Pavel Vladimirovich Filatov

Introduction. Highly pathogenic for humans orthopoxviruses (OPV) – Variola virus (VARV) and Monkeypox virus (MPXV) can cause systemic diseases characterized by high contagiousness and often leading to death. In previous publications (https://doi.org/10.3390/v14112580), we reported on the development of a sensitive, rapid and easy-to-use immunochemical test potentially suitable for use in the infection foci and in laboratory conditions with a high level of biosecurity. The prototype of the diagnostic kit was tested on non-pathogenic and low-pathogenic for humans OPV and specifically detected them with a sensitivity within the range of 103 to 104 PFU/mL. The aim of this work was to evaluate the sensitivity of this assay in detecting highly pathogenic for humans MPXV and VARV, as well as the applicability of the assay in a laboratory with a high level of biosafety (BSL-4). Materials and methods. The efficiency of virus detection in cryolysates of CV-1 cell culture samples infected with VARV and MPXV was assessed using a one-step dot immunoassay method in a laboratory with biosafety level BSL-4. Results. It was shown that the one-step dot assay allows detection of MPXV at a concentration of 2.5 × 103 PFU/mL, and VARV at a concentration of 1.0 × 104 PFU/mL. It was noted that vapors from disinfectants (hydrogen peroxide/formaldehyde) applied in biosafety cabinet decontamination may interfere with assay performance and affect result interpretation. Conclusion. The one-step dot immunoassay can be performed in a BSL-4 laboratory. When limiting the contact of the detecting system with formaldehyde vapors, the sensitivity of the test for detection of VARV and MPXV falls within the previously declared range of 103–104 PFU/mL.

Tick-borne encephalitis is a flavivirus disease, endemic to vast areas of Europe and Asia, caused by the tick-borne encephalitis virus from the genus Orthoflavivirus, transmitted through the bite of a tick. The course of infection is characterized by fever followed by the development of potentially fatal encephalitis. Serological diagnosis of tick-borne encephalitis is complicated by severe cross-reactivity with antibodies to other endemic and imported flavivirus infections. The aim of the study was to obtain a recombinant analogue of domain III of the surface protein E of the tick-borne encephalitis virus (EDIII-TBEV) and assess its ability to detect antibodies to the tick-borne encephalitis virus and differentiate them from antibodies to other flavivirus infections. Materials and methods. A comparative study was performed using panels of sera containing and not containing antibodies to the tick-borne encephalitis virus, and panels of samples with antibodies to yellow fever, dengue, and Zika viruses. The study was conducted using commercial tests and experimental kits based on EDIII-TBEV in the ELISA format and in the dot analysis format. Results and discussion. It has been shown that the use of experimental kits based on EDIII-TBEV makes it possible to effectively detect IgG antibodies to tick-borne encephalitis virus and, in contrast to commercial tests, to differentiate them from antibodies to the pathogens of dengue, yellow and Zika tropical fevers. A recombinant analogue of domain III of the surface protein E in the tick-borne encephalitis virus can be a useful capture reagent when creating diagnostic test systems in the format of enzyme-linked immunosorbent assay and dot-immunoassay for the differentiated detection of IgG antibodies to the tick-borne encephalitis virus.

Оценка эффективности применения конъюгатов куриных IgY антител к IgG человека в иммуноферментном анализе и дот-иммуноанализе

Introduction. Following the successful eradication of smallpox, mass vaccination against this disease was discontinued in 1980. The unvaccinated population continues to be at risk of infection due to military use of variola virus or exposure to monkeypox virus in Africa and non-endemic areas. In cases of these diseases, rapid diagnosis is of great importance, since the promptness and effectiveness of therapeutic and quarantine measures depend on it. The aim of work is to develop a kit of reagents for enzyme-linked immunosorbent assay (ELISA) for fast and highly sensitive detection of orthopoxviruses (OPV) in clinical samples. Materials and methods. The efficiency of virus detection was evaluated by single-stage ELISA in the cryolisate of CV-1 cell culture samples infected with vaccinia, cowpox, rabbitpox, and ectromelia viruses, as well as in clinical samples of infected rabbits and mice. Results. The method of rapid ELISA was shown to allow the detection of OPV in crude viral samples in the range of 5.0 1025.0 103 PFU/ml, and in clinical samples with a viral load exceeding 5 103 PFU/ml. Conclusions. The assay involves a minimum number of operations and can be performed within 45 minutes, which makes it possible to use it in conditions of a high level of biosecurity. Rapid ELISA method was developed using polyclonal antibodies, which significantly simplifies and reduces the cost of manufacturing a diagnostic system.

Разработка метода одновременного выявления серологических маркеров лихорадки денге

The aim of the work was an experimental evaluation of the characteristics of the kit for the rapid immunochemical detection of orthopoxviruses (OPV). The kit is based on the method of one-stage dot-immunoassay on flat protein arrays using gold conjugates and a silver developer. Rabbit polyclonal antibodies against the vaccinia virus were used as capture and detection reagents. The sensitivity of detection of OPV and the specificity of the analysis were assessed using culture crude preparations (monkeypox virus, vaccinia virus, rabbitpox virus, cowpox virus, and ectromelia virus), a suspension from a crust from a human vaccination site as well as blood and tissue suspensions of infected rabbits. It has been shown that the assay using the kit makes it possible to detect OPV within 36 min at a temperature of 18–40 °C in unpurified culture samples of the virus and clinical samples in the range of 10³–10⁴ PFU/mL. Tests of the kit did not reveal cross-reactivity with uninfected cell cultures and viral pathogens of exanthematous infections (measles, rubella and chicken pox). The kit can be used to detect or exclude the presence of a virus threat in samples and can be useful in various aspects of biosecurity. The simplicity of analysis, the possibility of visual accounting the and interpretation of the results make it possible to use the test in laboratories with a high level of biological protection and in out-of-laboratory conditions.

The aim of the study was to develop a sensitive and fast immunochemical test for the detection of orthopoxviruses (OPXV) in the “point of care” format.Materials and methods. The analyses were performed in cultured crude and purifed preparations of vaccinia virus, cowpoxvirus, rabbitpoxvirus and ectromelia virus, as well as in the blood and tissue suspensions of infected mice and rabbits. OPXV-antigen was detected by one-stage and two-stage protocols of dot-immunoassay based on flat protein arrays using rabbit polyclonal antibodies as capture and detection reagents.Results and discussion. The results show that the detection limit of OPXV is inversely related to the degree of their purifcation. The one-stage (rapid) protocol is specifc and allows detecting OPXV in crude culture samples of the virus and in clinical samples in the range of 104–103 PFU/ml within 39 minutes. Rapid dot-immunoassay can be applied to detect or exclude the presence of a viral threat in samples and can be useful in various aspects of biosafety provision. The simplicity of the one-stage protocol, the possibility to visually account the results and easy interpretation of the results allow the rapid test to be used in the “point of care” format.

Introduction. The abolition of smallpox vaccination has led to the disappearance of population immunity to pox viruses. However, the threat of infection by pathogenic orthopoxviruses persists and determines the need to develop sensitive and operational methods for indicating pathogens.Objectives and purposes. Development of a sensitive, fast and easy-to-use immunochemical test for the detection of orthopoxviruses in the «point of care» format.Material and methods. We used preparations of cultural vaccinia virus (VV) with varying degrees of purification, polyclonal antibodies from hyperimmune rabbit serum, and equipment from a previously developed autonomous kit for dot-immunoassay on flat protein arrays.Results and discussion. It has been established that rabbit polyclonal antibodies can be used in a single-stage dotanalysis, both as a capture agent immobilized on a substrate and as a detection reagent bound with colloidal gold particles. It is shown that the effectiveness of the detection of VV is inversely related to the degree of purification of viruses from sub-viral structures. The sensitivity of the rapid detection of viruses in a crude preparation was about 30 times higher than in pure viral material. The increase in sensitivity, presumably, occurs due to binding to the capture antibodies of subviral structures, which form large aggregates of sensitized gold particles. The test does not detect cross-reactions with heterogeneous viruses (measles, rubella and chickenpox) that cause exantematous diseases.Conclusion. The one-stage variant of the dot-immunoassay reduces the analysis time to 40 minutes and improves the detection sensitivity of orthopoxviruses in crude viral preparations to the range of 105-104 PFU / ml. Full makeup, ease of analysis and the ability to visually accounting for results allow the test to be used outside of laboratories.