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PubMed1990

Inactivation and stability of viral diagnostic reagents treated by gamma radiation.

The objective of this study was to apply the pertinent findings from gamma inactivation of virus infectivity to the production of high quality diagnostic reagents. A Gammacell 220 (Atomic Energy of Canada, Ltd., Ottawa, Canada) was used to subject 38 viruses grown in either susceptible tissue cultures or embryonated chicken eggs to various doses of gamma radiation from a cobalt-60 source. The radiation required to reduce viral infectivity was 0.42 to 3.7 megarads (Mrad). The effect of gamma treatment on the antigenic reactivity of reagents for the complement fixation (CF), hemagglutination (HA) and neuraminadase assays was determined. Influenza antigens inactivated with 1.7 Mrad displayed comparable potency, sensitivity, specificity and stability to those inactivated by standard procedures with beta-propiolactone (BPL). Significant inactivation of influenza N1 and B neuraminidase occurred with greater than 2.4 Mrad radiation at temperatures above 4 degrees C. All 38 viruses were inactivated, and CF or HA antigens were prepared successfully. Antigenic potency remained stable with all antigens for 3 years and with 83% after 5 years storage. Influenza HA antigens evaluated after 9 years of storage demonstrated 86% stability. Gamma radiation is safer than chemical inactivation procedures and is reliable and effective replacement for BPL in preparing diagnostic reagents.

Animals↗
PubMed2006

Validation of syndromic surveillance for respiratory infections.

STUDY OBJECTIVE: A key public health question is whether syndromic surveillance data provide early warning of infectious outbreaks. One cause for skepticism is that biological correlates of the administrative and clinical data used in these systems have not been rigorously assessed. This study measures the value of respiratory data currently used in syndromic surveillance systems to detect respiratory infections by comparing it against criterion standard viral testing within a pediatric population. METHODS: We conducted a longitudinal study with prospective validation in the emergency department (ED) of a tertiary care children's hospital. Children aged 7 years or younger who presented with a respiratory syndrome or who were tested for respiratory syncytial virus (RSV), influenza virus, parainfluenza virus, adenovirus, or enterovirus between January 1993 and June 2004 were included. We assessed the predictive ability of the viral tests by fitting generalized linear models to respiratory syndrome counts. RESULTS: Of 582,635 patient visits, 89,432 (15.4%) were for respiratory syndromes, and of these, 7,206 (8.1%) patients were tested for the viruses of interest. RSV was significantly related to respiratory syndrome counts (adjusted rate ratio [RR] 1.33; 95% confidence interval [CI] 1.04 to 1.71). In multivariate models including all viruses tested, influenza virus was also a significant predictor of respiratory syndrome counts (RR 1.47; 95% CI 1.03 to 2.10). This model accounted for 81.6% of the observed variability in respiratory syndrome counts. CONCLUSION: Respiratory syndromic surveillance data strongly correlate with virologic test results in a pediatric population, providing evidence of the biologic validity of such surveillance systems. Real-time outbreak detection systems relying on syndromic data may be an important adjunct to the current set of public health systems for the detection and surveillance of respiratory infections.

Boston↗
PubMed2004

Cell culture propagation and biochemical analysis of the Ljungan virus prototype strain.

Ljungan virus (LV) is proposed as a potentially important rodent harbored viral human pathogen. Little is known about the biophysical nature of the virus and despite being molecularly characterized, progress in epidemiological and basic biological studies of LV has been hampered by the lack of a robust and reliable cell culture propagation system. Here we report the first description of an efficient lytic multi-cycle cell culture propagation of the LV prototype strain (87-012). Biophysical analysis of gradient purified LV virions generated by this system identified mature infectious virions to possess a sedimentation coefficient of 160S and in agreement with previous molecular prediction, polyprotein analysis suggests that the native virion is composed of only three major structural proteins. The nucleotide composition of the complete genome of the LV cell culture adapted virus was determined and compared to that of the parental prototype LV. Numerous mutations were observed scattered throughout the viral genome and particularly in VP1. The development of this cell culture system for LV should open new avenues in the study of LV biology, structure, pathogenesis, and prevalence of natural infection in the wider community.

Animals↗
PubMed2005

Compatibility of lyotropic liquid crystals with viruses and mammalian cells that support the replication of viruses.

We report a study that investigates the biocompatibility of materials that form lyotropic liquid crystals (LCs) with viruses and mammalian cells that support the replication of viruses. This study is focused on aqueous solutions of tetradecyldimethyl-amineoxide (C(14)AO) and decanol (D), or disodium cromoglycate (DSCG; C(23)H(14)O(11)Na(2)), which can form optically birefringent, liquid crystalline phases. The influence of these materials on the ability of vesicular stomatitis virus (VSV) to infect human epitheloid cervical carcinoma (HeLa) cells was examined by two approaches. First, VSV was dispersed in aqueous C(14)AO+ D or DSCG, and then HeLa cells were inoculated by contacting the cells with the aqueous C(14)AO + D or DSCG containing VSV. The infectivity of VSV to the HeLa cells was subsequently determined. Second, VSV was incubated in LC phases of either C(14)AO + D or DSCG for 4 h, and the concentration (titer) of infectious virus in the LC was determined by dilution into cell culture medium and subsequent inoculation of HeLa cells. Using these approaches, we found that the LC containing C(14)AO + D caused inactivation of virus as well as cell death. In contrast, we determined that VSV retained its infectivity in the presence of aqueous DSCG, and that greater than 74-82% of the HeLa cells survived contact with aqueous DSCG (depending on concentration of DSCG). Because VSV maintained its function (and we infer structure) in LCs formed from DSCG, we further explored the influence of the virus on the ordering of the LC. Whereas the LC formed from DSCG was uniformly aligned on surfaces prepared from self-assembled monolayers (SAMs) of HS(CH(2))(11)(OCH(2)CH(2))(4)OH on obliquely deposited films of gold in the absence of VSV, the introduction of 10(7)-10(8) infectious virus particles per milliliter caused the LC to assume a non-uniform orientation and a colorful appearance that was readily distinguished from the uniformly aligned LCs. Control experiments using cell lysates with equivalent protein concentrations but no virus did not perturb the uniform alignment of the LC.

Biocompatible Materials↗
PubMed2004

Routine use of a highly automated and internally controlled real-time PCR assay for the diagnosis of herpes simplex and varicella-zoster virus infections.

BACKGROUND: Detection of herpes viruses can be significantly improved by PCR. The development of real-time PCR, which has overcome several limitations of conventional PCR, improved the prospects for implementation of PCR-based assays in diagnostic laboratory. OBJECTIVES: To compare the diagnostic performance of an automated sample extraction procedure in combination with an internally controlled real-time PCR assay for detection of herpes simplex virus (HSV) and varicella-zoster virus (VZV) to conventional shell vial culture. STUDY DESIGN: One hundred eighty-two consecutive specimens from patients suspected of HSV or VZV infection were examined by internally controlled PCR and shell vial culture. An internal control consisting of phocine herpes virus was processed along with the specimens during the entire procedure and permitted to monitor extraction and amplification efficiency, including inhibition. RESULTS: A total of 48 (26.4%) specimens were positive for HSV or VZV by culture, and 77 (42.3%) by real-time PCR. Thus, overall sensitivity increased by 60.4%. All culture-positive specimens were detected and typed correctly by PCR, except for a single specimen that contained PCR inhibitors. Specifically, the real-time PCR assay increased the detection rate for HSV-1 and HSV-2 by 43.9% and 62.5%, respectively. In PCR-positive specimens, lower levels of viral DNA were found in culture-negative than in culture-positive specimens. The increase of HSV detection rates by PCR varied with the origin of specimen and was particularly significant for skin specimens (7/14 versus 3/14 detected by culture) and bronchoalveolar lavages (8/8 versus 1/8). In addition, real-time PCR significantly increased the detection rate for VZV. CONCLUSIONS: Compared to shell vial culture, our real-time PCR assay demonstrated a superior sensitivity and an added value of using internal control for checking the quality of examination of each specimen. These results provide a solid basis for implementation of real-time PCR in the routine diagnosis of HSV and VZV infections in various clinical specimens.

Automation↗
PubMed2006

Stable HepG2- and Huh7-based human hepatoma cell lines for efficient regulated expression of infectious hepatitis B virus.

BACKGROUND/AIMS: Hepatitis B virus (HBV) cannot be propagated in cultured cells but two human hepatoma cell lines, HepG2 and Huh7, support virus replication when transfected with HBV DNA. If standardization is required stably transfected cell lines provide distinct advantages. One such line, HepG2.2.15, is widely used in antiviral research but HBV production is limited and difficult to control. Our aim was to establish stable, inducibly HBV producing HepG2 and Huh7 cell lines that overcome these limitations. METHODS: Based on the tetracycline (Tet)-regulated TetOFF system, a Tet-responsive promoter-controlled HBV genome was introduced into separately established, well-regulatable HepG2 and Huh7 lines expressing Tet-responsive trans-activators (tTAs). Stable clones were analyzed for regulatability and levels of HBV expression, quality of the virus produced, and responsiveness towards antivirals. RESULTS: HepG2- and Huh7-based cell lines were established which, Tet-controllably, produce more HBV than HepG2.2.15 cells. The secreted virions were infectious for primary tupaia hepatocytes, and the cell lines responded as well as HepG2.215 cells to different antivirals. CONCLUSIONS: The new HBV cell lines should be valuable tools for academic and pharmaceutical HBV research. The parental tTA-cells will facilitate the generation of additional lines, producing HBV variants, or other genes, in an identical host cell background.

Antiviral Agents↗
PubMed2005

A model of the dynamics of insect cell infection at low multiplicity of infection.

In the present paper, we offer a preliminary mathematical model that describes the dynamic process of cell infection with baculovirus at low multiplicity of infection (MOI). The model accounts for the chain of events that follow the infection of insect cells, namely the eclipse period, the budding of viral particles from those cells, their attachment to non-infected cells and the initiation of a new infection cycle. These cycles appear as fluctuations in the viral concentration of actual cell culture media. The potential of the present approach in simulating the in vitro production of biological insecticides is demonstrated. The influence of the shape of the virus-budding function is shown, and parameter sensitivity analysis is carried out. The model provides a quantitative tool for the analysis of this complex dynamic system.

Animals↗
PubMed2004

Viral growth assay to evaluate the replicative capacity of HIV-1 isolates.

The replicative capacity of HIV is studied by carrying out replication-competition experiments with the insertion of the gene of interest. These assays cannot capture the complicated patterns of mutations of different genes.A cross sectional study was carried out on 10 HIV-infected nai;ve patients and on 15 patients failing HAART. The CD8-depleted PBMCs, with known proviral DNA and cellular HIV-RNA copy numbers, were cultured. A reference curve was determined using the data obtained from 10 nai;ve patients. The replicative capacity was calculated as the ratio multiplied by 100 of the p24 antigen level of isolates over the p24 antigen level determined on the reference curve.A linear correlation between p24 antigen level and the infectious doses of HIV-DNA alone or plus cellular RNA copy number of PBMCs was found in naive patients (r=0.63, P<0.001 and r=0.67, P<0.001, respectively). Although all patients failing therapy had strains with impaired replicative capacity, a wide range of values (0.1-74.5%) was detected. All strains with a replicative capacity above 10% had non-nucleoside reverse transcriptase inhibitors related mutations.A viral assay to evaluate the HIV replicative capacity is described. The high variability of replicative capacity confirms the need to undertake replicative capacity assay using the whole virus.

Antiretroviral Therapy, Highly Active↗
PubMed2004

Comparative study of influenza virus replication in Vero and MDCK cell lines.

The choice of a cell line for the production of influenza vaccines is determined by how well the virus is able to replicate and how easily the cell line can be maintained. Madin-Darby canine kidney (MDCK) cells have long been known to successfully support influenza growth. Vero cells are also another well studied candidate cell line. In this work, we have compared these two cell lines for their ability to propagate type A and type B cold-adapted and wild type influenza viruses. The growth of these viruses has been measured as plaque forming units (via plaque assay) as well as viral particle formation (via a novel quantitative RT-PCR assay) to assess the suitability of these cell lines to support the development of live attenuated influenza vaccines. The novel qRT-PCR assay outlined in this work was demonstrated to be an efficient, sensitive and reproducible method for measuring wild type (wt) and cold-adapted (ca) influenza strains. Replicates of six per sample consistently showed an average variation around +/-10%. In this study we have also found qRT-PCR to be a useful method for differentiating between wt and ca influenza strains based on their differing growth characteristics at varying temperatures. This can subsequently be used to assess reassortants prepared from ca donor strains for the purposes of live viral vaccine development. For type A and B influenza viruses studied in this work, MDCK cells supported a more rapid viral growth (measured in terms of genome copies) compared with Vero cells. For the type A viruses studied here, the genome copies: infectious unit (genome copy, gc:infectious unit, iu) ratio was found to be more favorable for Vero cells compared with MDCK cells. For the type B viruses studied in this work, the gc:iu was equivalent in both cell lines tested. Ultimately, however, the use of any new cell line would need to be approved by regulatory agencies prior to its commercial application.

Animals↗
PubMed2004

Large-scale production, purification and crystallization of wild-type adeno-associated virus-2.

Adeno-associated virus-2 (AAV-2) has long been recognized as a potential vector for human gene therapy. Although much progress has been made in the molecular virology of AAV-2, structural studies of AAV-2 have been hampered by the low efficiency of virus production in culture, the low purity of preparations, and the low solubility of pure virus particles in solution. Methods of larger scale AAV-2 production have been developed through adaptation to suspension culture and re-optimization of the times of infection and transfection with respect to particle production. The methods allow the purification of 10mg ( approximately 10(15) particles) of AAV-2 per preparation at approximately 99% purity as judged by SDS-PAGE. This was sufficient for the screening of conditions for the formation of diffraction-grade crystals, ultimately leading to an atomic structure for AAV-2.

Crystallization↗
PubMed2005

The growth of attenuated influenza vaccine donor strains in continuous cell lines.

The growth of the Russian live attenuated influenza vaccine donor strains A/Leningrad/134/17/57, A/Leningrad/134/47/57 and B/USSR/60/69 was studied in cells of the VERO and Madin-Darby canine kidney (MDCK) lines as six-well cultures and cell factories infected at different multiplicities of infection. Yields for A/Leningrad/134/17/57 and A/Leningrad/134/47/57 were comparable in either cell line over a range of multiplicities but were about 10-fold lower than in the allantoic fluids of infected chicken embryos. For both A/Leningrad/134/47/57 and B/USSR/60/69, yields from the MDCK line were about 10-fold higher than for the VERO line. For B/USSR/60/69, yields in eggs were approximately 100-fold higher than those obtained in the MDCK line. A feature of the growth of B/USSR/60/69 was its reduced capacity to produce infectious progeny in either cell line at multiplicities of infection of 2.0 or 1.0 pfu/cell. Inhibition was due probably due to the presence of defective-interfering particles and was not detected with A/Leningrad/134/17/57 or A/Leningrad/134/47/57 in cultures of either line infected at the same multiplicities. Yields for both A/Leningrad/134/47/57 and B/USSR/60/69 in cells of the MDCK line were comparable when grown in six-well cultures or cell factories.

Animals↗
PubMed2006

A novel method for cloning of non-cytolytic viruses.

Hantaviruses are rodent-borne pathogens with a segmented single-stranded RNA genome of negative polarity. Spontaneous occurrence of variants with genetic heterogeneity have been observed both in vivo and in vitro. The objective of this study was to establish a method for the cloning of genetically homogenous hantaviruses which can be used for subsequent functional studies. Infected VeroE6 cells were incubated with an agarose/medium overlay to prevent uncontrolled distribution of de novo synthesized virus. Thereafter, the overlay was removed and stored for isolation of the diffused virus. The cell layer was fixed and viral antigen-containing foci were detected by immunochemistry. The relative location of the foci on the culture dish was used to trap individual virus clones in the corresponding overlay. The clones were picked and used for re-infection. According to this novel protocol three different hantaviruses, i.e. Hantaan, Puumala, and Tula virus, were purified. In the course of purification the titers of the resulting virus stocks were increased by 10-1000-fold. In addition, this method was used to purify a minor Puumala virus variant from a parental stock containing a mixture of two variants. Taken together, the method presented is well suited to isolate genetically homogenous hantaviruses and might also be applicable for other non-cytolytic viruses.

Animals↗
PubMed2006

Identification of cell lines permissive for human coronavirus NL63.

Six cell lines routinely used in laboratories were tested for permissiveness to the infection with the newly identified human coronavirus NL63. Two monkey epithelial cell lines, LLC-MK2 and Vero-B4, showed a cytopathic effect (CPE) and clear viral replication, whereas no CPE or replication was observed in human lung fibroblasts MRC-5s. In Rhabdomyosarcoma cells, Madin-Darby-Canine-kidney cells and in an undefined monkey kidney cell line some replication was observed but massive exponential rise in virus yield lacked The results will lead to an improved routine diagnostic algorithm for the detection of the human coronavirus NL63.

Animals↗
PubMed2004

Metabolism of MDCK cells during cell growth and influenza virus production in large-scale microcarrier culture.

The production of equine influenza in Madin-Darby canine kidney (MDCK) cells in large-scale microcarrier culture is described with detailed on- and off-line analytical data during cell growth and virus replication. Metabolite concentration profiles for glucose, glutamine, lactate and ammonium are shown. Lactate and ammonium concentrations were always below inhibiting levels. Concentration profiles for essential and non-essential amino acids of the cell culture medium are discussed. During cell growth proline was released into the medium with a significant rate while two amino acids, serine and methionine were almost depleted. After infection, virus titer increased after a delay of 10-16 h whereas first changes in amino acid metabolism could be observed within 4h post-infection. Here, glutamate and aspartate increase correlated to virus release kinetics, indicating cell disruption and apoptosis. Starting with a moi of 0.025 resulted in a maximum virus yield of 2.4 log HA/100 microl at 44 h post-infection.

Amino Acids↗
PubMed2004

In vitro growth and stability of recombinant rabies viruses designed for vaccination of wildlife.

Three live rabies virus (RV) recombinant vaccine candidates, SPBNGA, SPBNGA-Cyto c (+), and SPBNGA-GA, were examined for their production levels and stability. Maximum production levels up to 10(10) infectious particles/mL were achieved using bioreactor technology. All virus lots exhibited thermostability profiles typical for RV vaccines and were non-pathogenic for intracranially inoculated immunocompetent mice. Moreover, sequence analysis indicated high genetic stability in all three RVs during 10 consecutive passages in newborn mice. This analysis revealed no change in the extra RV G gene in the SPBNGA-GA vaccine or in the cytochrome c gene in the SPBNGA-Cyto c (+) vaccine. Moreover, no changes were detected in the G gene codon for Glu333, which renders the virus non-pathogenic. However, after the fifth passage, a mutation resulting in an Asn194 --> Lys194 exchange emerged in the G genes of all three RVs. This mutation was associated with a modest increase in pathogenicity in SPBNGA and SPBNGA-Cyto c (+), but not in SPBNGA-GA, which contained the mutation in only one of its two G genes and which remained non-pathogenic. These results demonstrate the feasibility of producing RV vaccines that remain highly stable even after multiple passages.

Animals↗
PubMed2006

Single amino acid substitutions in the hemagglutinin of influenza A/Singapore/21/04 (H3N2) increase virus growth in embryonated chicken eggs.

Most of the recently circulating H3N2 influenza A strains do not replicate well in embryonated chicken eggs and had to be isolated by cell culture, which presents a great challenge for influenza vaccine production using embryonated chicken eggs. We previously reported that a human H3N2 virus, A/Fujian/411/02, which replicates poorly in eggs, could be improved by changing a minimum of two HA residues (G186V/V226I or H183L/V226A). Here, we extended our work to the A/Singapore/21/04 strain that was also unable to grow in eggs. We showed that a single amino acid substitution of either G186V or A196T in the HA resulted in significantly increased virus replication in eggs without affecting virus antigenicity.

Amino Acid Substitution↗