Cultured Monkey Cells Research Articles

Ex vivo and in vivo suppression of SARS-CoV-2 with combinatorial AAV/RNAi expression vectors

Despite rapid development and deployment of vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), clinically relevant modalities to curb the pandemic by directly attacking the virus on a genetic level remain highly desirable and are urgently needed. Here we comprehensively illustrate the capacity of adeno-associated virus (AAV) vectors co-expressing a cocktail of three short hairpin RNAs (shRNAs; RNAi triggers) directed against the SARS-CoV-2 RdRp and N genes as versatile and effective antiviral agents. In cultured monkey cells and human gut organoids, our most potent vector, SAVIOR (SARS virus repressor), suppressed SARS-CoV-2 infection to background levels. Strikingly, in control experiments using single shRNAs, multiple SARS-CoV-2 escape mutants quickly emerged from infected cells within 24–48 h. Importantly, such adverse viral adaptation was fully prevented with the triple-shRNA AAV vector even during long-term cultivation. In addition, AAV-SAVIOR efficiently purged SARS-CoV-2 in a new model of chronically infected human intestinal cells. Finally, intranasal AAV-SAVIOR delivery using an AAV9 capsid moderately diminished viral loads and/or alleviated disease symptoms in hACE2-transgenic or wild-type mice infected with human or mouse SARS-CoV-2 strains, respectively. Our combinatorial and customizable AAV/RNAi vector complements ongoing global efforts to control the coronavirus disease 2019 (COVID-19) pandemic and holds great potential for clinical translation as an original and flexible preventive or therapeutic antiviral measure.

Sequence amplification via cell passaging creates spurious signals of positive adaptation in influenza virus H3N2 hemagglutinin.

Clinical influenza A virus isolates are frequently not sequenced directly. Instead, a majority of these isolates (∼70% in 2015) are first subjected to passaging for amplification, most commonly in non-human cell culture. Here, we find that this passaging leaves distinct signals of adaptation, which can confound evolutionary analyses of the viral sequences. We find distinct patterns of adaptation to Madin–Darby (MDCK) and monkey cell culture absent from unpassaged hemagglutinin sequences. These patterns also dominate pooled datasets not separated by passaging type, and they increase in proportion to the number of passages performed. By contrast, MDCK–SIAT1 passaged sequences seem mostly (but not entirely) free of passaging adaptations. Contrary to previous studies, we find that using only internal branches of influenza virus phylogenetic trees is insufficient to correct for passaging artifacts. These artifacts can only be safely avoided by excluding passaged sequences entirely from subsequent analysis. We conclude that future influenza virus evolutionary analyses should appropriately control for potentially confounding effects of passaging adaptations.

Segmentation of Microscopic Images of Living Cells: A Study

In this paper, three different segmentation techniques are used, namely, region growing segmentation technique, watershed segmentation and texture segmentation, to accurately extract the shapes of membranes and nuclei from the inverted microscopic images, taken throughout the monolayer formation of BGM-70 (Baby Grevit Monkey) cell culture. This strategy is a prerequisite for an accurate quantitative analysis of cell shape and morphodynamics during organogenesis and is the basis for an integrated understanding of biological processes. The segmentation of cellular structures is achieved by first normalizing the microscopic images by performing CLAHE operation followed by de-noising by morphological opening and filling of the holes and then applying segmentation algorithm for cell shape reconstruction.

Raf/MEK/ERK pathway activation is required for Junín virus replication

In the present work we investigated the importance of the Raf/MEK/ERK signalling pathway in the multiplication of the arenavirus Junín (JUNV) in monkey and human cell cultures. We established that JUNV induces a biphasic activation of ERK and we proved that a specific inhibitor of the ERK pathway, U0126, impairs viral replication. Furthermore, U0126 exerted inhibitory action against the arenaviruses Tacaribe and Pichinde. Moreover, treatment with known ERK activators such as phorbol 12-myristate 13-acetate and serum increased viral yields whereas ERK silencing by small interfering RNAs caused the inhibition of viral multiplication. Therefore, activation of the Raf/MEK/ERK signalling pathway is required to ensure efficient JUNV replication and may constitute a host target for the development of novel effective therapeutic strategies to deal with arenavirus infections.

GeXPS multiplex PCR analysis of the simian varicella virus transcriptome in productively infected cells in culture and acutely infected ganglia

Simian varicella zoster virus (SVV) infection of non-human primates serves as a model to study varicella zoster virus (VZV) infection and pathogenesis in humans. While macroarray analysis detected all 69 predicted unique open reading frames (ORFs) in SVV-infected cells in culture, it lacked the sensitivity to detect the low-abundance transcripts expressed in latently infected monkey ganglia. Recently, a multiplex RT-PCR assay using the GenomeLab Genetic Analysis System (GeXPS) identified 10 VZV transcripts in latently-infected human ganglia. GeXPS was used to analyze the SVV transcriptome in SVV-infected monkey cells in culture as well as in acutely infected ganglia from African green monkeys. Oligonucleotide primers containing both SVV ORF- and cell-specific sequences linked to universal DNA sequences were used in RT-PCR to produce products of predetermined sizes. Amplification products were resolved by capillary gel electrophoresis and detected by fluorescence spectrophotometry. Transcripts corresponding to all 69 predicted SVV ORFs, in addition to transcripts within the leftward end region and ORF 61 antisense transcripts were detected in virus-infected cells in culture. Except for two transcripts (ORFs 14 and 35), all transcripts found in infected tissue culture cells were also found in acutely infected monkey ganglia.

Varicella-zoster virus–induced apoptosis in MeWo cells is accompanied by down-regulation of Bcl-2 expression

Varicella-zoster virus infects multiple human and monkey cells in culture. The mode of cell death appears to be autophagy or apoptosis. Analysis of VZV-infected human melanoma (MeWo) cells revealed that Bcl-2 mRNA and protein levels were decreased significantly 64 and 72 hpi (hours post infection), accompanied by the release of cytochrome c from mitochondria into the cytoplasm. Western blot analysis of virus-infected cells revealed activation of caspase-8, a marker for the extrinsic pathway of apoptosis, and caspase-9, a marker for the intrinsic pathway of apoptosis at 64 and 72 hpi. Significant increases in the levels of cleaved caspase-3 and cleaved poly (ADP) ribose polymerase (PARP) were also seen at the height of cytopathic effect. Thus VZV induces apoptosis in MeWo cells in which Bcl-2 is down-regulated. Future studies will determine differences in the cascade of apoptotic events in non-neuronal cells compared to neurons that allow VZV to become latent.

Transformation of Anaplasma phagocytophilum

BackgroundTick-borne pathogens cause emerging zoonoses, and include fastidious organisms such as Anaplasma phagocytophilum. Because of their obligate intracellular nature, methods for mutagenesis and transformation have not been available.ResultsTo facilitate genetic manipulation, we transformed A. phagocytophilum (Ap) to express a green fluorescent protein (GFP) with the Himar1 transposase system and selection with the clinically irrelevant antibiotic spectinomycin.ConclusionThese transformed bacteria (GFP/Ap) grow at normal rates and are brightly fluorescent in human, monkey, and tick cell culture. Molecular characterization of the GFP/Ap genomic DNA confirmed transposition and the flanking genomic insertion locations were sequenced. Three mice inoculated with GFP/Ap by intraperitoneal injection became infected as demonstrated by the appearance of morulae in a peripheral blood neutrophil and re-isolation of the bacteria in culture.

Construction and in vitro characterization of a chimeric simian and human immunodeficiency virus with the RANTES gene

Chimeric simian–human immunodeficiency virus (SHIV) containing the env gene of HIV-1 infects macaque monkeys and provides basic information that is useful for the development of HIV-1 vaccines. Regulated-on-activation-normal-T-cell-expressed-and-secreted (RANTES), a CC-chemokine, enhances antigen-specific T helper type-1 responses against HIV-1. With the final goal of testing the adjuvant effects of RANTES in SHIV-macaque models, we constructed a SHIV having the RANTES gene (SHIV-RANTES) and characterized its properties in vitro. SHIV-RANTES replicated both in human and monkey T cell lines. Along with SHIV-RANTES replication, RANTES was detected in the supernatant of human and monkey cell cultures, at maximal levels of 98.5 and 4.1 ng/ml, respectively. A flow cytometric analysis showed that the expressed RANTES down-modulated CC-chemokine receptor 5 (CCR5) on PM1 cells, which was restored by adding anti-RANTES antibody. UV-irradiated culture supernatants from the SHIV-RANTES-infected cells suppressed replication of CCR5-tropic HIV-1 BaL in PM-1 cells. Differentiating real-time RT-PCR showed that pre-infection of SHIV-RANTES in C8166 cells expressing CCR5 suppressed the replication of HIV-1 BaL. Biological activity of the expressed RANTES and the inserted RANTES gene in SHIV-RANTES remained stable after 10 passages. These results suggest that SHIV-RANTES is worth testing in macaque models.

Quantitative detection of enteroviruses in activated sludge by cell culture and real-time RT-PCR using paramagnetic capturing

We have compared in extracts of activated sludge the number of enteroviruses detectable with buffalo green monkey (BGM) cell-cultures versus the number of enteroviral genomes determined by reverse-transcription quantitative real-time PCR (RT-qPCR). In order to find conditions adequate for quantifying enteroviral RNA isolated from (waste)water we have investigated affinity capture of RNA with polystyrene beads (Dynabeads). The capture efficiency strongly depended on the genomic region chosen for the affinity binding. Capture of the RNA by its 3'-tail was most efficient (almost 100%); other regions within the genome yielded variable but lower results. Indirect capture (first hybridization of the RNA to the oligonucleotides, then attachment of the duplex molecules to the beads) was much more efficient than direct capture (attachment of the oligonucleotides to the beads first, then binding of the RNA), and resulted in RNA capture of maximally 60-80%. At least partly, this was due to incomplete hybridization of the RNA to the complementary oligonucleotides. No correlation was found between the number of cytopathic effects (CPE) determined by cell culture and the number of genomes quantified by RT-qPCR; RT-qPCR values were consistently much higher than the number of CPE. This points to overestimation of infectious enteroviruses by RT-qPCR and/or underestimation by the cell culture approach.

Nuclear Localization Signals in p170, the Large Subunit of Translation Initiation Factor 3

Apart from their role in translation, eukaryotic translation factors or their individual subunits may perform other functions, in particular, regulating nuclear processes. Primary structure analysis revealed four potential nuclear localization signals (NLS) in the human eIF3 large subunit, p170. NLS were tested for ability to direct p170 into the nucleus. For this purpose, cDNAs coding for p170 fragments fused with the green fluorescent protein were expressed in CV-1 and Cos-1 cultured monkey cells. The location of the expression product was studied by fluorescence microscopy. At least two of the four putative bipartite NLS proved to direct the corresponding p170 fragments into the nucleus. Larger p170 fragments with the same NLS were retained in the cytoplasm. It was assumed that, with the help of some specific factors or after limited proteolysis, p170 enters the nucleus and participates in regulating genome expression. Alternatively, the cytoplasmic function of p170 might be regulated via a reversible binding of integrins to NLS.

Cell cycle dependence of radiation-induced homologous recombination in cultured monkey cells

A lacZ transgene recombination system that reports homologous recombination events involving duplicated lacZ segments was used to study recombination in monkey cells exposed to ionizing radiation at different points in the cell cycle. With this system, recombination events can be detected in single cells by histochemical staining soon after exposure of cells to DNA-damaging treatment. Ionizing radiation rapidly induced recombination 5–10-fold in cells that were at the mitosis stage of the cell cycle. Irradiation either of cells at other points in the cell cycle or of nonsynchronized cells had less of an effect on recombination between lacZ segments.

Isolation of Coxiella burnetii from dairy cattle and ticks, and some characteristics of the isolates in Japan.

Coxiella burnetii was isolated from raw milk (36/214, 16.8%) and uterus swab samples (13/61, 21.3%) originating from dairy cattle with reproductive disorders, aborted bovine fetus samples (2/4, 50%), mammary gland samples (4/50, 8%) originating from healthy dairy cattle, and tick samples (4/15, 26.7%) originating from 2 pastures. Fifty-nine strains had various degrees of pathogenicity, high (8; 13.6%), moderate (28; 47.5%) and low (23; 39%), for guinea pigs. The results of isolation suggested a high prevalence of Coxiella infection in dairy cattle with reproductive problems in Japan. Twelve strains (7, 2 and 3 strains from cattle, ticks and humans, respectively) and the reference Nine Mile strain of phases I and II were propagated in both yolk sacs of embryonated hen eggs and Buffalo green monkey (BGM) cell cultures. Protein profiles of these strains were similar to those of the reference strain of phase I. Lipopolysaccharide (LPS) profiles of 12 strains were similar to those of the reference strain of phase I and different from those of the reference strain of phase II. The LPS profiles of 12 strains suggested that these strains are associated with an acute form of Q fever.

Analysis of mutations induced by replication of UV‐damaged plasmid DNA in hela cell extracts

We have used an SV40-based shuttle vector, pZ189, to investigate the capacity of HeLa cell extracts to reproduce the in vivo process of mutation fixation. We showed previously that when UV-irradiated pZ189 is replicated in these extracts, bypass of UV photoproducts occurs, resulting in base substitution mutations in the supF gene of the vector. Here we report the DNA sequence characterization of a collection of 60 of these UV-induced mutants. Most of the mutations observed are single or tandem double base substitutions at dipyrimidine sites; of these, approximately 90% are G:C-->A:T transitions. Mutations are observed predominantly at a few sites, in particular at positions 155 and 156 in the supF sequence. No dramatic differences in the mutation spectrum were observed when the orientation of the supF gene was reversed with respect to the SV40 origin of replication, suggesting that mutation fixation occurs similarly on both the leading and the lagging strands for DNA replication. Generally, the mutational hot spots observed in vitro are at the same sites as those observed when UV-irradiated pZ189 was passaged in human or monkey cells in culture. Thus, it appears that the replication and mutagenesis of UV-damaged templates in HeLa cell extracts accurately reflects these processes in the intact cell.

Effects of clofibric and beclobric acid in rat and monkey hepatocyte primary culture: influence on peroxisomal and mitochondrial beta-oxidation and the activity of catalase, glutathione S-transferase and glutathione peroxidase.

The effect of hypolipidaemic compounds on peroxisomal fatty acid beta-oxidation and on peroxisome morphology in the liver differs widely between rodent and primate species. We studied the relative importance of peroxisomal and mitochondrial beta-oxidation of palmitate in primary cultures of hepatocytes isolated from rat and monkey liver in the absence or presence of clofibric acid or beclobric acid. It was demonstrated that it is possible to differentiate between peroxisomal and mitochondrial beta-oxidation activities in intact cells. Overall beta-oxidation of palmitate was ca. 30% higher in rat hepatocytes than in monkey liver cells. In both monkey and rat cell cultures the mitochondrial component was over 90% of the total palmitate beta-oxidation. In rat hepatocyte culture clofibric acid and beclobric acid caused a 5- to 8-fold stimulation of peroxisomal beta-oxidation, while in monkey cells this activity was not significantly increased. However, in cells derived from both species mitochondrial palmitate beta-oxidation was increased (rat 2.5-fold; monkey 1.5-fold). These results indicate that the species differences in the increase in peroxisomal fatty acid oxidation are not a result of an inability to metabolize fatty acids in rat liver cell mitochondria. A comparison of the activity of enzymes involved in the detoxification of hydrogen peroxide showed that catalase and glutathione-S-transferase activity is 2.9-fold higher in monkey hepatocytes than in rat liver cells, while glutathione peroxidase activity was 1.6-fold higher in rat cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Primary pathogenicity of an European isolate of Chlamydia psittaci from turkey poults

Chlamydia psittaci was isolated as the sole pathogenic agent from a severe outbreak of respiratory disease in a commercial broiler turkey farm in the Netherlands. The mortality rate in the flocks was 35%. Clinical signs included conjunctivitis, swelling of the sinus infraorbitalis and sneezing. Cloacal excretion of chlamydia was demonstrated in twelve out of fifteen birds examined by a direct immunofluorescence test. In all the fifteen birds antibodies against Chlamydia psittaci were detected in the sera by a competitive ELISA. At necropsy sinusitis, rhinitis, airsacculitis, pneumonia, pericarditis and enlargement of the liver and spleen were found. Chlamydiae were demonstrated in the sinus material of all and in conjunctival smears of eight of the fifteen examined birds. Chlamydiae were isolated from all the examined birds after one to three passages on Buffalo Green Monkey (BGM) cell cultures using samples taken from lung, liver and spleen. No other pathogens were isolated. The chlamydia isolate was typed using a panel of serovar-specific monoclonal antibodies in a micro-immunofluorescence test. The isolate belonged to the avian Chlamydia psittaci serovar D. Experimental inoculation with this isolate of 7-day-old specific pathogen free (SPF) turkeys resulted in severe clinical signs, with mortality and extensive pathological lesions, similar to those seen in turkeys from the examined broiler turkey farm. From the data it was concluded that this Chlamydia psittaci isolate can cause severe disease in turkeys.

Modulation of adjuvant arthritis in Lewis rats by recombinant vaccinia virus expressing the human 60-kilodalton heat shock protein.

The immune response to the mycobacterial 65-kDa heat shock protein (hsp65) is considered an important event in the induction of adjuvant arthritis (AA) in rats; this induction probably occurs through a molecular mimicry mechanism involving cross-reactivity against the rat homolog hsp60. To analyze the role of mammalian molecule hsp60 in arthritis, we generated a recombinant vaccinia virus (hsp60-VV) carrying the human hsp60 gene inserted into the thymidine kinase locus under the control of the 7.5k vaccinia virus promoter. Human hsp60 is almost identical to its rat homolog (97.4% linear amino acid homology) and shares about 50% of amino acid positions with Mycobacterium tuberculosis hsp65. The latter supposedly carries a critical epitope for AA induction that is not present in human hsp60. Infections with hsp60-VV of monkey cell cultures led to the expression of the human hsp60 molecule, as evidenced by immunoblotting analysis with specific monoclonal antibodies. Also, Lewis rats infected with hsp60-VV produced specific antibodies, demonstrating the in vivo expression of human hsp60 in the infected animals. Therefore, we used hsp60-VV to analyze whether the delivery of hsp60 could affect the induction of AA in Lewis rats. hsp60-VV clearly reduced and retarded arthritic symptoms when administered to rats at day 7 after AA induction. In contrast, inoculation of rats with a control recombinant vaccinia virus did not affect the course of the disease. The improvement in AA with hsp60-VV administration was associated with a specific immune response, as determined by the presence of antibodies to hsp60 in the sera and the proliferation induced by hsp60 of T cells from popliteal lymph nodes. These results support a critical role for immunity to heat shock proteins in AA. Since the protective construct is virtually identical to rat homolog hsp60, we conclude that immunity directed to conserved areas of this family of proteins is directly involved in the pathogenesis of AA.

A cytoplasmically anchored nuclear protein interferes specifically with the import of nuclear proteins but not U1 snRNA.

A cytoplasmically anchored mutant SV40 T antigen, FS T antigen, was shown previously to interfere specifically with the nuclear import of a heterologous nuclear protein, adenovirus 5 fiber protein, in cultured monkey cells (Schneider, J., C. Schindewolf, K. van Zee, and E. Fanning. 1988. Cell. 54:117-125; van Zee, K., F. Appel, and E. Fanning. 1991. Mol. Cell. Biol. 11:5137-5146). In this report, we demonstrate that FS T antigen also interferes with the nuclear import of adenovirus E1A and a peptide-albumin conjugate bearing multiple copies of the T antigen nuclear localization signal, but not with the import of U1 snRNA. A kinetic analysis indicates that nuclear import of the albumin-peptide conjugate is inhibited only when high intracellular concentrations of FS T antigen are reached. After microinjection into the cytoplasm of cultured cells, purified FS T antigen protein does not accumulate at the nuclear periphery, but rather is distributed in a punctate pattern throughout the cytoplasm. These data support a model in which cytoplasmic anchoring of FS T antigen enables the mutant protein to sequester and titrate out a cellular factor which is required for nuclear protein but not U1 snRNA import.

Site-specific proteolytic cleavage of Ku protein bound to DNA.

Ku protein, a relatively abundant nuclear protein associated with DNA of mammalian cells, is known to be a heterodimer with subunits of 85 and 72 kDa which binds in vitro to DNA ends and subsequently translocates along the molecule. The functional role played by this protein in the cell, however, remains to be elucidated. We have observed here that Ku protein, purified from cultured monkey cells, is the target of specific endoproteolysis in vitro, by which the 85 kDa subunit is cleaved at a precise site while the 72 kDa subunit remains intact. This cleavage releases an 18 kDa polypeptide and converts Ku protein into a heterodimer composed of the 72 kDa subunit associated with a 69 kDa fragment from the 85 kDa subunit. The proteolyzed form of Ku protein, denoted Ku', has DNA binding properties similar to those of Ku protein. The proteolytic mechanism, which is inhibited by leupeptin and chymostatin, is extremely sensitive to ionic conditions, in particular to pH, being very active at pH 7.0 and completely inhibited at pH 8.0. In addition, cleavage occurs only when Ku protein is bound to DNA, not free in solution. We suggest that in vivo, such proteolysis might be necessary for Ku protein function at some stage of the cell cycle.

Analysis of the mechanism of interaction of simian Ku protein with DNA

Ku protein is a relatively abundant DNA-binding protein which was first detected as the autoantigen in a patient with scleroderma-polymyositis overlap syndrome (hence the name 'Ku'). It is a heterodimer of two polypeptide chains of molecular weights 85,000 and 72,000, and it characteristically binds, in vitro, to the ends of DNA fragments, and translocates to form regular multimeric complexes, with one protein bound per 30 bp of DNA. We have studied the mechanism of interaction of Ku protein with DNA in vitro, using protein extracted from cultured monkey cells. We find that the precise structure of the DNA ends is not important for binding, as Ku protein can bind to hairpin loops and to mononucleosomes. Bound protein also does not require DNA ends for continued binding, since complexes formed with linear DNAs can be circularized by DNA ligase. Dissociation of the complex also appears to require DNA ends, since ligase closed circular complexes were found to be extremely stable even in the presence of 2 M NaCl. We also found that Ku molecules slide along DNA, with no preferential binding to specific sequences. Thus, Ku protein behaves like a bead threaded on a DNA string, a binding mechanism which allows us to make a new hypothesis concerning the function of this protein in the nucleus.

Sequence-specific single-strand-binding protein for the simian virus 40 early promoter stimulates transcription in vitro

We have detected, in nuclear extracts of non-infected cultured monkey cells, a protein (protein H16) that binds a specific single-stranded DNA sequence in the early promoter of simian virus 40 (SV40). This protein does not bind double-stranded DNA, nor RNA. In the present paper, the DNA-binding properties of protein H16 and its effects on transcription by RNA polymerase II in vitro have been investigated. The protein binds only to the late strand of the early promoter, within the region of the 21 base-pair repeats, and shows no affinity for any other SV40 sequence. The high percentage of cytosine residues in the late strand in this region appears to be important for recognition by the protein. Protein H16 does not bind the control region of SV40 in negatively supercoiled DNA circles. When bound to the late strand, the protein is displaced from its binding site by reassociation of the early strand with the late strand. Its binding to DNA is not sensitive to methylation of the dinucleotide CG in its binding site. The protein has been purified to near homogeneity by preparative gel retardation, and has an apparent molecular weight of 70,000. Purified protein H16 stimulates transcription by purified RNA polymerase II in vitro. The possible role of sequence-specific single-strand-binding proteins in transcription is discussed.