Three-dimensional Culture of Human Airway Epithelium in Matrigel for Evaluation of Human Rhinovirus C and Bocavirus Infections / 生物医学与环境科学（英文）

OBJECTIVE@#Newly identified human rhinovirus C (HRV-C) and human bocavirus (HBoV) cannot propagate in vitro in traditional cell culture models; thus obtaining knowledge about these viruses and developing related vaccines are difficult. Therefore, it is necessary to develop a novel platform for the propagation of these types of viruses.@*METHODS@#A platform for culturing human airway epithelia in a three-dimensional (3D) pattern using Matrigel as scaffold was developed. The features of 3D culture were identified by immunochemical staining and transmission electron microscopy. Nucleic acid levels of HRV-C and HBoV in 3D cells at designated time points were quantitated by real-time polymerase chain reaction (PCR). Levels of cytokines, whose secretion was induced by the viruses, were measured by ELISA.@*RESULTS@#Properties of bronchial-like tissues, such as the expression of biomarkers CK5, ZO-1, and PCK, and the development of cilium-like protuberances indicative of the human respiration tract, were observed in 3D-cultured human airway epithelial (HAE) cultures, but not in monolayer-cultured cells. Nucleic acid levels of HRV-C and HBoV and levels of virus-induced cytokines were also measured using the 3D culture system.@*CONCLUSION@#Our data provide a preliminary indication that the 3D culture model of primary epithelia using a Matrigel scaffold in vitro can be used to propagate HRV-C and HBoV.

Effect of the somatic proteins from Trichinella spiralis muscle larvae on apoptosis of H446 small cell lung cancer cells / 中国人兽共患病学报

To probe the effects of Trichinella spiralis muscle larval somatic proteins on small cell lung cancer H446 cells and the possible mechanism of anti-tumor,H446 cells were culture with 0.2 mg/mL,0.4 mg/mL,0.6 mg/mL,0.8 mg/mL,1.0 mg/mL,and 1.2 mg/mL somatic proteins respectively.The experimental group was set and no dosing as control group.MTT colorimetric assay was used to test the effects of T.spiralis muscle larval somatic proteins on the proliferative activity of H446 cells.We used flow cytometry (FCM) to test the influence of T.spiralis muscle larval somatic proteins induced H446 cells apoptosis.The real-time PCR and Western blot methods were used to detect the expression of Cyt-C and apoptotic protease activating factor 1(Apaf-1) mRNA and protein.The MTT colorimetric assay showed that T.spiralis muscle larval somatic proteins could inhibit the proliferation of H446 cells;the flow cytometry showed that polypide proteins acted on H446 cells after 24 h appeared an obvious effect on promoting apoptosis.Results of real-time PCR and Western blot analysis indicated that compared with the control group,Cyt-C and Apaf-1 showed up-regulated expression.T.spiralis muscle larval somatic proteins could inhibit proliferation activity and induce the apoptosis of H446 cells,and its effects may be related to up-regulated expression of Cyt-C and Apaf-1.

Gene Knockdown in Human Rhinovirus 1B Using 2'-OMe-modified siRNAs Results in the Reactivation of the Interferon Response / 生物医学与环境科学（英文）

The aim of this study was to investigate the knockdown efficiency of 2'-O-methylated (2'-OMe)-modified small interfering RNAs (siRNAs) on human rhinovirus 1B (HRV1B) replication and the interferon response. Thus, 24 2'-OMe-modified siRNAs were designed to target HRV1B. The RNA levels of HRV1B, Toll-like receptor 3, melanoma differentiation-associated gene 5, retinoic acid inducible gene-I, and interferons were determined in HRV1B-infected HeLa and BEAS-2B epithelial cells transfected with 2'-OMe-modified siRNAs. The results revealed that all 2'-OMe-modified siRNAs interfered with the replication of HRV1B in a cell-specific and transfection efficiency-dependent manner. Viral activation of Toll-like receptor 3, melanoma differentiation-associated gene 5, retinoic acid inducible gene-I, and the interferon response was detected. In conclusion, the 2'-OMe-modified siRNAs used in this study could interfere with HRV1B replication, possibly leading to the reactivation of the interferon response.

Norovirus Infection and Histo-blood Group Antigens in Children Hospitalized with Diarrhea in Lulong and Chenzhou in China / 生物医学与环境科学（英文）

Norovirus (NoV) is a pathogen that commonly causes viral diarrhea in children. Studies indicate that NoV recognizes human histo-blood group antigens (HBGAs) as cell attachment factors. In order to explore the correlation between of NoV infection and HBGAs, a cross-sectional study was conducted in children less than five years old who were hospitalized with diarrhea in two areas of China between November 2014 and February 2015. Of the paired stool and saliva samples taken from 424 children, NoV was detected in 24 (6%) children, with viral genotypes GII.3 (n=5), GII.4 (n=14), GII.12 (n=1), and GII.17 (n=4). All of the individuals having NoV infection were either secretors (Lea-b+/Lex-y+) or partial secretors (Lea+b+/Lex+y+) except one GII.3 infection of a non-secretor (Lea+b-/Lex+y-). These results suggest that secretor positive is associated with NoV infection, although non-secretors are not absolutely protected from NoV infection.

Progress on development and research of human bocavirus 1 / 病毒学报

Human bocavirus 1 (HBoV1) is a novel virus that mainly causes respiratory tract infection, and it has the characteristic of genome of Parvovirus, containing three open reading frames that encode non-structural proteins NS1 and NP1 and structural proteins VP1 and VP2. Circular episome is present during the rolling circle replication of HBoV1, which provides the possibility of full genome amplification and infectious clone construction to save HBoV1. The recombination between HBoV1 and HBoV2-4 occurs frequently. With the three-dimensional culture, in vitro culture of HBoV1 provides a powerful tool for research on the pathogenesis of HBoV1. This review focuses on the molecular characteristics, association with diseases, in vitro culture, diagnosis and treatment of HBoV1.

Genome amplification and sequence analysis of human bocavirus 2 / 病毒学报

To obtain the genome sequence of human bocavirus 2 (HBoV2), different regions of HBoV2 genome were amplified through PCR in fecal specimens which had been identified as single-positive for HBoV2 in 2010. A genome sequence of HBoV2 (HBoV2-NC, 5444 bp) was obtained after sequence assembly. The phylogenetic analysis showed that HBoV2-NC had the closest evolutionary relationship with HBoV2 Lanzhou strain. The predication of inverted terminal repeats of HBoV2-NC by DINAMelt showed that inverted terminal repeats were contained in HBoV2-NC 5' terminal, which had the typical stem-loop structure in other parvoviruses. Finally, some flanking sequences of HBoV2-NC were amplified by linker-PCR.

Current research on picornavirus 3C protease / 病毒学报

The picornavirus family comprises many small viruses, several of which are important pathogens of humans and livestock. The 3C protease (3Cpro) of different species and genera of picornavirus contains the classic G-X-C-G motif and Cys-His-Asp/Glu catalytic triad. 3Cpro conducts maturation cleavage in the regions of VP2-VP3 and VP3-VP1 in P1, 2A-2B and 2B-2C in P2 and the whole P3. Picornavirus 3Cpro has been shown to have significant substrate preference in Q-G/S/A/V/H/R and E-S/G/R/M as well as species and genera specificity through analyses of the maturation cleavage of picornavirus polyproteins. Innate immune adaptors such as TRIF, MAVS, IRF3, IRF7 and NEMO have various potential cleavage sites in picornavirus 3Cpro (TRIF and NEMO show considerable diversity in their cleavage sites). Useful information will be provided for the development of broad-spectrum antiviral agents as well as evasion mechanisms of the innate immune system against picornavirus 3Cpro through continued research of picornavirus 3Cpro.

The effects of recombinant human beta-defensin-3 on expression of interleukin-17A and interleukin-22 in BEAS-2B cell / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To research the effects of recombinant human beta-defensin-3 (hBD-3) on expression of interleukin-17A (IL-17A) and interleukin-22 (IL-22) in BEAS-2B cell.</p><p><b>METHODS</b>The BEAS-2B cells were stimulated with different concentrations of hBD-3 for 6 hours and 24 hours, respectively. Toll-like receptor 2 (TLR2), IL-17A and IL-22 mRNA expression levels were determined by real-time PCR, and the expression levels of IL-17A and IL-22 protein were examined by enzyme linked immune-sorbent assay.</p><p><b>RESULTS</b>TLR2 mRNA in BEAS-2B cells were significantly increased in a concentration-and time-dependent manner after stimulating by hBD-3 for 24 hours compared to 6 hours. The IL-17A has significantly increased in mRNA and protein levels stimulated 24 hours in a concentration of 100 ng/ml, however, IL-17A mRNA expression has increased while protein didn't change stimulated 6 hours in a concentration of 50 ng/ml. The IL-22 mRNA and protein expression reached peak levels after stimulating in a concentration of 50 ng/ml of hBD-3 while IL-22 expression declined in mRNA and protein levels as the concentration of hBD-3 increased.</p><p><b>CONCLUSIONS</b>Recombinant hBD-3 can up-regulated the expression of TLR2, IL-17A and IL-22, lower concentration of hBD-3 mainly increased the expression of IL-22 while higher concentration of hBD-3 mainly increased the expression of IL-17A. These results show that different concentrations of hBD-3 maybe activate different transcription factors which was mediated by TLR2, initiating host immune response.</p>

Signal transduction of innate immunity to virus infection / 病毒学报

The innate immune system is essential for the initial detection of invading viruses and subsequent activation of adaptive immunity. Three types pattern recognition receptors (PRRs) in innate immune cells play a pivotal role in the first line of host defense system. PRRs include Toll-like receptors (TLRs), RIG-I-like receptors(RLRs) and nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs). PRRs recognize pathogen-associated molecular patterns(PAMPs) or danger-associated molecular patterns (DAMPs) to initiate and regulate innate and adaptive immune responses. Three types PRRs have their own features in ligand recognition and cellular location. Activated PRRs deliver signals to adaptor molecules (MyD88, TRIF, IRAK, IPS-1), which act as important messengers to activate downstream kinases (IKK complex, MAPKs, TBK1, RIP-1) and transcription factors (NF-kappaB, AP-1, IRF3), which produce effected molecules including cytokines, chemokines, inflammatory enzymes, and type I interferons. This review focuses on discussing PRRs signaling pathways and achievements in this field in order to provide beneficial strategies for human life and immune diseases prevention.

Molecular and epidemiological study on among children under 5 years old in Nanjing / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To study the infected information, clinical symptom and molecular epidemiological characteristics of HuCV infection among children under 5 years old in Nanjing.</p><p><b>METHODS</b>In Nanjing Children's Hospital of Nanjing Medical University from July 2010 to June 2011, we collected 428 stool specimens from children with diarrhea and 428 asymptomatic controls. Human Calicivirus were tested by using RT-PCR. Then we sequenced the nucleic acid of PCR amplifications and identified the genotype and gene group of prevalent strains.</p><p><b>RESULTS</b>63 (14.72%) out of 428 stool samples were detected as HuCV. 58 were norovirus and 5 were sapovirus, while GII-4 2006b was the predominant strain of NoV. In the 428 control samples, 19 samples were positive for calicivirus, there were 8 NoV and 13 SaV (Including 3 co-infection cases).</p><p><b>CONCLUSION</b>Human caliciviruses with different genotypes circulated among children in Nanjing,and GII. 2006b is the dominant genotype.</p>

Study on concentration of nuorovirus genegroup II from environmental water / 病毒学报

A new viral sampling concentration device was designed which was equipped with a new cationic filter membrane-Nanoceram suitable for field sampling. Norovirus Genegroup II was detected from environmental water with the aid of this device. The effects on virus recovery of prefiltration, various second-concentration methods, and different eluants were investigated through pre-experiment. The concentration optimized process, and the optimal concentration process were then determined. The results showed that the prefiltration had a profound effect on virus recovery, and two second-concentration method: PEG-NaC1 precipitation and celite adsorption, had almost the same concentration effects. The Na2 HPO4 solution of 0.15 mol/L was selected as the final eluant to elute the adsorbed Nuorovirus from the celite. The virus recovery of Nanoceram was determined to be 3.02%. Finally, successful detection of Norovirus GII in sewage from Yangqiao River, Fengtai District, Beijing was acheived. All these data had shown that the Naneceram filter concentration method could concentrate Norovirus from environmental water with a steady effects.