Lamb Kidney Cells Research Articles

Lumpy skin disease virus isolation, experimental infection, and evaluation of disease development in a calf

Lumpy skin disease (LSD) is one of the most economically significant viral diseases of cattle caused by the Lumpy Skin Disease Virus (LSDV), classified as a member of the genus Capripoxvirus and belongs to the family Poxviridae. Nodular skin samples were collected from clinically sick cattle in the districts of Amuru and Wara Jarso Ethiopia to isolate LSD virus. The virus was isolated using primary lamb testis and kidney cells. The isolated LSDV was infected into a healthy calf while maintaining the necessary biosecurity measures to generate skin lesions and to assess disease progression using postmortem examinations. On the fourth day after virus inoculation, the calf developed typical LSD skin nodules with increased rectal temperature, which lasted until the 12th day, when they began to decrease. Viral shedding was detected in nasal, oral, and conjunctival swabs from 6 to 14 days after infection using real-time PCR. Post-mortem tissue specimens tested positive for LSD virus using real-time PCR and virus isolation. This study showed that LSDV were responsible for the LSD outbreaks, and the appearance of typical skin nodules accompanied by fever (> 39.5 °C) defined the virus’s virulent status. The experimental infection with the isolated infectious LSDV could serve as a platform for future vaccine evaluation study using an LSDV challenge model.

Exploring the presence of bovine leukemia virus among breast cancer tumors in a rural state.

The bovine leukemia virus (BLV) is a deltaretrovirus that causes malignant lymphoma and lymphosarcomas in cattle globally and has high prevalence among large scale U.S. dairy herds. Associations between presence of BLV DNA in human mammary tissue and human breast cancer incidence have been reported. We sought to estimate the prevalence of BLV DNA in breast cancer tissue samples in a rural state with an active dairy industry. We purified genomic DNA from 56 fresh-frozen breast cancer tissue samples (51 tumor samples, 5 samples representing adjacent normal breast tissue) banked between 2016 and 2019. Using nested PCR assays, multiple BLV tax sequence primers and primers for the long terminal repeat (LTR) were used to detect BLV DNA in tissue samples and known positive control samples, including the permanently infected fetal lamb kidney cell line (FLK-BLV) and blood from BLV positive cattle. The median age of patients from which samples were obtained at the time of treatment was 60 (40-93) and all were female. Ninety percent of patients had invasive ductal carcinoma. The majority were poorly differentiated (60%). On PCR assay, none of the tumor samples tested positive for BLV DNA, despite having consistent signals in positive controls. We did not find BLV DNA in fresh-frozen breast cancer tumors from patients presenting to a hospital in Vermont. Our findings suggest a low prevalence of BLV in our patient population and a need to reevaluate the association between BLV and human breast cancer.

Development of an Inactivated Camelpox Vaccine from Attenuated Camelpox Virus Strain: Safety and Protection in Camels.

This article describes the preparation of an inactivated vaccine from an attenuated strain of camelpox. The attenuated camelpox virus (CMLV) was grown in lamb kidney cells and in Vero cells. CMLV was accumulated to a significantly higher (p ≤ 0.05) titer in lamb kidney cells (7.75 ± 0.08 log TCID50/mL) than in Vero cells (4.00 ± 0.14 log TCID50/mL). During virus inactivation, a concentration of 0.05% beta-propiolactone (BPL) completely inactivated the virus in 6 h at a temperature of 22 ± 1 °C, while a concentration of 0.2% formaldehyde inactivated the virus in 8 h. However, a viral antigen inactivated by BPL was used for vaccine preparation. The inactivated viral antigen was adsorbed with aluminum hydroxide gel, and as a result, an inactivated candidate vaccine was prepared. While the safety of the candidate vaccine was tested in camels and white mice, the protective efficacy of the vaccine was tested only in camels. In the safety evaluation of the inactivated vaccine, the vaccine was not observed to cause any adverse effects in mice and camels. During the immunogenicity study in camels, antibody formation started (0.2 ± 0.16 log2) at Day 21 post-vaccination (PV), and the antibody titer peaked (1.33 ± 0.21 log2) at Day 60 PV and decreased at Day 90 PV (0.50 ± 0.22 log2). Furthermore, no antibodies were detected in vaccinated camels from Days 180 to 365 PV. Camels that received vaccination and were subsequently exposed to wild-type virus evinced a healthy state despite lacking antibodies. In contrast, unvaccinated camels exhibited susceptibility to camelpox upon challenge.

Characterization of the camel pox virus strain used in producing camel pox virus vaccine

Background: The camel pox virus is a widespread infectious viral disease of camels. It is necessary to conduct research on new strains for the development of vaccines. Aim: The research aims to characterize a novel strain isolated from the camel pox virus used to produce a camel pox virus vaccine. Methods: The objects of the study were the “M-0001” strain isolated from a sample of animals infected with the camel pox virus during the epidemic. The cultural and reproductive properties of the virus isolate were studied using primary cell lines from primary trypsinized lamb kidney and testicular cell cultures (LK and LT). Other samples included kidney cell lines from transplanted sheep) as well as a kidney cell line from transplanted cattle, Vero (transplanted green monkey kidney cell line), and calf trachea. The strain was PCR-tested and sequenced for characterization purposes. Results: The PCR results show that the study sample is species specific and corresponds to the camel pox virus by the size of the cumulative amplifications, which is 241 bp. Given the maximum percentage of a sequence match analyzed by the BLAST algorithm based on the international database and the results of phylogenetic analysis, the M0001 sample was determined to belong to the Camel pox virus (gene bank inventory number KP768318.1). Conclusion: The sample “M0001” is located on the same branch with a representative from CMLV (Camel pox virus). Among the cell cultures tested, the LK and LT cell lines were the most sensitive to the isolated CMLV isolate. Reproducing the virus in these cell cultures remains stable even after 15 consecutive passes. The cytopathic effect of the virus was less pronounced and low in transplanted cell lines, and the cytopathic effect was no longer apparent in the third passage. A genome alignment of the virus has identified potentially conserved sites, and analysis of loci in different virus types revealed one maximally conserved locus. An epizootic strain of the camelina virus “M-0001” candidate to produce vaccines for the camels was obtained. An experimental vaccine sample based on an isolated and charred camellia virus will be created in the future.

Comparison and efficacy of two different sheep pox vaccines prepared from the Bakırköy strain against lumpy skin disease in cattle.

PurposeLumpy skin disease (LSD) is a highly contagious and economically important viral infection of cattle, which leads to financial losses in the livestock industry of affected countries. Vaccination is the most effective control measure to prevent the disease. Heterologous sheep pox (SP) vaccine was used against LSD in Turkey. In this research, it was aimed to adapt SP Bakırköy vaccine strain attenuated in lamb kidney cells to Madin-Darby bovine kidney (MDBK) cells to provide better protection than commercial SP vaccine in cattle.Materials and MethodsTo evaluate safety and efficacy of vaccines, while animals were immunized with 10 doses (104.75 50% tissue culture infectious dose [TCID50]) and 5 doses of SP vaccine (104 TCID50) produced in MDBK cells, others were immunized with commercial Penpox-M vaccine (103.9 TCID50). Two cattle were kept as unvaccinated. At day 31 post-vaccination, all animals were challenged with the virulent LSD virus. Blood and swab samples were taken on certain days post-inoculation. Logarithmic differences challenge virus titers between vaccinated and unvaccinated animals were calculated.ResultsThe clinical sign was not observed in animals immunized with 10 doses of SP vaccine. The differences between the animals immunized with SP vaccine and control group was less than log 2.5 and the viremia occurred in immunized animals. The difference in titer was higher than log 2.5 in animals immunized with the Penpox-M, and viremia did not occur.ConclusionSP vaccine strain propagated in MDBK cells and can be used for immunization to prevent LSD infections. However, SP vaccine strain propagated in MDBK showed poor protection as compared to Penpox-M.

A target enrichment high throughput sequencing system for characterization of BLV whole genome sequence, integration sites, clonality and host SNP

Bovine leukemia virus (BLV) is an oncogenic retrovirus which induces malignant lymphoma termed enzootic bovine leukosis (EBL) after a long incubation period. Insertion sites of the BLV proviral genome as well as the associations between disease progression and polymorphisms of the virus and host genome are not fully understood. To characterize the biological coherence between virus and host, we developed a DNA-capture-seq approach, in which DNA probes were used to efficiently enrich target sequence reads from the next-generation sequencing (NGS) library. In addition, enriched reads can also be analyzed for detection of proviral integration sites and clonal expansion of infected cells since the reads include chimeric reads of the host and proviral genomes. To validate this DNA-capture-seq approach, a persistently BLV-infected fetal lamb kidney cell line (FLK-BLV), four EBL tumor samples and four non-EBL blood samples were analyzed to identify BLV integration sites. The results showed efficient enrichment of target sequence reads and oligoclonal integrations of the BLV proviral genome in the FLK-BLV cell line. Moreover, three out of four EBL tumor samples displayed multiple integration sites of the BLV proviral genome, while one sample displayed a single integration site. In this study, we found the evidence for the first time that the integrated provirus defective at the 5′ end was present in the persistent lymphocytosis cattle. The efficient and sensitive identification of BLV variability, integration sites and clonal expansion described in this study provide support for use of this innovative tool for understanding the detailed mechanisms of BLV infection during the course of disease progression.

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

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

Indirect ELISA (iELISA) standardization for the diagnosis of bovine enzootic leukosis

ABSTRACT: Enzootic bovine leukosis (EBL) is an infectious disease caused by bovine leukemia virus (BLV) that affects cattle worldwide. Agar gel immunodiffusion (AGID) was the reference test for EBL diagnosis for many years, but enzyme-linked immunosorbent assay (ELISA) showed higher sensitivity, was faster to perform, and resulted in an objective reading. However, the importation of ELISA kits is lengthy and expensive, and currently, no AGID kits are available in Brazil. The aim of this work was to standardize an indirect ELISA (iELISA) for EBL diagnosis using BLV antigens produced in Tadarida brasiliensis lung (Tb1Lu) cells, which are Bovine viral diarrhea virus (BVDV) free, unlike fetal lamb kidney (FLK) cells, currently used for this purpose. Following standardization, iELISA results were compared with those obtained by AGID and the commercial Chekit Leucose-Serum ELISA. Compared to AGID, iELISA had 94,44% sensitivity, 75.68% specificity, 79.10% positive predictive value (PPV) and 93.30% negative predictive value (NPV), with 84% concordance and a Kappa index of 0.699. Compared to the Chekit Leucose-Serum ELISA, iELISA showed 92.60% sensitivity, 87.09% specificity, 90.27% PPV and 90,00% NPV, with 90.27% concordance and a Kappa index of 0.801. Taking into account the high agreement with the traditional tests and the absence of non-specific reactions with BVDV, the developed assay could be used as diagnostic method to control EBL in Brazil.

Development of an in situ hybridization assay using an AS1 probe for detection of bovine leukemia virus in BLV-induced lymphoma tissues.

Enzootic bovine leukosis (EBL) is a malignant B cell lymphoma caused by infection with bovine leukemia virus (BLV). Histopathological examination is commonly used for diagnosis of the disease, but observation of lymphoma alone does not confirm EBL because cattle may be affected by sporadic forms of lymphoma that are not associated with BLV. Detection of BLV in tumor cells can be definitive evidence of EBL, but currently, there is no technique available for such a purpose. In this study, we focused on a viral non-coding RNA, AS1, and developed a novel in situ hybridization assay for the detection of BLV from formalin-fixed paraffin-embedded (FFPE) tissues. RNA-seq analysis revealed that all examined B lymphocytes derived from clinical EBL abundantly expressed AS1 RNA, indicating a possible target for detection. The in situ hybridization assay using an AS1 probe clearly detected AS1 RNA in fetal lamb kidney cells persistently infected with BLV. The utility of this assay in clinical samples was assessed using three EBL-derived lymph node specimens and one BLV-negative specimen, and AS1 RNA was detected specifically in the EBL-derived tissues. These results suggest that AS1 RNA is a useful target for the detection of BLV from FFPE specimens of tumor tissues. This technique is expected to become a powerful tool for EBL diagnosis.

A lumpy skin disease virus which underwent a recombination event demonstrates more aggressive growth in primary cells and cattle than the classical field isolate.

Genomic changes by recombination have been recently observed in lumpy skin disease viruses circulating in Russia. The first characterized naturally occurring recombinant lumpy skin disease virus Saratov/2017 occurred through recombination between a live attenuated virus vaccine and the Southern African lumpy skin disease virus. Understanding if recombination can increase or decrease virulence of viruses through changes in different gene regions is required to improve the understanding of capripoxvirus biology. In this study, the in vitro and in vivo growth of the recombinant Saratov/2017 and the classical field isolate Dagestan/2015 was compared. Primary lamb kidney and lamb testis cells as well as the goat ovarian cell line were used to assess virus replication. In the goat ovarian cell line, Saratov/2017 and Dagestan/2015 induced comparable cytopathic activity and virus titres. In contrast, in primary lamb kidney and lamb testis cells, Saratov/2017 grew more aggressively causing more massive rounding up of cells, detachment and agglomeration compared to Dagestan/20152015. Growth curves of Saratov/2017 and Dagestan/2015 were assessed in primary lamb testis cells using different multiplicities of infection (MOI), with Saratov/2017 demonstrating faster replication at the different MOI and time points evaluated post-infection. In cattle, Saratov/2017 demonstrated more pronounced skin reactions when titrated by skin inoculation of serially diluted virus. In both primary cells and cattle, the titre of Saratov/2017 was significantly higher compared to Dagestan/2015 (p≤.05). These results demonstrate recombinant Saratov/2017 exhibits more aggressive replication properties.

Assessment of the sensitivity of primary cells and cell lines to the Southern African Territories (SAT) serotypes in the diagnosis of foot-and-mouth disease virus

Virus isolation is the gold standard for foot-and-mouth disease virus (FMDV) detection in diagnostic procedures. This technique is heavily reliant on the use of sensitive cells for rapid and accurate detection of FMDV. To investigate the sensitivity of RM (primary lamb kidney cells), BHK-21 (baby hamster kidney cells) and IR-P1 (a derivative of female pig kidney cells) to infection with FMDV of the Southern African Territories (SAT) serotypes, we examined the virus concentration required to induce cytopathic effect (CPE) on each cell type. The results suggested that sensitivity of RM and IR-P1 cells was high and not significantly different (P < 0.05). BHK-21 however, exhibited low sensitivity to the strains used. Comparisons of three batches of each cell type were also done to establish the consistency of the sensitivity of these cells to FMDV infection. IR-P1 and BHK-21 cell batches gave consistent results for all samples used whereas RM cells showed significant differences (P > 0.05) between batches. TCID 50/ml was used to determine the viral titre required to induce CPE. IR-P1 cell line proved to have consistently higher TCID50/mL for all cell batches while RM cell batches displayed a difference in TCID50/mL values. The IR-P1 cell line was concluded to be a good cell culture system for virus isolation as it showed relatively high and reproducible sensitivity to all the FMDV strains used. The findings of this study indicate that the use of IR-P1 cell line could be considered for FMDV diagnostic work.

Detection and molecular characterization of bovine leukemia virus in various regions of Iran.

Purpose. Bovine leukemia virus (BLV) infects cattle worldwide, imposing an economic impact on the dairy cattle industry. The purpose of this study was to evaluate the molecular epidemiology of BLV in Iran.Methodology. Blood samples taken from 280 cows aged over 2 years old from 13 provinces of Iran were used for leukocyte count and blocking ELISA. Genomic DNA was extracted from the peripheral blood leukocytes of BLV-infected samples and fetal lamb kidney cells to perform PCR of partial env, rex and tax genes and long-terminal-repeat region. The PCR products were sequenced, the phylogenetic tree of each gene was constructed, and nucleotide and amino acid sequence pair distances were calculated.Results. The frequency of BLV infection was 32.8 % among animals and was 80 % among provinces. In BLV seropositive animals, the rate of persistent lymphocytosis was 36.9 %. The constructed phylogenetic trees showed the presence of two BLV genotypes (1 and 4) in Iranian strains. As previous studies, our results showed that the env gene was more variable than previously thought, the Rex protein could withstand more amino acid changes compared to the Tax protein, and no significant differences were observed in average changes of the nucleotide of these genes between clinical stages.Conclusions. Our data indicates an increase in the frequency of this infection in Iran. This is the first study report of the presence of BLV genotype 4 in Iranian farms. These findings may have an important role in the control and prevention of BLV infection in Iran and other countries.

TESTING SHEEP AND GOAT POX VIRUSES FOR THEIR REPRODUCTION IN PRIMARY AND SUBCULTURED CELLS

Results of tests of sheep and goat poxviruses for their reproduction in primary and subcultured cell cultures derived from lamb and goat kid kidneys and testicles are presented. Monolayer cultures were subcultured by 5 passages in plastic vials and infected with sheep and goat poxviruses. It was shown that production ARRIAH strain of sheep pox virus and ARRIAH 2003 strain of goat pox virus successfully propagated both in primary lamb and goat kid kidney and testicle cell cultures and lamb and goat kid kidney and testicle cell subcultures. Activity of sheep and goat poxviruses passaged 5 times was 5.5–6.0 lg TCID50/cm3. Taking into account that modern cell cultivation conditions allow primary trypsinized cell populations subcultivation up to 25–30th passage, subcultures together with continuous cell lines can be used for large-scale sheep and goat poxvirus production and research purposes.

Molecular characterization of Peste des Petits ruminants virus isolated from four outbreaks occurred in southern Iran

BackgroundPeste des petits ruminants (PPR) is a severe infectious disease in both domestic and wild small ruminants. Due to its heavy economic burden and hence social and health impacts on human populations, Food and Agriculture Organization of the United Nations (FAO) and The World Organization for Animal Health (OIE) have targeted PPR for eradication by 2030. In order to plan and implement a successful eradication program, factual status assessments prior to devising disease control strategies is a vital criterion. The aim of this study was to isolate and characterize PPR virus from a rising wave of outbreaks in southern Iran.ResultsTwenty-one clinical samples, including blood as well as oral, nasal and ocular swabs were collected from ten sick animals in 4 various herds and were examined with ELISA and RT-PCR for the presence of PPR virus antigen and genome, respectively. The virus was successfully isolated in primary lamb kidney cell culture and identified by RT-PCR. Phylogenetic analysis of the sequenced N genes revealed that, while the earliest reports of Iran’s outbreaks were grouped into clusters with Saudi Arabia, Turkey and Africa, in this study reported sequences were grouped with samples from Pakistan, Tajikistan and China in particular. This observation suggests a shift in PPRV flow from the western borders of the country to the eastern neighboring countries.ConclusionsLineage IV of PPR virus is presently circulating in Iran, with certain levels of genetic diversity. Present study along with previous reports demonstrates the dispersal patterns and movements of PPR virus, which highlights the reversal pattern of virus flow in recent years. Such information is necessary to understand PPRV molecular epidemiology and to develop more proper control strategies to eradicate the disease in the planned time.

Experimental infection of sheep with Bovine leukemia virus (BLV): Minimum dose of BLV-FLK cells and cell-free BLV and neutralization activity of natural antibodies

Bovine leukemia virus (BLV) is an important cattle pathogen that causes major economic losses worldwide, especially in dairy farms. The use of animal models provides valuable insight into the pathogenesis of viral infections. Experimental infections of sheep have been conducted using blood from BLV-infected cattle, infectious BLV molecular clones or tumor-derived cells. The Fetal Lamb Kidney cell line, persistently infected with BLV (FLK-BLV), is one of the most commonly used long-term culture available for the permanent production of virus. FLK-BLV cells or the viral particles obtained from the cell-free culture supernatant could be used as a source of provirus or virus to experimentally infect sheep. In this report, we aimed to determine the minimum amount of FLK-BLV cells or cell-free supernatant containing BLV needed to produce infection in sheep. We also evaluated the amount of antibodies obtained from a naturally-infected cow required to neutralize this infection. We observed that both sheep experimentally inoculated with 5000 FLK-BLV cells became infected, as well as one of the sheep receiving 500 FLK-BLV cells. None of the animals inoculated with 50 FLK-BLV cells showed evidence of infection. The cell-free FLK-BLV supernatant proved to be infective in sheep up to a 1:1000 dilution. Specific BLV antibodies showed neutralizing activity as none of the sheep became infected. Conversely, the animals receiving a BLV-negative serum showed signs of BLV infection. These results contribute to the optimization of a sheep bioassay which could be useful to further characterize BLV infection.

Replacement of foot-and-mouth disease virus cattle tongue titration by in vitro titration

Titration of foot-and-mouth disease cattle challenge virus in cattle tongue has been the standard for many years in many countries, although titration in animals has been replaced by in vitro methods for all other applications. The objective of the analysis was the replacement of in vivo titration of cattle challenge virus by in vitro titration. Using data from 32 in vivo titration experiments together with the in vitro titration results of the same samples obtained by plaque count on primary lamb or pig kidney cells, as well as data from the virus isolation control chart used in the laboratory, we show that the reproducibility of the in vitro titration is much higher than that of the in vivo titration. The titer on primary kidney cells was on average 1.4 log10 higher than the titer determined by titration in cattle tongue (PFU/ml compared to bovine ID50/ml), but the difference varied among different strains. The study also shows that the probability of infection in cattle tongue is high even when a lower challenge dose is used, which makes the variability between strains less important. Based on these results, we propose to change the standard dose for cattle challenge from 104 bovine ID50 to 105.4 PFU, and to replace the in vivo cattle tongue titration method with the in vitro titration method.

Isolation and molecular diagnosis of Peste des petits ruminants (PPR) virus from contaminated areas in Iran

Due to the numerous reports concerning the Peste des petits ruminants (PPR) in different regions of Iran, the isolation and genetic characterization of native isolates is very important. During 20132014, 168 samples were collected from whole blood, eye, nasal and oral swabs, and lymph nodes of sheep and goats with clinical signs in nine provinces with infected focal zones in Iran. Vero cell line and primary Lamb Kidney cells were inoculated with isolates and observed at least in 5 consecutive passages for cytopathic effects (CPE). The sheep samples from two provinces, created CPE in both kinds of cell cultures. Observation by electron microscopy and confirmation by RT-PCR was used to confirm PPR.

Recombinant Sheep Pox Virus Proteins Elicit Neutralizing Antibodies.

The aim of this work was to evaluate the immunogenicity and neutralizing activity of sheep pox virus (SPPV; genus Capripoxvirus, family Poxviridae) structural proteins as candidate subunit vaccines to control sheep pox disease. SPPV structural proteins were identified by sequence homology with proteins of vaccinia virus (VACV) strain Copenhagen. Four SPPV proteins (SPPV-ORF 060, SPPV-ORF 095, SPPV-ORF 117, and SPPV-ORF 122), orthologs of immunodominant L1, A4, A27, and A33 VACV proteins, respectively, were produced in Escherichia coli. Western blot analysis revealed the antigenic and immunogenic properties of SPPV-060, SPPV-095, SPPV-117 and SPPV-122 proteins when injected with adjuvant into experimental rabbits. Virus-neutralizing activity against SPPV in lamb kidney cell culture was detected for polyclonal antisera raised to SPPV-060, SPPV-117, and SPPV-122 proteins. To our knowledge, this is the first report demonstrating the virus-neutralizing activities of antisera raised to SPPV-060, SPPV-117, and SPPV-122 proteins.

Process intensification for an enhanced replication of a newly adapted RM-65 sheep pox virus strain in Vero cells grown in stirred bioreactor

Sheep pox virus initially adapted to replicate in primary lamb kidney cells was adapted to Vero cells by serial passages in monolayer cultures. After nine passages the virus was able to correctly replicate in Vero cells, virus titer achieved was 105.875 TCID50 (median tissue culture infective dose)ml−1.To optimize the production process, the effects of MOI (multiplicity of infection), TOI (time of infection) and the culture medium were investigated. Cell infection at a MOI of 0.005 concurrently with cell seeding showed the best results in terms of specific virus productivity. The effect of MEM enrichment with several components was investigated using the experimental design approach. 67 experiments were performed in 6-well plates to select the best combination. The highest titer was achieved when MEM was supplemented with 5mM glucose, 5mM fructose and 25mM sucrose. Spinner culture confirms these data; virus titer was 107.375 TCID50ml−1.In addition Vero cells were cultivated in a 7-l bioreactor in batch mode on 3gl−1 Cytodex1, and infected at cell seeding at a MOI of 0.005. Maximal virus titer was 107.275 TCID50ml−1. This corresponds to 44-fold factor enhancement compared to spinner cultures conducted in MEM+2% FCS.

Fabrication and characterization of chitosan–gelatin/nanohydroxyapatite–polyaniline composite with potential application in tissue engineering scaffolds

In this study to further improve surface characteristics of tissue engineering scaffolds, a novel composite based on chitosan–gelatin/nanohydroxyapatite–polyaniline (CS-Gel/nHA-PANI) was prepared by blending the synthesized nHA and PANI with chitosan and gelatin solution followed by lyophilization technique. The prepared composite scaffold was characterized using Fourier transform infrared spectroscopy, X-ray diffraction, and scanning electron microscopy techniques. The effects of nHA and PANI on the physicochemical properties of the scaffold including swelling ratio, density, biodegradation, biomineralization, and mechanical behavior were investigated. The composite scaffold was highly porous with the mean pore size of 200 μm. Compared with CS-Gel and CS-Gel/nHA scaffolds, the CS-Gel/nHA-PANI scaffold exhibited lower degradation rate and higher biomineralization and mechanical strength. Preliminary evaluation of the biocompatibility and cytotoxicity of the CS-Gel/nHA-PANI composite scaffold was performed by MTT assay using bovine leukemia virus fetal lamb kidney cell line (BLV-FLK) and then cell attachment studies were assessed using dental pulp stem cells. Results indicated no toxicity, and cells attached and proliferated on the pore surfaces of the scaffold. These findings suggested that the developed scaffold possess the prerequisites and can be used as a scaffold for hard tissues regeneration.