Biological and physicochemical properties of cymbidium mosaic virus

The isolated cymbidium mosaic virus (CMV) is one of the most wide-spread and dangerous pathogens that infects promising varieties of orchids. It causes characteristic symptoms on orchid plants, which are manifested in the form of a mosaic. Over time, these areas are necrotized, leading to the stop of flowering the plants and reducing their decorative value. The CyMV is not spread by insects-carriers, but is transmitted by the mechanical inoculation with juice. Electron microscopy revealed flexible filamentous viral particles with a length of about 500 nm. The purified viral preparation is sedimented with a single peak with a sedimentation coefficient of 142S. The floating density of the virus in the preformed CsCl gradient corresponded to 1.3 g/cm3. The electrophoretic analysis of proteins in polyacrylamide gel under denatured conditions showed the presence of two polypeptides with molecular weights of 27 and 31 kDa. RNA CyMV has a molecular weight of 2 · 106 Da. In the translation system of rabbit reticulocytes in vitro, a protein with a molecular weight of about 27 kDa is synthesized. The obtained data allow us to refer CyMV to the group of potexviruses.

Antigenic properties of sARs-CoV-2/human/RUs/nsk-FRCFtM-1/2020 coronavirus isolate from a patient in novosibirsk

Objective: isolation of coronavirus SARS-CoV-2 from clinical sample of patient with COVID-19 in Novosibirsk; obtaining a purified and inactivated viral antigen and study of its antigenic properties. Materials and methods: virus isolation was carried out in Vero cell culture from nasopharyngeal swab positive on SARS-CoV-2 RNA. The efficiency of SARSCoV-2 replication in cell culture was assessed on the appearance of cytopathic effect (CPE) and the presence of viral RNA in cultural medium with reverse transcription – polymerase chain reaction (RT-PCR). Purification, concentration and inactivation of the viral preparation were carried out according to standard methods. The purity of the purified preparation and the profile of viral proteins were determined by electrophoresis in 10% polyacrylamide gel (PAG) with the addition of sodium dodecyl sulfate (SDS). The presence and specificity of viral proteins were detected using COVID-19 convalescent’s sera with enzyme-linked immunosorbent assay (ELISA) and immunoblotting. Results: SARS-CoV-2/human/ RUS/Nsk-FRCFTM-1/2020 isolate was obtained after passage on Vero cells from a virus-containing clinical sample. A purified, concentrated, inactivated, whole-virion antigen was obtained. It contains three structural proteins: glycoprotein S (approximately 200 kDa), nucleoprotein N (48 kDa), and matrix protein M (20-25 kDa). All viral proteins were detected with serum antibodies of COVID-19 convalescents. Conclusion: SARS-CoV-2 coronavirus can be isolated in Vero cell culture. The antigenic specificity of the three structural viral proteins (S, N, and M) is preserved in the purified inactivated viral preparation. The inactivated whole-virion antigen of SARS-CoV-2/human/RUS/Nsk-FRCFTM-1/2020 isolate can be used to study the antigenic immunomodulating properties of viral proteins, to obtain immune sera of laboratory animals, and also as a component of test systems for the detection of specific antibodies with ELISA and immunoblotting.

PROSPECTIVE POTATO VARIETIES’ RECOVERY IN VITRO

As research shows, the most effective concentration of the Aciclovir anti-viral preparation is the dose of 15 mg/l, which definitely removes potato plants of the M. virus.

The efficiency of use lentiviral vectors for the transformation of rooster spermatogenic cells in vivo

Male gonad cells are considered as promising target cells for the introduction of recombinant DNA within obtaining genetically modified individuals with given characteristics. The use of testicular spermatogonial stem cells is of the greatest interest. In the process of differentiation, this type of cell gives rise to a significant population of mature male germ cells. In the case of their genetic transformation, differentiated cells can be used to inseminate females in order to produce transgenic progeny. The aim of the research was to study the efficiency of using lentiviral vectors for the local transformation of roosters’ testicular spermatogenic cells. We used a lentiviral vector containing the ZsGreen reporter gene under the control of the CMV promoter. In vitro transformation of rooster spermatogenic cells was carried out by infection with a viral preparation, in vivo through multiple injections of the viral preparation into the testicular parenchyma of roosters ( n = 5). The efficiency of transformation was assessed by expression of the reporter ZsGreen gene in transfected spermatogenic cells. The success of using lentiviral vectors for the genetic transformation of rooster spermatogenic cells was shown in experiments in vitro and in vivo . The transformation efficiency of this cells types in an in vitro culture varied from 45 to 57% and averaged 48 ± 4%. The expression of the ZsGreen reporter gene in the cells of the spermatogenic epithelium of the testes was established in almost all experimental roosters in the in vivo experiments. The number of seminiferous tubules with transformed spermatogenic cells varied in the studied experimental roosters from 10 to 22%. The effectiveness of genetic transformation of the testes spermatogenic cells was 1.8 ± 0.2%. The obtained results indicate to the success of using lentiviral vectors for the genetic transformation of spermatogenic cells of rooster testes in vivo in order to create individuals with genetically transformed germ cells for the further production of transgenic offspring with given characteristics.

Improved yield of AAV2 and rAAV2-retro serotypes following sugar supplementation during the viral production phase

Hyperosmolarity has been used as a means to improve yields in various processes such as antibody production in hybridoma cells and retroviral and adenoviral production systems. In this study we demonstrate that treatment of AAVpro 293T packaging cells with the sugars sucrose or sorbitol during adeno-associated virus (AAV) production improves the yields of AAV2 and rAAV2-retro serotypes 1.8-fold and 1.5-fold, respectively. Using an iodixanol gradient centrifugation strategy followed by a column-based buffer exchange, we show that the sugar supplements are not carried over to the final viral preparation and have no effect on the purity or infectivity of the virus. Given the wide availability, low cost, and ease of use of these sugars, we believe that this strategy can be easily adopted by vector cores and laboratories to improve yields of their AAV preparations.

Current and Emerging Cell Culture Manufacturing Technologies for Influenza Vaccines

Annually, influenza virus infects millions of people worldwide. Vaccination programs against seasonal influenza infections require the production of hundreds of million doses within a very short period of time. The influenza vaccine is currently produced using a technology developed in the 1940s that relies on replicating the virus in embryonated hens’ eggs. The monovalent viral preparation is inactivated and purified before being formulated in trivalent or tetravalent influenza vaccines. The production process has depended on a continuous supply of eggs. In the case of pandemic outbreaks, this mode of production might be problematic because of a possible drastic reduction in the egg supply and the low flexibility of the manufacturing process resulting in a lack of supply of the required vaccine doses in a timely fashion. Novel production systems using mammalian or insect cell cultures have emerged to overcome the limitations of the egg-based production system. These industrially well-established production systems have been primarily selected for a faster and more flexible response to pandemic threats. Here, we review the most important cell culture manufacturing processes that have been developed in recent years for mass production of influenza vaccines.