Infected Cell Culture Supernatant Research Articles

Purification of Epizootic Hemorrhagic Disease Virus (and Other Orbiviruses) Particles from Infected Mammalian or Insect Cells.

Epizootic hemorrhagic disease virus (EHDV), like other orbiviruses, infects and replicates in mammalian and insect vector cells. Within its ruminant hosts EHDV, like bluetongue virus (BTV), it has mainly been associated with infection of endothelial cells of capillaries as well as leukocyte subsets. Furthermore, EHDV infects and replicates within its biological vector, Culicoides biting midges and Culicoides-derived cells. A wide range of common laboratory cell lines such as BHK, BSR, and Vero cells are susceptible to infection with certain EHDV strains. Cell culture supernatants of infected cells are commonly used for both in vivo and in vitro infection studies. For specific virological or immunological studies, using highly purified virus particles, however, might be beneficial or even required. Here we describe a purification method for EHDV particles, which had been originally developed for certain strains of BTV.

Virus purification highlights the high susceptibility of SARS-CoV-2 to a chlorine-based disinfectant, chlorous acid.

Chlorous acid water (HClO2) is known for its antimicrobial activity. In this study, we attempted to accurately assess the ability of chlorous acid water to inactivate SARS-CoV-2. When using cell culture supernatants of infected cells as the test virus, the 99% inactivation concentration (IC99) for the SARS-CoV-2 D614G variant, as well as the Delta and Omicron variants, was approximately 10ppm of free chlorine concentration with a reaction time of 10 minutes. On the other hand, in experiments using a more purified virus, the IC99 of chlorous acid water was 0.41-0.74ppm with a reaction time of 1 minute, showing a strong inactivation capacity over 200 times. With sodium hypochlorite water, the IC99 was 0.54ppm, confirming that these chlorine compounds have a potent inactivation effect against SARS-CoV-2. However, it became clear that when using cell culture supernatants of infected cells as the test virus, the effect is masked by impurities such as amino acids contained therein. Also, when proteins (0.5% polypeptone, or 0.3% BSA + 0.3% sheep red blood cells, or 5% FBS) were added to the purified virus, the IC99 values became high, ranging from 5.3 to 76ppm with a reaction time of 10 minutes, significantly reducing the effect. However, considering that the usual usage concentration is 200ppm, it was shown that chlorous acid water can still exert sufficient disinfection effects even in the presence of proteins. Further research is needed to confirm the practical applications and effects of chlorous acid water, but it has the potential to be an important tool for preventing the spread of SARS-CoV-2.

Abstract 1885: Selection of an optimal anti-PD-L1 single domain antibody format for the vectorization into oncolytic vaccinia virus and the generation of bispecific immunomodulators

Abstract Background: Single domain antibodies (sdAbs) isolated after immunization of camelids are particularly attractive formats for their high modularity and small size allowing a better diffusion into tumors. However, the short in vivo half-life of sdAbs, related to the lack of a Fc domain, limits their clinical application. By replicating specifically into tumor cells, the oncolytic vaccinia virus (VACV) is an optimal vector to deliver and maintain high intra-tumoral concentrations of therapeutic sdAb. Moreover, sdAb targeting immunological targets, such as PD-L1, may synergize the anti-tumoral activity of VACV. Randox and Transgene report the selection and characterization of a sdAb targeting the human PD-L1 and the design of optimal formats, including bispecific anti-PD-L1-CD40 ligand, for vectorization into VACV. Methods: Alpacas were immunized with human PD-L1 protein and sdAb coding sequences were isolated by PCR. Anti-PD-L1 sdAb binders were selected by phage display and sdAb blockers of PD-L1/PD-1 interaction were identified by ELISA. The ability of the selected sdAb to disrupt the PD-L1/PD-1 interaction was verified on transformed and primary cells. To fine-tune an optimal anti-PD-L1, several sdAb formats were designed and vectorized into VACV. The sdAb format exhibiting the best PD-L1/PD-1 blocking activity was selected by the screening of culture supernatants of several VACV-sdAb infected tumor cells. Finally, anti-PD-L1 sdAb-CD40L fusions were designed to generate a strong CD40 agonist active only in a PD-L1 positive environment. Results: SdAb clone 1A1 exhibited the best PD-L1/PD-1 blocking activity which remained unchanged after extensive humanization (latterly becoming named GS542). GS542 was vectorized in VACV as monomeric single chain homodimer, and fused to Fc domain or antibody hinge domain to foster dimerization together with full length IgG1 avelumab as anti-PD-L1 benchmark. All constructs were expressed by infected tumor cells. The single chain homodimer displayed the best PD-L1/PD-1 blocking activity, superior to that of avelumab. Moreover, GS542-CD40L fusions were designed to take advantage of the natural trimerization of CD40L to increase the avidity for PD-L1 while clustering CD40L at the surface of PD-L1+ cells to trans-activate the CD40 pathway. Evaluation of these GS542-CD40L fusions showed strong CD40 agonist activity depending on the presence of PD-L1+ cells making these constructs safer CD40 agonists. Conclusion: An anti-PD-L1 sdAb with a strong blocking activity was selected, humanized and evaluated under different VACV-vectorized formats. The single chain homodimeric sdAb expressed by VACV was identified as the best PD-L1/PD-1 blocking format. Furthermore, bispecific anti-PD-L1 sdAb-CD40L fusions that exhibited strong CD40 agonist activity was charactered within a PD-L1+ environment. Citation Format: Jean-Baptiste Marchand, Elodie Pintado, Marshall Dunlop, Christelle Remy, Patricia Kleinpeter, Shirley Shön, Fend Laetitia, Renée Brandely, Delphine Suhner, Eline Winter, Nathalie Silvestre, Claire Huguet, Peter Fitzgerald, Eric Quéméneur. Selection of an optimal anti-PD-L1 single domain antibody format for the vectorization into oncolytic vaccinia virus and the generation of bispecific immunomodulators [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1885.

First report of Duck Hepatitis A virus genotype 2 in India.

Sudden death of ducklings was reported in a duck farm located at Tiruvallur district in Tamil Nadu, India. Disease investigation began with post mortem findings of dead birds revealing enlarged pale-pink / pale-yellow liver with multifocal petechiae and ecchymosis. A positive amplification with duck hepatitis A virus specific primers by reverse transcription-polymerase chain reaction (RT-PCR) on the tissue samples collected from dead birds indicated infection by duck hepatitis A virus (DHAV), an avian picornavirus, known to cause acute and high-mortality in ducklings. The virus isolation was successful in 9-days old embryonated chicken eggs, in primary chicken embryo fibroblast (CEF) cells and from experimentally infected ducklings. The embryonic death on day 5 to 7 post inoculation in chicken embryos with signs of cutaneous hemorrhage, edema and greenish yellow liver together with histopathology of embryonic liver and kidney further confirmed DHAV infection. TEM analysis of the infected allantoic fluid and infected CEF cell culture supernatant showed the presence of spherical shaped, non-enveloped virion particles of ~ 20-38nm diameter, typical for DHAV. Experimental infection of ducklings with RT-PCR positive tissue supernatant caused 40% to 50% mortality with typical petechial hemorrhages on the surface of liver. Further, histopathological analysis and RT-PCR of the inoculated duckling's tissues confirmed the presence of DHAV. Nucleotide sequencing of the 5'UTR region and VP1 region confirmed duck hepatitis A virus genotype 2 (DHAV-2). To the best of our knowledge, this is the first report of laboratory confirmation of DHAV-2 in India. This study warrants the need for the extensive epidemiological surveillance to understand the prevalence of DHAV-2 in India and to take appropriate control measures to curtail the disease spread.

Limited environmental stability of infectious porcine endogenous retrovirus type C; Usage of reverse transcriptase in combination with viral RNA as markers for infectious virus.

Porcine endogenous retroviruses (PERVs) are an integral part of the pig genome with infectious potential, as shown in vitro. In view of nonclinical and clinical xenotransplantation, data are essential that give an insight into viral pathogenicity. This includes PERV's environmental stability and environmental risk. We analyzed two ecotropic PERV-C (PERV-C[1312] and -[5683]), monitoring cell-free culture supernatants of infected ST-IOWA cells at various time intervals at room temperature (22°C +/-1°C). The virus was stored in the presence or absence of sterile wood litter, as used for large animal husbandry. This approach was set to determine the environmental stability of exogenous PERV-C at defined conditions for the first time. Reverse transcriptase (RT) activity and viral RNA were monitored for up to 57 days and remaining infectivity of supernatant without wood litter was tested from day 7 onwards on naïve ST-IOWA cells. Results show that viral RNA decreases but remains detectable over the whole observation period, whereas RT activity showed 83%-96% reduction from day 7 on. This effect was stronger in the presence of wood litter and fresh harvested virus was more stable than frozen virus stocks. Even under these optimal conditions, no infectivity was shown for viral RNA-positive and RT-reduced supernatant harvested at day7. The results confirm that PERV-C is less stable and the reduction of RT activity is accompanied by reduced infectivity, independently of existing viral RNA. The combination of both RT and viral RNA measurement is a suitable method to differentiate infectious PERV-C.

Emerging SARS-CoV-2 Genotypes Show Different Replication Patterns in Human Pulmonary and Intestinal Epithelial Cells.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) quickly spread worldwide following its emergence in Wuhan, China, and hit pandemic levels. Its tremendous incidence favoured the emergence of viral variants. The current genome diversity of SARS-CoV-2 has a clear impact on epidemiology and clinical practice, especially regarding transmission rates and the effectiveness of vaccines. In this study, we evaluated the replication of different SARS-CoV-2 isolates representing different virus genotypes which have been isolated throughout the pandemic. We used three distinct cell lines, including Vero E6 cells originating from monkeys; Caco-2 cells, an intestinal epithelium cell line originating from humans; and Calu-3 cells, a pulmonary epithelium cell line also originating from humans. We used RT-qPCR to replicate different SARS-CoV-2 genotypes by quantifying the virus released in the culture supernatant of infected cells. We found that the different viral isolates replicate similarly in Caco-2 cells, but show very different replicative capacities in Calu-3 cells. This was especially highlighted for the lineages B.1.1.7, B.1.351 and P.1, which are considered to be variants of concern. These results underscore the importance of the evaluation and characterisation of each SARS-CoV-2 isolate in order to establish the replication patterns before performing tests, and of the consideration of the ideal SARS-CoV-2 genotype–cell type pair for each assay.

Validation of Inactivation Methods for Arenaviruses.

Several of the human-pathogenic arenaviruses cause hemorrhagic fever and have to be handled under biosafety level 4 conditions, including Lassa virus. Rapid and safe inactivation of specimens containing these viruses is fundamental to enable downstream processing for diagnostics or research under lower biosafety conditions. We established a protocol to test the efficacy of inactivation methods using the low-pathogenic Morogoro arenavirus as surrogate for the related highly pathogenic viruses. As the validation of chemical inactivation methods in cell culture systems is difficult due to cell toxicity of commonly used chemicals, we employed filter devices to remove the chemical and concentrate the virus after inactivation and before inoculation into cell culture. Viral replication in the cells was monitored over 4 weeks by using indirect immunofluorescence and immunofocus assay. The performance of the protocol was verified using published inactivation methods including chemicals and heat. Ten additional methods to inactivate virus in infected cells or cell culture supernatant were validated and shown to reduce virus titers to undetectable levels. In summary, we provide a robust protocol for the validation of chemical and physical inactivation of arenaviruses in cell culture, which can be readily adapted to different inactivation methods and specimen matrices.

Characterization of red-spotted grouper nervous necrosis virus isolated from ovarian fluids of asymptomatic wild Asian seabass, Lates calcarifer

Viral nervous necrosis (VNN) caused by nervous necrosis virus is a serious disease affecting a wide range of marine, brackishwater and freshwater fishes. The disease causes acute mortalities in larval and early juvenile stages while adult fishes are asymptomatic and remain as carriers of the virus. In the present study, nervous necrosis virus was isolated from ovarian fluids of asymptomatic wild-collected Asian seabass, Lates calcarifer in SSN-1 cell line. The complete genome of the virus was amplified by PCR from plaque purified virus and sequenced. Bioinformatic analysis of the sequence revealed that the isolate belonged to red-spotted grouper nervous necrosis virus. The virus purified from infected SSN-1 cell culture supernatants by cesium chloride gradient ultracentrifugation resulted in two bands in the gradient. Both the bands on SDS-PAGE analysis and western blot revealed a single 40 kDa capsid protein. The virus replication was optimum at 26–28 °C in vitro and the virus can be completely inactivated using 3 mM binary ethylenimine in 32 h at 25 °C. The isolated virus was infectious and caused 100% mortality in Asian seabass larvae. The inactivated virus when injected to Asian seabass fingerlings produced neutralizing antibody titre ranging from 1:320 to 1: 1280. The isolate is a potential candidate for production of an inactivated vaccine against VNN.

Isolation of peste des petits ruminants virus using primary goat kidney cell culture from kidneys obtained at slaughter.

BackgroundTraditionally isolation of peste des petits ruminant virus (PPRV) is performed in Vero cells that takes several blind passages before observing typical cytopathic effects (CPEs). As an alternate, researchers have been using lamb kidney (LK) cells but day‐old lambs are difficult to obtain and requires animal sacrifice.ObjectiveWe established a primary goat kidney (GK) cell culture from the kidneys obtained at slaughter.MethodsThe kidney of Black Bengal goats were collected from slaughter house and processed to make single cell suspension. The cells were resuspended in appropriate culture medium and maintained under optimum culture condition.ResultsThe 80% confluent monolayer of GK cells was obtained after 15–20 days post seeding. Upon infection with a field isolate of PPRV, the well‐developed CPEs characterized by cell rounding, vacuolation in the cytoplasm and fusion of cells were observed after 48 hr post infection. Virus quantification in the culture supernatant revealed more viral RNA in GK cells than LK cells. The multicycle growth analysis of PPRV showed a steady increase in the virus loads in the culture supernatant of infected GK cells, suggesting an adaptation of the PPRV in GK cells.ConclusionsThe findings suggest that primary GK cells can be successfully prepared from the mature kidney cortical tissues and can be used for the isolation of PPRV. This system could reduce the unnecessary sacrifice of lambs or kids. Since kidneys of slaughtered goats are available throughout the year, using this protocol primary cell culture from mature goat kidney can provide primary cells to the laboratory throughout the year.

Heat Inactivation of Different Types of SARS-CoV-2 Samples: What Protocols for Biosafety, Molecular Detection and Serological Diagnostics?

Standard precautions to minimize the risk of SARS-CoV-2 transmission implies that infected cell cultures and clinical specimens may undergo some sort of inactivation to reduce or abolish infectivity. We evaluated three heat inactivation protocols (56 °C-30 min, 60 °C-60 min and 92 °C-15 min) on SARS-CoV-2 using (i) infected cell culture supernatant, (ii) virus-spiked human sera (iii) and nasopharyngeal samples according to the recommendations of the European norm NF EN 14476-A2. Regardless of the protocol and the type of samples, a 4 Log10 TCID50 reduction was observed. However, samples containing viral loads > 6 Log10 TCID50 were still infectious after 56 °C-30 min and 60 °C-60 min, although infectivity was < 10 TCID50. The protocols 56 °C-30 min and 60 °C-60 min had little influence on the RNA copies detection, whereas 92 °C-15 min drastically reduced the limit of detection, which suggests that this protocol should be avoided for inactivation ahead of molecular diagnostics. Lastly, 56 °C-30 min treatment of serum specimens had a negligible influence on the results of IgG detection using a commercial ELISA test, whereas a drastic decrease in neutralizing titers was observed.

Production and characterization of a monoclonal antibody against the sialidase of Gardnerella vaginalis using a synthetic peptide in a MAP8 format.

Bacterial vaginosis is one of the most frequent vaginal infections. Its main etiological agent is Gardnerella vaginalis, which produces several virulence factors involved in vaginal infection and colonization, in particular, sialidase (SLD), a potential clinical biomarker that participates in immune response modulation and mucus degradation. The main objective of this work was the production and evaluation of a monoclonal antibody against G. vaginalis sialidase and its validation in immunoassays. For immunization of mice, a synthetic multiantigenic peptide was used, and hybridomas were generated. After fusion, hybridomas were evaluated for antibody production and cloned by limited dilution. One clone producing IgG1 was selected and characterized by indirect ELISA, dot blot, and Western blot, and we also tested clinical isolates and HeLa cells infected with G. vaginalis. The results showed that the anti-SLD antibody recognized a single protein of ~90 kDa that correlated with the estimated molecular weight of SLD. In addition, anti-SLD antibody recognized SLD from complete bacteria and from culture supernatants of infected Hela cells. In conclusion, our results showed that the anti-SLD antibody recognized SLD from different sources and could be considered a new tool for the diagnosis of bacterial vaginosis.Key Points • Anti-sialidase mAb was generated using a synthetic peptide • The mAb recognizes synthetic peptide and intact protein from multiple sources • The antibody was characterized by several immunological methods

Development and characterization of a skin cell line (SGA) from the mosquitofish Gambusia affinis and its susceptibility to fish Betanodavirus

A new continuous cell line designated as SGA has been developed from the skin tissue of the freshwater mosquitofish Gambusia affinis. The cell line grew well in Leibovitz's L-15 medium supplemented with 15% fetal bovine serum at 28 °C. Immunophenotyping of the cell line showed the epithelial nature of the cells. Chromosome number analysis showed that SGA cells have a modal diploid chromosome number of 48. Replication of the two different strains of betanodavirus (RGNNV and SJNNV) in SGA cell line showed the maximum virus titre of 108.82 TCID50 mL−1 for RGNNV and 107.2 TCID50 mL−1 for SJNNV. The cytopathic effect in the cell line was observed at 3 days post-infection (dpi) and multiple vacuolations were observed at 7 dpi. Further, DsRed2 plasmid was efficiently delivered into SGA cell line and was found that these cells were transfectable and can be used for assessing gene promoter activity. In vivo challenge experiments using the RGNNV infected cell culture supernatant showed signs of the disease in healthy mosquitofish with mortality commencing from 15 dpi. The above results suggest that the cell line is permissive for propagating betanodavirus and also could be an essential tool for studying the molecular pathogenesis of betanodavirus infection.

Characterization of a novel rhabdovirus isolated from a stranded harbour porpoise (Phocoena phocoena)

An adult male harbour porpoise (Phocoena phocoena) stranded off the coast of Alaska displaying poor body condition, scattered mild ulcerative dermatitis, and necrotizing balanoposthitis. Necropsy findings included severe verminous panniculitis, pneumonia, hepatitis, and enteritis. Histopathological examination of skin lesions revealed a pustular epidermitis and dermatitis, with ballooning degeneration of keratinocytes and occasional amphophilic intracytoplasmic inclusion bodies. A swab sample collected from the ulcerative penile lesions was processed for virus isolation resulting in cytopathic effects observed in primary beluga whale kidney (BWK) cells. Transmission electron microscopy revealed bullet-shaped virions budding from the cell surface of infected BWK cells consistent with a rhabdovirus. A cDNA library was prepared using RNA extracted from infected cell culture supernatant and sequenced on an Illumina MiSeq sequencer. The near-complete genome of a novel rhabdovirus was recovered. Genetic and phylogenetic analyses based on the complete L gene supported the harbour porpoise rhabdovirus (HPRV) as a new species. HPRV clustered phylogenetically with dolphin rhabdovirus (DRV) and this cetacean rhabdovirus clade was found to be the sister group to members of the genus Perhabdovirus that infect fish. A specific nested RT-PCR assay detected HPRV RNA in the epaxial musculature of the harbour porpoise. Our results are consistent with a previous hypothesis that cetacean rhabdoviruses may have arisen following a host jump from fish and suggest that DRV and HPRV represent separate species belonging in a new genus within the family Rhabdoviridae. Further research is needed to determine the health impact of HPRV in harbour porpoise populations, its prevalence, and route of transmission.

Measuring Dengue Virus RNA in the Culture Supernatant of Infected Cells by Real-time Quantitative Polymerase Chain Reaction

At present, real-time polymerase chain reaction (PCR) technology is an indispensable tool for the detection and quantification of viral genomes in research laboratories, as well as for molecular diagnosis, because of its sensitivity, specificity, and convenience. However, in most cases, the quantitative PCR (qPCR) assay generally used to detect virus infection has relied on the purification of viral nucleic acid prior to the PCR step. In this study, the fluorescence-based reverse transcription qPCR (RT-qPCR) assay is developed through the combination of a processing buffer and a one-step RT-PCR reagent so that the whole process, from the harvest of the culture supernatant of virus-infected cells until real-time detection, can be performed without viral RNA purification. The established protocol enables the quantification of a wide range of RNA concentrations of dengue virus (DENV) within 90 min. In addition, the adaptability of the direct RT-qPCR assay to the evaluation of an antiviral agent is demonstrated by an in vitro experiment using a previously reported DENV inhibitor, mycophenolic acid (MPA). Moreover, other RNA viruses, including yellow fever virus (YFV), Chikungunya virus (CHIKV), and measles virus (MeV), can be quantified by direct RT-qPCR with the same protocol. Therefore, the direct RT-qPCR assay described in this report is useful for monitoring RNA virus replication in a simple and rapid manner, which will be further developed into a promising platform for a high-throughput screening study and clinical diagnosis.

Measuring Dengue Virus RNA in the Culture Supernatant of Infected Cells by Real-time Quantitative Polymerase Chain Reaction.

At present, real-time polymerase chain reaction (PCR) technology is an indispensable tool for the detection and quantification of viral genomes in research laboratories, as well as for molecular diagnosis, because of its sensitivity, specificity, and convenience. However, in most cases, the quantitative PCR (qPCR) assay generally used to detect virus infection has relied on the purification of viral nucleic acid prior to the PCR step. In this study, the fluorescence-based reverse transcription qPCR (RT-qPCR) assay is developed through the combination of a processing buffer and a one-step RT-PCR reagent so that the whole process, from the harvest of the culture supernatant of virus-infected cells until real-time detection, can be performed without viral RNA purification. The established protocol enables the quantification of a wide range of RNA concentrations of dengue virus (DENV) within 90 min. In addition, the adaptability of the direct RT-qPCR assay to the evaluation of an antiviral agent is demonstrated by an in vitro experiment using a previously reported DENV inhibitor, mycophenolic acid (MPA). Moreover, other RNA viruses, including yellow fever virus (YFV), Chikungunya virus (CHIKV), and measles virus (MeV), can be quantified by direct RT-qPCR with the same protocol. Therefore, the direct RT-qPCR assay described in this report is useful for monitoring RNA virus replication in a simple and rapid manner, which will be further developed into a promising platform for a high-throughput screening study and clinical diagnosis.

Derivation of two tilapia (Oreochromis niloticus) cell lines for efficient propagation of Tilapia Lake Virus (TiLV)

Tilapia Lake Virus (TiLV) has been associated with disease outbreaks in cultured tilapia worldwide. In this study, we developed and characterized two cell lines from the brain (OnlB) and liver (OnlL) of the Nile tilapia, Oreochromis niloticus for the efficient propagation of TiLV. Both the cells grew well in Leibovitz's – 15 (L-15) medium supplemented with 20% fetal bovine serum (FBS) and have been sub-cultured more than 45 times. Chromosome analysis of the cells revealed that both lines had normal diploid number (2n = 44). TiLV was isolated from diseased tilapia and continuously propagated for 20 passages in these cell lines. The maximum TiLV titer was 107.3±0.05 and 107.0±0.96 TCID50/ml, in OnlL and OnlB respectively. The TiLV isolate consistently produced the same CPE in all passages. In vivo challenge experiments using the TiLV infected cell culture supernatant reproduced symptoms of the disease in healthy tilapia, with mortality commencing 10 days post-infection and we were able to isolate TiLV from the challenged fish The above results suggest both cell lines are highly permissive for propagating TiLV and could be important tools for studying the molecular pathogenesis of TiLV infection.

Method for simple and rapid concentration of Zika virus particles from infected cell-culture supernatants

Experimental studies on Zika virus (ZIKV) may require improvement of infectious titers in viral stocks obtained by cell culture amplification. The use of centrifugal filter devices to increase infectious titers of ZIKV from cell-culture supernatants is highlighted here. A mean gain of 2.33 ± 0.12 log10 DICT50/mL was easily and rapidly obtained with this process. This efficient method of ultrafiltration may be applied to other viruses and be useful in various experimental studies requiring high viral titers.

A replication-competent foot-and-mouth disease virus expressing a luciferase reporter

Bioluminescence is a powerful tool in the study of viral infection both in vivo and in vitro. Foot-and-mouth disease virus (FMDV) has a small RNA genome with a limited tolerance to foreign RNA entities. There has been no success in making a reporter FMDV expressing a luciferase in infected cell culture supernatants. We report here for the first time a replication-competent FMDV encoding Nanoluciferase, named as Nano-FMDV. Nano-FMDV is genetically stable during serial passages in cells and exhibits growth kinetics and plaque morphology similar to its parental virus. There are applications for the use of Nano-FMDV such as real-time monitoring of FMDV replication in vitro and in vivo.

Productive and Penicillin-Stressed Chlamydia pecorum Infection Induces Nuclear Factor Kappa B Activation and Interleukin-6 Secretion In Vitro.

Nuclear factor kappa B (NFκB) is an inflammatory transcription factor that plays an important role in the host immune response to infection. The potential for chlamydiae to activate NFκB has been an area of interest, however most work has focused on chlamydiae impacting human health. Given that inflammation characteristic of chlamydial infection may be associated with severe disease outcomes or contribute to poor overall fitness in farmed animals, we evaluated the ability of porcine chlamydiae to induce NFκB activation in vitro. C. pecorum infection induced both NFκB nuclear translocation and activation at 2 hours post infection (hpi), an effect strongly enhanced by suppression of host de novo protein synthesis. C. suis and C. trachomatis showed less capacity for NFκB activation compared to C. pecorum, suggesting a species-specific variation in NFκB activation. At 24 hpi, C. pecorum induced significant NFκB activation, an effect not abolished by penicillin (beta lactam)-induced chlamydial stress. C. pecorum-dependent secretion of interleukin 6 was also detected in the culture supernatant of infected cells at 24 hpi, and this effect, too, was unchanged by penicillin-induced chlamydial stress. Taken together, these results suggest that NFκB participates in the early inflammatory response to C. pecorum and that stressed chlamydiae can promote inflammation.

Inhibition of Respiratory Syncytial Virus Replication by Simultaneous Targeting of mRNA and Genomic RNA Using Dual-Targeting siRNAs.

We attempted to generate siRNAs with two active strands, which can simultaneously knock down the expression of mRNA and viral genomic RNA. In this study, short hairpin RNAs (shRNAs) against N and F genes were used. Expression of F and N mRNA transcripts as well as genomic RNA was determined with relative real-time RT-PCR. The RSV load in infected cell culture supernatant was determined by absolute quantitative real-time PCR. We found that (i) in the presence of shRNA-N, a greater reduction in viral genomic RNA was found; (ii) the level of expression at MOI 0.01 was reduced more than MOI 0.1; (iii) reduction in N transcript was greater than F; and (iv) finally, in combination pre-treatment with two shRNAs, the reduction was not significant as compared to single shRNA transfection. shRNAs also inhibited the production of RSV progeny as shown by viral load in infected HEp-2 cells. (i) Virus load reduction was greater at MOI 0.01 than 0.1 and (ii) significant load reduction was not seen with combination shRNA pre-treatment. The antiviral potency was also confirmed by plaque assay and western blot analysis. Our results provided further evidence that RNAi could be a powerful treatment option against respiratory viruses.