Cytopathic Effect Formation Research Articles

A Screening Study Identified Decitabine as an Inhibitor of Equid Herpesvirus 4 That Enhances the Innate Antiviral Response.

Equid herpesvirus 4 (EHV-4) is a common respiratory pathogen in horses. It sporadically induces abortion or neonatal death. Although its contribution in neurological disorders is not clearly demonstrated, there is a strong suspicion of its involvement. Despite preventive treatments using vaccines against EHV-1/EHV-4, the resurgence of alpha-EHV infection still constitutes an important threat to the horse industry. Yet very few studies have been conducted on the search for antiviral molecules against EHV-4. A screening of 42 antiviral compounds was performed in vitro on equine fibroblast cells infected with the EHV-4 405/76 reference strain (VR2230). The formation of cytopathic effects was monitored by real-time cell analysis (RTCA), and the viral load was quantified by quantitative PCR. Aciclovir, the most widely used antiviral against alpha-herpesviruses in vivo, does not appear to be effective against EHV-4 in vitro. Potential antiviral activities were confirmed for eight molecules (idoxuridine, vidarabine, pritelivir, cidofovir, valganciclovir, ganciclovir, aphidicolin, and decitabine). Decitabine demonstrates the highest efficacy against EHV-4 in vitro. Transcriptomic analysis revealed the up-regulation of various genes implicated in interferon (IFN) response, suggesting that decitabine triggers the immune antiviral pathway.

Vero CCL-81 and Calu-3 Cell Lines as Alternative Hosts for Isolation and Propagation of SARS-CoV-2 Isolated in Malaysia.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been identified as the etiologic agent for the pneumonia outbreak that started in early December 2019 in Wuhan City, Hubei Province, China. To date, coronavirus disease (COVID-19) has caused almost 6 million deaths worldwide. The ability to propagate the virus into a customizable volume will enable better research on COVID-19 therapy, vaccine development, and many others. In the search for the most efficient replication host, we inoculated three (3) local SARS-CoV-2 isolates of different lineages (Clade L/Lineage B Wuhan, Clade GR/Lineage B.1.1.354, and Clade O/Lineage B.6.2) into various clinically important mammalian cell lines. The replication profile of these isolates was evaluated based on the formation of cytopathic effects (CPE), viral load (Ct value and plaque-forming unit (pfu)), as well as observation by electron microscopy (EM). Next-generation sequencing (NGS) was performed to examine the genomic stability of the propagated SARS-CoV-2 in these cell lines. Our study found that Vero E6 and Vero CCL-81 cell lines posed similar capacities in propagating the local isolates, with Vero CCL-81 demonstrating exceptional potency in conserving the genomic stability of the Lineage B Wuhan isolate. In addition, our study demonstrated the utility of Calu-3 cells as a replication host for SARS-CoV-2 without causing substantial cellular senescence. In conclusion, this study provides crucial information on the growth profile of Malaysian SARS-CoV-2 in various mammalian cell lines and thus will be a great source of reference for better isolation and propagation of the SARS-CoV-2 virus isolated in Malaysia.

Effect of farnesyltransferase inhibitors on SARS-CoV-2

The emergence of SARS-CoV-2 in 2019 led to a severe pandemic situation. Treatment options are limited, and the efficacy of vaccines decreases due to mutations in SARS-CoV-2 strains. Therefore, new treatment options are urgently needed, and computational compound screenings are used to predict drugs quickly. One of these screenings revealed farnesyltransferase inhibitors (FTIs) as potential candidates. SARS-CoV-2 infected Calu-3 cells were treated with lonafarnib and tipifarnib and fold change viral replication of SARS-CoV-2 was measured using RT-qPCR. Furthermore, morphological changes, like CPE formation, were evaluated. Effects on Calu-3 cells were analyzed using MTT assay. We demonstrated that the FTIs lonafarnib and tipifarnib have an effect on SARS-CoV-2 Wildtype and the Delta variant. Both FTIs dose-dependently reduced morphological changes and the formation of cytopathic effects in SARS-CoV-2 infected Calu-3 cells. The effect of the FTIs on Omicron needs to be further elucidated because of inefficient viral replication. The FTI lonafarnib and tipifarnib might be effective drugs against different SARS-CoV-2 strains.

THE EFFECTS OF ORIENTIN ON THP-1 CELLS INFECTED BY DENV2

Orientin, a flavonoid known for its significant antioxidative properties, has also shown to possess antiviral properties against some viruses. Nevertheless, up to now, there was no report on its ability against dengue virus. This study investigated the effects of orientin against DENV2 infecting human monocyte, THP-1 cells. THP-1 cells infected with DENV2 (3.16×102 TCID50/mL) were treated with a maximum non-toxic dose (MNTD) of orientin (0.053 ± 0.006 µM) for two days. Cytopathic effect (CPE) formation was observed after two days, followed by measurement of the percentage of cell viability using 3-(4,5- Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The treatment was compared to cells infected with DENV2 only, cells treated with orientin at MNTD only, and control cells. The MNTD of orientin on THP-1 cells were determined to be 0.053 ± 0.006 µM. Treatment of DENV2 infected THP-1 cells with the MNTD of orientin showed CPE formation similar to the THP-1 cells infected with DENV2 only. The MTT assay showed that orientin did not exhibit antiviral activity against DENV2 infecting THP-1 cells. Percentage of cell viability of the orientin treated cells and DENV2-infected cells were not significantly different. Orientin at MNTD does not exhibit antiviral effects on THP-1 cells infected with DENV2.

Molecular characterization of fowl adenovirus isolate of Malaysia attenuated in chicken embryo liver cells and its pathogenicity and immunogenicity in chickens.

Fowl adenovirus (FAdV) is the causative agent of inclusion body hepatitis (IBH) in chickens with significant economic losses due to high mortality and poor production. It was objectives of the study to attenuate and determine the molecular characteristic of FAdV isolate (UPM1137) of Malaysia passages in primary chicken embryo liver (CEL) cells. The cytopathic effect (CPE) was recorded and the present of the virus was detected by polymerase chain reaction (PCR). Nucleotide and amino acid changes were determined and a phylogenetic tree was constructed. The pathogenicity and immunogenicity of the virus at passage 35 (CEL35) with virus titre of 106.7TCID50/mL was determined in day old specific pathogen free (SPF) chicks via oral or subcutaneous route of inoculation. The study demonstrated that the FAdV isolate was successfully propagated and attenuated in CEL cells up to 35th consecutive passages (CEL35) with delayed of CPE formation within 48 to 72 post inoculation (pi) from CEL20 onwards. The virus caused typical CPE with basophilic intranuclear inclusion bodies, refractile and clumping of cells. The virus is belong to serotype 8b with substitution of amino acid at position 44, 133 and 185 in L1 loop of hexon gene and in knob of fiber gene at position 348 and 360 at CEL35. It is non-pathogenic, but immunogenic in SPF chickens. It was concluded that the FAdV isolate was successfully attenuated in CEL cells with molecular changes in major capsid proteins which affect its infectivity in cell culture and SPF chickens.

Synergistic effects of acyclovir and 3, 19-isopropylideneandrographolide on herpes simplex virus wild types and drug-resistant strains.

BackgroundAn andrographolide analogue, 3, 19-isopropylideneandrographolide (IPAD), exerts an inhibitory effect on replication of wild-type herpes simplex virus serotype 1 (HSV-1). In this study, we examined the anti-viral activity of IPAD on HSV wild types (HSV-1 strain KOS and HSV-2 clinical isolate) and HSV-1 drug-resistant strains (DRs). Synergistic effects of IPAD with acyclovir (ACV) were also evaluated.MethodsMTT and cytopathic effect (CPE) reduction assays were performed to determine cytotoxicity and anti-viral activities, respectively. A combination assay was used to determine synergistic effects of IPAD and ACV. Presence of viral DNA and protein in experimental cells was investigated using the polymerase chain reaction and western blotting, respectively.ResultsA non-cytotoxic concentration of IPAD (20.50 μM) completely inhibited CPE formation induced by HSV wild types and HSV-1 DRs after viral entry into the cells. The anti-HSV activities included inhibition of viral DNA and protein synthesis. The minimum inhibitory concentrations of ACV for HSV wild types and HSV-1 DRs were 20.20 and 2,220.00 μM, respectively. Combination of ACV with IPAD showed synergistic effects in inhibition of CPE formation, viral DNA and protein synthesis by HSV wild types as well as HSV-1 DRs. For the synergistic effects on HSV wild types and HSV-1 DRs, the effective concentrations of ACV were reduced.ConclusionsThese results showed the inhibitory potential of IPAD on HSV wild types and HSV-1 DRs and suggested that IPAD could be used in combination with ACV for treatment of HSV-1 DRs infections.

Over-expression of mitochondrial antiviral signaling protein inhibits coxsackievirus B3 infection by enhancing type-I interferons production

BackgroundRecent studies have revealed that Mitochondrial Antiviral Signaling (MAVS) protein plays an essential role in the inhibition of viral infection through type I interferon (IFN) pathway. It has been shown that 3C (pro) cysteine protease of coxsackievirus B3 (CVB3) cleaves MAVS to inhibit type I IFNs induction. Other workers also found that MAVS knock-out mice suffered CVB3 susceptibility and severe histopathological change. Accordingly,our experiments were designed to explore the protection of over-expressing MAVS against CVB3 infection and the possible mechanism.ResultsIn this study, HeLa cells (transfected with MAVS constructs pre- or post- exposure to CVB3) were used to analyze the function of exogenous MAVS on CVB3 infection. The results revealed that though CVB3 infection induced production of type I IFNs, viral replication and cell death were not effectively inhibited. Similarly, exogenous MAVS increased type I IFNs moderately. Morever, we observed robust production of type I IFNs in CVB3 post-infected HeLa cells thereby successfully inhibiting CVB3 infection, as well formation of cytopathic effect (CPE) and cell death. Finally, introduction of exogenous MAVS into CVB3 pre-infected cells also restricted viral infection efficiently by greatly up-regulating IFNs.ConclusionsIn summary, exogenous MAVS effectively prevents and controls CVB3 infection by modulating and promoting the production of type I IFNs. The IFNs level in MAVS over-expressing cells is still tightly regulated by CVB3 infection. Thus, the factors that up-regulate MAVS might be an alternative prescription in CVB3-related syndromes by enhancing IFNs production.

Isolation and molecular characterization of type I and type II feline coronavirus in Malaysia

BackgroundFeline infectious peritonitis virus (FIPV) and feline enteric coronavirus (FECV) are two important coronaviruses of domestic cat worldwide. Although FCoV is prevalent among cats; the fastidious nature of type I FCoV to grow on cell culture has limited further studies on tissue tropism and pathogenesis of FCoV. While several studies reported serological evidence for FCoV in Malaysia, neither the circulating FCoV isolated nor its biotypes determined. This study for the first time, describes the isolation and biotypes determination of type I and type II FCoV from naturally infected cats in Malaysia.FindingsOf the total number of cats sampled, 95% (40/42) were RT-PCR positive for FCoV. Inoculation of clinical samples into Crandell feline kidney cells (CrFK), and Feline catus whole fetus-4 cells (Fcwf-4), show cytopathic effect (CPE) characterized by syncytial cells formation and later cell detachment. Differentiation of FCoV biotypes using RT-PCR assay revealed that, 97.5% and 2.5% of local isolates were type I and type II FCoV, respectively. These isolates had high sequence homology and phylogenetic similarity with several FCoV isolates from Europe, South East Asia and USA.ConclusionsThis study reported the successful isolation of local type I and type II FCoV evident with formation of cytopathic effects in two types of cell cultures namely the CrFK and Fcwf-4 , where the later cells being more permissive. However, the RT-PCR assay is more sensitive in detecting the antigen in suspected samples as compared to virus isolation in cell culture. The present study indicated that type I FCoV is more prevalent among cats in Malaysia.

Screening of Brazilian medicinal plants for antiviral activity against rotavirus

Ethnopharmacological relevanceBrazilian medicinal plants traditionally used for the treatment of diarrhoea were investigated for their in vitro antiviral activity against the simian rotavirus SA11. Materials and methodsThe ethanolic crude extracts of plants collected in the cerrado of Minas Gerais, Brazil were submitted to phytochemical screening. The cytotoxicity of the extracts was inferred by cellular morphologic alterations. Antiviral activity was assessed by the ability of the extracts to inhibit the cytopathic effect (CPE) of rotavirus on the treated cells. RT-PCR was performed to confirm and/or confront antiviral assay data. ResultsThe maximum non-toxic concentration ranged from 50 to 500μg/mL. All extracts were toxic at a concentration of 5000μg/mL but no extract showed cytotoxicity at 50μg/mL. The species Byrsonima verbascifolia, Myracrodruon urundeuva, Eugenia dysenterica and Hymenaea courbaril exhibited the strongest in vitro activity against rotavirus. Their extracts prevented the formation of CPE, and RT-PCR analysis detected no amplification of genetic material from rotavirus. Tannins, flavonoids, saponins, coumarins and terpenes were the major classes of natural products found in the leaf extracts that showed antiviral activity. ConclusionAmong the species studied, Byrsonima verbascifolia, Eugenia dysenterica, Hymenaea courbaril and Myracrodruon urundeuva showed potential activity against rotavirus and are worthy of further study. The present study corroborates ethnopharmacological data as a valuable source in the selection of plants with antiviral activity and to some extent validates their traditional uses.

239 EMBRYOTOXICITY ASSAY FOR ANTIVIRAL COMPOUND BPIP (5-[(4-BROMOPHENYL)METHYL]-2-PHENYL-5H-IMIDAZO[4,5-c]PYRIDINE) IN BOVINE IN VITRO-PRODUCED EMBRYOS

Bovine viral diarrhea virus (BVDV) causes serious economic losses in the cattle industry. Evidence exists that only zona pellucida (ZP)-free bovine embryos are susceptible to BVDV infection (Vanroose et al. 1998 Biol. Reprod. 58, 857–866); however, BVDV can adhere to and therefore ‘infect’ both in vivo-(Waldrop et al. 2004 Theriogenology 62, 387–397) and in vitro-produced ZP intact embryos (Stringfellow et al. 2000 Theriogenology 53, 827–839). To eliminate these sanitary risks, pre-treatment of embryos with antiviral compounds may be a promising approach (Givens et al. 2006 Theriogenology 65, 344–355). BPIP (5-[(4-bromophenyl)methyl]-2-phenyl-5H-imidazo[4,5-c]pyridine) has been reported to display antiviral activity against BVDV, with a 50% effective inhibition of BVDV-induced cytopathic effect formation at a concentration of 0.04 µM (Paeshuyse et al. 2006 J. Virol. 80, 149–160). However, since the short- and long-term effects of BPIP have not been described, the aim of the current study was to assess whether addition of BPIP for 2 days at a concentration of 5 µM is toxic for ZP-free cattle embryos. Oocytes were aspirated from 3–6-mm follicles of cattle ovaries, matured for 24 h, and subsequently co-incubated with 1 × 106 sperm cells mL−1 in IVF-TALP with 20 µg/mL−1 heparin for 24 h at 39°C and 5% CO2 in air. After fertilization, presumptive zygotes were put in groups of 25 into 50-µL droplets of SOF under oil in 5% CO2, 5% O2, and 90% N2 for 6 days. Afterwards, morulae and blastocysts were collected, rendered ZP-free by means of pronase treatment, and divided into 4 groups: (i) ZP-free control group, (ii) ZP-free control group treated with a volume of DMSO equal to condition (iv), (iii) ZP-free group treated with 5 µM BPIP in DMSO, and (iv) ZP-free group treated with 10 µM BPIP in DMSO. Because BPIP is a fat-soluble molecule, embryos were cultured in 0.5 mL SOF without oil for 2 days. At Day 8, all embryos were fixed, TUNEL-stained, and analyzed for total cell number and percentage of apoptotic cells. Three independent replicates were performed. Results are shown in Table 1 and were analyzed by means of ANOVA. Only group iv showed a significant decrease in total cell number, indicating that at 10 µM BPIP may negatively influence embryo development. At both 5 and 10 µM, BPIP treatment resulted in an increase in percentage of apoptotic cells compared to the control group. However, a similar increase was observed using DMSO alone (group ii), indicating that the apoptotic effect may be due solely to the DMSO. In conclusion, BPIP does not appear to cause embryo toxicity at 5 µM, but an alternative, less toxic, dissolving agent may be considered. Table 1.Embryotoxicity assay of BPIP

Potency estimation of measles, mumps and rubella trivalent vaccines with quantitative PCR infectivity assay

The quantitative PCR infectivity assay is a combination of virus propagation and quantitative PCR. Previously [Schalk JAC, van den Elzen C, Ovelgönne H, Baas C, Jongen PMJM. Estimation of the number of infectious measles viruses in live virus vaccines using quantitative real-time PCR. J Virol Methods 2004;117:179–87.], we used this assay to estimate the titer of infectious measles virus in trivalent, live, measles, mumps, rubella vaccines (MMR 1 In this paper ‘MMR’ is used as the generic shorthand for trivalent measles, mumps and rubella vaccines and should not be confused with ‘MMR II’, the trademark of the Merck, Sharp and Dohme vaccine. 1 ). Here we describe the further improvement and development of the assay for simultaneous potency estimation of measles, mumps and rubella viruses. The potency of measles and mumps virus is estimated within one assay after 1 day of cell culture. The potency of rubella virus is estimated in a separate assay after 2 days of cell culture. Compared to conventional CCID50 and plaque assays, the quantitative PCR infectivity assay has the advantage in being fast because the assay is not dependent on the formation of cytopathic effect. Furthermore assay design is simplified: serological neutralization can be omitted because PCR is virus-specific and, under the conditions used, the individual components of trivalent measles, mumps, rubella vaccines do not interfere with each other. The assay was validated and compared to the performance of a plaque assay.

Effective primary isolation of wild-type Canine distemper virus in MDCK, MV1 Lu and Vero cells without nucleotide sequence changes within the entire haemagglutinin protein gene and in subgenomic sections of the fusion and phospho protein genes

Canine distemper virus (CDV) is an important pathogen of many carnivores. We are developing a field-based model of morbillivirus virulence and pathogenesis through a study of distemper in naturally infected free-ranging raccoons. The isolation of CDV from raccoon tissues is essential for this work. CDV has often been isolated from animals only after co-cultivation of infected tissues with peripheral blood mononuclear cells derived from specific pathogen-free dogs or similar methods. We explored the utility and consequences of a simpler and cheaper alternative: CDV isolation in Vero, MDCK, and MV1 Lu cells. Virus growth was detected first in MDCK cells, whereas viral cytopathic effects were most obvious in Vero cells. CDV growth in MV1 Lu cells was relatively protracted and occurred without the formation of cytopathic effects. In primary CDV isolates, the entire nucleotide sequence of the receptorbinding haemagglutinin ( H) gene, and subgenomic fusion ( F) and phospho ( P) protein gene sequences corresponding to nt 5399–5733 and 2132–2563 of CDV reference strain Onderstepoort, respectively, were identical to those in matched infected tissues. Virus isolation confirmed the presence of CDV in instances where RT-PCR failed to detect CDV in infected tissues. Different viral phenotypes and genotypes were detected. The conservation of H gene sequences in primary CDV isolates suggests that MDCK, MV1 Lu, and Vero cells express proper receptors for wild-type CDV.

Influence of cell surface glycoprotein gC produced by pseudorabies virus on cytopathic effect.

The wild-type pseudorabies virus (WT-PRV) produced a round-type cytopathic effect (CPE) in PK-15 cell line of porcine kidney origin, while PRVgCs lacking in gC-transmembrane-anchor region and PRVgC-defecting in gC gene produced a syncytium-type CPE. The mouse embryo cell line (BALB/3T3 clone A31) were transfected with recombinant plasmid of pcDNA3 which incorporated with gC gene. The transfected A31/gC cells were stably expressing gC. Only a round-type CPE was observed in these cells infected with WT-PRV, while a syncytium-type CPE was observed in the cells infected with each of the PRVgCs and PRVgC-. Any viruses described above induced a syncytium-type CPE in A31/pcDNA cells transfected with a plasmid without gC gene. By WT-PRV infection, PK-15 cells generated about 2- or 8-fold more gC than the A31/gC and A31/pcDNA cells when gC was measured by hemagglutination test. Flowcytometric analysis revealed that amount of gC on the cell surface of A31/gC and PK-15 cells increased after infection with WT-PRV. Round-type CPE was observed with the increase of gC. These results suggest that the type of CPE formation induced by PRV is dominated by the amount of gC on the infected cell surface.