Passage In Cell Culture Research Articles

BIOLOGICAL, MOLECULAR AND IMMUNOCYTOCHEMICAL CHARACTERIZATION OF CELL CULTURE ADAPTED HUMAN ROTAVIRUS STRAINS DETECTED IN THE CITY OF PONTA GROSSA, PARANA, BRAZIL

Rotaviruses (RV) are the single most important cause of severe diarrheal illness in infants and young children in both developed and developing countries worldwide. They affect mainly children less than 24 months of age. Presently, we adapted human rotavirus strains from field materials in cell culture, as well as, studied their properties by the analyses of the cytopathic effect, immunofluorescence features, plaque properties and virus genome stability. The study was carried out with 53 rotavirus-positive fecal specimens, collected in the city of Ponta Grossa, Parana, before the introduction of vaccination program in Brazil. The samples were submitted to at least 3 blind passages in MA-104 cell culture before discarded as non-cultivable. The adaptation process was monitored by the development of the cytopathic effect, immunofluorescence assay , plaque assay and genome stability by reverse transcriptase-polymerase chain reactions amplification. Fourteen human rotavirus strains were adapted to grow in cell culture. The isolation was demonstrated by the development of typical cytopathic effect. Immunofluorescence assay revealed granular inclusions with specific fluorescence mainly at the perinuclear area of the infected cells. Plaque assay allowed the demonstration of plaques with varying diameter depending upon the strains. Reverse transcriptase-polymerase chain reactions resulted in amplification and genotyping, demonstrating the stability of the genome through cell culture adaptation. DOI: http://dx.doi.org/10.17525/vrr.v19i1.97

Malignant catarrhal fever in American bison (Bison bison) experimentally infected with alcelaphine herpesvirus 2

Malignant catarrhal fever (MCF), due to ovine herpesvirus 2 (OvHV-2), causes appreciable death loss in ranched bison (Bison bison) throughout North America. No vaccine exists to protect animals from disease. Since OvHV-2 has not been propagated in vitro, one strategy to develop a modified live vaccine is to use a closely related, non-pathogenic member of the malignant catarrhal fever virus family as a vector expressing potentially protective OvHV-2 epitopes. To date, no controlled experimental challenge studies with alcelaphine herpesvirus 2 (AlHV-2) derived from topi (Damaliscus lunatus jimela) have been reported The unique or light DNA segment of the AlHV-2 genome was sequenced and annotated and the virus was tested for its ability to infect and induce disease in American bison. Yearling bison were inoculated intranasally (n=4) or intramuscularly (n=3) with 2×10−4.7 TCID50 of AlHV-2, and monitored for infection and the development of disease. Six inoculated bison became infected with AlHV-2. Two of the six animals developed clinical signs and had gross and histological lesions consistent with terminal MCF, which differed in distribution from those in bison with MCF due to OvHV-2. One other animal developed minor clinical signs and had gross and histological pulmonary lesions consistent with early (pre-clinical) stages of MCF. Unmodified low cell culture passage AlHV-2 derived from topi is an unsuitable vaccine vector for the prevention of MCF. However, the annotated genome might be useful in identifying genes which could be deleted to potentially attenuate the virus for bison.

Pseudorabies Virus pUL46 Induces Activation of ERK1/2 and Regulates Herpesvirus-Induced Nuclear Envelope Breakdown

Herpesvirus capsid morphogenesis occurs in the nucleus, while final maturation takes place in the cytosol, requiring translocation of capsids through the nuclear envelope. The nuclear egress complex, consisting of homologs of herpes simplex virus pUL31 and pUL34, is required for efficient nuclear egress via primary envelopment and de-envelopment. Recently, we described an alternative mode of nuclear escape by fragmentation of the nuclear envelope induced by replication-competent pUL31 and pUL34 deletion mutants of the alphaherpesvirus pseudorabies virus (PrV), which had been selected by serial passaging in cell culture. Both passaged viruses carry congruent mutations in seven genes, including UL46, which encodes one of the major tegument proteins. Herpesvirus pUL46 homologs have recently been shown to activate the PI3K-Akt and ERK1/2 signaling pathways, which are involved in regulation of mitosis and apoptosis. Since in uninfected cells fragmentation of the nuclear envelope occurs during mitosis and apoptosis, we analyzed whether pUL46 of PrV is involved in signaling events impairing the integrity of the nuclear envelope. We show here that PrV pUL46 is able to induce phosphorylation of ERK1/2 and, thus, expression of ERK1/2 target genes but fails to activate the PI3K-Akt pathway. Deletion of UL46 from PrV-ΔUL34Pass and PrV-ΔUL31Pass or replacement by wild-type UL46 resulted in enhanced nuclear envelope breakdown, indicating that the mutations in pUL46 may limit the extent of NEBD. Thus, although pUL46 induces ERK1/2 phosphorylation, controlling the integrity of the nuclear envelope is independent of the ERK1/2 signaling pathway. Herpesvirus nucleocapsids can leave the nucleus by regulated, vesicle-mediated transport through the nuclear envelope, designated nuclear egress, or by inducing nuclear envelope breakdown (NEBD). The viral proteins involved in NEBD are unknown. We show here that the pseudorabies virus tegument protein pUL46 induces the ERK1/2 signaling pathway and modulates NEBD. However, these two processes are independent and ERK1/2 signaling induced by pUL46 is not involved in herpesvirus-induced NEBD.

Attenuation of Human Enterovirus 71 High-Replication-Fidelity Variants in AG129 Mice

In a screen for ribavirin resistance, a novel high-fidelity variant of human enterovirus 71 (EV71) with the single amino acid change L123F in its RNA-dependent RNA polymerase (RdRp or 3D) was identified. Based on the crystal structure of EV71 RdRp, L123 locates at the entrance of the RNA template binding channel, which might form a fidelity checkpoint. EV71 RdRp-L123F variants generated less progeny in a guanidine resistance assay and virus populations with lower mutation frequencies in cell culture passage due to their higher replication fidelity. However, compared with wild-type viruses, they did not show growth defects. In vivo infections further revealed that high-fidelity mutations L123F and G64R (previously reported) negatively impacted EV71 fitness and greatly reduced viral pathogenicity alone or together in AG129 mice. Interestingly, a variant with double mutations, RG/B4-G64R/L123F (where RG/B4 is an EV71 genotype B4 virus constructed by reverse genetics [RG])showed higher fidelity in vitro and less virulence in vivo than any one of the above two single mutants. The 50% lethal dose (LD50) of the double mutant increased more than 500 times compared with the LD50 of wild-type RG/B4 in mice. The results indicated that these high-fidelity variants exhibited an attenuated pathogenic phenotype in vivo and offer promise as a live attenuated EV71 vaccine. The error-prone nature of the RNA-dependent RNA polymerase (RdRp) of RNA viruses during replication results in quasispecies and aids survival of virus populations under a wide range of selective pressures. Virus variants with higher replication fidelity exhibit lower genetic diversity and attenuated pathogenicity in vivo. Here, we identified a novel high-fidelity mutation L123F in the RdRp of human enterovirus 71 (EV71). We further elucidated that EV71 variants with the RdRp-L123F mutation and/or the previously identified high-fidelity mutation RdRp-G64R were attenuated in an AG129 mouse model. As EV71 has emerged as a serious worldwide health threat, especially in developing countries in the Asia-Pacific region, we urgently need EV71 vaccines. Learning from the poliovirus vaccination, we prefer live attenuated EV71 vaccines to inactivated EV71 vaccines in order to effectively control EV71 outbreaks at low cost. Our results imply a new means of attenuating EV71 and reducing its mutation rate at the same time.

Proteolytic enzymes in embryonated chicken eggs sustain the replication of egg-grown low-pathogenicity avian influenza viruses in cells in the absence of exogenous proteases

Low pathogenic influenza viruses grow readily in embryonated chicken eggs but require the addition of exogenous proteases to grow in MDCK cell culture. In this study, we found that the influenza viruses propagated previously in eggs, can grow for up to two passages in cell culture without the addition of exogenous proteolytic enzymes. These results indicate that the reason for virus propagation in cells during the first two passages may be due to proteases from egg allantoic fluid carried over from egg culture. The ability of influenza viruses to grow in cells in the absence of trypsin is currently considered as a hallmark of highly pathogenic influenza viruses. Our data indicate that differentiating between high and low pathogenicity using cell culture only is not appropriate and other indicators such as sequence analysis and in vitro pathogenicity index should be performed.

Virulence-Associated Genome Mutations of Murine Rotavirus Identified by Alternating Serial Passages in Mice and Cell Cultures

Although significant clinical efficacy and safety of rotavirus vaccines were recently revealed in many countries, the mechanism of their attenuation is not well understood. We passaged serially a cell culture-adapted murine rotavirus EB strain in mouse pups or in cell cultures alternately and repeatedly and fully sequenced all 11 genes of 21 virus samples passaged in mice or in cell cultures. Sequence analysis revealed that mouse-passaged viruses that regained virulence almost consistently acquired four kinds of amino acid (aa) substitutions in VP4 and substitution in aa 37 (Val to Ala) in NSP4. In addition, they gained and invariably conserved the 3' consensus sequence in NSP1. The molecular changes occurred along with the acquisition of virulence during passages in mice and then disappeared following passages in cell cultures. Intraperitoneal injection of recombinant NSP4 proteins confirmed the aa 37 site as important for its diarrheagenic activity in mice. These genome changes are likely to be correlated with rotavirus virulence. Serial passage of a virulent wild-type virus in vitro often results in loss of virulence of the virus in an original animal host, while serial passage of a cell culture-adapted avirulent virus in vivo often gains virulence in an animal host. Actually, live attenuated virus vaccines were originally produced by serial passage in cell cultures. Although clinical efficacy and safety of rotavirus vaccines were recently revealed, the mechanism of their attenuation is not well understood. We passaged serially a murine rotavirus by alternating switch of host (mice or cell cultures) repeatedly and sequenced the eleven genes of the passaged viruses to identify mutations associated with the emergence or disappearance of virulence. Sequence analysis revealed that changes in three genes (VP4, NSP1, and NSP4) were associated with virulence in mice. Intraperitoneal injection of recombinant NSP4 proteins confirmed its diarrheagenic activity in mice. These genome changes are likely to be correlated with rotavirus virulence.

Deliberate Attenuation of Chikungunya Virus by Adaptation to Heparan Sulfate-Dependent Infectivity: A Model for Rational Arboviral Vaccine Design

Mosquito-borne chikungunya virus (CHIKV) is a positive-sense, single-stranded RNA virus from the genus Alphavirus, family Togaviridae, which causes fever, rash and severe persistent polyarthralgia in humans. Since there are currently no FDA licensed vaccines or antiviral therapies for CHIKV, the development of vaccine candidates is of critical importance. Historically, live-attenuated vaccines (LAVs) for protection against arthropod-borne viruses have been created by blind cell culture passage leading to attenuation of disease, while maintaining immunogenicity. Attenuation may occur via multiple mechanisms. However, all examined arbovirus LAVs have in common the acquisition of positively charged amino acid substitutions in cell-surface attachment proteins that render virus infection partially dependent upon heparan sulfate (HS), a ubiquitously expressed sulfated polysaccharide, and appear to attenuate by retarding dissemination of virus particles in vivo. We previously reported that, like other wild-type Old World alphaviruses, CHIKV strain, La Réunion, (CHIKV-LR), does not depend upon HS for infectivity. To deliberately identify CHIKV attachment protein mutations that could be combined with other attenuating processes in a LAV candidate, we passaged CHIKV-LR on evolutionarily divergent cell-types. A panel of single amino acid substitutions was identified in the E2 glycoprotein of passaged virus populations that were predicted to increase electrostatic potential. Each of these substitutions was made in the CHIKV-LR cDNA clone and comparisons of the mutant viruses revealed surface exposure of the mutated residue on the spike and sensitivity to competition with the HS analog, heparin, to be primary correlates of attenuation in vivo. Furthermore, we have identified a mutation at E2 position 79 as a promising candidate for inclusion in a CHIKV LAV.

Antigenic characterisation of lyssaviruses in South Africa

There are at least six Lyssavirus species that have been isolated in Africa, which include classical rabies virus, Lagos bat virus, Mokola virus, Duvenhage virus, Shimoni bat virus and Ikoma lyssavirus. In this retrospective study, an analysis of the antigenic reactivity patterns of lyssaviruses in South Africa against a panel of 15 anti-nucleoprotein monoclonal antibodies was undertaken. A total of 624 brain specimens, collected between 2005 and 2009, confirmed as containing lyssavirus antigen by direct fluorescent antibody test, were subjected to antigenic differentiation. The lyssaviruses were differentiated into two species, namely rabies virus (99.5%) and Mokola virus (0.5%). Furthermore, rabies virus was further delineated into two common rabies biotypes in South Africa: canid and mongoose. Initially, it was found that the canid rabies biotype had two reactivity patterns; differential staining was observed with just one monoclonal antibody. This difference was likely to have been an artefact related to sample quality, as passage in cell culture restored staining. Mongoose rabies viruses were more heterogeneous, with seven antigenic reactivity patterns detected. Although Mokola viruses were identified in this study, prevalence and reservoir host species are yet to be established. These data demonstrate the usefulness of monoclonal antibody typing panels in lyssavirus surveillance with reference to emergence of new species or spread of rabies biotypes to new geographic zones.

Stability of the parainfluenza virus 5 genome revealed by deep sequencing of strains isolated from different hosts and following passage in cell culture.

ABSTRACTThe strain diversity of a rubulavirus, parainfluenza virus 5 (PIV5), was investigated by comparing 11 newly determined and 6 previously published genome sequences. These sequences represent 15 PIV5 strains, of which 6 were isolated from humans, 1 was from monkeys, 2 were from pigs, and 6 were from dogs. Strain diversity is remarkably low, regardless of host, year of isolation, or geographical origin; a total of 7.8% of nucleotides are variable, and the average pairwise difference between strains is 2.1%. Variation is distributed unevenly across the PIV5 genome, but no convincing evidence of selection for antibody-mediated evasion in hemagglutinin-neuraminidase was found. The finding that some canine and porcine, but not primate, strains are mutated in the SH gene, and do not produce SH, raised the possibility that dogs (or pigs) may not be the natural host of PIV5. The genetic stability of PIV5 was also demonstrated during serial passage of one strain (W3) in Vero cells at a high multiplicity of infection, under conditions of competition with large proportions of defective interfering genomes. A similar observation was made for a strain W3 mutant (PIV5VΔC) lacking V gene function, in which the dominant changes were related to pseudoreversion in this gene. The mutations detected in PIV5VΔC during pseudoreversion, and also those characterizing the SH gene in canine and porcine strains, predominantly involved U-to-C transitions. This suggests an important role for biased hypermutation via an adenosine deaminase, RNA-specific (ADAR)-like activity.IMPORTANCE Here we report the sequence variation of 16 different isolates of parainfluenza virus 5 (PIV5) that were isolated from a number of species, including humans, monkeys, dogs, and pigs, over 4 decades. Surprisingly, strain diversity was remarkably low, regardless of host, year of isolation, or geographical origin. Variation was distributed unevenly across the PIV5 genome, but no convincing evidence of immune or host selection was found. This overall genome stability of PIV5 was also observed when the virus was grown in the laboratory, and the genome stayed remarkably constant even during the selection of virus mutants. Some of the canine isolates had lost their ability to encode one of the viral proteins, termed SH, suggesting that although PIV5 commonly infects dogs, dogs may not be the natural host for PIV5.

Early changes in cytokine expression in peste des petits ruminants disease

Peste des petits ruminants is a viral disease of sheep and goats that has spread through most of Africa as well as the Middle East and the Indian subcontinent. Although, the spread of the disease and its economic impact has made it a focus of international concern, relatively little is known about the nature of the disease itself. We have studied the early stages of pathogenesis in goats infected with six different isolates of Peste des petits ruminants virus representing all four known lineages of the virus. No lineage-specific difference in the pathogenicity of the virus isolates was observed, although there was evidence that even small numbers of cell culture passages could affect the degree of pathogenicity of an isolate. A consistent reduction in CD4+ T cells was observed at 4 days post infection (dpi). Measurement of the expression of various cytokines showed elements of a classic inflammatory response but also a relatively early induction of interleukin 10, which may be contributing to the observed disease.

TIN2 Mutations In Dyskeratosis Congenita Cause Telomere Shortening In Induced Pluripotent Stem Cells Through Potent Dominant Negative Inhibition Of Telomerase

Dyskeratosis congenita (DC) is a bone marrow failure syndrome characterized by widespread defects in diverse tissues and a strong predisposition to cancer. DC is caused by germline mutations in genes controlling maintenance of telomeres, nucleoprotein caps that protect chromosome ends. Mutations in components of the telomerase enzyme comprise a large share of cases, including in TERT, TERC, dyskerin, TCAB1, NOP10 and NHP2. These mutations compromise telomerase function leading to telomere shortening, which in turn impairs stem cell function. We previously created patient-derived iPS cells from patients with mutations in TERT, dyskerin or TCAB1 and analyzed these cells to understand the biochemical defects in the telomerase pathway. In each case we found a unique mechanism underlying these telomerase defects, including: reduced catalytic function (TERT mutations), impaired telomerase assembly (dyskerin mutations) and mislocalization of the enzyme to nucleoli (TCAB1 mutations). A six-member protein complex – shelterin - is essential for proper function of telomeres. Despite the critical importance of shelterin proteins in telomere regulation, only a single telomere binding protein – TIN2 – is mutated in DC. However, how these mutations compromise telomere maintenance remains poorly understood. TIN2 mutations occur in a common, autosomal dominant form of DC, presenting in early life, with particularly severe clinical manifestations and poor outcomes. Mutations in the TIN2 gene are clustered in exon 6a, which corresponds to a protein domain of unknown function. To understand how TIN2 mutations impair telomere maintenance and cause DC, we reprogrammed fibroblasts from patients with TIN2 mutations to iPS cells. We succeeded in generating pluripotent iPS cells from a patient with a frame shift mutation at position 284 of the protein. TIN2-mutant iPS cells expressed all the markers of wild-type iPS cells and human ES cells and could be differentiated to all three germ cell layers in culture. With reprogramming from fibroblasts to iPS cells, telomerase is upregulated and causes telomere elongation in wild-type cells. In analyzing telomeres from TIN2-mutant iPS cells, we found that telomere elongation was abrogated. Instead of telomere elongation, TIN2-mutant iPS cells showed telomere shortening with reprogramming and during passage in cell culture. After extended cell passage, TIN2-mutant iPS cells lost the ability to self-renew and differentiated, concomitant with the activation of the telomere surveillance checkpoint p53. To better understand how TIN2 mutant proteins interfere with telomere maintenance, we overexpressed GFP, wild-type TIN2, or TIN2 truncation mutants from DC patients into human, telomerase-positive cancer cells. Genomic DNA was collected from these cells during passage and analyzed for telomere lengths by Southern blot. Expression of GFP or wild-type TIN2 had no effect on telomere lengths, which were stably maintained during the experiment. In marked contrast, expression of the TIN2 truncation mutants from DC patients led to progressive and dramatic telomere shortening with cell passage. Together, these data in patient-derived iPS cells and in human cancer cells suggest that TIN2 mutants inhibit the action of telomerase at telomeres. These results constitute a new molecular mechanism at play in DC and yield new insight into one of the most common forms of DC. Disclosures:No relevant conflicts of interest to declare.

Isolation and Characterization of Porcine Epidemic Diarrhea Viruses Associated with the 2013 Disease Outbreak among Swine in the United States

Porcine epidemic diarrhea virus (PEDV) was detected in May 2013 for the first time in U.S. swine and has since caused significant economic loss. Obtaining a U.S. PEDV isolate that can grow efficiently in cell culture is critical for investigating pathogenesis and developing diagnostic assays and for vaccine development. An additional objective was to determine which gene(s) of PEDV is most suitable for studying the genetic relatedness of the virus. Here we describe two PEDV isolates (ISU13-19338E and ISU13-22038) successfully obtained from the small intestines of piglets from sow farms in Indiana and Iowa, respectively. The two isolates have been serially propagated in cell culture for over 30 passages and were characterized for the first 10 passages. Virus production in cell culture was confirmed by PEDV-specific real-time reverse-transcription PCR (RT-PCR), immunofluorescence assays, and electron microscopy. The infectious titers of the viruses during the first 10 passages ranged from 6 × 10(2) to 2 × 10(5) 50% tissue culture infective doses (TCID50)/ml. In addition, the full-length genome sequences of six viruses (ISU13-19338E homogenate, P3, and P9; ISU13-22038 homogenate, P3, and P9) were determined. Genetically, the two PEDV isolates were relatively stable during the first 10 passages in cell culture. Sequences were also compared to those of 4 additional U.S. PEDV strains and 23 non-U.S. strains. All U.S. PEDV strains were genetically closely related to each other (≥99.7% nucleotide identity) and were most genetically similar to Chinese strains reported in 2011 to 2012. Phylogenetic analyses using different genes of PEDV suggested that the full-length spike gene or the S1 portion is appropriate for sequencing to study the genetic relatedness of these viruses.

Further evidence for the association of distinct amino acid residues with in vitro and in vivo growth of infectious bursal disease virus

A cell-culture-adapted reverse genetics strain of very virulent infectious bursal disease virus (IBDV) of chickens, designated as BD-3tcC, having four amino acid substitutions (Gln253His, Asp279Asn, Ala284Thr and Ser330Arg) in the capsid protein VP2 was tested for its genetic stability during serial passage in chickens and chicken embryo fibroblast (CEF) cell culture. Results of in vitro and in vivo experiments demonstrated that all four introduced mutations in BD-3tcC remained stable during serial passage in CEF cell culture, but during passage in chickens, amino acid residues at position 253 and 284 reverted from histidine to glutamine and threonine to alanine, respectively. In a parallel experiment, the same substitutions also occurred in a conventionally attenuated vaccine strain D-78 on serial passage in chickens. However, no reversion or substitution took place at positions 279 and 330 during in vivo passage of the mutant virus BD-3tcC or vaccine virus D-78. The findings provide conclusive evidence that while IBDV requires histidine and threonine at positions 253 and 284 for cell culture adaptation, glutamine and alanine at these positions are selected preferentially during in vivo replication.

Immunogenicity in mice of human metapneumovirus with a truncated SH glycoprotein

The SH glycoprotein of human metapneumovirus (HMPV) is twice the size of that of human respiratory syncytial virus and possesses a large, hydrophilic luminal domain. The glycoprotein is located on the surface of the virion and of virus infected cells and, if immunogenic, might be expected to play a role in anti-viral immunity. Initial attempts to study anti-SH antibody immunogenicity were thwarted by the instability of the SH gene on passage both in human bronchial epithelial cells and in mice. Repeated passage of virus isolates in human bronchial epithelial cells in culture resulted in the appearance and eventual predominance of HMPV mutants lacking all or most of the luminal domain of SH coincidental with the loss of productive infection in mouse lungs. Where infection was established in mice with an early cell culture passage, the virus recovered from mouse lung differed markedly from the inoculum, carrying 19 coding mutations in the SH luminal domain. Immunization of mice with a mutant virus variant expressing only 14 amino acids of the luminal domain of SH induced a cross-reactive antibody response to both the F glycoprotein and the SH glycoprotein but a largely sub-group specific response to the G glycoprotein. Similar patterns of response were achieved by immunization with individual HMPV glycoproteins expressed from recombinant vaccinia viruses. Recombinant truncated SH glycoprotein induced sub-group cross-reactive antibodies capable of neutralizing wild-type virus. Recombinant F glycoprotein also induced cross-reactive neutralizing antibodies whilst recombinant G glycoprotein induced largely strain-specific, non-neutralizing antibodies.

Characterization of Synthetic Chikungunya Viruses Based on the Consensus Sequence of Recent E1-226V Isolates

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that re-emerged in 2004 and has caused massive outbreaks in recent years. The lack of a licensed vaccine or treatment options emphasize the need to obtain more insight into the viral life cycle and CHIKV-host interactions. Infectious cDNA clones are important tools for such studies, and for mechanism of action studies on antiviral compounds. Existing CHIKV cDNA clones are based on a single genome from an individual clinical isolate, which is expected to have evolved specific characteristics in response to the host environment, and possibly also during subsequent cell culture passaging. To obtain a virus expected to have the general characteristics of the recent E1-226V CHIKV isolates, we have constructed a new CHIKV full-length cDNA clone, CHIKV LS3, based on the consensus sequence of their aligned genomes. Here we report the characterization of this synthetic virus and a green fluorescent protein-expressing variant (CHIKV LS3-GFP). Their characteristics were compared to those of natural strain ITA07-RA1, which was isolated during the 2007 outbreak in Italy. In cell culture the synthetic viruses displayed phenotypes comparable to the natural isolate, and in a mouse model they caused lethal infections that were indistinguishable from infections with a natural strain. Compared to ITA07-RA1 and clinical isolate NL10/152, the synthetic viruses displayed similar sensitivities to several antiviral compounds. 3-deaza-adenosine was identified as a new inhibitor of CHIKV replication. Cyclosporin A had no effect on CHIKV replication, suggesting that cyclophilins -opposite to what was found for other +RNA viruses- do not play an essential role in CHIKV replication. The characterization of the consensus sequence-based synthetic viruses and their comparison to natural isolates demonstrated that CHIKV LS3 and LS3-GFP are suitable and representative tools to study CHIKV-host interactions, screen for antiviral compounds and unravel their mode of action.

Targeting the Giant Cell Tumor Stromal Cell: Functional Characterization and a Novel Therapeutic Strategy

Giant cell tumor of bone (GCTB) is a benign, locally destructive neoplasm, with tumors comprised of mesenchymal fibroblast-like stromal cells; monocytic, mononuclear cells of myeloid lineage; and the characteristic osteoclast-like, multinucleated giant cells. Hampering the study of the complex interaction of its constituent cell types is the propensity of longstanding, repeatedly passaged cell cultures to undergo phenotypic alteration and loss of osteoclast-inducing capacities. In this study, we employed a novel, single-step technique to purify freshly harvested stromal cells using a CD14-negative selection column. Using 9 freshly harvested GCTB specimens and the purified stromal cell component, we performed analyses for markers of osteoblast lineage and analyzed the capacity of the stromal cells to undergo osteoblastic differentiation and induce osteoclastogenesis in co-cultures with monocytic cells. Successful purification of the CD14-negative stromal cells was confirmed via flow cytometric analysis and immunocytochemistry. Osteogenic media upregulated the expression of osteocalcin, suggesting an osteoblastic lineage of the GCTB stromal cells. The effects of the Wnt pathway agonist, SB415286, and recombinant human bone morphogenetic protein (BMP)-2 on osteoblastogenesis varied among samples. Notably, osteogenic media and SB415286 reversed the receptor activator of NF-κB ligand (RANKL)/osteoprotegerin (OPG) expression ratio resulting in diminished osteoclastogenic capacity. Recombinant human BMP2 had the opposite effect, resulting in enhanced and sustained support of osteoclastogenesis. Targeting the giant cell tumor stromal cell may be an effective adjunct to existing anti-resorptive strategies.

Evaluation of live gumboro vaccine prepared from local variant strain for control of infectious bursal disease in Egypt

Aim: The present study was designed to evaluate live Gumboro (IBD) vaccine prepared from local variant isolated strain (Egy-IBD Var 2009 Vp2 gene-, partial cds submitted in gen bank at accession no. : JN 118617) for controlling IBD problem in Egypt. Material and Methods: Local isolated variant strain was adapted on Baby Grivet Monkey kidney cell line -70(BGM-70) 7.5 4.5 used for preparation of tissue culture (T.C) live vaccine. T.C IBDV had a titer of 10 TCID /ml (10 TCID per dose) after 50 50 five passages in BGM-70 cell culture. Evaluation of prepared vaccine was done in vitro by measuring ELISA, and in vivo by protection % against very virulent or variant field IBD isolated strains. Result: Evaluation revealed that the prepared vaccine was safe; sterile; pure; non-immunosuppressive; and efficient. The Geometric Mean Titer (GMT) of ELISA for the prepared vaccine was 8271 and more than 10000 in compared with different commercial IBD vaccines; while protection percentage gave 96-100%; 92-96% and 96-98% in groups vaccinated with commercial (intermediate; intermediate plus and classical) IBD vaccines; respectively in compared to 96-100% in group vaccinated with local prepared vaccine when challenged with very virulent or variant IBD isolated strains. Conclusion: We can use live T.C. IBD vaccine prepared from local variant isolated virus strain as method for control IBD disease in Egypt.

Schmallenberg virus experimental infection of sheep

Since late 2011, a novel orthobunyavirus, named Schmallenberg virus (SBV), has been implicated in many cases of severely malformed bovine and ovine offspring in Europe. In adult cattle, SBV is known to cause a mild transient disease; clinical signs include short febrile episodes, decreased milk production and diarrhoea for a few days. However, the knowledge about clinical signs and pathogenesis in adult sheep is limited. In the present study, adult sheep of European domestic breeds were inoculated with SBV either as cell culture grown virus or as virus with no history of passage in cell cultures. Various experimental set-ups were used. Sampling included blood collection at different time points during the experimental period and selected organ material at autopsy. Data from this study showed, that the RNAemic period in sheep was as short as reported for cattle; viral genome was detectable for about 3-5 days by real-time RT-PCR. In total, 13 out of 30 inoculated sheep became RNAemic, with the highest viral load in animals inoculated with virus from low cell culture passaged or the animal passaged material. Contact animals remained negative throughout the study. One RNAemic sheep showed diarrhoea for several days, but fever was not recorded in any of the animals. Antibodies were first detectable 10-14 days post inoculation. Viral RNA was detectable in spleen and lymph nodes up to day 44 post inoculation. In conclusion, as described for cattle, SBV-infection in adult sheep predominantly results in subclinical infection, transient RNAemia and a specific antibody response. Maintenance of viral RNA in the lymphoreticular system is observed for an extended period.

Establishment and serial passage of cell cultures derived from LuCaP xenografts

LuCaP serially transplantable xenografts derived from primary and metastatic human prostate cancer encompass the molecular and cellular heterogeneity of the disease and are an invaluable resource for in vivo preclinical studies. A limitation of this model, however, has been the inability to establish and passage cell cultures derived from the xenografts. Here, we describe a novel spheroid culture system that supports long-term growth of LuCaP cells in vitro. Xenografts were minced and digested with collagenase. Tissue dissociation was terminated while the majority of cells remained as clusters rather than single cells. The cell clusters were suspended in StemPro medium supplemented with R1881 and Y-27632, a Rho kinase inhibitor, and placed in ultralow attachment dishes for spheroid culture. Serial passage was achieved by partial digestion to small clusters with trypsin/EDTA in the presence of Y-27632. Cell viability, growth and phenotype were monitored with LIVE/DEAD®, MTS, qRT-PCR, and immunocytochemical assays. Cells from six LuCaP xenografts formed proliferating spheroids that were serially passaged a minimum of three times and cryopreserved. Two of the cell lines, LuCaP 136 and LuCaP 147, were further passaged and characterized. Both expressed biomarkers characteristic of the xenografts of origin, were determined to be of independent origin by STR fingerprinting, and were free of mycoplasma. LuCaP 147 formed tumors similar to the original xenograft when injected into mice. The ability to culture LuCaP cells affords new opportunities for fast, cheap, and efficient preclinical studies and extends the value of the LuCaP xenograft models.

The case for Zostavax vaccination in systemic lupus erythematosus

Data published thus far demonstrate that the HZ vaccine has been well tolerated by most individuals, immunocompetent and immunocompromised. It is currently licensed for healthy adults ≥ 50 years old. In the healthy population, advancing age is the greatest risk factor for the development of HZ, with highest rates seen in those ≥70 years of age, likely due to age-related diminution of CMI. HZ incidence is higher in SLE patients compared to healthy people at all ages. Hence, SLE patients, even as young adults, have higher reported HZ incidence than 80 year-old healthy adults, and should be considered a similar high-risk population who are most likely to derive meaningful benefit from vaccination. Even if the efficacy of vaccination in the SLE population proves to be less than seen in the immunocompetent elderly population, the reduction in incidence and severity of both acute HZ and PHN will provide benefit. Until results of randomized clinical trials of Zostavax in SLE patients are reported, we must rely on clinical assessment of potential risks and benefits of vaccinating this population. Risks of complications directly attributible to zoster vaccination, including the theoretic possibility of vaccine-induced infection, can be mitigated by several means. First, prior exposure to VZV can be easily confirmed by commercially available serology, thereby avoiding the introduction of live-attenuated VZV into naive patients. In these subjects, the varicella vaccine (Varivax (Merck) or Varilrix (GlaxoSmithKline)), which has significantly lower potency than Zostavax and is intended for primary prevention of varicella, may be considered. Vaccination should be delayed until acute flares have resolved and induction therapy has been completed and patients are receiving only maintenance immunosuppressive therapy. Discontinuing disease modifying therapy for the sole purpose of vaccination should only be undertaken after careful consideration of the risks of inducing flare of underlying disease after withdrawal of therapy. Finally, the OKA vaccine strain was attenuated by serial passages in cell cultures and is avirulant as compared to wild type strains. Furthermore, it is susceptible to standard antiviral therapy, shall any vaccine-induced infection occur. Clinical trials addressing the safety and efficacy of Zostavax in SLE will provide more definitive information upon which to base vaccination decisions. However, results remain years away. In the mean time, vaccination among stable SLE patients ≥50 years old should be strongly considered.