KMB17 Cells Research Articles

Characterization of coxsackievirus A10 strains isolated from children with hand, foot, and mouth disease.

Hand, foot, and mouth disease (HFMD) is a contagious disease that threatens the health of children under 5 years of age. Coxsackievirus A10 (CV-A10) is one of the main pathogens of HFMD. Currently, preventive vaccines and specific therapeutic drugs are not available for CV-A10. In this study, a total of 327 stool specimens were collected from pediatric patients from 2009 to 2017 during HFMD surveillance, among which 14 CV-A10 strains could only be isolated from rhabdomyosarcoma cells, but not from KMB17 and Vero cells. Through adaptive culture, 2 and 11 CV-A10 strains were recovered from Vero and KMB17 cell cultures, respectively. The growth of CV-A10 strains in Vero cells was better than that in KMB17 cells. The 14 CV-A10 strains belonged to the F genotype, and the nucleotides and amino acids of their complete genomes shared 92.6%-96.3% and 98.4%-98.9% identities, respectively. The different CV-A10 strains exhibited varying virulence in vivo, but had similar effects on tissue injury, with the hind limb muscles, kidneys, and lungs being severely affected. Additionally, the hind limb muscles had the highest viral loads. CV-A10 was found to exhibit a strong tropism to muscle tissue. The results of this study are critical to developing vaccines against CV-A10 infections.

A comparative study on biological characteristics of ten coxsackievirus A10 virus strains

In this study, we analyzed ten CVA10 strains were genotyped and cultured for 10 generations to detect plaque morphology, pathogenicity, growth and other characteristics. Mice were injected with live and inactivated virus to detect neutralizing antibody titers. The results suggested that all CVA10 strains fell into Genotype C. Each strain cultured on KMB17 and Vero cells, increased from 1st generation onwards to peak in the 3rd and 4th, and the titer at which each became infectious ranged from 5.0 to 6.5 and 6.0 to 7.0 lgCCID50/ml, respectively. Two-day-old BALB/c mice were selected and inoculated intracerebral with the CVA10 strains, Limb paralysis was significant as early as 3 d; paralysis of all limbs for 50% of affected mice. LT50 was approximately 6 d, all died within 8 d. Ten strains induced good immune response, the GMT value of booster immunizations was higher than that of initial immunization. This provide reference points for selecting CVA10 vaccine candidates.

Biological characteristics of coxsackievirus A6 clinical isolates

Objective To analyze the biological characteristics of clinical isolates of coxsackievirus A6 (CVA6), a pathogen of hand, foot and mouth disease (HFMD), and to provide reference for vaccine development. Methods CVA6 strains were isolated from 21 stool and throat swab specimens of patients with HFMD in Yunnan Province and then identified. Their growth characteristics, plaque morphology and virulence to suckling mice were analyzed. Results Five CVA6 strains, named CVA6-129, CVA6-113, CVA6-57, CVA6-94 and CVA6-162, were isolated and all belonged to D3 subtype. Only the CVA6-129 strain could proliferate rapidly in Vero and KMB17 cells and the proliferation peaked 30 h after inoculation. The infectious titer of the CVA6-129 strain was 7.54 lgCCID50 (50% cell culture infective dose)/ml in KMB17 cells. Different morphologies of plaques were formed by the CVA6-129 strain in Vero and KMB17 cells at the same time points, which were small and round with clear edges in Vero cells, and large and irregular with blurry edges in KMB17 cells. Suckling mice were susceptible to CVA6 via intramuscular and intraperitoneal injection. The most common symptoms in infected suckling mice were reduced mobility, hind limb paralysis and quadriplegia. CVA6 infection could result in death in severe cases. Conclusions This study isolated five CVA6 strains from a number of clinical samples of suspected HFMD cases, of which the CVA6-129 strain showed potential as a vaccine candidate. Key words: Coxsackievirus A6 (CVA6); Hand, foot and mouth disease (HFMD); Vaccine; Biological characteristics

Expression characteristics of signaling molecules associated with innate immune response induced by HSV1 and HSV2 in respiratory and vaginal epithelial cells

Objective To investigate the influences of herpes simplex virus 1 and 2(HSV1 and HSV2) infection on the expression of signaling molecules associated with innate immune response in respiratory and vaginal epithelial cells for bettering understanding of HSV infection and pathological characteristics in the primary infection site, namely mucosal epithelial tissues. Methods KMB17 and VK2 cells were infected with HSV. Changes in cell morphology and inner structure after HSV infection were observed under optical microscope and scanning electron microscope, respectively. Viral proliferation in KMB17 and VK2 cells was detected by plaque assay, microcytopathic assay and real-time quantitative PCR. Expression of signaling molecules associated with innate immune response in virus-infected KMB17 and VK2 cells were analyzed by real-time quantitative PCR. Results Both HSV1 and HSV2 could infect KMB17 and VK2 cells, and cause damage to cell morphology and inner structure after 12 hours. Both of the two viruses formed similar plaque on the single layer of KMB17 and VK2 cells, although HSV2 proliferated slower than HSV1. There were differences in the expression of signaling molecules associated with innate immune response induced by the two viruses in KMB17 and VK2 cells. Conclusion Both HSV1 and HSV2 could infect and proliferate in epithelial cells (KMB17 and VK2 cells). Although there were slight differences in viral proliferation between them, significant differences in the expression of signaling molecules associated with innate immune response induced by the two viruses were observed. Key words: Human herpes simplex virus; Epithelial cell; Signaling molecules associated with innate immune response

Identification and validation of a novel microRNA-like molecule derived from a cytoplasmic RNA virus antigenome by bioinformatics and experimental approaches.

BackgroundIt is generally believed that RNA virus replicating in the cell cytoplasm would not encode microRNAs (miRNAs) due to nucleus inaccessibility. Recent studies have described cytoplasmic RNA virus genome-derived miRNAs in West Nile virus (WNV) and Dengue virus (DENV). However, naturally occurring miRNAs derived from the antigenome of a cytoplasmic RNA virus have not been described.MethodsHepatitis A virus (HAV) was served as a model virus to investigate whether the antigenome of a cytoplasmic RNA virus would be processed into miRNAs or miRNA-like small RNAs upon infection. HAV antigenome was queried for putative miRNA precursors (pre-miRNA) with the VMir analyzer program. Mature miRNA prediction was performed using MatureBayes and Bayes-SVM-MiRNA web server v1.0. Finally, multiple experimental approaches, including cloning and sequencing-, RNAi-, plasmid-based miRNA expression- and luciferase reporter assays, were performed to identify and validate naturally occurring viral antigenome-derived miRNAs.ResultsUsing human HAV genotype IA (isolate H2) (HAVH2), a virally encoded miRNA-like small RNA was detected on the antigenome and named hav-miR-N1-3p. Transcription of viral pre-miRNA in KMB17 and HEK293T cells led to mature hav-miR-N1-3p production. In addition, silencing of the miRNA-processing enzyme Dicer or Drosha caused a dramatic reduction in miRNA levels. Furthermore, artificial target of hav-miR-N1-3p was silenced by synthesized viral miRNA mimics and the HAVH2 naturally-derived hav-miR-N1-3p.ConclusionThese results suggested that the antigenome of a cytoplasmic RNA virus could be processed into functional miRNAs. Our findings provide new evidence supporting the hypothesis that cytoplasmic RNA viruses naturally encode miRNAs through cellular miRNA processing machinery.

Transcriptional regulation by HSV-1 induced HTRP via acetylation system

The protein HTRP (human transcription regulator protein) is encoded by the differential gene htrp and induced by Herpes simplex virus type 1 (HSV-1) infection in KMB-17 cells. HTRP was found to interact with SAP30 (mSin3A Association Protein), one of the components of co-repressor complex mSin3A, which is part of the deacetylation transfer enzyme HDAC. To reveal the biological significance of the interaction between HTRP and SAP30, real- time PCR and a dual-luciferase detecting system was used. The results indicate that HTRP could inhibit the transcription of a viral promoter, whose interaction with SAP30 synergistically affects transcriptional inhibition of the viral genes, and is related to HDAC enzyme activity. ChIP experiments demonstrate that HTRP could promote HDAC activity by increasing the deacetylation level of lysine 14 and lysine 9 in histone H3.

The repairing effect of a recombinant human connective‐tissue growth factor in a burn‐wounded rhesus‐monkey (Macaca mulatta) model

CTGF (connective-tissue growth factor) has been characterized as an extracellular-matrix-associated protein that modulates basic-fibroblast-growth-factor signalling and angiogenesis. In the present paper, the cloning of the ctgf gene from human umbilical-vein endothelial cells and expression of the protein in Escherichia coli as an N-terminal hexahistidine fusion protein is described. Recombinant human CTGF (rhCTGF) was expressed and purified so that we could investigate its effect on the proliferation of human embryo fibroblast KMB-17 and NIH3T3 cells. The results indicated not only that the protein was properly folded, but also that it had the same specific activity and stability as the native protein. Furthermore, we administered this recombinant protein in a non-human primate [rhesus monkey (Macaca mulatta)] burn-wound model and report the clinical findings and structural effects. Epitheliotrophic effects were conspicuous in wounded tissues at 10-100 ng of CTGF/cm(2), suggesting that administered rhCTGF can play a normal physiological role in wound repairing in a non-human primate model.

Molecular evolution of hepatitis A virus in a human diploid cell line

To investigate the hotspots, direction, and the time course of evolution of hepatitis A virus in the process of consecutive cell culture passage in human KMB17 diploid cells. Wild type hepatitis A virus H2w was serially propagated in KMB17 cells until passage 30, and the full-length genomes of H2w and its six chosen progenies were determined by directly sequencing RT-PCR products amplified from viral genomic RNA. Alignment comparison of sequences from H2w with its six progenies and phylogenetic analysis of the whole VP1 region from H2w, progenies of H2w, and other cell culture adapted hepatitis A virus were then carried out to obtain data on the molecular evolution of hepatitis A virus in the process of consecutive passage in KMB17 cells. Most of the mutations occurred by passage 5 and several hotspots related to adaptation of the virus during cell growth were observed. After that stage, few additional mutations occurred through the remaining duration of passage in KMB17 cells except for mutation in the virulence determinants, which occurred in the vicinity of passage 15. The phylogenetic analysis of the whole VP1 region suggested that the progenies of H2w evolved closely to other cell culture adapted hepatitis A virus, i.e. MBB, L-A-1, other than its progenitor H2w. Hepatitis A virus served as a useful model for studying molecular evolution of viruses in a given environment. The information obtained in this study may provide assistance in cultivating the next generation of a seed virus for live hepatitis A vaccine production.

Mutational characteristics in consecutive passage of rapidly replicating variants of hepatitis A virus strain H2 during cell culture adaptation.

To investigate the molecular mechanism of cell adaptation and rapid replication of hepatitis A virus strain H2 in KBM17 cells. Virus of strain H2 at passage 7 was consecutively passaged in KBM17 cells for 22 passages, every passage was incubated for 14 days. Antigenic and infectious titers of every passage and one-step growth dynamics of passage 22 were determined with ELISA. Genomes of passage 6, passage 12, passage 18 and passage 22 were sequenced and compared with H2K7. During continuous passage of vaccine strain H2 at passage K7 in KMB17 cells, infectious and antigenic titers increased with the increase of passages, infectious titers at day 14 reached 6.77LgCCID(50)ml(-1) for passage 6 (P6), 7.0 LgCCID(50)ml(-1) for passage 12 (P12), 7.33 LgCCID(50)ml(-1) for passage 18 (P18) and 7.83 LgCCID(50)ml(-1) for passage 22 (P22), respectively. The one-step growth dynamics showed that replicating peak of P22 appeared at day 14 with infectious titers of 7.83 LgCCID(50)ml(-1) and antigenic titer of 1:1024. After passage 22 a new cell-adapted variant (P22) of H2K7 with rapid and shortened replication cycle from 28 days to 14 days was obtained. Sequencing and comparisons of genomes of P6, P12, P18 and P22 showed that mutational numbers in genomes of different passages increased with adaptive passages, and mutations scattered over the genome. In comparison with that of K7, P6 had only 6 nucleotides (nt) mutations, P12 had 7 mutational changes, in addition to 6 same mutations with P6, there appeared a new mutation in 5'NTR at nucleotide position 591 resulting in a nucleotide exchange from A to G. P18 had 10 nt mutations, among the 10 mutations, 7 mutational changes were same as with P12, three new mutational changes appeared in the genome, one in 5'NTR, one in 3C coding region, one in 3D coding region, at P22 there appeared 18 nucleotide changes in the genome, on the basis of P18,there occurred additional 8 nucleotide mutations, two in 5'NTR, three in 2C, one in 3A, one in 3C and one in 3D. The results suggested that although H2K7 was already an attenuated strain, the mutations of genome is not sufficient to completely adapt the KMB17, further mutations caused rapid replication adaptation. 18-nt changes scattering over the genome are cooperatively responsible for further adaptation characterized by rapid and shortened replication cycle from 28 days to 14 days in KMB17 cells. The mutations in 2C coding region play more important role in increase of infectious titer than other mutations, the mutations in 2B coding region show less important role than it usually does in cell adaptation, nucleotide changes in 5' NTR seem to be not relevant to cell adaptation during initial stages (before P6), but do in late stages.

Primary Study of Attenuated Live Hepatitis A Vaccine (H2 Strain) in Humans

Strain H2, an attenuated live hepatitis A virus (HAV), was derived from the fecal specimen of a patient with hepatitis A in Hangzhou, China. After isolation and passage in a culture of newborn monkey kidney cells, adaptation to grow in human lung diploid cells (KMB17), and serial passage at a low temperature (32 degrees C) in KMB17 cells, this strain became the master seed virus for H2-strain vaccine. Twelve human volunteers received the experimental vaccine subcutaneously and were closely observed for 20 w. None of the subjects developed any local or systemic reactions, and there were no elevations of serum glutamic-pyruvic transaminase, type 5 isoenzyme of lactate dehydrogenase, or isocitrate dehydrogenase. Seroconversion occurred in all subjects at a mean time of 3 w after inoculation. ELISA competitive test for titer of antibody to HAV showed values ranging from 1:2 to 1:8 with a geometric mean titer of 1:3.48 at 20 w after inoculation. No marked decrease in titer of HAV antibody was found in the subjects tested at 1 y. These antibodies were proved to be neutralizing antibodies.