serum samples.

探究柯萨奇病毒B3型(Coxsackie virus type B3,CVB3)感染的细胞是否诱导内源性小干扰RNA(small interfering RNA,siRNA)的产生.以CVB3接种HeLa细胞,在细胞培养箱(5％CO2、37℃)孵育1 h.随后加入含2％血清的细胞维持液,继续培养3h和6h后收集细胞.用高通量测序(二代测序)试剂盒提取细胞RNA,并反转录合成cDNA,构建文库,上机测序.过滤数据,去除插入片段过长的序列、低质量序列、poly A序列和小片段序列,与已知的小RNA数据库比对鉴定siRNA.通过茎-环反转录-聚合酶链反应,证实内源性siRNA在感染CVB3后的表达.结果显示,感染CVB33 h和6 h后,HeLa细胞产生了多种内源性siRNA.其中内源性siRNA(novel_sir3502和novel_sir2806)在感染后3 h和6 h均可持续表达.经比对,novel_sir3502和novel_sir2806均可以识别45S和28S核糖体前体RNA.结果提示,CVB3感染可能干扰核糖体成熟.

The capacity to accurately diagnosis COVID-19 is essential for effective public health measures to manage the ongoing global pandemic, yet no presently available diagnostic technologies or clinical protocols can achieve full positive predictive value (PPV) and negative predictive value (NPV) performance. Two factors prevent accurate diagnosis: the failure of sampling methods ( e.g ., 40% false negatives from PCR testing of nasopharyngeal swabs) and sampling-time-dependent failures reflecting individual humoral responses of patients (e.g., serological testing outside of the sero-positive stage). Here, we report development of a diagnostic protocol that achieves full PPV and NPV based on a cohort of 500 confirmed COVID-19 cases, and present several discoveries about the sero-conversion dynamics throughout the disease course of COVID-19. The fundamental enabling technology for our study and diagnostic protocol—termed SANE, for Symptom (dpo)-Antibody-Nucleic acid-Epidemiological history—is our development of a peptide-protein hybrid microarray (PPHM) for COVID-19. The peptides comprising PPHM covid-19 were selected based on clinical sample data, and give our technology the unique capacity to monitor a patient’s humoral response throughout the disease course. Among other assay-development related and clinically relevant findings, our use of PPHM covid-19 revealed that 5% of COVID-19 patients are from an “early sero-reversion” subpopulation, thus explaining many of the mis-diagnoses we found in our comparative testing using PCR, CLIA, and PPHM covid-19 . Accordingly, the full SANE protocol incorporates orthogonal technologies to account for these patient variations, and successfully overcomes both the sampling method and sampling time limitations that have previously prevented doctors from achieving unambiguous, accurate diagnosis of COVID-19.