Virus Titration Research Articles

Dual oxidase1 ameliorates survival, viral clearance and pulmonary pathology during influenza infection

Abstract Influenza virus infections cause severe morbidity and mortality, especially in high-risk patient populations. Bronchoepithelial cells orchestrate an oxidative antimicrobial system present in the airway liquid consisting of lactoperoxidase, the thiocyanate ion, and hydrogen peroxide. Dual Oxidase 1 (Duox1), an enzyme highly expressed in these cells, is the source of hydrogen peroxide. While in vitro studies suggest an anti-influenza, and a general antimicrobial role of Duox1, its antiviral role in vivo has not been addressed so far. We hypothesized that Duox1 has a protective effect in vivo against influenza. To test this hypothesis, we infected wild-type C57BL/6 and Duox1-deficient mice intranasally with the mouse-adapted A/Puerto Rico/8/1934 H1N1 (PR8) influenza virus strain. To evaluate the clinical and immunological significance of Duox1 in influenza infection, multicolor flow cytometry, multiplex ELISA, viral titration, histology and immunostaining techniques were used. Our results indicate that Duox1-deficient mice have increased mortality following influenza infection. Weight loss of Duox1-deficient mice post-infection was also significantly higher than in Duox1-expressing animals. Duox1 also limited influenza virus replication in the lung. Characterization of infiltrated leukocyte subsets following influenza infection revealed impaired natural killer cell recruitment in Duox1-deficient animals while other lymphoid or myeloid leukocyte subsets were not affected In summary, our results provide the first evidence for the in vivo antiviral role of Duox1, and also implicate that targeting the Duox1-based system has the potential to provide novel treatment or prophylactic strategies against influenza.

Zika Virus Infection Induces Elevation of Tissue Factor Production and Apoptosis on Human Umbilical Vein Endothelial Cells.

Zika virus (ZIKV) infection is typically characterized by a mild disease presenting with fever, maculopapular rash, headache, fatigue, myalgia, and arthralgia. A recent animal study found that ZIKV-infected pregnant Ifnar−/−mice developed vascular damage in the placenta and reduced amount of fetal capillaries. Moreover, ZIKV infection causes segmental thrombosis in the umbilical cord of pregnant rhesus macaques. Furthermore, several case reports suggest that ZIKV infection cause coagulation disorders. These results suggest that ZIKV could cause an alteration in the host hemostatic response, however, the mechanism has not been investigated thus far. This paper aims to determine whether ZIKV infection on HUVECs induces apoptosis and elevation of tissue factor (TF) that leads to activation of secondary hemostasis. We infected HUVECs with two ZIKV strains and performed virus titration, immunostaining, and flow cytometry to confirm and quantify infection. We measured TF concentrations with flow cytometry and performed thrombin generation test (TGT) as a functional assay to assess secondary hemostasis. Furthermore, we determined the amount of cell death using flow cytometry. We also performed enzyme-linked immunosorbent assay (ELISA) to determine interleukin (IL)-6 and IL-8 production and conducted blocking experiments to associate these cytokines with TF expression. Both ZIKV strains infected and replicated to high titers in HUVECs. We found that infection induced elevation of TF expressions. We also showed that increased TF expression led to shortened TGT time. Moreover, the data revealed that infection induced apoptosis. In addition, there was a significant increase of IL-6 and IL-8 production in infected cells. Here we provide in vitro evidence that infection of HUVECs with ZIKV induces apoptosis and elevation of TF expression that leads to activation of secondary hemostasis.

EHDV-2 Infection Prevalence Varies in Culicoides sonorensis after Feeding on Infected White-Tailed Deer over the Course of Viremia.

Epizootic hemorrhagic disease viruses (EHDVs) are arboviral pathogens of white-tailed deer and other wild and domestic ruminants in North America. Transmitted by various species of Culicoides, EHDVs circulate wherever competent vectors and susceptible ruminant host populations co-exist. The impact of variation in the level and duration of EHDV viremia in white-tailed deer (Odocoileus virginianus) on Culicoides infection prevalence is not well characterized. Here we examined how infection prevalence in a confirmed North American vector of EHDV-2 (Culicoides sonorensis) varies in response to fluctuations in deer viremia. To accomplish this, five white-tailed deer were experimentally infected with EHDV-2 and colonized C. sonorensis were allowed to feed on deer at 3, 5, 7, 10, 12, 14, 18, and 24 days post infection (dpi). Viremia profiles in deer were determined by virus isolation and titration at the same time points. Blood-fed Culicoides were assayed for virus after a 10-day incubation (27 °C) period. We found that increases in deer EHDV blood titers significantly increased both the likelihood that midges would successfully acquire EHDV and the proportion of midges that reached the titer threshold for transmission competence. Unexpectedly, we identified four infected midge samples (three individuals and one pool) after feeding on one deer 18 and 24 dpi, when viremia was no longer detectable by virus isolation. The ability of ruminants with low-titer viremia to serve as a source of EHDV for blood-feeding Culicoides should be explored further to better understand its potential epidemiological significance.

A flow cytometric granularity assay for the quantification of infectious virus

A flow cytometry-based assay was developed to assess the infective titer of two recombinant viruses: a recombinant herpes simplex type 2 (rHSV-2) and a recombinant canary pox (rALVAC.gfp). This method uses granularity of infected Vero and QT-35 cells, respectively, and correlates this to the infectious titer of virus samples. The percent of the cell populations with a high level of granularity could accurately be correlated to viral titers obtained through a traditional plaque assay, with R2 values greater than 0.8 using a semi-logarithmic scale. This approach offers a rapid, high-throughput method for infectious virus titration with similar accuracy to a traditional plaque assay.

Generation, Amplification, and Titration of Recombinant Respiratory Syncytial Viruses.

The use of recombinant viruses has become crucial in basic or applied virology. Reverse genetics has been proven to be an extremely powerful technology, both to decipher viral replication mechanisms and to study antivirals or provide development platform for vaccines. The construction and manipulation of a reverse genetic system for a negative-strand RNA virus such as a respiratory syncytial virus (RSV), however, remains delicate and requires special know-how. The RSV genome is a single-strand, negative-sense RNA of about 15 kb that serves as a template for both viral RNA replication and transcription. Our reverse genetics system uses a cDNA copy of the human RSV long strain genome (HRSV). This cDNA, as well as cDNAs encoding viral proteins of the polymerase complex (L, P, N, and M2-1), are placed in individual expression vectors under T7 polymerase control sequences. The transfection of these elements in BSR-T7/5 cells, which stably express T7 polymerase, allows the cytoplasmic replication and transcription of the recombinant RSV, giving rise to genetically modified virions. A new RSV, which is present at the cell surface and in the culture supernatant of BSRT7/5, is gathered to infect human HEp-2 cells for viral amplification. Two or three rounds of amplification are needed to obtain viral stocks containing 1 x 106 to 1 x 107 plaque-forming units (PFU)/mL. Methods for the optimal harvesting, freezing, and titration of viral stocks are described here in detail. We illustrate the protocol presented here by creating two recombinant viruses respectively expressing free green fluorescent protein (GFP) (RSV-GFP) or viral M2-1 fused to GFP (RSV-M2-1-GFP). We show how to use RSV-GFP to quantify RSV replication and the RSV-M2-1-GFP to visualize viral structures, as well as viral protein dynamics in live cells, by using video microscopy techniques.

High-Throughput Zika Viral Titer Assay for Rapid Screening of Antiviral Drugs.

Zika virus has recently emerged as a worldwide pathogen and public health burden due to its rapid spread and identification as a causative agent for multiple neurological defects, including congenital microcephaly. While there has been a flurry of recent research to address this emerging pathogen, there are currently no approved drug treatments for ZIKV infection. The gold standard for testing antiviral activity is to quantify infectious virion production. However, current infectious viral production assays, such as the plaque-forming or focus-forming unit assay, are tedious and labor intensive with a low-screening throughput. To facilitate drug development, we developed a Zika viral titration assay using an automated imaging system and an image analysis algorithm for viral colony quantification. This assay retained the principle of the classical virus titer assay, while improving workflow and offering higher screening throughput. In addition, this assay can be broadly adapted to quantification of other viruses.

Identification of the SAMHD1 gene in grass carp and its roles in inducing apoptosis and inhibiting GCRV proliferation

SAMHD1 is an innate immunity restriction factor that inhibits virus infection through IRF3-mediated antiviral and apoptotic responses. Fish SAMHD1 shares some similar properties with those in mammals. In this study, a SAMHD1 orthologue from grass carp (Ctenopharyngodon idellus) was cloned and characterized. The full-length cDNA of CiSAMHD1 is 2792 bp with an ORF of 1884 bp encoding a polypeptide of 627 amino acids. Multiple alignments showed that SAMHD1 is highly conserved among different species. Phylogenetic tree analysis revealed that CiSAMHD1 shared a high degree of homology with Sinocyclocheilus rhinocerous SAMHD1. Expression analysis indicated that CiSAMHD1 was widely expressed in all tissues tested including the brain, eyes, spleen, gill, intestine, liver, heart and kidney. It was significantly up-regulated in spleen, liver and intestines after treatment with poly I:C. Also, CiSAMHD1 can be induced following stimulation with recombinant IFN in CIK cells. The promoter sequence of CiSAMHD1 was identified to explore the mechanism underlying the transcriptional regulation of CiSAMHD1. The promoter sequence of CiSAMHD1 (1370 bp) consists of IRF1, IRF3, IRF9 and p65 binding elements. Gel mobility shift assay also showed that IRF1, IRF3, IRF9 and p65 prokaryotic proteins can separately interact with CiSAMHD1 promoter. Dual luciferase assay and q-PCR suggested that the promoter of CiSAMHD1 can be activated by the overexpression of CiIRF3 and CiIRF9, but cannot be triggered by CiIRF1 and Cip65. In contrast, knockdown of CiIRF3 or CiIRF9 inhibits the transcription of CiSAMHD1. Intriguingly, CCK assay suggested that CiSAMHD1 decreased cell viability. TUNEL apoptosis assay and Hoechst 33258 staining assay indicated that apoptosis is induced by the overexpression of CiSAMHD1. Crystal violet staining, detection of two GCRV genes (vp3 and vp5) and viral titration showed that CiSAMHD1 can suppress the proliferation of grass carp reovirus (GCRV) in CIK cells.

In vivo and ex vivo models of infectious bursal disease virus (IBDV) in inbred chicken lines differing in their resistance to the disease

Infectious bursal disease virus (IBDV) targets B cells in the bursa of Fabricius (BF), causing immunosuppression in chickens and mortality. Susceptibility differs between inbred chickens, with 0 % mortality in ‘resistant’ lines and up to 80 % mortality in ‘susceptible’ lines. However, the mechanism of disease resistance is not understood. In order to address this, chickens (n=18) from three ‘resistant’ lines (15, C and O) and one ‘susceptible’ line (W) were infected with the very virulent IBDV strain, UK661. Clinical scores were recorded and tissues harvested at necropsy on day one, two and three post-infection for RNA extraction and virus titration, compared to non-infected controls. Interestingly, within a given line, we observed a range of symptoms, with some individuals experiencing more severe disease than others, despite no difference in viral replication. Line 15 was the least susceptible to disease based on the average clinical scores (3.2 (15), 5.7 (C), 4.8 (O) and 4.7 (W)) and the percentage of birds with a clinical score of 2 or above (17 % (15), 100 % (C), 83 % (O) and 83 % (W)). The average peak virus replication was also significantly lower in line 15 birds (6.3 log10 fold change) compared to lines C or O (7.0 and 6.8 log10 fold change) (P<0.01). RNA-sequence analysis will be performed using BF samples to understand the biological pathways that confer IBDV resistance. Moreover, primary bursal cells harvested from resistant and susceptible lines were infected with IBDV ex vivo and ongoing work aims to quantify differentially expressed genes in these cells.

Viral replication and innate immunity of feline herpesvirus-1 virulence-associated genes in feline respiratory epithelial cells

Feline herpesvirus-1 (FHV-1) infection occurs worldwide and is a leading cause of respiratory and ocular diseases in cats. Current vaccines reduce the severity of symptoms but do not prevent infection and, therefore, do not provide defense against an establishment of latency and reactivation. We hypothesize that immunomodulation of FHV-1 is the cause of lack in protection and that deletion of virulence/immune modulatory genes of FHV-1 will enhance safety and immunogenicity. Our objective was to use feline respiratory epithelial cell (FREC) cultures to define in vitro growth characteristics and immunomodulation resulting from infection of FRECs with the virulent FHV-1 strain C27 (WT) and glycoprotein C-deletion (gC-), glycoprotein E-deletion (gE-), serine/threonine protein kinase-deletion (PK-), as well as gE and thymidine kinase-double-deletion (gE-TK-) mutants generated by bacterial artificial chromosome mutagenesis.Differentiated FRECs were mock inoculated or inoculated with WT, gC-, gE-, PK-, or gE-TK- mutants. Virus titration and real-time quantitative PCR assays were performed on samples collected at 1 hpi followed by 24 h intervals between 24 and 96 hpi to determine growth kinetics. Real-time PCR was used to quantitate IFNα, TNFα, IL-1β, IL-10, and TGFβ-specific mRNA levels. Immunoassays were performed to measure the protein levels of subsets of cytokines/chemokines secreted by FRECs.Inoculation of FRECs with gE-TK- resulted in significantly lower end-point titers than inoculation with WT or gE-. Both PK- and gC- inoculated FRECs also produced significantly lower end-point titers at 96 hpi than WT. Overall, intracellular virus titers were higher than those of extracellular virus. PCR results for viral DNA paralleled the virus titration results. Further, in contrast to WT inoculation, an increase in IFNα and IL-10 mRNA expression was not observed following inoculation with gE-TK- and PK-, but inoculation with gE-TK- and PK- did result in increased TGFβ expression in FRECs compared to responses following infection with WT. Moreover, gE-TK- and PK- blocked the inhibition of IL-8 and neutrophil chemoattractant (KC), which was observed following inoculation with WT.In summary, the results obtained in FRECs may be used to predict the safety and immunogenicity characteristics of these mutants in vivo. Our study highlights the value of the FREC system for studying replication kinetics/immune modulation factors of FHV-1 and screening prospective vaccine candidates before their use in experimental cats.

Unravelling the first key steps in equine herpesvirus type 5 (EHV5) pathogenesis using ex vivo and in vitro equine models

Equine herpesvirus type 5 (EHV5) is a ubiquitous, yet obscure pathogen in the horse population and is commonly associated with fatal equine multinodular pulmonary fibrosis (EMPF). To date, little is known about the precise pathogenesis of EHV5. Here, we evaluated the dynamics of EHV5 infection in representative ex vivo and in vitro equine models, using immunofluorescence staining and virus titration. EHV5 was unable to infect epithelial cells lining the mucosa of nasal and tracheal explants. Similarly, primary equine respiratory epithelial cells (EREC) were not susceptible to EHV5 following inoculation at the apical or basolateral surfaces. Upon direct delivery of EHV5 particles to lung explants, few EHV5-positive cell clusters were observed at 72 hours post-inoculation (hpi). These EHV5-positive cells were identified as cytokeratin-positive alveolar cells. Next, we examined the potential of EHV5 to infect three distinct equine PBMC populations (CD172a+ monocytes, CD3+ T lymphocytes and Ig light chain+ B lymphocytes). Monocytes did not support EHV5 replication. In contrast, up to 10% of inoculated equine T and B lymphocytes synthetized intracellular viral antigens 24 hpi and 72 hpi, respectively. Still, the production of mature virus particles was hampered, as we did not observe an increase in extracellular virus titer. After reaching a peak, the percentage of infected T and B lymphocytes decayed, which was partly due to the onset of apoptosis, but not necrosis. Based on these findings, we propose a model for EHV5 pathogenesis in the horse. Uncovering EHV5 pathogenesis is the corner step to finally contain or even eradicate the virus.

Differential susceptibility & replication potential of Vero E6, BHK-21, RD, A-549, C6/36 cells & Aedes aegypti mosquitoes to three strains of chikungunya virus.

Background & objectives:Chikungunya virus (CHIKV), a mosquito-borne arthritogenic virus causes infections ranging from febrile illness to debilitating polyarthralgia in humans. Re-emergence of the virus has affected millions of people in Africa and Asia since 2004. During the outbreak, a new lineage of the virus has evolved as an adaptation for enhanced replication and transmission by Aedes albopictus mosquito. A study was designed to compare the susceptibility of four vertebrate cell lines, namely Vero E6 (African green monkey kidney), BHK-21 (Baby hamster kidney), RD (human rhabdomyosarcoma), A-549 (human alveolar basal epithelial cell) and C6/36 (Ae. albopictus) to Asian genotype and two lineages of East, Central and South African (E1:A226 and E1:A226V) of CHIKV.Methods:One-step growth kinetics of different CHIKV strains was carried out in the above five cell lines to determine the growth kinetics and virus yield. Virus titre was determined by 50 per cent tissue culture infectious dose assay and titres were calculated by the Reed and Muench formula. Growth and virus yield of the three strains in Ae. aegypti mosquitoes was studied by intrathoracic inoculation and virus titration in Vero E6 cell line.Results:Virus titration showed Vero E6, C6/36 and BHK-21 cell lines are high virus yielding with all the three lineages while RD and A-549 yielded low virus titres. C6/36 cell line was the most sensitive and yielded the maximum titre. Ae. aegypti mosquitoes, when inoculated with high titre virus, yielded an almost equal growth with the three strains while rapid growth of E1:A226V and Asian strain was observed with 1 log virus.Interpretation & conclusions:C6/36 cell line was found to be the most sensitive and high yielding for CHIKV irrespective of lineages while Vero E6 and BHK-21 cell lines yielded high titres and may find application for vaccine/diagnostic development. Infection of Ae. aegypti mosquitoes with the three CHIKV strains gave almost identical pattern of growth.

Simultaneous zika and dengue serotype-4 viral detection and isolation from a donor plasma unit.

During zika and dengue viruse (ZIKV and DENV) outbreaks, the majority of the infected individuals remain clinically asymptomatic. Such asymptomatic individuals may occasionally acquire both arboviruses, donate blood, and contaminate haemoderivatives. The aim of this study was to characterize a ZIKV/DENV-4 coinfection in asymptomatic blood donor who donated blood during a large mixed ZIKV/DENV outbreak in the Säo Paulo State, Brazil. On the basis of post-donation information, one blood donor sample was found positive for ZIKV and DENV RNA. The DENV molecular serotyping was performed by molecular testing. The sample was also titrated on VERO E6 cells in order to define the presence of infectious arboviruses. The real-time PCR testing of the blood donor sample demonstrated very high viral load for both ZIKV and DENV. Further, molecular serotyping of DENV showed that the presence of DENV-4. The viral titration in cell culture indicated a titre of 2.75x10[6] PFU/ml which was concordant with the presence of infectious viruses in the blood donation. This is an interesting report for the simultaneous presence of infectious ZIKV and DENV-4 in asymptomatic blood sample. Special attention must be paid during mixed arboviral outbreaks for the possibility of transfusion-transmission of multiple arboviral agents.

Generation of Yellow Fever virus vaccine in skeletal muscle cells of chicken embryos

BACKGROUND The Yellow Fever (YF) vaccine is produced by the inoculation of embryonated chicken eggs with YF17DD virus on the ninth day of development. Full embryos are collected on the twelfth day of development for vaccine formulation. Skeletal muscle tissue is the main site where biosynthesis of viral particles occurs.OBJECTIVES This study aimed to analyse the experimental infection of skeletal muscle cells of chicken embryos by the 17DD Yellow Fever virus (YFV) in vivo and in vitro.METHODS Chicken embryos infected with YF17DD virus were analysed by immunofluorescence using confocal and super-resolution microscopes. Primary cultures of skeletal muscle cells of non-infected chicken embryos were evaluated for susceptibility and permissiveness to YF17DD virus using different protocols. This evaluation was performed based on morphological, viral titration, molecular biology, and colorimetric techniques.FINDINGS The present work phenotypically characterises embryonic chicken skeletal muscle cells as myogenic precursors expressing the Pax7 transcription factor in some cases. We demonstrated that these cells are susceptible to in vitro infection at different multiplicities of infection (MOIs), reproducing the same infection pattern observed in vivo. Furthermore, myogenic precursors and myoblasts are preferred infection targets, but establishment of infection does not depend on the presence of these cells. The peak of viral production occurred at 48 hpi, with decay occurring 72 hpi, when the cytopathic effect can be observed.MAIN CONCLUSIONS In conclusion, the primary culture of chicken skeletal muscle cells is a good model for studying muscle cells infected with YF17DD virus. This culture system displays satisfactory emulation of the in vitro phenomenon observed, contributing to our understanding of virus infection dynamics and leading to the development of alternative methods of vaccine production.

Growth and Purification of Vaccinia Virus Stocks for MPM Imaging.

This chapter provides methods for the propagation, purification, and titration of vaccinia virus (VACV) and the highly attenuated strain-modified vaccinia Ankara (MVA). Additionally, we provide information on VACV recombinants we have used for intravital imaging with multiphoton excitation.

ВИДІЛЕННЯ БАКТЕРІОФАГУ З ЛІТИЧНОЮ АКТИВНІСТЮ ПРОТИ НОВОЇ ІДЕНТИФІКОВАНОЇ PANTOEA AGGLOMERANS

In a consequence of agricultural human activity, a set of phytopathogenic bacteria gain new properties and ability to cause diseases in animal and human organisms. Moreover, bacterial loss of sensitivity to antibiotics becomes more increasing threat. The most effective alternative method of processing of plants are bacteriophages. The aim of this work is isolation and identification of a vegetable enterobacteria and search of its specific bacteriophages. Methods: biochemical identification of bacteria, analysis on sensitivity to antibiotics by means of disks, titration and accumulation of virus, electronic microscopy. Results: from onions samples with symptoms of a bacteriosis several bacteria were isolated. One of them was identified as Pantoea agglomerans. The sensitivity of this isolate to antibiotics was investigated, the resistance to cefalexin and norfloxacin is revealed. The bacteriophage specific to this bacteria is isolated from waste waters. The morphology of a bacteriophage is investigated by means of electronic microscopy, the virus belongs to the Myoviridae family. Phytopathogenic properties of bacteria and the antibacterial activity of phage isolate were investigated on potatoes in vitro. P. agglomerans led to development of a bacteriosis on potatoes cubes, and the isolated bacteriophage successfully inhibited its growth. Conclusions: This study demonstrated that common vegetables such as onions could be a source of human pathogenic bacteria. In this work, we isolated P.agglomerans, member of family Enterobacteriaceae. Taking into account that this bacteria was unsensitive to some antibiotics, it can be regarded as an alarming sign. The use of bacteriophages could solve problems of antimicriobial resistance and protecting of crops from bacterial infections. Isolated bacteriophage from waste waters inhibited growth of P.agglomerans in vitro showing that it could be considered as a part of phage drugs.

Development of reverse genetics systems and investigation of host response antagonism and reassortment potential for Cache Valley and Kairi viruses, two emerging orthobunyaviruses of the Americas.

Orthobunyaviruses such as Cache Valley virus (CVV) and Kairi virus (KRIV) are important animal pathogens. Periodic outbreaks of CVV have resulted in the significant loss of lambs on North American farms, whilst KRIV has mainly been detected in South and Central America with little overlap in geographical range. Vaccines or treatments for these viruses are unavailable. One approach to develop novel vaccine candidates is based on the use of reverse genetics to produce attenuated viruses that elicit immune responses but cannot revert to full virulence. The full genomes of both viruses were sequenced to obtain up to date genome sequence information. Following sequencing, minigenome systems and reverse genetics systems for both CVV and KRIV were developed. Both CVV and KRIV showed a wide in vitro cell host range, with BHK-21 cells a suitable host cell line for virus propagation and titration. To develop attenuated viruses, the open reading frames of the NSs proteins were disrupted. The recombinant viruses with no NSs protein expression induced the production of type I interferon (IFN), indicating that for both viruses NSs functions as an IFN antagonist and that such attenuated viruses could form the basis for attenuated viral vaccines. To assess the potential for reassortment between CVV and KRIV, which could be relevant during vaccination campaigns in areas of overlap, we attempted to produce M segment reassortants by reverse genetics. We were unable to obtain such viruses, suggesting that it is an unlikely event.

In-vitro inhibition of spring viremia of carp virus replication by RNA interference targeting the RNA-dependent RNA polymerase gene

Spring viremia of carp, a fatal viral disease, is caused by the spring viremia of carp virus (SVCV) and can result in up to 70% mortalities in common carps and significant economic losses in several other cyprinid aquaculture. The present study aimed to investigate the possible control of SVCV replication in Epithelioma papulosum cyprini (EPC) cells using the RNA interference technology targeting the RNA-dependent RNA polymerase (L) gene of the SVCV that is essential for its replication. Three stealth small interfering RNA (siRNA) sequences were designed to target three different regions on the SVCV-L gene. The specific siRNAs designed were investigated individually or in combinations to inhibit the SVCV-L gene expression and the virus replication. Results showed that the most effective siRNA sequence was the siRNA-602 that specifically reduced the SVCV replication by two logs as indicated by the virus titration and quantitative real-time PCR. Results, also, showed that the minimum effective concentration of siRNA-602 was 20 nM when used to transfect the EPC cells before the virus inoculation. Results of this study clearly indicate that targeting the SVCV-L gene by RNAi can reduce the SVCV replication in vitro, that may lead to the control of SVCV in fish.

A novel cell line established from caudal fin tissue of Carassius auratus gibelio is susceptible to cyprinid herpesvirus 2 infection with the induction of apoptosis

Herpesviral haematopoietic necrosis induced by cyprinid herpesvirus 2 (CyHV-2) in Carassius auratus gibelio has caused huge economic losses in aquaculture. To better characterize this pathogen at molecular and cellular levels, it is necessary to study its pathogenesis in a sustainable cell line. In this study, a new cell line (GiCF) from the caudal fin of Carassius auratus gibelio (C. a. gibelio) was established and characterised. Typical cytopathic effect represented by characteristic morphological changes was induced in the cells at the 7th day post infection with CyHV-2, which was isolated from diseased C. a. gibeli. The susceptibility of GiCF to CyHV-2 was further demonstrated by the presence of viral assembling factory in the cytoplasm unveiled by transmission electron microscopy, and viral protein synthesis through immunofluorescence and Western blot assays. Sustainable propagation of CyHV-2 in GiCF had been demonstrated by virus infection and titration assays in a consecutive manner. The virus titer reached 104.9±0.22 TCID50/ml, and infectious CyHV-2 was produced from the GiCF cells over 30 subcultures from the established cell lines. In addition, apoptosis was observed in the GiCF cells upon CyHV-2 infection by DAPI staining, TUNEL and Annexin V staining, and quantitative expression patterns of caspase genes suggested that caspase activation might involve in CyHV-2 induced apoptosis. In summary, a new cell line, GiCF, susceptible to CyHV-2 infection was established from caudal fin tissue of C. a. gibelio, and it should serve as an ideal infection platform to characterize CyHV-2 and its pathogenic effect including apoptosis.

Quantitative Flow Cytometry to Measure Viral Production Using Infectious Pancreatic Necrosis Virus as a Model: A Preliminary Study

In recent decades, flow cytometry (FCM) has become an important tool in virology, due to its applications in viral replication and viral-cell interactions, as well as its capacity to quantify proteins (qFCM). In the present study, we have designed and evaluated a qFCM procedure for the in vitro analysis and quantification of fish viral proteins, using the infectious pancreatic necrosis virus (IPNV) as a model. We have also tested its use for viral titration and adapted the MARIS (method for analysing RNA following intracellular sorting) method for simultaneous quantification of viral RNA expression in infected cells. The procedure has proved to be repeatable and reproducible to an acceptable level, although to ensure reproducibility, the repetition of standard curves is inevitable. Regarding its use for viral quantification, a direct relationship (by a second-degree polynomial regression) between viral titres and Molecules of Equivalent Soluble Fluorochrome (MESF) was observed. Finally, the results support the use of this technology, not only for virus quantification, but also to study viral replication from a quantitative approach.

Differential antiviral activity of European sea bass interferon-stimulated 15 protein (ISG15) against RGNNV and SJNNV betanodaviruses

ISG15 is an antiviral protein acting intracellularly, by conjugation to viral or cellular proteins, or extracellularly, as cytokine. In this work, an in vitro system, consisting of E-11 cells over-expressing European sea bass ISG15 (Dl_ISG15_E11 cells), has been developed to evaluate the European sea bass ISG15 protein activity against RGNNV and SJNNV isolates. Regarding RGNNV, RNA2 copy number and viral titres were similar in E-11 and Dl_ISG15_E11 cells, and the cellular survival analyses demonstrated that Dl_ISG15_E11 cells were not protected from this virus. In contrast, ISG15 compromises SJNNV replication, since a reduction of the SJNNV genome synthesis has been recorded. The ISG15 anti-SJNNV activity was confirmed by viral titration and survival assays. In addition, a role of the intracellular ISG15 in modulating the transcription of endogenous genes has being recorded, with tlr3 gene being knocked out and e3 gene being up-regulated in RGNNV-inoculated Dl_ISG15_E11 cells. Sea bass ISG15 has also been detected extracellularly, and its activity has been evaluated by co-culture. The survival rate of RGNNV-inoculated E-11 cells increased from 25% to 46% when they were co-cultured with ISG15-producing cells. Similarly, the survival rate of SJNNV-inoculated E-11 cells increased from 27% to 51% in co-culture with ISG15-producing cells. To our knowledge, this is the first description of a differential antiviral activity of an ISG15 protein against two betanodavirus species, and the first evaluation of the cytokine-like activity of a fish ISG15 protein on non-immune cells.