Observation Of Cytopathic Effect Research Articles

3D-printed airway model as a platform for SARS-CoV-2 infection and antiviral drug testing

We present a bioprinted three-layered airway model with a physiologically relevant microstructure for the study of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection dynamics. This model exhibited clear cell-cell junctions and mucus secretion with an efficient expression of angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2). Having infected air-exposed epithelial cells in the upper layer with a minimum multiplicity of infection of 0.01, the airway model showed a marked susceptibility to SARS-CoV-2 within one-day post-infection (dpi). Furthermore, the unique longevity allowed the observation of cytopathic effects and barrier degradation for 21 dpi. The in-depth transcriptomic analysis revealed dramatic changes in gene expression affecting the infection pathway, viral proliferation, and host immune response which are consistent with COVID-19 patient data. Finally, the treatment of antiviral agents, such as remdesivir and molnupiravir, through the culture medium underlying the endothelium resulted in a marked inhibition of viral replication within the epithelium. The bioprinted airway model can be used as a manufacturable physiological platform to study disease pathogeneses and drug efficacy.

Characterization of a New Toti-like Virus in Sea Bass, Dicentrarchus labrax.

The European sea bass Dicentrarchus labrax is the main species reared in Mediterranean aquaculture. Its larval stage, which is very sensitive and highly affected by sanitary and environmental conditions, is particularly scrutinized in hatcheries. Recently, a Mediterranean sea bass farm had to deal with an abnormal increase in mortality, especially between 20 and 35 days post-hatching (dph). Biological investigations led to the observation of cytopathic effects on three different fish cell lines after almost 3 weeks of culture at 14 °C in contact with homogenized affected larvae, suggesting the presence of a viral agent. High-throughput sequencing revealed a 6818-nucleotide-long RNA genome with six putative ORFs, corresponding to the organization of viruses belonging to the Totiviridae family. This genome clustered with the newly described and suggested Pistolvirus genus, sharing 45.5% to 37.2% nucleotide identity with other piscine toti-like viruses such as Cyclopterus lumpus toti-like virus (CLuTLV) or piscine myocarditis virus (PMCV), respectively. Therefore, we propose to name this new viral agent sea bass toti-like virus (SBTLV). Specific real-time RT-PCR confirmed the presence of the viral genome in the affected larval homogenate from different production batches and the corresponding cell culture supernatant. Experimental infections performed on sea bass fingerlings did not induce mortality, although the virus could be detected in various organs and a specific immune response was developed. Additional studies are needed to understand the exact involvement of this virus in the mortality observed in hatcheries and the potential associated cofactors.

A nano-luciferase expressing human coronavirus OC43 for countermeasure development

The genetic diversity of the coronavirus (CoV) family poses a significant challenge for drug discovery and development. Traditional antiviral drugs often target specific viral proteins from specific viruses which limits their use, especially against novel emerging viruses. Antivirals with broad-spectrum activity overcome this limitation by targeting highly conserved regions or catalytic domains within viral proteins that are essential for replication. For rapid identification of small molecules with broad antiviral activity, assays with viruses representing family-wide genetic diversity are needed. Viruses engineered to express a reporter gene (i.e. luminescence, fluorescence, etc.) can increase the efficiency, sensitivity or precision of drug screening over classical measures of replication like observation of cytopathic effect or measurement of infectious titers. We have previously developed reporter virus systems for multiple other endemic, pandemic, epidemic and enzootic CoV. Human CoV OC43 (HCoV-OC43) is a human endemic CoV that causes respiratory infection with age-related exacerbations of pathogenesis. Here, we describe the development of a novel recombinant HCoV-OC43 reporter virus that expresses nano-luciferase (HCoV-OC43 nLuc), and its potential application for screening of antivirals against CoV.

5-Nitrobenzo[c][1, 2, 5]selenadiazole as therapeutic agents in the regulation of oxidative stress and inflammation induced by influenza A(H1N1)pdm09 in vitro and in vivo.

Currently, various problems are being faced in the treatment of influenza, so the development of new safe and effective drugs is crucial. Selenadiazole, an important component of selenium heterocyclic compounds, has received wide attention for its biological activity. This study aimed to verify the antiviral activity of 5-nitrobenzo[c][1,2,5]selenadiazole (SeD-3) in vivo and in vitro. The cell counting kit-8 assay and observation of cytopathic effect verified that SeD-3 could improve the survival of influenza A(H1N1)pdm09-infected Madin-Darby canine kidney cells. Polymerase chain reaction quantification and neuraminidase assay showed that SeD-3 could inhibit the proliferation of H1N1 virus. The time of addition assay demonstrated that SeD-3 may have a direct effect on virus particles and block some stages of H1N1 life cycle after virus adsorption. Cell cycle, JC-1, Annexin V, and terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling-4',6-diamidino-2-phenylindole (TUNEL-DAPI) assays showed that SeD-3 inhibited H1N1 infection-induced apoptosis. Cytokine detection demonstrated SeD-3 inhibited the production of proinflammatory factors after infection, including tumor necrosis factor-α (TNF-α), TNF-β, interferon-γ, interleukin 12 (IL-12), and IL-17F. In vivo experiments suggested that the pathological damage in the lungs was significantly alleviated after treatment with SeD-3 by hematoxylin and eosin staining. The TUNEL assay of lung tissues indicated that SeD-3 inhibited DNA damage during H1N1 infection. Immunohistochemical assays were performed to further explore the mechanism that SeD-3 inhibited H1N1-induced apoptosis via reactive oxygen species-mediated MAPK, AKT, and P53 signaling pathways. In conclusion, SeD-3 may become a new potential anti-H1N1 influenza virus drug due to its antiviral and anti-inflammatory activity.

Differential Cell Line Susceptibility to the SARS-CoV-2 Omicron BA.1.1 Variant of Concern.

The unique mutations of the SARS-CoV-2 Omicron variant are associated with increased transmissibility, immune escape, increased binding affinity to ACE-2, and increased viral load. Omicron exhibited a shift in tropism infecting the upper respiratory tract compared to other variants of concern which have tropism for the lower respiratory tract. The tropism of omicron variants in cell lines of different hosts and tissue origins still remains unclear. Considering this, we assessed the susceptibility of different cell lines to the SARS-CoV-2 omicron BA.1.1 variant and permissiveness among different cell lines for omicron replication. Susceptibility and permissiveness of a total of eleven cell lines, including six animal cell lines and five human cell lines for omicron BA.1.1 infection, were evaluated by infecting individual cell lines with omicron BA.1.1 isolate at a 0.1 multiplicity of infection. Virus replication was assessed by observation of cytopathic effects followed by viral load determination by real-time PCR assay and virus infectivity determination by TCID50 assay. The characteristic cytopathic effect, increased viral load, and productive omicron replication was detected in Vero CCL-81, Vero E6, Vero/hSLAM, MA-104, and Calu-3 cells. Although LLC MK-2 cells showed an increased TCID50 titer at the second infection, the viral load did not show much difference in both infections. Caco-2 cells did not show evident CPE, but they supported omicron replication at a low level. A549, RD, MRC-5, and BHK-21 cells supported omicron BA.1.1 replication without the CPE. This is the first study on the comparison of susceptibility of different cell lines to Omicron variant BA.1.1, which might be useful for future studies on emerging SARS-CoV-2 variants.

Development of rapid neutralization assay of viral hemorrhagic septicemia virus (VHSV) based on chimeric rhabdovirus expressing heterologous glycoprotein

The titer of neutralizing antibodies (NAbs) against viral hemorrhagic septicemia virus (VHSV) has been determined by conventional neutralization assay based on the observation of cytopathic effect (CPE) and plaque formation in cultured cells. However, this method requires several days for the determination and can be affected by operator bias. To develop a rapid and high-throughput neutralization assay against VHSV, we rescued a surrogate chimeric snakehead rhabdovirus, rSHRV-Gvhsv-eGFP, which has the enhanced green fluorescent protein (eGFP) gene between N and P genes and has VHSV G gene instead of SHRV G gene in the genome. The efficacy of rSHRV-Gvhsv-eGFP to determine serum neutralization activity was evaluated using various serum samples derived from New Zealand white rabbits and olive flounder (Paralichthys oliavaceus). Although neutralization titers analyzed using rSHRV-Gvhsv-eGFP were similar to the titers measured using rVHSV-A-eGFP, the time needed for the determination of neutralization titer was much shortened (24 h for rSHRV-Gvhsv-eGFP and 48 h for rVHSV-A-eGFP), proving the usefulness of rSHRV-Gvhsv-eGFP for the neutralization assay against VHSV. In addition, as the neutralization activities using rSHRV-Gvhsv-eGFP could be well-observed without adding fresh serum as a complement source, no preparation is required for the optimization of control fresh serum from naïve fish. The present results suggest that the rapid neutralization assay using rSHRV-Gvhsv-eGFP can be used to investigate neutralization activities against VHSV.

Isolation and identification of monkeypox virus MPXV-ROK-P1-2022 from the first case in the Republic of Korea.

ObjectivesMonkeypox outbreaks in nonendemic countries have been reported since early May 2022. The first case of monkeypox in the Republic of Korea was confirmed in a patient who traveled to Europe in June 2022, and an attempt was made to isolate and identify the monkeypox virus (MPXV) from the patient’s specimens.MethodsClinical specimens from the patient were inoculated in Vero E6 cells. The isolated virus was identified as MPXV by the observation of cytopathic effects on Vero E6 cells, transmission electron microscopy, conventional polymerase chain reaction (PCR), and sequencing of PCR products.ResultsCytopathic effects were observed in Vero E6 cells that were inoculated with skin lesion swab eluates. After multiple passages from the primary culture, orthopoxvirus morphology was observed using transmission electron microscopy. In addition, both MPXV-specific (F3L and ATI) and orthopoxvirus-specific genes (A39R, B2R, and HA) were confirmed by conventional PCR and Sanger sequencing.ConclusionThese results indicate the successful isolation and identification of MPXV from the first patient in the Republic of Korea. The isolated virus was named MPXV-ROK-P1-2022.

Development of two cell lines from yellowfin seabream Acanthopagrus latus fin and brain suitable for propagating SDDV but not for ISKNV, MRV and GIV

Scale drop disease virus (SDDV), a very distinct member of genus Megalocytivirus in family Iridoviridae, was recently confirmed as the most alarming causative agent associated with yellowfin seabream Acanthopagrus latus ascites diseases in South China. To better understand how host-pathogen interacting and developing cell-culture based vaccine, establishment of highly susceptible cell line generating from the natural host fish for SDDV infection will be a top priority. Here, two novel cell lines from yellowfin seabream fin and brain were established and characterized, and designated as YFSBF and YFSBB, respectively. Morphologically, both cell lines predominantly consisted of fibroblast-like cells and grew well in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum. Karyotype analysis revealed that chromosome numbers of YFSBF and YFSBB were wildly distributed without obvious featured chromosomic modes. Viral susceptibility tests showed that SDDV could propagate well in both YFSBF and YFSBB, and YFSBF showed more susceptible to SDDV than YFSBB did, being confirmed by cytopathic effect observation, absolute real-time quantitative PCR determination, immunofluorescence assay, Western blot analysis and transmission electron microscopy examination. Viral susceptibility tests also confirmed that SDDV could effectively propagate in MFF-1 cell, a highly susceptible host cell line for infectious spleen and kidney necrosis virus (ISKNV), whereas either YFSBF or YFSBB was refractory to ISKNV infection. Moreover, neither YFSBF nor YFSBB could support the effective propagations of mandarin ranavirus (MRV) and grouper ranavirus (GIV-R). Collectedly, this study provides some important information for better understanding the differential infection profile among SDDV, ISKNV and other fish iridovirus.

Isolation of Giant Viruses of Acanthamoeba castellanii.

This article describes a practical method for prospecting and isolating giant viruses based on direct inoculation of environmental samples into amoeba cultures of Acanthamoeba castellanii. The giant viruses that infect amoebas have already been isolated from various environmental samples in several countries worldwide, including in extreme environments. Here we describe the methodologic procedures regarding the prospecting of giant viruses in A. castellanii, including the preparation of environmental samples, the culture of amoebas, and the observation of cytopathic effects that can indicate the presence and potential isolation of giant viruses. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Sample collection Support Protocol: Propagation of Acanthamoeba castellanii Basic Protocol 2: Prospecting of giant viruses in environmental samples by cytopathic effect analysis.

First isolation and whole genome characterization of porcine deltacoronavirus from pigs in Peru.

Porcine deltacoronavirus is a newly emergent enteric pathogen affecting swine farms worldwide. It has been detected in several countries in Europe, Asia and North America; yet, it has not been reported in South America. In November 2019, an enteric disease outbreak in a pig farm located in San Martin, Peru, was reported along with submission of three intestinal samples from pigs who succumbed to the disease. Samples were processed for molecular detection by qRT-PCR, viral isolation and further sequencing analysis. A taqman-based RT-PCR was performed to differentiate among the most relevant swine enteric coronaviruses described to date. All samples were positive to porcine deltacoronavirus with a cycle threshold (Ct) value between 9 and 14, revealing a high viral load, while testing negative to porcine epidemic diarrhea and transmissible gastroenteritis viruses. Following detection, viral isolation was performed using PK-15 and Vero cell lines. After 5 days of inoculation, no cytopathic effect was observed. A second blind passage allowed the observation of cytopathic effect on PK-15 cells, while it remained absent in Vero cells. A fluorescence test using an anti-N monoclonal antibody confirmed viral replication. One sample was processed for whole genome sequencing (WGS). In short, raw reads were imported into CLC genomics and assembled de novo. Out of 479k reads generated from the sample, 436k assembled into a 25,501bp contig which was 99.5% identical to a reference porcine deltacoronavirus strain from the USA within the North American phylogroup. Yet, there are relevant differences at the nucleotide and amino acid levels compared with previously described porcine deltacoronavirus strains. Altogether, our findings represent the first report of porcine deltacoronavirus in South America, which provides information of its evolutionary origin. Thus, this study offers new insights into the molecular epidemiology of porcine deltacoronavirus infections in the swine industry.

Replication of SARS-CoV-2 in cell lines used in public health surveillance programmes with special emphasis on biosafety.

Background & objectives:Polio, measles, rubella, influenza and rotavirus surveillance programmes are of great public health importance globally. Virus isolation using cell culture is an integral part of such programmes. Possibility of unintended isolation of SARS-CoV-2 from clinical specimens processed in biosafety level-2 (BSL-2) laboratories during the above-mentioned surveillance programmes, cannot be ruled out. The present study was conducted to assess the susceptibility of different cell lines to SARS-CoV-2 used in these programmes.Methods:Replication of SARS-CoV-2 was studied in RD and L20B, Vero/hSLAM, MA-104 and Madin–Darby Canine Kidney (MDCK) cell lines, used for the isolation of polio, measles, rubella, rotavirus and influenza viruses, respectively. SARS-CoV-2 at 0.01 multiplicity of infection was inoculated and the viral growth was assessed by observation of cytopathic effects followed by real-time reverse transcription–polymerase chain reaction (qRT-PCR). Vero CCL-81 cell line was used as a positive control.Results:SARS-CoV-2 replicated in Vero/hSLAM, and MA-104 cells, whereas it did not replicate in L20B, RD and MDCK cells. Vero/hSLAM, and Vero CCL-81 showed rounding, degeneration and detachment of cells; MA-104 cells also showed syncytia formation. In qRT-PCR, Vero/hSLAM and MA-104 showed 106 and Vero CCL-81 showed 107 viral RNA copies per µl. The 50 per cent tissue culture infectious dose titres of Vero/hSLAM, MA-104 and Vero CCL-81 were 105.54, 105.29 and 106.45/ml, respectively.Interpretation & conclusions:Replication of SARS-CoV-2 in Vero/hSLAM and MA-104 underscores the possibility of its unintended isolation during surveillance procedures aiming to isolate measles, rubella and rotavirus. This could result in accidental exposure to high titres of SARS-CoV-2, which can result in laboratory acquired infections and community risk, highlighting the need for revisiting biosafety measures in public health laboratories.

Virologic Features of Severe Acute Respiratory Syndrome Coronavirus 2 Infection in Children.

Data on pediatric coronavirus disease 2019 (COVID-19) has lagged behind adults throughout the pandemic. An understanding of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral dynamics in children would enable data-driven public health guidance. Respiratory swabs were collected from children with COVID-19. Viral load was quantified by reverse-transcription polymerase chain reaction (RT-PCR); viral culture was assessed by direct observation of cytopathic effects and semiquantitative viral titers. Correlations with age, symptom duration, and disease severity were analyzed. SARS-CoV-2 whole genome sequences were compared with contemporaneous sequences. One hundred ten children with COVID-19 (median age, 10 years [range, 2 weeks-21 years]) were included in this study. Age did not impact SARS-CoV-2 viral load. Children were most infectious within the first 5 days of illness, and severe disease did not correlate with increased viral loads. Pediatric SARS-CoV-2 sequences were representative of those in the community and novel variants were identified. Symptomatic and asymptomatic children can carry high quantities of live, replicating SARS-CoV-2, creating a potential reservoir for transmission and evolution of genetic variants. As guidance around social distancing and masking evolves following vaccine uptake in older populations, a clear understanding of SARS-CoV-2 infection dynamics in children is critical for rational development of public health policies and vaccination strategies to mitigate the impact of COVID-19.

Characterization of SARS-CoV-2 East Java isolate, Indonesia

Background: Incidents of SARS-CoV-2 in East Java increased steadily, and it became the second epicenter in Indonesia. The COVID-19 pandemic caused a dire multisectoral crisis all around the world. This study investigates and characterizes local isolates from East Java, Indonesia. Methods: There were 54 patients suspected with SARS-COV-2 infection and 27 patients were COVID-19 positive. Virus isolates were obtained from COVID-19 inpatients’ nasopharyngeal swabs at the Dr Soetomo Teaching Hospital, Surabaya. There were only three isolates (#6, #11, #35) with good growth characteristics. Serial blind passage and cytopathic effect observation in the Vero E6 cell line were performed for virus isolation. Confirmation of the SARS-CoV-2 infection was proven by means of reverse transcriptase-polymerase chain reactions using SARS-CoV-2 specific primers, scanning electron microscopy, and scanning transmission electron microscopy examination. Whole genome sequencing was performed using ARTIC protocol. Furthermore, SARS-CoV-2 characterization was identified through a western blot using rabbit serum immunized with inactive SARS-CoV-2 vaccine and human natural COVID-19 infection serum. Results: Spike gene analysis of three samples (#6, #11, #35) found that the D614G mutation was detected in all isolates, although one isolate exhibited the D215Y and E484D mutation. Based on whole genome analysis, those three isolates were included in clade 20A, and two isolates were included in lineage B.1.6 with one isolate belongs to lineage B.1.4.7. Conclusion: Based on molecular characterization and immunogenicity of SARS-CoV-2 East Java, Indonesia showed high titer and it has mutation in some regions.

Characterization ofSARS-CoV-2 East Javaisolate, Indonesia.

Background:Incidentsof SARS-CoV-2 inEast Javaincreased steadily, and itbecame the second epicenter in Indonesia.TheCOVID-19 pandemic caused a dire multisectoral crisis all around the world.Thisstudyinvestigates and characterizeslocal isolates from East Java,Indonesia. Methods:There were54patients suspected with SARS-COV-2 infectionand27 patients were COVID-19 positive.Virus isolateswereobtained fromCOVID-19 inpatients'nasopharyngeal swabsat theDrSoetomoTeaching Hospital, Surabaya.There were only threeisolates(#6, #11, #35) withgoodgrowth characteristics.Serial blind passage and cytopathic effect observation intheVeroE6 cell line wereperformed for virus isolation. Confirmation ofthe SARS-CoV-2 infection was proven by means of reverse transcriptase-polymerase chain reactions using SARS-CoV-2 specific primers, scanning electron microscopy, and scanning transmission electron microscopy examination. Whole genome sequencing was performed using ARTIC protocol. Furthermore, SARS-CoV-2 characterization wasidentifiedthroughawestern blot using rabbit serum immunized with inactive SARS-CoV-2 vaccine andhuman natural COVID-19 infection serum. Results:Spike gene analysis of threesamples (#6, #11, #35)found thattheD614G mutationwasdetected inallisolates, although one isolate exhibited theD215Y and E484D mutation. Based on whole genome analysis,those three isolates were included in clade 20A, and two isolates were included in lineage B.1.6 with one isolate belongs to lineage B.1.4.7. Conclusion:Based onmolecular characterizationand immunogenicityof SARS-CoV-2 East Java, Indonesiashowed hightiter and it has mutation in some regions.

First Isolation of a Novel Aquatic Flavivirus from Chinook Salmon (Oncorhynchus tshawytscha) and Its In Vivo Replication in a Piscine Animal Model.

The first isolation of a flavivirus from fish was made from moribund Chinook salmon (Oncorhynchus tshawytscha) from the Eel River, California, USA. Following the observation of cytopathic effect in a striped-snakehead fish cell line, 35-nm virions with flaviviral morphology were visualized using electron microcopy. Next-generation sequencing and rapid amplification of cDNA ends obtained the complete genome. Reverse transcriptase quantitative PCR (RT-qPCR) confirmed the presence of viral RNA in formalin-fixed tissues from the wild salmon. For the first time, in vivo replication of an aquatic flavivirus was demonstrated following intracoelomic injection in a Chinook salmon model of infection. RT-qPCR demonstrated viral replication in salmon brains up to 15 days postinjection. Infectious virus was then reisolated in culture, fulfilling Rivers' postulates. Only limited replication occurred in the kidneys of Chinook salmon or in tissues of rainbow trout (Oncorhynchus mykiss). The proposed salmon flavivirus (SFV) has a 10.3-kb genome that encodes a rare dual open reading frame, a feature uncharacteristic of classical flaviviruses. Phylogenetic analysis places SFV in a basal position among a new subgroup of recently recognized aquatic and bat flaviviruses distinct from the established mosquito-borne, tick-borne, insect-only, and unknown-vector flavivirus groups. While the pathogenic potential of the virus remains to be fully elucidated, its basal phylogeny and the in vivo infection model will allow SFV to serve as a prototype for aquatic flaviviruses. Ongoing field and laboratory studies will facilitate better understanding of the potential impacts of SFV infection on ecologically and economically important salmonid species.IMPORTANCE Chinook salmon are a keystone fish species of great ecological and commercial significance in their native northern Pacific range and in regions to which they have been introduced. Threats to salmon populations include habitat degradation, climate change, and infectious agents, including viruses. While the first isolation of a flavivirus from wild migrating salmon may indicate an emerging disease threat, characterization of the genome provides insights into the ecology and long evolutionary history of this important group of viruses affecting humans and other animals and into an expanding group of recently discovered aquatic flaviviruses.

Functional analysis of Epinephelus coioides peroxisome proliferative–activated receptor α (PPARα): Involvement in response to viral infection

Peroxisome proliferative–activated receptor α (PPARα) belongs to the superfamily of nuclear receptors (NR). Studies have demonstrated that PPARα functions in energy metabolism, hepatic function, immune response, cell cycle, and apoptosis. In teleost fish, few studies have investigated the role of PPARα in the immune response. In this study, the grouper PPARα gene (EcPPARα) was investigated for its role in viral infection. The open reading frame of EcPPARα encoded a protein of 469 amino acids and contained an N-terminal domain (NTD), a DNA-binding domain (DBD), a hinge region, and a C-terminal ligand-binding domain (LBD). Phylogenetic analysis revealed that EcPPARα was most closely related to homologous genes in Sander lucioperca and Perca flavescens. Upon challenge with SGIV (Singapore grouper iridovirus) and RGNNV (Red-spotted grouper nervous necrosis virus), EcPPARα expression levels were significantly upregulated in different tissues. Subcellular localization analysis showed that the EcPPARα protein localized throughout the cytoplasm and nucleus with diffuse intracellular expression patterns, which is consistent with the localization pattern of mammalian PPARs. Based on morphological observation of cytopathic effect (CPEs), viral gene expression mRNAs, and virus titer assays, the results presented here showed that an overexpression of EcPPARα promoted SGIV production in grouper spleen cells. Overexpression of EcPPARα significantly inhibited the expression of several cytokines, including interferon-related genes (IFN-γ, ISG15, MXI, MXII, MAVS and MDA5), inflammatory cytokines (IL-1β, IL-6, IL-8, TNF-α) and Toll like receptor adaptors (TRAF6 and MyD88). Luciferase activity of IFN-α, IFN-γ, ISRE and NF-κB promoters was also significantly decreased in EcPPARα overexpression cells. Due to these detected interferon-related genes and inflammatory cytokines play important antiviral effect against SGIV in grouper, we speculated that the promotion effect of EcPPARα on SGIV replication may be caused by down-regulation of interferon and inflammatory response. In addition, through apoptotic body observation, capspase-3 activity detection, and flow cytometry analysis, it was found that overexpression of EcPPARα promoted SGIV-induced apoptosis in fathead minnow (FHM) cells. These data may increase an understanding of the role of PPARα in fish antiviral immune responses and apoptosis.

Development of a High-Throughput Serum Neutralization Test Using Recombinant Pestiviruses Possessing a Small Reporter Tag.

A serum neutralization test (SNT) is an essential method for the serological diagnosis of pestivirus infections, including classical swine fever, because of the cross reactivity of antibodies against pestiviruses and the non-quantitative properties of antibodies in an enzyme-linked immunosorbent assay. In conventional SNTs, an immunoperoxidase assay or observation of cytopathic effect after incubation for 3 to 7 days is needed to determine the SNT titer, which requires labor-intensive or time-consuming procedures. Therefore, a new SNT, based on the luciferase system and using classical swine fever virus, bovine viral diarrhea virus, and border disease virus possessing the 11-amino-acid subunit derived from NanoLuc luciferase was developed and evaluated; this approach enabled the rapid and easy determination of the SNT titer using a luminometer. In the new method, SNT titers can be determined tentatively at 2 days post-infection (dpi) and are comparable to those obtained by conventional SNTs at 3 or 4 dpi. In conclusion, the luciferase-based SNT can replace conventional SNTs as a high-throughput antibody test for pestivirus infections.

Characteristic of slow growth in cell culture of adenovirus type 54 causing nationwide outbreak epidemic keratoconjunctivitis in Japan

To characterize the virological features of adenovirus type 54 (Ad54) causing nationwide outbreak of severe epidemic keratoconjunctivitis (EKC) in Japan, we comparatively analysed the viral propagation phenotype of Ad54 and other Ads: type 37 (Ad37), 64 (Ad64), and 5 (Ad5), in A549 cells quantitatively. Laboratory investigation. We compared the growth rate of Ads using copy numbers and cytopathic effect observation during propagation in A549 cell lines. Expressions of mRNA of E1 gene were also calculated and compared. Phylogenetic analysis of the region, including putative promoter of E1 gene and E1 open reading frame (ORF), were performed. Increases in viral loads, growth rate, and viral propagation were slower for Ad54 than for other Ads. The expression level of the E1 gene per infected cell was lower for Ad54 than for other Ad types on post-infection day 1. Phylogenetic analysis of the E1 gene putative promoter and ORF revealed Ad54 was the closest to Ad type 8. The propagation of Ad54 in A549 is slow compared with Ad37, Ad64 and Ad5. This slow propagation could have been caused by slow genomic replication resulting from delayed viral entry or E1 transcription initiation. The EKC caused by Ad54 needs more attention because the slow propagation of Ad54 may contribute to prolonged disease duration.

Fetal Calf Serum Exerts an Inhibitory Effect on Replication of Duck Hepatitis A Virus Genotype 1 in Duck Embryo Fibroblast Cells.

Among the causative agents of duck viral hepatitis, duck hepatitis A virus genotype 1 (DHAV-1) is the most common virus reported in most outbreaks worldwide. How to propagate DHAV-1 in cell cultures efficiently remains a problem to be explored. Here, we aimed to test the effect of serum type on DHAV-1 replication in duck embryo fibroblast (DEF) cells. Comparative studies involved virus culture and passage, observation of cytopathic effect (CPE), virus quantification, and plaque formation assay. From the results of these investigations, we conclude that use of chicken serum (CS) in maintenance medium allows DHAV-1 to establish productive, cytocidal infection in DEF cells, whereas FCS exerts inhibitory effects on DHAV-1 replication, CPE development, and plaque formation. By using a neutralization test, we found that the direct action of FCS on virions is likely to play a key role in inhibiting DHAV-1 replication in DEF cells. Mechanism analyses revealed that FCS inhibits DHAV-1 replication at virus adsorption and reduces extracellular virus yields. The present work may shed light on a new perspective for antiviral agent development, and have provided a virus–host cell system for further studies on molecular mechanism involved DHAV-1 replication and pathogenesis.

Antiviral activity of nano-realgar against herpes simplex virus Type II in vitro

To explore the antiviral activity of nano-realgar against herpes simplex virus Type II (HSV-2) in vitro. Methods: Acyclovir (ACV) as a positive control, the cytotoxicity of nano-realgar at different concentrations (including 200.00, 150.00, 100.00, 50.00, 25.00, 12.50, 6.25, 3.13, 1.54, 0.78, 0.39 and 0 mg/L) on normal Vero cells were determined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay. HSV-2 virus titer was determined by plaque assay, and the Vero cells model of HSV-2 infection was established. Subsequently, the antiviral effects of nano-realgar at different concentrations (including 20.00, 10.00, 5.00, 2.50, 1.25, 0.63, 0.31, 0.15, 0.08, 0.04 and 0 mg/L) on infected cells model were evaluated by the observation of cytopathic effect (CPE) and MTT method under the 3 modes including pre-treatment, treatment and direct inactivation. Results: The 50% cytotoxic concentration (CC50) of nano-realgar on Vero cells was 37.15 mg/L. The titer of HSV-2 was 7.30 log PFUs/mL. In the 3 modes, the half-maximal effective concentration (EC50) of nano-realgar on HSV-2 infected Vero cells were 0.13, 1.80 and 0.52 mg/L, and the corresponding therapeutic index (TI) were 285.77, 20.64, 71.44, respectively. The TI value of nano-realgar on pre-treatment mode was higher than that of nano-realgar on treatment and direct inactivation modes. Conclusion: Nano-realgar can play a good anti-HSV-2 activity in the 3 modes (pre-treatment, treatment and direct inactivation), and the anti-HSV-2 efficacy of nano-realgar on pre-treatment mode is better than that of nano-realagr on other 2 modes.