Rapid, simple, and cost-effective plaque assay for murine norovirus using microcrystalline cellulose.

Murine norovirus (MNV) is used widely as a practical alternative to human norovirus (HuNoV). Plaque-forming assays for MNV are important for developing therapeutic agents against HuNoV infections. Although agarose-overlay MNV assays have been reported, recent improvements in cellulose derivatives suggest that they could be optimized further, particularly with respect to improving the overlay material. To determine which overlay material is optimal for the MNV plaque assay, we compared four typical cellulose derivatives [microcrystalline cellulose (MCC), hydroxyethyl cellulose (HEC), hydroxypropyl methylcellulose (HPMC), and carboxymethyl cellulose (CMC)] with conventional agarose. We found that 3.5% (w/v) MCC-containing medium provided clear round-shaped plaques on RAW 264.7 cells 1 day after inoculation; the visibility of plaques was comparable with that of the original agarose-overlay assay. Removing residual MCC powder from the MCC-overlay assay before fixing was important for obtaining distinct plaques that are clearly countable. Finally, after calculating the plaque diameter as a percentage of well diameter, we found that 12- and 24-well plates were better than other plates for accurate plaque counting. The MCC-based MNV plaque assay is cost-effective and rapid, and produces plaques that are easy to count. Accurate virus quantification using this optimized plaque assay will enable reliable estimation of norovirus titers.

A rigorous assessment and comparison of enumeration methods for environmental viruses.

Determining exact viral titers in a given sample is essential for many environmental and clinical applications, e.g., for studying viral ecology or application of bacteriophages for food safety. However, virus quantification is not a simple task, especially for complex environmental samples. While clonal viral isolates can be quantified with relative high accuracy using virus-specific methods, i.e., plaque assay or quantitative real-time PCR, these methods are not valid for complex and diverse environmental samples. Moreover, it is not yet known how precisely laser-based methods, i.e., epifluorescence microscopy, flow cytometry, and nanoparticle tracking analysis, quantify environmental viruses. In the present study, we compared five state-of-the-art viral quantification methods by enumerating four model viral isolates of different genome and size characteristics as well as four different environmental water samples. Although Nanoparticle tracking analysis combined with gentle staining at 30 °C could be confirmed by this study to be a reliable quantification technique for tested environmental samples, environmental samples still lack an universally applicable and accurate quantification method. Special attention has to be put on optimal sample concentrations as well as optimized sample preparations, which are specific for each method. As our results show the inefficiency when enumerating small, or single-stranded DNA or RNA viruses, the global population of viruses is presumably higher than expected.

Assessment of Prolonged Dengue Virus Infection in Dermal Fibroblasts and Hair-Follicle Dermal Papilla Cells.

Dengue virus (DENV)-mediated hair loss is one of the post-dengue fatigue syndromes and its pathophysiology remains unknown. Whether long-term or persistent infection with DENV in the scalp results in hair loss is unclear. In this study, we cultured human dermal fibroblasts (WS1 cells) and primary human hair-follicle dermal papilla cells (HFDPCs) in the long term with DENV-2 infection. The production of virion, the expression of inflammatory and anti-virus genes, and their signaling transduction activity in the infected cells were analyzed. DENV-2 NS3 protein and DENV-2 5' UTR RNA were detected in fibroblasts and HFDPCs that were subjected to long-term infection with DENV-2 for 33 days. A significant amount of DENV-2 virion was produced by both WS1 cells and HFDPCs in the first two days of acute infection. The virion was also detected in WS1 cells that were infected in the long term, but HFDPCs failed to produce DENV-2 after long-term culture. Type I and type III interferons, and inflammatory cytokines were highly expressed in the acute phase of DENV infection in HFPDC and WS1 cells. However, in the long-term cultured cells, modest levels of anti-viral protein genes were expressed and we observed reduced signaling activity, which was correlated with the level of virus production changes. Long-term infection of DENV-2 downregulated the expression of hair growth regulatory factors, such as Rip1, Wnt1, and Wnt4. This in vitro study shows that the long-term infection with DENV-2 in dermal fibroblasts and dermal papilla cells may be involved with the prolonged-DENV-infection-mediated hair loss of post-dengue fatigue syndrome. However, direct evidence for viral replication in the human hair of a dengue victim or animal infection model is required.

Assessment of Mumps Virus-Specific Antibodies: Comparison of Plaque Reduction Neutralization Test and Enzyme-Linked Immunosorbent Assay Estimates.

BACKGROUND: The plaque reduction neutralization test (PRNT), which measures a subset of immunoglobulin antibodies (functional neutralizing antibodies), and the enzyme-linked immunosorbent assay (ELISA), which measures total immunoglobulin (neutralizing and nonneutralizing antibodies), characterize different aspects of the anti-mumps virus antibody response after vaccination. METHODS: Data from a recent phase 3 clinical trial (NCT01681992) of 2 measles-mumps-rubella vaccines were used to compare anti-mumps antibody responses measured using an unenhanced PRNT (GSK; seropositivity cutoff and threshold, 2.5 and 4 times the 50% end-point dilution, respectively) with those estimated using an ELISA (thresholds, 5 and 10 ELISA units/mL, respectively). RESULTS: Of 3990 initially seronegative samples, 3284 (82.3%) were seropositive after vaccination for anti-mumps antibodies in both assays. The Pearson correlation coefficient for double-positive samples was 0.57, indicative of a moderate correlation. Receiver operating characteristic curve analysis showed that an ELISA threshold of 51.7 ELISA units/mL best corresponded to the PRNT seroresponse threshold. There was no obvious vaccine brand effect on the correlation between assays. CONCLUSIONS: The moderate correlation between the anti-mumps antibody measurements obtained with PRNT and ELISA reflects different aspects of the serological response. In the absence of a well-defined protective serological threshold, PRNT provides complementary information on the antibody response, whereas ELISA remains a critically useful measurement of vaccine immunogenicity.

Simple drop cast method for enumeration of bacteriophages.

Phage enumeration is a basic prerequisite for application of phages in industrial, medical and other processes. Double layer agar (DLA) plaque assay is the classical method employed for isolation, detection as well as enumeration of phage particles in a solution. However, DLA method is considered cumbersome due to its specific temperature requirements and need for one petriplate with two agar layers for each phage sample. We are proposing a drop cast method for enumeration of phages which is comparatively easier and cost effective than classical DLA method as single layer of agar without any specific temperature condition is required. Added advantage of this method is that 7-10 dilutions of phage suspension can be enumerated on a single agar plate in contrast to one dilution per plate as required in DLA method. Although standard deviation in phage count was higher in the proposed method than DLA method, still drop cast method provided first-approximation phage titer which can be further validated by DLA method for more accuracy. Hence, the present method can be considered reliable, easy and cost effective for determining approximate phage count in an unknown phage suspension.

Ectromelia Virus Disease Characterization in the BALB/c Mouse: A Surrogate Model for Assessment of Smallpox Medical Countermeasures.

In 2007, the United States- Food and Drug Administration (FDA) issued guidance concerning animal models for testing the efficacy of medical countermeasures against variola virus (VARV), the etiologic agent for smallpox. Ectromelia virus (ECTV) is naturally-occurring and responsible for severe mortality and morbidity as a result of mousepox disease in the murine model, displaying similarities to variola infection in humans. Due to the increased need of acceptable surrogate animal models for poxvirus disease, we have characterized ECTV infection in the BALB/c mouse. Mice were inoculated intranasally with a high lethal dose (125 PFU) of ECTV, resulting in complete mortality 10 days after infection. Decreases in weight and temperature from baseline were observed eight to nine days following infection. Viral titers via quantitative polymerase chain reaction (qPCR) and plaque assay were first observed in the blood at 4.5 days post-infection and in tissue (spleen and liver) at 3.5 days post-infection. Adverse clinical signs of disease were first observed four and five days post-infection, with severe signs occurring on day 7. Pathological changes consistent with ECTV infection were first observed five days after infection. Examination of data obtained from these parameters suggests the ECTV BALB/c model is suitable for potential use in medical countermeasures (MCMs) development and efficacy testing.

Assessment of plaque assay methods for alphaviruses.

Viruses from the Alphavirus genus are responsible for numerous arboviral diseases impacting human health throughout the world. Confirmation of acute alphavirus infection is based on viral isolation, identification of viral RNA, or a fourfold or greater increase in antibody titers between acute and convalescent samples. In convalescence, the specificity of antibodies to an alphavirus may be confirmed by plaque reduction neutralization test. To identify the best method for alphavirus and neutralizing antibody recognition, the standard solid method using a cell monolayer overlay with 0.4% agarose and the semisolid method using a cell suspension overlay with 0.6% carboxymethyl cellulose (CMC) overlay were evaluated. Mayaro virus, Una virus, Venezuelan equine encephalitis virus (VEEV), and Western equine encephalitis virus (WEEV) were selected to be tested by both methods. The results indicate that the solid method showed consistently greater sensitivity than the semisolid method. Also, a "semisolid-variant method" using a 0.6% CMC overlay on a cell monolayer was assayed for virus titration. This method provided the same sensitivity as the solid method for VEEV and also had greater sensitivity for WEEV titration. Modifications in plaque assay conditions affect significantly results and therefore evaluation of the performance of each new assay is needed.

Swine influenza virus infection dynamics in two pig farms; results of a longitudinal assessment.

In order to assess the dynamics of influenza virus infection in pigs, serological and virological follow-ups were conducted in two whole batches of pigs from two different farms (F1 and F2), from 3 weeks of age until market age. Anti-swine influenza virus (SIV) antibodies (measured by ELISA and hemagglutination inhibition) and nasal virus shedding (measured by RRT-PCR and isolation in embryonated chicken eggs and MDCK cells) were carried out periodically. SIV isolates were subtyped and hemagglutinin and neuraminidase genes were partially sequenced and analyzed phylogenetically. In F1, four waves of viral circulation were detected, and globally, 62/121 pigs (51.2%) were positive by RRT-PCR at least once. All F1 isolates corresponded to H1N1 subtype although hemagglutination inhibition results also revealed the presence of antibodies against H3N2. The first viral wave took place in the presence of colostral-derived antibodies. Nine pigs were positive in two non-consecutive sampling weeks, with two of the animals being positive with the same isolate. Phylogenetic analyses showed that different H1N1 variants circulated in that farm. In F2, only one isolate, H1N2, was detected and all infections were concentrated in a very short period of time, as assumed for a classic influenza outbreak. These findings led us to propose that influenza virus infection in pigs might present different patterns, from an epidemic outbreak to an endemic form with different waves of infections with a lower incidence.

A quantitative comet infection assay for influenza virus.

The virus comet assay is a cell-based virulence assay used to evaluate an antiviral drug or antibody against a target virus. The comet assay differs from the plaque assay in allowing spontaneous flows in 6-well plates to spread virus. When implemented quantitatively the comet assay has been shown to have an order-of-magnitude greater sensitivity to antivirals than the plaque assay. In this study, a quantitative comet assay for influenza virus is demonstrated, and is shown to have a 13-fold increase in sensitivity to ribavirin. AX4 cells (MDCK cells with increased surface concentration of α2-6 sialic acid, the influenza virus receptor) have reduced the comet size variability relative to MDCK cells, making them a better host cell for use in this assay. Because of enhanced antiviral sensitivity in flow-based assays, less drug is required, which could lead to lower reagent costs, reduced cytotoxicity, and fewer false-negative drug screen results. The comet assay also serves as a readout of flow conditions in the well. Observations from comets formed at varying humidity levels indicate a role for evaporation in the mechanism of spontaneous fluid flow in wells.

Assessment of the efficacy of the neuraminidase inhibitor oseltamivir against 2009 pandemic H1N1 influenza virus in ferrets.

Pandemic 2009 influenza A (H1N1) virus (H1N1pdm) is different from contemporary seasonal human viruses in that it can cause infection deep in the lungs of critical care patients. Here we establish a mammalian animal model and assessed the efficacy of the neuraminidase (NA) inhibitor oseltamivir treatment against H1N1pdm virus infection. Oseltamivir (25 mg/kg/day twice daily for 5 days) was orally administered to groups of ferrets, starting either 2 or 24 h after inoculation with 10(6)PFU of A/California/04/2009 (H1N1) influenza virus. We determined that virus replication was restricted to 1 or 2 of 4 lung lobes in oseltamivir-treated animals, while virus was consistently isolated from 4 of 4 lung lobes in control animals (1.5-3.8log(10)PFU/g). Analysis of arterial blood oxygenation revealed less pronounced changes in partial oxygen and carbon dioxide pressure in oseltamivir-treated ferrets, and histologic examination confirmed reduced pneumonia. Treated animals had significantly decreased inflammatory responses in the upper respiratory tract (P < 0.05), less fever and weight loss, and less reduction of activity. Virus titers in the nasal washes of treated and control ferrets did not differ significantly. NA sequencing and fluorescence-based phenotypic assays identified no oseltamivir-resistant variants. Overall, oseltamivir treatment decreases the signs of infection and reduced the spread of H1N1pdm influenza virus in the lungs of ferrets and therefore impeded the development of viral pneumonia.

Effects of ionic strength on bacteriophage MS2 behavior and their implications for the assessment of virus retention by ultrafiltration membranes.

Bacteriophage MS2 is widely used as a surrogate to estimate pathogenic virus elimination by membrane filtration processes used in water treatment. Given that this water technology may be conducted with different types of waters, we focused on investigating the effects of ionic strength on MS2 behavior. For this, MS2 was analyzed while suspended in solutions of various ionic strengths, first in a batch experiment and second during membrane ultrafiltration, and quantified using (i) quantitative reverse transcriptase PCR (qRT-PCR), which detects the total number of viral genomes, (ii) qRT-PCR without the RNA extraction step, which reflects only particles with a broken capsid (free RNA), and (iii) the PFU method, which detects only infectious viruses. At the beginning of the batch experiments using solutions containing small amounts of salts, losses of MS2 infectivity (90%) and broken particles (20%) were observed; these proportions did not change during filtration. In contrast, in high-ionic-strength solutions, bacteriophage kept its biological activity under static conditions, but it quickly lost its infectivity during the filtration process. Increasing the ionic strength decreased both the inactivation and the capsid breakup in the feed suspension and increased the loss of infectivity in the filtration retentate, while the numbers of MS2 genomes were identical in both experiments. In conclusion, the effects of ionic strength on MS2 behavior may significantly distort the results of membrane filtration processes, and therefore, the combination of classical and molecular methods used here is useful for an effective validation of the retention efficiency of ultrafiltration membranes.

Resistance of herpes simplex viruses to nucleoside analogues: mechanisms, prevalence, and management.

Herpes simplex viruses (HSV) type 1 and type 2 are responsible for recurrent orolabial and genital infections. The standard therapy for the management of HSV infections includes acyclovir (ACV) and penciclovir (PCV) with their respective prodrugs valacyclovir and famciclovir. These compounds are phosphorylated by the viral thymidine kinase (TK) and then by cellular kinases. The triphosphate forms selectively inhibit the viral DNA polymerase (DNA pol) activity. Drug-resistant HSV isolates are frequently recovered from immunocompromised patients but rarely found in immunocompetent subjects. The gold standard phenotypic method for evaluating the susceptibility of HSV isolates to antiviral drugs is the plaque reduction assay. Plaque autoradiography allows the associated phenotype to be distinguished (TK-wild-type, TK-negative, TK-low-producer, or TK-altered viruses or mixtures of wild-type and mutant viruses). Genotypic characterization of drug-resistant isolates can reveal mutations located in the viral TK and/or in the DNA pol genes. Recombinant HSV mutants can be generated to analyze the contribution of each specific mutation with regard to the drug resistance phenotype. Most ACV-resistant mutants exhibit some reduction in their capacity to establish latency and to reactivate, as well as in their degree of neurovirulence in animal models of HSV infection. For instance, TK-negative HSV mutants establish latency with a lower efficiency than wild-type strains and reactivate poorly. DNA pol HSV mutants exhibit different degrees of attenuation of neurovirulence. The management of ACV- or PCV-resistant HSV infections includes the use of the pyrophosphate analogue foscarnet and the nucleotide analogue cidofovir. There is a need to develop new antiherpetic compounds with different mechanisms of action.

Comprehensive assessment of 2009 pandemic influenza A (H1N1) virus drug susceptibility in vitro.

BACKGROUND: Antiviral drugs are an important option for managing infections caused by influenza viruses. This study assessed the drug susceptibility of 2009 pandemic influenza A (H1N1) viruses collected globally between April 2009 and January 2010. METHODS: Virus isolates were tested for adamantane susceptibility, using pyrosequencing to detect the S31N marker of adamantane resistance in the M2 protein and biological assays to assess viral replication in cell culture. To assess neuraminidase (NA) inhibitor (NAI) susceptibility, virus isolates were tested in chemiluminescent NA inhibition assays and by pyrosequencing to detect the H275Y (H274Y in N2 numbering) marker of oseltamivir resistance in the NA. RESULTS: With the exception of three, all viruses that were tested for adamantane susceptibility (n=3,362) were resistant to this class of drugs. All viruses tested for NAI susceptibility (n=3,359) were sensitive to two US Food and Drug Administration-approved NAIs, oseltamivir (mean ±sd 50% inhibitory concentration [IC(50)] 0.25 ±0.12 nM) and zanamivir (mean IC(50) 0.29 ±0.09 nM), except 23 (0.7%), which were resistant to oseltamivir, but sensitive to zanamivir. Oseltamivir-resistant viruses had the H275Y mutation in their NA and were detected in patients exposed to the drug through prophylaxis or treatment. NA activity of all viruses was inhibited by the NAIs peramivir, laninamivir (R-125489) and A-315675, except for H275Y variants, which exhibited approximately 100-fold reduction in peramivir susceptibility. CONCLUSIONS: This report provides data regarding antiviral susceptibility of 2009 pandemic influenza A (H1N1) surveillance viruses, the majority of which were resistant to adamantanes and sensitive to NAIs. These findings provide information essential for antiviral resistance monitoring and development of novel diagnostic tests for detecting influenza antiviral resistance.

Assessment of in vitro prophylactic and therapeutic efficacy of chloroquine against Chikungunya virus in vero cells.

The resurgence of Chikungunya virus (CHIKV) in the form of unprecedented and explosive epidemics in India and the Indian Ocean islands after a gap of 32 years is a major public health concern. Currently, there is no specific therapy available to treat CHIKV infection. In the present study, the in vitro prophylactic and therapeutic effects of chloroquine on CHIKV replication in Vero cells were investigated. Inhibitory effects were observed when chloroquine was administered pre-infection, post-infection, and concurrent with infection, suggesting that chloroquine has prophylactic and therapeutic potential. The inhibitory effects were confirmed by performing a plaque reduction neutralization test (PRNT), real-time reverse transcriptase (RT)-PCR analysis of viral RNA levels, and cell viability assays. Chloroquine diminished CHIKV infection in a dose-dependent manner, with an effective concentration range of 5-20 microM. Concurrent addition of drug with virus, or treatment of cells prior to infection drastically reduced virus infectivity and viral genome copy number by >/=99.99%. The maximum inhibitory effect of chloroquine was observed within 1-3 hr post-infection (hpi), and treatment was ineffective once the virus successfully passed through the early stages of infection. The mechanism of inhibition of virus activity by chloroquine involved impaired endosomal-mediated virus entry during early stages of virus replication, most likely through the prevention of endocytosis and/or endosomal acidification, based on a comparative evaluation using ammonium chloride, a known lysosomotropic agent.

Rapid assessment of the viability of Mycobacterium avium subsp. paratuberculosis cells after heat treatment, using an optimized phage amplification assay.

Thermal inactivation experiments were carried out to assess the utility of a recently optimized phage amplification assay to accurately enumerate viable Mycobacterium avium subsp. paratuberculosis cells in milk. Ultra-heat-treated (UHT) whole milk was spiked with large numbers of M. avium subsp. paratuberculosis organisms (10(6) to 10(7) CFU/ml) and dispensed in 100-microl aliquots in thin-walled 200-microl PCR tubes. A Primus 96 advanced thermal cycler (Peqlab, Erlangen, Germany) was used to achieve the following time and temperature treatments: (i) 63 degrees C for 3, 6, and 9 min; (ii) 68 degrees C for 20, 40, and 60 s; and (iii) 72 degrees C for 5, 10, 15, and 25 s. After thermal stress, the number of surviving M. avium subsp. paratuberculosis cells was assessed by both phage amplification assay and culture on Herrold's egg yolk medium (HEYM). A high correlation between PFU/ml and CFU/ml counts was observed for both unheated (r(2) = 0.943) and heated (r(2) = 0.971) M. avium subsp. paratuberculosis cells. D and z values obtained using the two types of counts were not significantly different (P > 0.05). The D(68 degrees C), mean D(63 degrees C), and D(72 degrees C) for four M. avium subsp. paratuberculosis strains were 81.8, 9.8, and 4.2 s, respectively, yielding a mean z value of 6.9 degrees C. Complete inactivation of 10(6) to 10(7) CFU of M. avium subsp. paratuberculosis/ml milk was not observed for any of the time-temperature combinations studied; 5.2- to 6.6-log(10) reductions in numbers were achieved depending on the temperature and time. Nonlinear thermal inactivation kinetics were consistently observed for this bacterium. This study confirms that the optimized phage assay can be employed in place of conventional culture on HEYM to speed up the acquisition of results (48 h instead of a minimum of 6 weeks) for inactivation experiments involving M. avium subsp. paratuberculosis-spiked samples.

Error assessment in recombinant baculovirus titration: evaluation of different methods.

The success of baculovirus/insect cells system in heterologous protein expression depends on the robustness and efficiency of the production workflow. It is essential that process parameters are controlled and include as little variability as possible. The multiplicity of infection (MOI) is the most critical factor since irreproducible MOIs caused by inaccurate estimation of viral titers hinder batch consistency and process optimization. This lack of accuracy is related to intrinsic characteristics of the method such as the inability to distinguish between infectious and non-infectious baculovirus. In this study, several methods for baculovirus titration were compared. The most critical issues identified were the incubation time and cell concentration at the time of infection. These variables influence strongly the accuracy of titers and must be defined for optimal performance of the titration method. Although the standard errors of the methods varied significantly (7-36%), titers were within the same order of magnitude; thus, viral titers can be considered independent of the method of titration. A cost analysis of the baculovirus titration methods used in this study showed that the alamarblue, real time Q-PCR and plaque assays were the most expensive techniques. The remaining methods cost on average 75% less than the former methods. Based on the cost, time and error analysis undertaken in this study, the end-point dilution assay, microculture tetrazolium assay and flow cytometric assay were found to be the techniques that combine all these three main factors better. Nevertheless, it is always recommended to confirm the accuracy of the titration either by comparison with a well characterized baculovirus reference stock or by titration using two different methods and verification of the variability of results.

Development of a 1-step cell-based assay for cost-effective screening of antiviral drugs for vaccinia virus.

We report a 1-step assay to screen antiviral substances combining the simultaneous application of cells, virus sample, and drug to 96-well plates. The results were obtained within 26 h when vaccinia virus plaques were counted or virus-induced cytopathic effect was measured. This fast cost-effective procedure may be used for other cytopathic viruses and is suitable to 1st screening tests.

Assessment of a vesicular stomatitis virus-based vaccine by use of the mouse model of Ebola virus hemorrhagic fever.

BACKGROUND: In humans and nonhuman primates, Ebola virus causes a virulent viral hemorrhagic fever for which no licensed vaccines or therapeutic drugs exist. In the present study, we used the mouse model for Ebola hemorrhagic fever to assess the safety and efficacy of a vaccine based on a live attenuated vesicular stomatitis virus expressing the Zaire ebolavirus (ZEBOV) glycoprotein. METHODS: Healthy mice were given the vaccine in various doses, decreasing from 2 x 10(4) to 2 plaque-forming units (pfu), with both systemic and mucosal vaccination routes used. Mice were challenged with 10(3) to 10(6) lethal doses of mouse-adapted ZEBOV. Severely immunocompromised mice were injected with 2 x 10(5) pfu, which is 10 times greater than the immunization dose normally used, to test vaccine safety. RESULTS: Two plaque-forming units of the vaccine protected against lethal challenge, and mucosal immunization was found to be as protective as systemic injection. The replicating vaccine was never detected in the immunized animals, nor were there clinical signs after immunization. Immunization of severely immunocompromised mice with 200,000 pfu of vaccine resulted in no clinical symptoms. CONCLUSIONS: Our data suggest that the vaccine is highly potent and safe and that it very rapidly induces "sterile" immunity in mice. The potential for mucosal delivery, if confirmed in nonhuman primates, makes it an excellent candidate for mass immunization during outbreaks or in the event of intentional release.

Immunocompetent syngeneic cotton rat tumor models for the assessment of replication-competent oncolytic adenovirus.

Oncolytic adenoviruses as a treatment for cancer have demonstrated limited clinical activity. Contributing to this may be the relevance of preclinical animal models used to study these agents. Syngeneic mouse tumor models are generally non-permissive for adenoviral replication, whereas human tumor xenograft models exhibit attenuated immune responses to the vector. The cotton rat (Sigmodon hispidus) is susceptible to human adenovirus infection, permissive for viral replication and exhibits similar inflammatory pathology to humans with adenovirus replicating in the lungs, respiratory passages and cornea. We evaluated three transplantable tumorigenic cotton rat cell lines, CCRT, LCRT and VCRT as models for the study of oncolytic adenoviruses. All three cells lines were readily infected with adenovirus type-5-based vectors and exhibited high levels of transgene expression. The cell lines supported viral replication demonstrated by the induction of cytopathogenic effect (CPE) in tissue culture, increase in virus particle numbers and assembly of virions seen on transmission electron microscopy. In vivo, LCRT and VCRT tumors demonstrated delayed growth after injection with replicating adenovirus. No in vivo antitumor activity was seen in CCRT tumors despite in vitro oncolysis. Adenovirus was also rapidly cleared from the CCRT tumors compared to LCRT and VCRT tumors. The effect observed with the different cotton rat tumor cell lines mimics the variable results of human clinical trials highlighting the potential relevance of this model for assessing the activity and toxicity of oncolytic adenoviruses.

A simplified PCR methodology for semiquantitatively analyzing dengue viruses.

The standard methodology for titrating dengue viruses, the plaque assay, is slow, time consuming and relatively expensive. Other methods require machinery that may not be routinely accessible to all researchers, particularly those in developing nations. We therefore sought to develop a rapid, simplified semiquantitative polymerase chain reaction (PCR) methodology based on the use of a template mimic. In particular, it was desired that the mimic should be applicable for use a DNA template to avoid the requirement for producing an in vitro RNA transcript. A 511 base pair fragment of the capsid-PrM junction of dengue serotype 4 was cloned into pGEM-T Easy vector and subjected to splicing overlap extension-PCR to generate a 160 base pair deletion. The deleted plasmid mimic competed competitively against the parent plasmid as well as the first strand cDNA of all four dengue viruses. The primers used are specific for the dengue virus, and no product was seen with first strand cDNA from a closely related flavivirus, Japanese encephalitis virus. Under the conditions used, accurate quantitation of the dengue viruses in the range of 10(3) to 10(6) pfu can be achieved in a single day, as opposed to the 7 days required for conventional plaque assay.