Infected Tissue Culture Fluids Research Articles

Cytopathic effect of Vero cells adapted Bangladeshi strain of peste des petits ruminants (PPR) virus in cell culture.

The present study described the cytopathic effect of PPR virus presently being used in serial passages at the level of 60th in Vero cells and infected tissue culture fluid was used in this study as viral inoculum. Vero cells were grown on cover slip & were infected with tissue culture fluid at a fixed multiplicity of infection (MOI) 0.01. The infected cover slip along with control were stained with H&E stain at periodic intervals and cytopathic effect was studied with microscope. The cytopathic effect (CPE) was visible at first from 24 hpi and the Vero cells showed initial cell rounding, aggregation, and syncytial development. Development of inclusion bodies and cell degradation was noticed by 72 hpi. Complete detachment of the cell monolayer was observed by 84 hpi. It is concluded that, development of numerous inclusion bodies are the indication of well adaptation & extensive multiplication of PPRV in Vero cells.

Evaluation of a field-deployable reverse transcription-insulated isothermal PCR for rapid and sensitive on-site detection of Zika virus

BackgroundThe recent emergence of Zika virus (ZIKV) in Brazil and its precipitous expansion throughout the Americas has highlighted the urgent need for a rapid and reliable on-site diagnostic assay suitable for viral detection. Such point-of-need (PON), low-cost diagnostics are essential for ZIKV control in vulnerable areas with limited resources.MethodsWe developed and evaluated a ZIKV-specific field-deployable RT-iiPCR reagent set targeting the E gene for rapid detection of ZIKV in ZIKV-spiked human and mosquito specimens, and compared its performance to the Center for Disease Control and Prevention (CDC) and Pan American Health Organization (PAHO) RT-qPCR assays targeting the E and NS2B genes, respectively.ResultsThese assays demonstrated exclusive specificity for ZIKV (African and Asian lineages), had limits of detection ranging from 10 to 100 in vitro transcribed RNA copies/μl and detection endpoints at 10 plaque forming units/ml of infectious tissue culture fluid. Analysis of human whole blood, plasma, serum, semen, urine, and mosquito pool samples spiked with ZIKV showed an agreement of 90% (k = 0.80), 92% (k = 0.82), 95% (k = 0.86), 92% (k = 0.81), 90% (k = 0.79), and 100% (k = 1), respectively, between the RT-iiPCR assay and composite results from the reference RT-qPCR assays. Overall, the concurrence between the ZIKV RT-iiPCR and the reference RT-qPCR assays was 92% (k = 0.83).ConclusionsThe ZIKV RT-iiPCR has a performance comparable to the reference CDC and PAHO RT-qPCR assays but provides much faster results (~1.5 h) with a field-deployable system that can be utilized as a PON diagnostic with the potential to significantly improve the quality of the health care system in vulnerable areas.

Characterization of Recent Isolates of Newcastle Disease virus

Fifty five tissue samples were collected aseptically from 5 flocks suspected to be naturally infected with NDV in EL- Ismalia, Al-sharkia, El- Gharbia, kafr-Elshiekh & El-Geza and subjected to trials of virus isolation. Nine field samples from 2 flocks (Kafr-Elshiekh and Giza) were positive for virus isolation. Inoculation of SPF chicken eggs induced death of the embryo within 48-72 hours post inoculation. HA test applied on the obtained allantoic fluids showed positive results suggesting that inoculated samples contained NDV. It was noticed that highest infectivity titer of the virus was 9.5 log10 EID50/ml by the 5th passage in SPF chicken eggs. Cell culture passage of the obtained virus isolate from the allantoic fluid through ten consecutive passages in BHK cell line. the CPE developed at the 3rd passage within 24-48 hours post cell infection where polykaryocytosis, cell syncitia, rounding of cells. Tissue cultured fluids (TCF) were found to be direct HA possitive. The virus titer was 3 log10 TCID50 /ml at the 3rd passage and reach to 7 log10 TCID50 /ml by the 6th passage. At the 9th passage, the virus titer was reached to 8.5 log10 TCID50 /ml. Identification of the obtained virus isolate was confirmed by VNT using specific anti-NDV in BHK cell. The isolate of NDV with HA-positive allantoic fluids of El-Giza flockes were virulent NDVand the F gene give expected size of 724 bp. The isolate of this study also compared against the reference and vaccinal strains from gene bank and the results revealed that the isolate NDV/chicken/Egypt/Giza/2015 was velogenic type resembling the genotype VIId strains. F gene analysis of the NDV/chicken/Egypt/Giza/2015 revealed that the most nucleotides identity from gene bank with this isolate were NDV/Chicken/GC/IS/2010/1224 and NDV/buzzard/Israel/714/2011 with a nucleotide identity of 92.7%. It was also closely related to Chicken/China/SDWF07/2011; Chicken/China/Shandong/01/2012 and NDV/Turkey/Israel/111/2011 with a nucleotide identity of 92.5%. Otherwise nucleotides identity between the selected NDV field isolate in this study and NDV/Lasota was 72.4%. Phylogenetic analysis of NDV/chicken/Egypt/Giza/2015 with other reference and vaccinal strains of NDV revealed that this isolate was related to NDV/B7/RLQP/CH/EG/12 and present in the same group with NDV/F388/RLQP/CH/EG/14 and NDV/F460/RLQP/CH/EG/13. Regarding evaluation of the prepared experimental vaccines, it was found that the NDV infected tissue culture fluid, were completely inactivated with 3% BEI at 30 °C for 18 hrs post treatments showing no pathological changes in the ECE and no CPE in the BHK cell culture. Humoral immune response of chickens to the prepared vaccines showed that vaccinated chicks with NDV-Montanide ISA 70 adjuvanted vaccine exhibited antibody titer of 8.3 log2 three weeks post vaccination recording the higher titer of (10.6 log2) at the 6th week post vaccination with and 3rd week post challenge test. The high antibodies values were persist till the 12th weeks post NDV-Montanide ISA 70 vaccination followed by gradual decrease till the end of the experiment (24th weeks). On the other hand, vaccination of chicks with inactivated NDV isolate without adjuvant failed to induce protective HI antibodies (3 log2) all over the experiment. Challenge test showed that chickens vaccinated with ISA-70 adjuvant vaccine showed no sign of disease and no mortalities while the antigen without adjuvant could not provide any protective efficiency to SPF chickens.

Specific Detection of Rinderpest Virus by Real-Time Reverse Transcription-PCR in Preclinical and Clinical Samples from Experimentally Infected Cattle

A highly sensitive detection test for Rinderpest virus (RPV), based on a real-time reverse transcription-PCR (rRT-PCR)system, was developed. Five different RPV genomic targets were examined, and one was selected and optimized to detect viral RNA in infected tissue culture fluid with a level of detection ranging from 0.59 to 87.5 50% tissue culture infectious doses (TCID(50)) per reaction depending on the viral isolate. The strain sensitivity of the test was validated on 16 RPV strains belonging to all three phylogenetic branches described for RPV. No cross-reactivity was detected with closely related peste des petit ruminants or with symptomatically similar viruses, including all seven serotypes of foot-and-mouth disease virus, two serotypes of vesicular stomatitis virus, bluetongue virus, and bovine herpes virus type 2. In samples from experimentally infected cattle, our real-time RT-PCR test was significantly more sensitive than the gold standard test of virus isolation, allowing the detection of the disease 2 to 4 days prior to the appearance of clinical signs. The comparison of clinical samples with putative diagnostic value from live animals showed that conjunctival swabs and blood buffy coat were the samples of choice for epidemiological surveillance, while lymph nodes performed the best as postmortem specimens. This portable and rapid real-time RT-PCR has the capability of the preclinical detection of RPV and provides differential diagnosis from look-alike diseases of cattle. As RPV is declared globally eradicated, this test provides an important rapid virus detection tool that does not require the use of infectious virus and allows the processing of a large number of samples.

Highly sensitive detection of swine vesicular disease virus based on a single tube RT-PCR system and DIG-ELISA detection

A highly sensitive detection of swine vesicular disease virus (SVDV) based on a single tube RT-PCR system and digoxigenin (DIG)-PCR-ELISA detection was developed. Using a one tube RT-PCR system, optimisation of the PCR conditions and optimisation of the microwell hybridisation and colourimetric detection of the amplicons resulted in a method that could detect viral RNA in infected tissue culture fluid with a titre as low as 0.1 TCID 50/100 μl. The same sensitivity was obtained with SVDV-spiked faeces, if the samples were pre-treated with 1,1,2-trichlorotrifluoroethane/chloroform and subsequently concentrated using an ultrafiltration system and RNA extracted with the Purescript kit. The specificity of the test was validated on 27 SVDV strains belonging to four different groups. No cross-reactivity with genetically and symptomatically related viruses was detected using RNA of foot-and-mouth disease virus (FMDV), porcine enterovirus (PEV), vesicular stomatitis virus (VSV), Coxsackie B5 virus (CV-B5) and encephalomyocarditis virus (EMCV). The test was validated successfully on clinical samples, being slightly more sensitive and much faster than virus isolation on cell cultures. Moreover the possibility of automating the procedure will allow the processing of large numbers of clinical samples.

Detection of swine vesicular disease virus RNA by reverse transcription-polymerase chain reaction

Two polymerase chain reaction (PCR) assays are described for the detection of swine vesicular disease virus (SVDV) RNA, a reverse transcription PCR (RT-PCR) and a reverse transcription nested PCR (RT-nPCR). Both the RT-PCR and RT-nPCR were able to detect representative members of each of seven phylogenetically distinct groups of SVDV and gave negative results with a range of porcine enteroviruses and of viruses responsible for vesicular conditions in pigs. When combined with a commercial kit for rapid RNA extraction, the RT-PCR was useful for the detection of SVDV in samples of epithelium and faeces from animals with clinical SVD. The addition of a second amplification step to create a nested PCR (RT-nPCR) increased the sensitivity of the technique for the detection of viral RNA (vRNA) in SVDV infected tissue culture fluid by a factor of approximately 1000, from 100 TCID 50 for the RT-PCR to 0.1 TCID 50 for RT-nPCR. When combined with a more elaborate extraction procedure for RNA, the RT-nPCR was considerably more sensitive than virus isolation in tissue culture for detecting SVDV in nasal swabs, tissues, and faeces collected from pigs between 7 days and 176 days after infection with a recent European isolate of SVDV. However, stringent conditions are necessary for carrying out the RT-nPCR to minimise the possibility of contamination.

Rapid diagnosis of poliovirus infection by PCR amplification.

A single-tube, single-primer-set reverse transcription-PCR assay was developed for the rapid detection of polioviruses in infected tissue culture fluids and clinical materials. The poliovirus-specific PCR primers are located in the VP1-2A region of the poliovirus genome. They generate a 290-bp product and can be used in duplex reactions with general enterovirus primers. The primers span the region used for genotype determination, so that genotype analysis of wild-type polioviruses can be performed by direct sequencing of the PCR products. Of 125 virus isolates typed as polioviruses by neutralization assays, 125 (100%) were also positive by PCR, and of 38 isolates typed as non-polio enteroviruses by conventional techniques, 38 (100%) were also negative by PCR. The assay described here is rapid, highly sensitive, and specific and has clinical applicability in the diagnosis of poliovirus infections.

Evaluation of Chlamydia psittaci subfraction and subunit preparations for their protective capacities

A mouse toxicity potency test was used to evaluate the protective capacity of Chlamydia psittaci (Baker strain) subfraction and subunit preparations. The elementary body (EB), Chlamydial outer membrane complex (COMC), major outer membrane protein (MOMP), lipopolysaccharide (LPS), and MOMP mixed together with LPS were utilized as antigens in vaccine preparation. The EBs and COMCs were prepared by centrifugation and detergent extraction as previously described for Chlamydia trachomatis. Isolation and purification of MOMP and LPS were achieved by subjecting infected tissue culture fluids to polyacrylamide gel electrophoresis and subsequent elution from the gel. Chromatographic purification of the MOMP from EBs resulted in a preparation which also contained minor quantities of LPS. After vaccination and intraperitoneal challenge with C. psittaci (Cello strain), the MOMP presented in EBs, COMCs or in a purified form, was capable of protecting mice from death, whereas LPS did not demonstrate this capability. Further comparison of the protective capacity of these antigens indicated that the electrophoretically purified MOMP was the least protective when suboptimal levels of antigens were administered to mice. However, the level of protection afforded by electrophoretically purified MOMP could be increased to that observed with the chromatographically purified MOMP by the addition of electrophoretically purified LPS to the vaccine. These results suggest that the MOMP is the major protective antigen of C. psittaci and that the LPS component, although not protective on its own, may play a role in the potentiation of a protective immune response.

Haemagglutination-inhibition test for haemorrhagic fever with renal syndrome using virus antigen prepared from infected tissue culture fluid.

Haemagglutinating (HA) antigens of four strains of virus related to that causing haemorrhagic fever with renal syndrome (HFRS) were prepared from infected tissue culture fluids by ultracentrifugation. The titres of the precipitated antigens were increased considerably by acetone extraction and sonication. Acetone extraction completely inactivated infectious virus in the antigen preparations. The antigens were pH-dependent, with pH optima at 5.8. Good correlations were observed in human and rat sera between the titres obtained by the haemagglutination-inhibition (HI) test and an indirect fluorescent antibody test. Moreover, strong cross-reactions among these strains were demonstrated by the HI test. The HI test has not been used previously with HFRS viruses because of the danger involved in preparing HA antigen, but these results indicate that a safe method is available for serological identification of HFRS.

Evaluation of immunoadsorbent electron microscopic techniques for detection of sindbis virus

Two immunosorbent electron microscopic techniques (ISEM), the protein A coated grid technique (PA-CGT) and the antibody coated grid technique (AB-CGT) were applied and evaluated for the detection of Sindbis virus from infected tissue culture fluids. At optimal conditions, the efficiency of trapping the virions was only about 1.5 higher with the PA-CGT as compared to the AB-CGT, but the PA-CGT was less dependent on the antiserum dilution used in the test. Both methods were suitable for quantitation experiments, since the number of virions trapped was proportional to the virus concentration. The influence of virus incubation time and temperatures, staining solutions, buffers and washing procedures on the trapping efficiency and specificity was further studied with the PA-CGT. Maximal trapping on coated grids was obtained after 3 h incubation of the virus. At room temperature, less debris was found on the grids, as compared to 37°C, and the numbers of virions counted were only slightly lower. The optimal staining solution was alcohol uranyl acetate. The specificity of the PA-CGT was dependent on washing steps with phosphate buffered saline containing bovine serum albumin. With the standard procedure, at room temperature around 3 × 10 7 virions/ml (1 × 10 6 PFU/ml) were specifically detected in about 1.5 h.

Rapid diagnosis of parainfluenza virus infection in children.

The indirect immunofluorescent-antibody (IFA) assay for detection of parainfluenza virus antigen was performed on samples of nasopharyngeal secretions obtained from infants and children with various respiratory illnesses to determine the usefulness of the IFA assay for rapid diagnosis of parainfluenza virus infection. Ninety-four samples of nasopharyngeal secretions were obtained from 78 children during a community outbreak of parainfluenza virus infection. Application of the IFA assay revealed the presence of parainfluenza antigen in 67 of the 94 samples. When these same specimens were inoculated into tissue culture, the presence of parainfluenza virus was confirmed in 62 (93%) of the 67 IFA-positive specimens by hemadsorption on tissue culture monolayers (31 cases), presence of parainfluenza hemagglutinin in infected tissue culture fluids (23 cases), or by other methods (8 cases). Only four IFA-negative specimens were subsequently shown to be positive for parainfluenza virus by tissue culture infectivity. The IFA assay provided a more rapid and more accurate method for diagnosis of parainfluenza infection in children than did routine tissue culture methods employed currently.

Concentration and purification of rubella virus hemagglutinin by hollow fiber ultrafiltration and sucrose density centrifugation

Large volumes of rubella virus were produced in Vero cell monolayers which were grown in the Corbeil-Bellco TM system. Infectious tissue culture fluids were concentrated at least 600 times in less than 4h by ultrafiltration on hollow fibers with a molecular weight cutoff of 100 000. Recovery of the hemagglutinating activity was 75%. Rubella virus was purified by three successive sucrose density gradient centrifugations using a combination and discontinuous and linear gradients. Specific activity was increased 1000-fold.

Detection of enterovirus specific IgG and IgM antibodies in humans by an indirect solid phase radioimmunoassay.

The development of a solid phase radioimmunoassay which is able to detect virus-specific IgG and IgM antibodies in serum specimens from patients with enterovirus infections is described. Viral antigen partially purified from infected tissue culture fluid was absorbed passively to individual polystyrene microtiter wells. Dilutions of sera were incubated on these antigens and bound anti-viral antibodies were monitored by the addition of 125-iodine labeled anti-human-IgG or anti-human-IgM antibody. Specificity of the assay to detect virus-specific IgM antibody was ensured by highly specific anti-IgM antibody which did not cross react with IgG and 2-mercaptoethanol sensitivity of IgM antibody titers. Changes of IgM antibody titers clearly indicated a current infection by that virus strain which was isoalted as etiological agent. Advantages and restrictions of the introduced radioimmunoassay are discussed.

Erythroid bursts produced by Friend leukaemia virus in vitro.

WHEN mouse haematopoietic cells are incubated in plasma clot or methyl cellulose cultures in the presence of erythropoietin (EP), at least two kinds of colonies are observed1–4. After 2 days, randomly distributed erythroid colonies (defined as a clone of eight or more benzidine-positive cells) appear1,2, and after 3–8 d of culture with higher concentrations of EP, larger colonies arise in clusters or ‘bursts’3,4. The cells of origin have been termed the colony-forming unit-erythroid (CFU-E)1 and burst-forming unit-erythroid (BFU-E)3,4, respectively. Erythropoiesis in mice is also increased considerably by the Friend virus complex5,6, which has been studied mostly in vivo although there are systems for the study of erythroid transformation in vitro soon after infection7–10. Nooter and Bentvelzen11, using methyl cellulose cultures, showed that mouse marrow cells, previously incubated in vitro with purified Rauscher leukaemia virus, develop more erythroid colonies than untreated control cells. Clarke et al.12, using the plasma clot method, observed erythroid colonies 2 d after incubation of mouse marrow cells with culture fluid from Friend virus infected monolayers of fibroblasts. These colonies were similar to EP-induced 2-d colonies in gross morphology and random distribution throughout the clot. Virus-induced bursts were not present in later stages of culture and erythroid colonies were not induced after incubation of marrow cells with plasma from Friend virus infected mice. We now report that when mouse marrow cells are incubated in vitro with infectious plasma or tissue culture fluids containing the Friend virus complex, a substantial number of erythroid bursts appear 5 d after the initiation of the cultures. The Friend virus complex consists of at least two biologically active viruses—the spleen focus-forming virus (SFFV) which is replication defective and essential for increased erythropoiesis in vivo, and a helper murine leukaemia virus (MuLV-F)13,14.

Temperature- and UV-light resistance of rubella virus infectivity.

The thermal and UV-light sensitivity of a laboratory-adapted rubella virus (strain Wright) was assayed quantitatively and by electron microscopic observation. As judged by the appearence of cytopathic effects in cultures of African green monkey kidney cells (Vero) and/or by the presence of rubella virus particles in electron microscopic preparations, infectivity was found to withstand inactivation temperatures up to 70° C for 30 minutes. Infectious tissue culture fluid treated by UV-irradiation for 60 minutes also produced cytopathic effects in Vero cells. Ultrastructurally, however, only a few rubella virus-like particles could be detected. Virus suspensions filtered through 0.22 μ membrane filters prior to heat-or UV-treatment failed to reveal residual infectivity. Some of the possible mechanisms responsible for this marked thermal and UV-resistance of the rubella virus strain used are discussed.

Preservation of enteroviruses by freeze-drying.

A method was developed for freeze-dry stabilization of poliovirus type 3. An ultrafiltration procedure was used to remove salts from infected tissue culture fluid, and the virus was freeze-dried after suspension in an alkaline organic buffer. This method was further tested with other picornaviruses including poliovirus types 1 and 2, coxsackieviruses A9, A20, B2, and B5, echovirus 11, and the encephalomyocarditis virus. Freeze-dried preparations of the poliovirus could be shipped to distant laboratories at ambient temperature with excellent retention of infectivity. Data are presented showing effects of freeze-drying as well as results of exposure to temperatures to 37 C.

Inactivated complement fixing antigen from Venezuelan equine encephalitis virus grown in tissue culture.

SummaryNoninfectious complement fixing antigens of Venezuelan equine encephalitis virus were prepared by chromatography of infected tissue culture fluids and subsequent inactivation of the purified product by exposure to ionizing radiation. The product was highly active, stable, and free of anti-complementary activity.

Purification of arboviruses grown in tissue culture.

Arboviruses (Chikungunya, dengue type 1, and Japanese encephalitis) cultivated in monolayer cultures of BHK-21 cells grown with Eagle's MEM supplemented with bovine serum albumin were purified by the following procedures: The viruses were precipitated from infectious tissue culture fluids by zinc acetate (0.05 M) and were resuspended in saturated ethylenediaminetetraacetate. The suspension was filtered through a Sephadex G200 [(lower) 10 cm]-Sepharose 6B [(upper) 40 cm] column (2.5 cm in diam) by upward elution. The filtrate was concentrated in a collodion bag in vacuo. The final step was sucrose density gradient centrifugation. The procedures were comparatively short and straightforward, and about 100 to 500 times purification factors were obtained consistently. The CHIK virus preparation after the sucrose density gradient centrifugation was demonstrated as a single peak, the infectivity and HA activity coinciding with each other; whereas the DEN-1 and JE viruses were shown to be heterogeneous. The problems of whether such a difference reflects some basic characteristics of groups A and B viruses need further investigation. Electron microscopic pictures of the preparations revealed spherical particles of (mμ): 50 to 60 (CHIK), 50 to 55 (DEN-1), and 45 to 50 (JE). These images are compatible, in general, with those previously reported by Igarashi et al. (CHIK virus) (13, 14); Matsumura and Hotta (DEN-1 virus) (15); Smith et al. (DEN-2 virus) (16); Takaku et al. (JE virus) (17); and Nozima et al. (JE virus) (18); although the origin of the viruses and the purification methods are not necessarily the same. The present purification procedures can probably be applied with success to other arboviruses in general. Further studies on the biochemical and biophysical nature of the purified virions are underway and will be reported later.

Effect of the purification of virus antigens on the production of specific complement-fixing antibodies.

The effect of viral purification procedures on the antibody response of guinea pigs to immunization with reovirus type 2 and echovirus type 19 was investigated. Three grades of antigens were employed: (i) infectious monkey kidney tissue culture fluid (TCF), (ii) virus sedimented in the ultracentrifuge and suspended in phosphate-buffered saline, and (iii) virus purified by centrifugation in CsCl density gradients. The antibody response of the guinea pigs was studied by the hemagglutination inhibition, complement fixation, and serum neutralization tests. Only sera produced from virus purified by CsCl density gradients reacted specifically with homologous antigen in the complement fixation test. Sera from animals receiving tissue culture fluid virus or sedimented virus cross-reacted with heterologous antigens such as tissue culture fluid from uninfected monkey kidney cells. All sera, however, reacted specifically in hemagglutination inhibition and serum neutralization tests. Sera from intranasally infected animals (reovirus type 2), even though reacting specifically in the complement fixation test, had much lower titers than sera from animals inoculated intramuscularly.

Preservation of Enteroviruses by Freeze-Drying

A method was developed for freeze-dry stabilization of poliovirus type 3. An ultrafiltration procedure was used to remove salts from infected tissue culture fluid, and the virus was freeze-dried after suspension in an alkaline organic buffer. This method was further tested with other picornaviruses including poliovirus types 1 and 2, coxsackieviruses A9, A20, B2, and B5, echovirus 11, and the encephalomyocarditis virus. Freeze-dried preparations of the poliovirus could be shipped to distant laboratories at ambient temperature with excellent retention of infectivity. Data are presented showing effects of freeze-drying as well as results of exposure to temperatures to 37 C.