Alternatives for obtaining a continuous cell line from Apis mellifera

ABSTRACT: In worldwide there are reports of a significant decrease in colonies of the species Apis mellifera, caused by several factors, including viral infections. In order to study and diagnose illnesses caused by viruses, in vitro cell culture is used as a valuable tool. Yet, there are still no immortalized cell lines of honey bee Apis mellifera. Primary cell cultures are promising for this purpose and can supply the lack of continuous strains, but their establishment is difficult and laborious, which often makes them unfeasible for many research centers. Through the use of cell immortalization techniques, it is possible to develop continuous cell lines and thus benefit, in different ways, research related to different species of bees. The choice of technique is challenging, since in addition to the ability to remain viable for countless passages, cells must keep the genotype and phenotype similar or identical to the original tissue. This review intends to present methodologies that can be used to immortalize Apis mellifera cells, aiming to establish a cell line. The genotypic and phenotypic implications of each technique are evaluated, and the purpose of the cell line to be developed.

Attempt to Isolate Elephant Endotheliotropic Herpesvirus (EEHV) Using a Continuous Cell Culture System

Elephant endotheliotropic herpesvirus (EEHV) infection is known to cause acute fatal hemorrhagic disease, which has killed many young Asian elephants (Elephas maximus). Until recently, in vitro isolation and propagation of the virus have not been successful. This study aimed to isolate and propagate EEHV using continuous cell lines derived from human and/or animal origins. Human cell lines, including EA. hy926, A549, U937, RKO, SW620, HCT-116 and HT-29, and animal cell lines, including CT26.CL25 and sp2/0-Ag14, were investigated in this study. Mixed frozen tissue samples of the heart, lung, liver, spleen and kidney obtained from fatal EEHV1A- or EEHV4-infected cases were homogenized and used for cell inoculation. At 6, 24, 48 and 72 h post infection (hpi), EEHV-inoculated cells were observed for cytopathic effects (CPEs) or were assessed for EEHV infection by immunoperoxidase monolayer assay (IPMA) or quantitative PCR. The results were then compared to those of the mock-infected controls. Replication of EEHV in the tested cells was further determined by immunohistochemistry of cell pellets using anti-EEHV DNA polymerase antibodies or re-inoculated cells with supernatants obtained from passages 2 or 3 of the culture medium. The results reveal that no CPEs were observed in the tested cells, while immunolabeling for EEHV gB was observed in only U937 human myeloid leukemia cells. However, quantitation values of the EEHV terminase gene, as well as those of the EEHV gB or EEHV DNA polymerase proteins in U937 cells, gradually declined from passage 1 to passage 3. The findings of this study indicate that despite poor adaptation in U937 cells, this cell line displays promise and potential to be used for the isolation of EEHV1 and EEHV4 in vitro.

SARS-CoV-2 does not replicate in embryonated hen’s eggs or in MDCK cell lines

The advent of COVID-19, has posed a risk that human respiratory samples containing human influenza viruses may also contain SARS-CoV-2. This potential risk may lead to SARS-CoV-2 contaminating conventional influenza vaccine production platforms as respiratory samples are used to directly inoculate embryonated hen’s eggs and continuous cell lines that are used to isolate and produce influenza vaccines. We investigated the ability of these substrates to propagate SARS-CoV-2 and found that neither could support SARS-CoV-2 replication.

Continuous Cell Lines from the European Biting Midge Culicoides nubeculosus (Meigen, 1830)

Culicoides biting midges (Diptera: Ceratopogonidae) transmit arboviruses of veterinary or medical importance, including bluetongue virus (BTV) and Schmallenberg virus, as well as causing severe irritation to livestock and humans. Arthropod cell lines are essential laboratory research tools for the isolation and propagation of vector-borne pathogens and the investigation of host-vector-pathogen interactions. Here we report the establishment of two continuous cell lines, CNE/LULS44 and CNE/LULS47, from embryos of Culicoides nubeculosus, a midge distributed throughout the Western Palearctic region. Species origin of the cultured cells was confirmed by polymerase chain reaction (PCR) amplification and sequencing of a fragment of the cytochrome oxidase 1 gene, and the absence of bacterial contamination was confirmed by bacterial 16S rRNA PCR. Both lines have been successfully cryopreserved and resuscitated. The majority of cells examined in both lines had the expected diploid chromosome number of 2n = 6. Transmission electron microscopy of CNE/LULS44 cells revealed the presence of large mitochondria within cells of a diverse population, while arrays of virus-like particles were not seen. CNE/LULS44 cells supported replication of a strain of BTV serotype 1, but not of a strain of serotype 26 which is not known to be insect-transmitted. These new cell lines will expand the scope of research on Culicoides-borne pathogens.

Propagation of the Aujeszky’s disease virus isolate in continuous cell lines

The objective of this study was to investigate the cultural properties of Aujeszky's disease virus isolate and the optimization of the conditions of obtaining virus-containing material in the most prospective cell cultures. As a result, it was established that the study virus isolate is highly pathogenic for laboratory animals: rabbits, guinea pigs, and rats. The following continuous cell lines were sensitive to the virus isolate: MDBK, Taurus-1 and ВНК-21. CPE (cytopathogenic effect of viruses) nature was similar in different cell cultures. The ability for propagation in cultures of continuous cell lines of the test virus isolates reached the maximum value to 4-6 passage, equaling to 6.85-7.65 lgTCD50/cm3. The obtained results show that the dynamics of the virus isolate dynamics had no significant differences, and following the adaptation, the virus isolate maintained the stable inherent propagation activity level throughout the study. As the study result, the most sensitive cell culture Taurus-1 was selected at passaging, with the highest virus accumulation observed, the infection activity was 7.55±0.12 lg TCD50/cm3. The presence of Aujeszky's disease virus was confirmed by the bioassay and the other laboratory methods, including PCR (polymerase chain reaction). The PCR method allowed defining the virus genome in the virus-containing cultural and organ and tissue material. PCR allowed amplifying the target gene fragment with the size of 194 base pairs (b.p.) in samples containing Aujeszky's disease virus DNA. No non-specific reactions were observed; PCR products did not exhibit the expected size

A Deleted Deletion Site in a New Vector Strain and Exceptional Genomic Stability of Plaque-Purified Modified Vaccinia Ankara (MVA)

Vectored vaccines based on highly attenuated modified vaccinia Ankara (MVA) are reported to be immunogenic, tolerant to pre-existing immunity, and able to accommodate and stably maintain very large transgenes. MVA is usually produced on primary chicken embryo fibroblasts, but production processes based on continuous cell lines emerge as increasingly robust and cost-effective alternatives. An isolate of a hitherto undescribed genotype was recovered by passage of a non-plaque-purified preparation of MVA in a continuous anatine suspension cell line (CR.pIX) in chemically defined medium. The novel isolate (MVA-CR19) replicated to higher infectious titers in the extracellular volume of suspension cultures and induced fewer syncytia in adherent cultures. We now extend previous studies with the investigation of the point mutations in structural genes of MVA-CR19 and describe an additional point mutation in a regulatory gene. We furthermore map and discuss an extensive rearrangement of the left telomer of MVA-CR19 that appears to have occurred by duplication of the right telomer. This event caused deletions and duplications of genes that may modulate immunologic properties of MVA-CR19 as a vaccine vector. Our characterizations also highlight the exceptional genetic stability of plaque-purified MVA: although the phenotype of MVA-CR19 appears to be advantageous for replication, we found that all genetic markers that differentiate wildtype and MVA-CR19 are stably maintained in passages of recombinant viruses based on either wildtype or MVA-CR.

KCTD15 is overexpressed in human childhood B-cell acute lymphoid leukemia

Leukemic cells originate from the malignant transformation of undifferentiated myeloid/lymphoid hematopoietic progenitors normally residing in bone marrow. As the precise molecular mechanisms underlying this heterogeneous disease are yet to be disclosed, the identification and the validation of novel actors in leukemia is of extreme importance. Here, we show that KCTD15, a member of the emerging class of KCTD ((K)potassium Channel Tetramerization Domain containing) proteins, is strongly upregulated in patients affected by B-cell type acute lymphoblastic leukemia (B-ALL) and in continuous cell lines (RS4;11, REH, TOM-1, SEM) derived from this form of childhood leukemia. Interestingly, KCTD15 downregulation induces apoptosis and cell death suggesting that it has a role in cellular homeostasis and proliferation. In addition, stimulation of normal lymphocytes with the pokeweed mitogen leads to increased KCTD15 levels in a fashion comparable to those observed in proliferating leukemic cells. In this way, the role of KCTD15 is likely not confined to the B-ALL pathological state and extends to activation and proliferation of normal lymphocytes. Collectively, data here presented indicate that KCTD15 is an important and hitherto unidentified player in childhood lymphoid leukemia, and its study could open a new scenario for the identification of altered and still unknown molecular pathways in leukemia.

Developing a primary Paralichthys olivaceus gill epithelial cells as an in vitro model for propagation of VHSV show a corresponding increase in cell viability with increase in protein concentration in growth media

BackgroundViral Hemorrhagic Septicemia Virus (VHSV) is a rhabdovirus that causes high mortalities linked to high economic losses in aquaculture. It has been grouped in four genotypes of which some do not easily propagate on continuous cell lines. As an alternative, the objectives of this study was to develop a primary gill epithelial cell (GEC) model from olive flounder (Paralichthys olivaceus) as an in vitro model for the propagation of VHSV.ResultsOur findings show that the primary GECs developed herein are highly permissive to replication of the JF-09 genotype IVa strain leading to high cytopathic effect observed within 96 hours post virus inoculation. Our findings also show that the viability GECs produced herein corresponded with increase in the concentration fetal bovine serum in growth medium. We envision that GECs produced herein will heighten our understanding of immune mechanisms associated with virus entry on gill mucosal surfaces in flounder.

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.

Cultivation of potato leafroll virus (PLRV) in mammalian continuous cell lines

Aim. To use the ability of potato leafroll virus (PLRV) to infect and multiply in mammalian continuous cell lines to purify PLRV isolates from the vegetative plant material, and to study the pathogenicity of those isolates for plants (after culturing in mammalian continuous cell line), to investigate morphological, physical-chemical, biological and antigen properties of PLRV isolates from mammalian cells and to study an alternative diagnostic method – the neutralization test in the mammalian continuous cell lines. Methods. The methods of cultivating animal viruses in the mammalian continuous cell line, microscopical biochemical, and serological methods, the method of artifi cial nutrition of aphids are detailed under Material and Methods. Results. It was demonstrated that successful cultivation of PLRV in mammalian continuous cell line allowed obtaining pure virus isolates from potato plants and aphids and preserving them for a long time (over a period of 7 years). The cultivation of PLRV in the mammalian continuous cell line did not impact its pathogenic properties and allowed transmitting the virus to plants. Continuous cells lines of pig embryonic kidney (PEKV), of kidney Syrian hamster (BHK- 21), of testicles of piglets (PTP), of kidneys of the bull (MDBC), and of carcinoma rabbit kidney (RK-13) were found to be sensitive to PLRV, Con tinuous cell lines of human (HeLa, Hep-2 and of African green monkey kidney (Vero) were not infected by the virus. The infectious activity of PLRV in the sensitive continuous cell lines was 20–8.5 lg TCD 50 /ml depending on the cell line. The isolates of PLRV were resistant to lipid- dissolving solvents, multiplied in a pH range from 4.0 till 10.0 and were thermoresistant at 50 oС in the absence of bivalent ions of magnesium, ТIP was in the range of 60–65 oС under our experimental conditions. The optimal temperature for the reproduction of PLRV in the cell culture was c. 24 °С. The use of neutralization test in the mammalian continuous cell line allowed isolation in pure culture and identifi cation of PLRV reliably in a time span of c. 14 days. Conclusions. It was proven that PLRV can be cultivated in the mammalian continuous cell lines of PEKV, ВНК-21, PTV, MDВК and RK-13. It was established that the cultivation of PLRV in these continuous cell lines did not impact its biological, pathogenic, antigenic and physical-chemical properties. The identifi cation of pure cultures of PLRV obtained in mammalian cells can be reliably performed by the use of neutralization reaction.