Continuous cell lines are practical models that are widely used in the study of disease mechanisms and particularly cancers. However, the issue of cell line cross-contamination has existed since the 1960s, despite repeated advocation for cell line authentication by many experts. Furthermore, cell line abuse has been underestimated and underreported. The China Center for Type Culture Collection (CCTCC) received 1373 cell samples for authentication from 2010 to 2019, and has found that the quality of cell lines has improved during this time, offering a positive outlook for the future.

Foot-and-mouth disease virus (FMDV) rapidly causes cytopathic effects in susceptible cells. Incomplete viral clearance during the acute infection leads to persistent infection. The relationship between host gene expression and the persistent infection remains unclear. In this study, we analyzed the transcriptome profiles of BHK-21 cells acutely and persistently infected with FMDV to identify differences in gene expression. GO and KEGG enrichment analyses showed that the 8,378 differentially expressed genes were significantly enriched in categories including metabolism, biosynthesis, ribosome function, and endocytosis. In persistently infected BHK-21 cells, ribosome- and translation-related genes were significantly down-regulated. There were more differentially expressed immune-related genes during persistent infection than during acute infection. Two hundred and seventy-four genes were differentially expressed in both acutely and persistently infected BHK-21 cells. Among these genes, heat shock protein family B member 1 (Hspb1) knockdown significantly inhibited FMDV replication. Our research provides a basis for further research to understand the mechanisms of persistent FMDV infection including the genes involved in FMDV replication.

Human cytomegalovirus (HCMV) is a widespread virus that can establish life-long latent infection in large populations. The establishment of latent infection prevents HCMV from being cleared by host cells, and HCMV reactivation from latency can cause severe disease and death in people with immature or compromised immune systems. To establish persistent and latent infection in healthy individuals, HCMV encodes a large array of proteins that can modulate different components and pathways of host cells. It has been reported that pUL138 encoded by the UL133-UL138 polycistronic locus promotes latent infection in primary CD34+ hematopoietic progenitor cells (HPCs) infected in vitro. In this study, recombinant HCMV Han(UL138del) was constructed by deleting the UL138 locus of Han, a clinical HCMV strain. Then, a comparative quantitative proteomic analysis of Han- and Han(UL138del)-infected MRC5 cells was performed to study the effect of pUL138 on host cells in the context of HCMV infection. Our results indicated that, during the early phase of HCMV infection, the innate immune response was differentially activated, while during the late phase of HCMV infection, multiple host proteins were differentially expressed between Han- and Han(UL138del)-infected cells, and these proteins are involved in the oxidation-reduction process, ER to Golgi vesicle-mediated transport, and extracellular matrix organization. Among these proteins, STEAP3, BORCS7, FAM172A, RELL1, and WDR48 were further demonstrated to affect HCMV infection. Our study provides a systematic view of the effect of pUL138 on the host cell proteome and highlights the proposition that multiple biological processes or host factors may be involved in the overall role of the UL133-UL138 polycistronic locus in HCMV persistence.

Objectives Cryptococcus heimaeyensisS20 is found in Antarctica and can produce exopolysaccharides (CHEPS). Here, we explore the anti-tumour effects of CHEPS on non-small cell lung cancer (NSCLC). Materials and methods Cell viability was assessed by CCK8 and colony formation assays. Flow cytometry was used to analyse the cell cycle, cell apoptosis and reactive oxygen species (ROS). Cell autophagy was detected by EGFP-LC3 puncta assay, Lyso-Tracker Red staining and transmission electron microscopy. mRNA and protein levels were analysed by qRT-PCR and Western blot. Related mechanisms were confirmed using appropriate inhibitors or shRNA. In vitro results were further confirmed by a tumour xenograft study. Results CHEPS inhibited the proliferation of NSCLC cells by inducing S- and G2/M-phase arrest and autophagic cell death, but not apoptosis. CHEPS was less toxic to normal human embryonic lung fibroblasts. CHEPS activated the MAPK pathway in NSCLC cells, and p38 and ERK promoted CHEPS-induced cell death. Further studies showed that p38 and ERK promoted CHEPS-induced NSCLC cell autophagy and ERK promoted CHEPS-induced S- and G2/M-phase arrest. ROS were induced by CHEPS. A ROS scavenger attenuated CHEPS-induced p38 and ERK activation, autophagy and cell death. Finally, CHEPS reduced orthotopic lung tumour growth without organ-related toxicity. CHEPS also induced ROS, activated p38 and ERK, and triggered autophagy in vivo. Conclusions CHEPS induces autophagic cell death and S- and G2/M-phase arrest in NSCLC cells via ROS/p38 and ROS/ERK signalling.

With the rapid development of single-cell technologies, the mechanisms underlying viral infections and the interactions between hosts and viruses are starting to be explored at the single-cell level. The foot-and-mouth-disease (FMD) virus (FMDV) causes an acute and persistent infection that can result in the break-out of FMD, which can have serious effects on animal husbandry. Single-cell techniques have emerged as powerful approaches to analyze virus infection at the resolution of individual cells. In this review, the existing single-cell studies examining FMDV will be systematically summarized, and the central themes of these studies will be presented.

Hepatitis C virus (HCV) replication involves many viral and host factors. Host factor SPRY domain- and SOCS box-containing protein 2(SPSB2) belongs to SPSB family, and it recruits target proteins by the SPRY domain and forms E3 ubiquitin ligase complexes by the SOCS box. As an adaptor protein, it can regulate the host’s response to infection, but little is known about whether SPSB2 plays a role in HCV replication. In the present study, we found that HCV infection significantly upregulated the mRNA and protein levels of SPSB2 in HCVcc-infected cells. Exogenous expression of SPSB2 in hepatoma cells decreased HCV RNA and protein levels which depended on the SOCS box, while knockdown of endogenous SPSB2 increased HCV RNA and protein levels. Additionally, we demonstrated that SPSB2 interacted with HCV structural protein E1 and nonstructural protein protein 5A (NS5A) via the C-terminal portion of the SPSB2 SPRY domain. Furthermore, SPSB2 induced NS5A ubiquitination and mediated NS5A degradation. Collectively, this study discovered host factor SPSB2 significantly inhibits HCV replication by interacting and degrading NS5A.

Food-and-mouth disease virus (FMDV) is a highly contagious virus that seriously threatens the development of animal husbandry. Although persistent FMDV infection can dramatically worsen the situation, the mechanisms involved in persistent FMDV infection remain unclear. In the present study, we identified the presence of evolved cells in the persistently FMDV-infected cell line. These cells exhibited resistance to the parent FMDV and re-established persistent infection when infected with FMDV-Op (virus supernatant of persistent infection cell lines), emphasizing the decisive role of evolved host cells in the establishment of persistent FMDV infection. Using RNA-seq, we identified the gene expression profiles of these evolved host cells. In total, 4,686 genes were differentially expressed in evolved cells compared with normal cells, with these genes being involved in metabolic processes, cell cycle, and cellular protein catabolic processes. In addition, 1,229 alternative splicing events, especially skipped exon events, were induced in evolved cells. Moreover, evolved cells exhibited a stronger immune defensive response and weaker MAPK signal response than normal cells. This comprehensive transcriptome analysis of evolved host cells lays the foundation for further investigations of the molecular mechanisms of persistent FMDV infection and screening for genes resistant to FMDV infection.

The interferon-inducible dynamin-like GTPase myxovirus resistance protein A (MxA) exhibits activity against multiple viruses. However, its role in the life cycle of hepatitis C virus (HCV) is unclear, and the mechanisms underlying the anti-HCV activity of MxA require further investigation. In this study, we demonstrated that exogenous MxA expression in the Huh7 and Huh7.5.1 hepatoma cell lines significantly decreased the levels of HCV RNA and core proteins, whereas MxA knockdown exerted the opposite effect. MxA-mediated inhibition of HCV replication was found to involve the JAK-STAT pathway: STAT1 phosphorylation and the expression of IFN-stimulated genes (ISGs) such as guanylate-binding protein 1 and 2′-5′-oligoadenylate synthetase 1 were augmented by MxA overexpression and reduced by endogenous MxA silencing. Treatment with the JAK inhibitor ruxolitinib abrogated the MxA-mediated suppression of HCV replication and activation of the JAK-STAT pathway. Additionally, transfection with an MxA mutant with disrupted GTP-binding consensus motifs abrogated activation of the JAK-STAT pathway and resistance to HCV replication. This study shows that MxA inhibits HCV replication by activating the JAK-STAT signaling pathway through a mechanism involving its GTPase function.

ABSTRACT Viral infection and replication are affected by host cell heterogeneity, but the mechanisms underlying the effects remain unclear. Using single-cell analysis, we investigated the effects of host cell heterogeneity, including cell size, inclusion, and cell cycle, on foot-and-mouth disease virus (FMDV) infection (acute and persistent infections) and replication. We detected various viral genome replication levels in FMDV-infected cells. Large cells and cells with a high number of inclusions generated more viral RNA copies and viral protein and a higher proportion of infectious cells than other cells. Additionally, we found that the viral titer was 10- to 100-fold higher in cells in G2/M than those in other cell cycle phases and identified a strong correlation between cell size, inclusion, and cell cycle heterogeneity, which all affected the infection and replication of FMDV. Furthermore, we demonstrated that host cell heterogeneity influenced the adsorption of FMDV due to differences in the levels of FMDV integrin receptors expression. Collectively, these results further our understanding of the evolution of a virus in a single host cell. IMPORTANCE It is important to understand how host cell heterogeneity affects viral infection and replication. Using single-cell analysis, we found that viral genome replication levels exhibited dramatic variability in foot-and-mouth disease virus (FMDV)-infected cells. We also found a strong correlation between heterogeneity in cell size, inclusion number, and cell cycle status and that all of these characteristics affect the infection and replication of FMDV. Moreover, we found that host cell heterogeneity influenced the viral adsorption as differences in the levels of FMDV integrin receptors' expression. This study provided new ideas for the studies of correlation between FMDV infection mechanisms and host cells.

Increasing attention has been paid to the hazard of cell line cross-contamination. More journals are requiring that cell lines be authenticated prior to manuscript submission. China Center for Type Culture Collection (CCTCC) has authenticated cell lines for publications in the International Journal of Cancer since 2009. Between 2010 and 2016, we authenticated 485 cell lines from 66 Chinese universities and research institutes. Almost 50% of cell lines were misidentified, most were completely replaced by other cell lines (mainly HeLa and its sublines). The number of cell lines we received for authentication increased dramatically after 2013, but the proportion of misidentified cell lines remained elevated (over 50%). We strongly recommend that researchers take authentication of cell lines seriously and routinely perform authentication of the cell lines used in their laboratories. This article is protected by copyright. All rights reserved.

In recent years, biological research involving human cell lines has been rapidly developing in China. However, some of the cell lines are not authenticated before use. Therefore, misidentified and/or cross-contaminated cell lines are unfortunately commonplace. In this study, we present a comprehensive investigation of cross-contamination and misidentification for a panel of 278 cell lines from 28 institutes in China by using short tandem repeat profiling method. By comparing the DNA profiles with the cell bank databases of ATCC and DSMZ, a total of 46.0% (128/278) cases with cross-contamination/misidentification were uncovered coming from 22 institutes. Notably, 73.2% (52 out of 71) of the cell lines established by the Chinese researchers were misidentified and accounted for 40.6% of total misidentification (52/128). Further, 67.3% (35/52) of the misidentified cell lines established in laboratories of China were HeLa cells or a possible hybrid of HeLa with another kind of cell line. Furthermore, the bile duct cancer cell line HCCC-9810 and degenerative lung cancer Calu-6 exhibited 88.9% match in the ATCC database (9-loci), indicating that they were from the same origin. However, when we used 21-loci to compare these two cell lines with the same algorithm, the percent match was only 48.2%, indicating that these two cell lines were different. The SNP profiles of HCCC-9810 and Calu-6 also revealed that they were different cell lines. 150 cell lines with unique profiles demonstrated a wide range of in vitro phenotypes. This panel of 150 genomically validated cancer cell lines represents a valuable resource for the cancer research community and will advance our understanding of the disease by providing a standard reference for cell lines that can be used for biological as well as preclinical studies.

Foot-and-mouth disease virus (FMDV) infects host cells in either an acute or persistent manner. In this study, we examined the relevance of the establishment of FMDV persistence to the expression of the emopamil-binding protein (EBP) gene in 231 individual persistently infected baby hamster kidney (BHK-21) cells after passages 28, 38, and 68 (PI28, PI38, and PI68). At PI28, the stage at which persistent infection of FDMV becomes unstable, the percentage of cells carrying FMDV was 66.7%, while 80.2% of cells were EBP positive. Additionally, in 55.6% of the EBP-positive cells at PI28, EBP expression was upregulated approximately 149.9% compared to uninfected BHK-21 cells. This was the highest expression level among all cell passages measured. Interestingly, in a parallel experiment, the average EBP expression level in the whole cell population at PI28 was only slightly higher (108.2%) than that in uninfected BHK-21 cells. At PI38, 98.7% of the cells were positive for FMDV 3D (an RNA-dependent RNA polymerase enzyme gene), and its maximum expression level observed at this passage. The expression level of EBP in 78.2% of the total cells, however, was reduced significantly. At PI68, 95.8% of the cells were 3D positive, and the expression of both the EBP and 3D genes were at the lowest levels of all the passages. Our studies using single cells yielded data that are otherwise inaccessible a using whole cell population. These results suggest that the establishment of persistent infection by FMDV is a dynamic process that results from the continuous adaptation and coevolution of viruses and cells to reach an equilibrium.

Increasing attention has been paid to the hazard of cell line cross-contamination. More journals are requiring that cell lines be authenticated prior to manuscript submission. China Center for Type Culture Collection (CCTCC) has authenticated cell lines for publications in the International Journal of Cancer since 2009. Between 2010 and 2016, we authenticated 485 cell lines from 66 Chinese universities and research institutes. Almost 50% of cell lines were misidentified, most were completely replaced by other cell lines (mainly HeLa and its sublines). The number of cell lines we received for authentication increased dramatically after 2013, but the proportion of misidentified cell lines remained elevated (over 50%). We strongly recommend that researchers take authentication of cell lines seriously and routinely perform authentication of the cell lines used in their laboratories. This article is protected by copyright. All rights reserved.

Increasing attention has been paid to the hazard of cell line cross-contamination. More journals are requiring that cell lines be authenticated prior to manuscript submission. China Center for Type Culture Collection (CCTCC) has authenticated cell lines for publications in the International Journal of Cancer since 2009. Between 2010 and 2016, we authenticated 485 cell lines from 66 Chinese universities and research institutes. Almost 50% of cell lines were misidentified, most were completely replaced by other cell lines (mainly HeLa and its sublines). The number of cell lines we received for authentication increased dramatically after 2013, but the proportion of misidentified cell lines remained elevated (over 50%). We strongly recommend that researchers take authentication of cell lines seriously and routinely perform authentication of the cell lines used in their laboratories. This article is protected by copyright. All rights reserved.

An external guide sequence (EGS) is a RNA sequence which can interact with a target mRNA to form a tertiary structure like a pre-tRNA and recruit intracellular ribonuclease P (RNase P), a tRNA processing enzyme, to degrade target mRNA. Previously, an in vitro selection procedure has been used by us to engineer new EGSs that are more robust in inducing human RNase P to cleave their targeted mRNAs. In this study, we constructed EGSs from a variant to target the mRNA encoding herpes simplex virus 1 (HSV-1) major transcription regulator ICP4, which is essential for the expression of viral early and late genes and viral growth. The EGS variant induced human RNase P cleavage of ICP4 mRNA sequence 60 times better than the EGS generated from a natural pre-tRNA. A decrease of about 97% and 75% in the level of ICP4 gene expression and an inhibition of about 7,000- and 500-fold in viral growth were observed in HSV infected cells expressing the variant and the pre-tRNA-derived EGS, respectively. This study shows that engineered EGSs can inhibit HSV-1 gene expression and viral growth. Furthermore, these results demonstrate the potential for engineered EGS RNAs to be developed and used as anti-HSV therapeutics.

A pale pink, Gram-reaction-negative, non-motile, aerobic bacterium, designated MC 3624(T), was isolated from a tundra soil near Ny-angstrom lesund, Svalbard Archipelago, Norway (78 degrees N). Growth occurred at 10-37 degrees C (optimum 25-30 degrees C) and at pH 6.0-9.0 (optimum pH 8.0). The predominant fatty acids were C-16 : (0) (17.7 %), C-18 (:) (1)omega 7c 11-methyl (13.4 %), summed feature3 (C-16:1 omega 7c and/or C-16 (:) (1)omega 6c) (10.1 %) and summed feature 8 (C-18 (:) (1)omega 6c and/or C-18 (:) (1)omega 7c) (38.3 %). The major respiratory quinone was ubiquinone-10, and the main polar lipids were phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylcholine and an unidentified aminolipids. The DNA G+C content was 68.9 mol%. Carotenoids of the spirilloxanthin series were produced. The nearest neighbour to the novel strain was Roseomonas wooponensis WW53(T) (94.36% 16S rRNA gene sequence similarity). On the basis of phenotypic, chemotaxonomic and phylogenetic data, strain MC 3624(T) represents a novel species of the genus Roseomonas, for which the name Roseomonas arcticisoli sp. nov. is proposed. The type strain is MC 3624(T) (=CCTCC AB 2014278(T)=LMG 28637(T)).

Cell lines are widely used as in vitro model systems in biologic and medical research. However, much of the research has been invalidated by the unwitting use of false cell lines. A significant proportion of the research involving human cell lines was initiated in China. Paradoxically, the cell lines used in China have never been authenticated. Here, we present a comprehensive survey of cross-contamination in 380 samples from 113 independent sources in China using short tandem repeat profiling methods. High levels of cross-contamination were uncovered (95 of 380, 25%). Notable false cell lines (e.g., KB and WISH) are still actively used under their false identity and tissue attributions. Most strikingly, 85.51% of lines established in China were misidentified (59 of 69) and accounted for over half of the misidentifications (59 of 95, 62.11%). Further, 93.22% of the contaminants in cell lines established in laboratories of China were HeLa cells or a possible hybrid of HeLa with an unknown cell line. Results from these misidentified lines have been published in thousands of potentially erroneous articles and may have distorted the findings visible to the scientific community. False lines have been used in drug screening, potentially leading to unusable or even harmful therapeutic strategies. We also noted the causes of contamination and provided suggestions for remediation.

A yellow, Gram-reaction-negative, non-motile, aerobic bacterium, designated D07T, was isolated from a tundra soil near Ny-Ålesund, Svalbard archipelago, Norway (78° N). Growth occurred at 4-37 °C (optimum 28-30 °C) and at pH 6.0-9.0 (optimum pH 7.0-8.0). The strain produced flexirubin-type pigments. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain D07T belonged to the genus Chryseobacterium in the family Flavobacteriaceae. The 16S rRNA gene sequence of this strain showed 93.83 and 93.31 % sequence similarity, respectively, to those of Chryseobacterium contaminans C26T and Chryseobacterium taklimakanense X-65T. Strain D07T contained anteiso-C15 : 0 (25.91 %), iso-C15 : 0 (16.05 %), iso-C16 : 0 3-OH (9.64 %), iso-C16 : 0 (9.42 %) and iso-C14 : 0 (7.36 %) as the predominant cellular fatty acids, MK-6 as the major respiratory quinone and phosphatidylethanolamine, five unknown aminolipids and three unknown lipids as the main polar lipids. The DNA G+C content was 49.3 mol%. On the basis of phenotypic, chemotaxonomic and phylogenetic data, strain D07T is considered to represent a novel species of the genus Chryseobacterium, for which the name Chryseobacterium frigidum sp. nov. is proposed. The type strain is D07T ( = CCTCC AB 2011160T = KCTC 42897T). Emended descriptions of Chryseobacterium bernardetii and Chryseobacterium taklimakanense are also provided.

Human diploid cell strains (HDCSs), possessing identical chromosome sets known to be free of all known adventitious agents, are of great use in developing human vaccines. However it is extremely difficult to obtain qualified HDCSs that can satisfy the requirements for the mass production of vaccines. We have developed a new HDCS, Walvax-2, which we derived from the lung tissue of a 3-month-old fetus. We established primary, master and working cell banks successfully from reconstituted frozen cells. Observations during the concurrent propagation of Walvax-2 and MRC-5 cells revealed differences in terms of growth rate, cell viability and viral sensitivities. Specifically, Walvax-2 cells replicated more rapidly than MRC-5 cells, with Walvax-2 cells attaining the same degree of confluence in 48 hours as was reached by MRC-5 cells in 72 hours. Moreover, Walvax-2 cells attained 58 passages of cell doublings whereas MRC-5 reached 48 passages during this period. We also assessed the susceptibility of these cells to rabies, hepatitis A, and Varicella viruses. Analysis of virus titers showed the Walvax-2 cells to be equal or superior to MRC-5 cells for cultivating these viruses. Furthermore, in order to characterize the Walvax-2 cell banks, a series of tests including cell identification, chromosomal characterization, tumorigenicity, as well as tests for the presence of microbial agents, exogenous viruses, and retroviruses, were conducted according to standard international protocols. In conclusion, results from this study show that Walvax-2 cell banks are a promising cell substrate and could potentially be used for the manufacturing of HDCVs.

ABSTRACT Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) is essential for HCV genome replication and virion production and is involved in the regulation of multiple host signaling pathways. As a proline-rich protein, NS5A is capable of interacting with various host proteins containing Src homology 3 (SH3) domains. Previous studies have suggested that vinexin, a member of the sorbin homology (SoHo) adaptor family, might be a potential binding partner of NS5A by yeast two-hybrid screening. However, firm evidence for this interaction is lacking, and the significance of vinexin in the HCV life cycle remains unclear. In this study, we demonstrated that endogenously and exogenously expressed vinexin β coimmunoprecipitated with NS5A derived from different HCV genotypes. Two residues, tryptophan (W307) and tyrosine (Y325), in the third SH3 domain of vinexin β and conserved Pro-X-X-Pro-X-Arg motifs at the C terminus of NS5A were indispensable for the vinexin-NS5A interaction. Furthermore, downregulation of endogenous vinexin β significantly suppressed NS5A hyperphosphorylation and decreased HCV replication, which could be rescued by expressing a vinexin β short hairpin RNA-resistant mutant. We also found that vinexin β modulated the hyperphosphorylation of NS5A in a casein kinase 1α-dependent on manner. Taken together, our findings suggest that vinexin β modulates NS5A phosphorylation via its interaction with NS5A, thereby regulating HCV replication, implicating vinexin β in the viral life cycle. IMPORTANCE Hepatitis C virus (HCV) nonstructural protein NS5A is a phosphoprotein, and its phosphorylation states are usually modulated by host kinases and other viral nonstructural elements. Additionally, cellular factors containing Src homology 3 (SH3) domains have been reported to interact with proline-rich regions of NS5A. However, it is unclear whether there are any relationships between NS5A phosphorylation and the NS5A-SH3 interaction, and little is known about the significance of this interaction in the HCV life cycle. In this work, we demonstrate that vinexin β modulates NS5A hyperphosphorylation through the NS5A-vinexin β interaction. Hyperphosphorylated NS5A induced by vinexin β is casein kinase 1α dependent and is also crucial for HCV propagation. Overall, our findings not only elucidate the relationships between NS5A phosphorylation and the NS5A-SH3 interaction but also shed new mechanistic insight on Flaviviridae NS5A (NS5) phosphorylation. We believe that our results may afford the potential to offer an antiviral therapeutic strategy.