Search results for: viral infectivity

Authors: Raquel Amanda Villamizar Gallardo, Oscar Orlando Ortíz Rodríguez

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Enteric viruses are cosmopolitan agents present in several environments including water. These viruses can cause different diseases including gastroenteritis, hepatitis, conjunctivitis, respiratory problems among others. Although in Colombia there are not regulations concerning to routine viral analysis of drinking water, an enhanced understanding of viral pollution and resistance to treatments is desired in order to assure pure water to the population. Viral detection is often complex due to the need of specialized and time-consuming procedures. In addition, viruses are highly diluted in water which is a drawback from the analytical point of view. To this end, a fast and selective detection method for detection enteric viruses (i.e. Hepatitis A and Rotavirus) were applied. Micro- magnetic particles were functionalized with monoclonal antibodies anti-Hepatitis and anti-Rotavirus and they were used to capture, concentrate and separate whole viral particles in raw water and drinking water samples from four treatment plants identified as CAR-01, MON-02, POR-03, TON-04 and located in the Northeastern Colombia. Viruses were molecularly by using RT-PCR One Step Superscript III. Each plant was analyzed at the entry and exit points, in order to determine the initial presence and eventual reduction of Hepatitis A and Rotavirus after disinfection. The results revealed the presence of both enteric viruses in a 100 % of raw water analyzed in all plants. This represents a potential health hazard, especially for those people whose use this water for agricultural purposes. However, in drinking water analysis, enteric viruses was only positive in CAR-01, where was found the presence of Rotavirus. As a conclusion, the results confirm Rotavirus as the best indicator to evaluate the efficacy of potable treatment plant in eliminating viruses. CAR potable water plant should improve their disinfection process in order to remove efficiently enteric viruses.

Keywords: drinking water, hepatitis A, rotavirus, virus removal

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Authors: S. Essa, H. Safar, R. Raghupathy

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Human cytomegalovirus (CMV) continues to be a source of severe complications to immunologically immature and immune-compromised hosts. Effective CMV vaccine that diminishes CMV disease in transplant patients and avoids congenital infection remains of high importance as no approved vaccines exist. Though the exact links of defense mechanisms are unidentified, viral-specific antibodies and Th1/Th2 cytokine responses have been involved in controlling viral infections. CMV envelope glycoprotein B (UL55/gB), the matrix proteins (UL83/pp65, UL99/pp28, UL32/pp150), and the assembly protein UL80a/pp38 are known to be targets of antiviral immune responses. In this study, mice were immunized with five HCMV antigens (UL32/pp150, UL80a/pp38, UL99/pp28, and UL83/pp65), and serum samples were collected and evaluated for eliciting viral-specific antibody responses. Moreover, Splenocytes were collected, stimulated, and assessed for cytokine responses. The results demonstrated a CMV-antigen-specific antibody response to pp38 and pp65 (E/C >2.0). The highest titers were detected with pp38 (average E/C 16.275) followed by pp65 (average E/C 7.72). Compared to control cells, splenocytes from PP38 antigen immunized mice gave a significantly higher concentration of GM-CSF, IFN-γ, IL-2 IL-4, IL-5, and IL-17A (P<0.05). Also, splenocytes from pp65 antigen immunized mice resulted in a significantly higher concentration of GM-CSF, IFN-γ, IL-2 IL-4, IL-10, IL-12, IL-17A, and TNF- α. The designation of target CMV peptides by identifying viral-specific antibodies and cytokine responses is vital for understanding the protective immune mechanisms during CMV infection and identifying appropriate viral antigens to develop novel vaccines.

Keywords: hepatitis C virus, peripheral blood mononuclear cells, neutrophils, cytokines

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Authors: Hajar Hosseini Khorami

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Porphyrins are organic, aromatic compounds found in heme, cytochrome, cobalamin, chlorophyll , and many other natural products with essential roles in biological processes that their cationic forms have been used as groups of favorable non-viral vectors recently. Cationic porphyrins are self-chromogenic reagents with a high capacity for modifications, great interaction with DNA and protection of DNA from nuclease during delivery of it into a cell with low toxicity. In order to have high efficient gene transfection into the cell while causing low toxicity, genetically manipulations of the non-viral vector, cationic porphyrin, would be useful. In this study newly modified cationic porphyrin derivative, 5, 10-dihexyl-15, 20bis (N-methyl-4-pyridyl) porphyrin was applied. Cytotoxicity of synthesized cationic porphyrin on Chinese Hamster Ovarian (CHO) cells was evaluated by using MTT assay. This cationic derivative is dose-dependent, with low cytotoxicity at the ranges from 100 μM to 0.01μM. It was uptake by cells at high concentration. Using direct non-viral gene transfection method and different concentration of cationic porphyrin were tested on transfection of CHO cells by applying derived transfection reagent with X-tremeGENE HP DNA as a positive control. However, no transfection observed by porphyrin derivative and the parameters tested except for positive control. Results of this study suggested that applying different protocol, and also trying other concentration of cationic porphyrins and DNA for forming a strong complex would increase the possibility of efficient gene transfection by using cationic porphyrins.

Keywords: cationic porphyrins, gene delivery, non-viral vectors, transfection reagents

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Authors: N. M. Sani, E. D. Jatau, O. S. Olonitola, M. Y. Gwarzo, P. Moodley, N. S. Mujahid

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Haemoglobin (HB) indicates anaemia level and by extension may reflect the nutritional level and perhaps the immunity of an individual. Some antiretroviral drugs like zidovudine are known to cause anaemia in People living with HIV/AIDS (PLWHA). A cross-sectional study using demographic data and blood specimen from 218 female commercial sex workers attending antiretroviral therapy (ART) clinics was conducted between December 2009 and July 2011 to assess the effect of zidovudine on haematologic and RNA viral load of female sex workers receiving antiretroviral treatment in north-western Nigeria. Anaemia is a common and serious complication of both HIV infection and its treatment. In the setting of HIV infection, anaemia has been associated with decreased quality of life, functional status, and survival. Antiretroviral therapy, particularly the highly active antiretroviral therapy (HAART), has been associated with a decrease in the incidence and severity of anaemia in HIV-infected patients who have received a HAART regimen for at least 1 year. In this study, result has shown that out of 218 patients, 26 with haemoglobin count between 5.1–10 g/dl were observed to have the highest viral load count of 300,000–350,000 copies/ml. It was also observed that most patients (190) with HB of 10.1–15.0 g/dl had viral load count of 200,000–250,000 copies/ml. An inverse relationship therefore exists, i.e. the lower the haemoglobin level, the higher the viral load count, even though the test statistics did not show any significance between the two (P=0.206). This shows that multivariate logistic regression analysis demonstrated that anaemia was associated with a CD4+ cell count below 50/µL in female sex workers with a viral load above 100,000 copies/mL who use zidovudine. Severe anaemia was less prevalent in this study population than in historical comparators; however, mild to moderate anaemia rates remain high. The study, therefore, recommends that hematological and virologic parameters be monitored closely in patients receiving first line ART regimen.

Keywords: anaemia, female sex worker, haemoglobin, Zidovudine

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Authors: Diana M. Torres, Anngie K. Hernandez, Andrea Villareal, Magda R. Gomez, Sadao Kobayashi

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Colombian Garlic crops exhibited mild mosaic, yellow stripes, and deformation. This group of symptoms suggested a viral infection. Several viruses belonging to the genera Potyvirus, Carlavirus and Allexivirus are known to infect garlic and lower their yield worldwide, but in Colombia, there are no studies of viral infections in this crop, only leek yellow stripe virus (LYSV) has been reported to our best knowledge. In Colombia, there are no management strategies for viral diseases in garlic because of the lack of information about viral infections on this crop, which is reflected in (i) high prevalence of viral related symptoms in garlic fields and (ii) high dispersal rate. For these reasons, the purpose of the present study was to evaluate the viral status of garlic in Colombia, which can represent a major threat on garlic yield and quality for this country 55 symptomatic leaf samples were collected for virus detection by RT-PCR and mechanical inoculation. Total RNA isolated from infected samples were subjected to RT-PCR with primers 1-OYDV-G/2-OYDV-G for Onion yellow dwarf virus (OYDV) (expected size 774pb), 1LYSV/2LYSV for LYSV (expected size 1000pb), SLV 7044/SLV 8004 for Shallot latent virus (SLV) (expected size 960pb), GCL-N30/GCL-C40 for Garlic common latent virus (GCLV) (expected size 481pb) and EF1F/EF1R for internal control (expected size 358pb). GCLV, SLV, and LYSV were detected in infected samples; in 95.6% of the analyzed samples was detected at least one of the viruses. GCLV and SLV were detected in single infection with low prevalence (9.3% and 7.4%, respectively). Garlic generally becomes coinfected with several types of viruses. Four viral complexes were identified: three double infection (64% of analyzed samples) and one triple infection (15%). The most frequent viral complex was SLV + GCLV infecting 48.1% of the samples. The other double complexes identified had a prevalence of 7% (GCLV + LYSV and SLV + LYSV) and 5.6% of the samples were free from these viruses. Mechanical transmission experiments were set up using leaf tissues of collected samples from infected fields, different test plants were assessed to know the host range, but it was restricted to C. quinoa, confirming the presence of detected viruses which have limited host range and were detected in C. quinoa by RT-PCR. The results of molecular and biological tests confirm the presence of SLV, LYSV, and GCLV; this is the first report of SLV and LYSV in garlic plants in Colombia, which can represent a serious threat for this crop in this country.

Keywords: SLV, GCLV, LYSV, leek yellow stripe virus, Allium sativum

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Authors: Dmitry Nosik, Nickolay Nosik, Elli Kaplina, Olga Lobach, Marina Chataeva, Lev Rasnetsov

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Background and aims: Human Immunodeficiency Virus (HIV) infection is very often associated with Herpes Simplex Virus (HSV) infection but HIV patients are treated with a cocktail of antiretroviral drugs which are toxic. The use of an antiviral drug which will be active against both viruses like ferrovir found in our previous studies is rather actual. Earlier we had shown that Fullerene poly-amino capronic acid (FPACA) was active in case of monoinfection of HIV-1 or HSV-1. The aim of the study was to analyze the efficiency of FPACA against mixed infection of HIV and HSV. Methods: The peripheral blood lymphocytes, CEM, MT-4 cells were simultaneously infected with HIV-1 and HSV-1. FPACA was added 1 hour before infection. Cells viability was detected by MTT assay, virus antigens detected by ELISA, syncytium formation detected by microscopy. The different multiplicity of HIV-1/HSV-1 ratio was used. Results: The double viral HIV-1/HSV-1 infection was more cytopathic comparing with monoinfections. In mixed infection by the HIV-1/HSV-1 concentration of HIV-1 antigens and syncytium formations increased by 1,7 to 2,3 times in different cells in comparison with the culture infected with HIV-1 alone. The concentration of HSV-1 increased by 1,5-1,7 times, respectively. Administration of FPACA (1 microg/ml) protected cells: HIV-1/HSV-1 (1:1) – 80,1%; HIV-1/HSV-1 (1:4) – 57,2%; HIV-1/HSV-1 (1:8) – 46,3 %; HIV-1/HSV-1 (1:16) – 17,0%. Virus’s antigen levels were also reduced. Syncytium formation was totally inhibited in all cases of mixed infection. Conclusion: FPACA showed antiviral activity in case of mixed viral infection induced by Human Immunodeficiency Virus and Herpes Simplex Virus. The effect of viral inhibition increased with the multiplicity of HIV-1 in the inoculum. The mechanism of FPACA action is connected with the blocking of the virus particles adsorption to the cells and it could be suggested that it can have an antiviral activity against some other viruses too. Now FPACA could be considered as a potential drug for treatment of HIV disease complicated with opportunistic herpes viral infection.

Keywords: antiviral drug, human immunodeficiency virus (hiv), herpes simplex virus (hsv), mixed viral infection

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Authors: Azizollah Khodakaram- Tafti, Ali Mohammadi, Ghasem Farjanikish

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Bovine viral diarrhea virus (BVDV) is one of the most important viral pathogens of cattle worldwide caused by Pestivirus genus, Flaviviridae family.The aim of the present study was to comparison several diagnostic methods and determine the prevalence of BVDV infection for the first time in dairy herds of Fars province, Iran. For initial screening, a total of 400 blood samples were randomly collected from 12 industrial dairy herds and analyzed using reverse transcription (RT)-PCR on the buffy coat. In the second step, blood samples and also ear notch biopsies were collected from 100 cattle of infected farms and tested by antigen capture ELISA (ACE), RT-PCR and immunohistochemistry (IHC). The results of nested RT-PCR (outer primers 0I100/1400R and inner primers BD1/BD2) was successful in 16 out of 400 buffy coat samples (4%) as acute infection in initial screening. Also, 8 out of 100 samples (2%) were positive as persistent infection (PI) by all of the diagnostic tests similarly including RT-PCR, ACE and IHC on buffy coat, serum and skin samples, respectively. Immunoreactivity for bovine BVDV antigen as brown, coarsely to finely granular was observed within the cytoplasm of epithelial cells of epidermis and hair follicles and also subcutaneous stromal cells. These findings confirm the importance of monitoring BVDV infection in cattle of this region and suggest detection and elimination of PI calves for controlling and eradication of this disease.

Keywords: antigen capture ELISA, bovine viral diarrhea virus, immunohistochemistry, RT-PCR, cattle

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Authors: Sirikwan Ponprateep, Anchalee Tassanakajon, Vichien Rimphanitchayakit

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A Kazal-type serine proteinase inhibitor, SPIPm2, was abundantly expressed in the hemocytes and secreted into shrimp plasma has anti-viral property against white spot syndrome virus (WSSV). To discover the molecular mechanism of antiviral activity, the binding assay showed that SPIPm2 bind to the components of viral particle and shrimp hemocyte. From our previous report, viral target protein of SPIPm2 was identified, namely WSV477 using yeast two-hybrid screening. WSV477 is an early gene product of WSSV and involved in viral propagation. In this study, the co-immunoprecipitation technique and Tandem Mass Spectrometry (LC-MS/MS) was used to identify the target protein of SPIPm2 from shrimp hemocyte. The target protein of SPIPm2 was cyclophilin A. In vertebrate, cyclophilin A or peptidylprolyl isomerase A was reported to be the immune suppressor interacted with cyclosporin A involved in immune defense response. The recombinant cyclophilin A from Penaeus monodon (rPmCypA) was produced in E.coli system and purified using Ni-NTA column to confirm the protein-protein interaction. In vitro pull-down assay showed the interaction between rSPIPm2 and rPmCypA. To study the biological function of these proteins, the expression analysis of immune gene in shrimp defense pathways will be investigated after rPmCypA administration.

Keywords: cyclophilin A, protein-protein interaction, Kazal-type serine proteinase inhibitor, Penaeus monodon

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Authors: Barun K. De

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Acquired immunodeficiency syndrome (AIDS) is caused by two types of human immunodeficiency viruses, collectively designated HIV. HIV infection is spreading globally particularly in developing countries. Before an individual is diagnosed with HIV, the disease goes through different phases. First there is an acute early phase that is followed by an established or chronic phase. Subsequently, there is a latency period after which the individual becomes immunodeficient. It is in the acute phase that an individual is highly infectious due to a high viral load. Presently, HIV diagnosis involves use of tests that do not detect the acute phase infection during which both the viral RNA and p24 antigen are expressed. Instead, these less sensitive tests detect antibodies to viral antigens which are typically sero-converted later in the disease process following acute infection. These antibodies are detected in both asymptomatic HIV-infected individuals as well as AIDS patients. Studies indicate that early diagnosis and treatment of HIV infection can reduce medical costs, improve survival, and reduce spreading of infection to new uninfected partners. Newer 4th generation combination antigen/antibody tests are highly sensitive and specific for detection of acute and established HIV infection (HIV1 and HIV2) enabling immediate linkage to care. The CDC (Center of Disease Control, USA) recently recommended an algorithm involving three different tests to screen and diagnose acute and established infections of HIV-1 and HIV-2 in a general population. Initially a 4th generation combo test detects a viral antigen p24 and specific antibodies against HIV -1 and HIV-2 envelope proteins. If the test is positive it is followed by a second test known as a differentiation assay which detects antibodies against specific HIV-1 and HIV-2 envelope proteins confirming established infection of HIV-1 or HIV-2. However if it is negative then another test is performed that measures viral load confirming an acute HIV-1 infection. Screening results of a Phoenix area population detected 0.3% new HIV infections among which 32.4% were acute cases. Studies in the U.S. indicate that this algorithm effectively reduces HIV infection through immediate treatment and education following diagnosis.

Keywords: new algorithm, HIV, diagnosis, infection

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Authors: Siobhan O’Shea, Sangeetha Vijaysri Nair, Hee Cheol Kim, Charles Thomas Nugent, Cheuk Yan William Tong, Sam Douthwaite, Andrew Worlock

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The Aptima® HIV-1 Quant Dx Assay is a fully automated assay on the Panther system. It is based on Transcription-Mediated Amplification and real time detection technologies. This assay is intended for monitoring HIV-1 viral load in plasma specimens and for the detection of HIV-1 in plasma and serum specimens. Nine-hundred and seventy nine specimens selected at random from routine testing at St Thomas’ Hospital, London were anonymised and used to compare the performance of the Aptima HIV-1 Quant Dx assay and Roche COBAS® AmpliPrep/COBAS® TaqMan® HIV-1 Test, v2.0. Two-hundred and thirty four specimens gave quantitative HIV-1 viral load results in both assays. The quantitative results reported by the Aptima Assay were comparable those reported by the Roche COBAS AmpliPrep/COBAS TaqMan HIV-1 Test, v2.0 with a linear regression slope of 1.04 and an intercept on -0.097. The Aptima assay detected HIV-1 in more samples than the Roche assay. This was not due to lack of specificity of the Aptima assay because this assay gave 99.83% specificity on testing plasma specimens from 600 HIV-1 negative individuals. To understand the reason for this higher detection rate a side-by-side comparison of low level panels made from the HIV-1 3rd international standard (NIBSC10/152) and clinical samples of various subtypes were tested in both assays. The Aptima assay was more sensitive than the Roche assay. The good sensitivity, specificity and agreement with other commercial assays make the HIV-1 Quant Dx Assay appropriate for both viral load monitoring and detection of HIV-1 infections.

Keywords: HIV viral load, Aptima, Roche, Panther system

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Authors: Monika Soni, Chandra Bhattacharya, Siraj Ahmed Ahmed, Prafulla Dutta

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Dengue is now a globally important arboviral disease. Extensive vector surveillance has already established A.aegypti as a primary vector, but A.albopictus is now accelerating the situation through gradual adaptation to human surroundings. Global destabilization and gradual climatic shift with rising in temperature have significantly expanded the geographic range of these species These versatile vectors also host Chikungunya, Zika, and yellow fever virus. Biggest challenge faced by endemic countries now is upsurge in co-infection reported with multiple serotypes and virus co-circulation. To foster vector control interventions and mitigate disease burden, there is surge for knowledge on vector susceptibility and viral tolerance in response to multiple infections. To address our understanding on transmission dynamics and reproductive fitness, both the vectors were exposed to single and dual combinations of all four dengue serotypes by artificial feeding and followed up to third generation. Artificial feeding observed significant difference in feeding rate for both the species where A.albopictus was poor artificial feeder (35-50%) compared to A.aegypti (95-97%) Robust sequential screening of viral antigen in mosquitoes was followed by Dengue NS1 ELISA, RT-PCR and Quantitative PCR. To observe viral dissemination in different mosquito tissues Indirect immunofluorescence assay was performed. Result showed that both the vectors were infected initially with all dengue(1-4)serotypes and its co-infection (D1 and D2, D1 and D3, D1 and D4, D2 and D4) combinations. In case of DENV-2 there was significant difference in the peak titer observed at 16th day post infection. But when exposed to dual infections A.aegypti supported all combinations of virus where A.albopictus only continued single infections in successive days. There was a significant negative effect on the fecundity and fertility of both the vectors compared to control (PANOVA < 0.001). In case of dengue 2 infected mosquito, fecundity in parent generation was significantly higher (PBonferroni < 0.001) for A.albopicus compare to A.aegypti but there was a complete loss of fecundity from second to third generation for A.albopictus. It was observed that A.aegypti becomes infected with multiple serotypes frequently even at low viral titres compared to A.albopictus. Possible reason for this could be the presence of wolbachia infection in A.albopictus or mosquito innate immune response, small RNA interference etc. Based on the observations it could be anticipated that transovarial transmission may not be an important phenomenon for clinical disease outcome, due to the absence of viral positivity by third generation. Also, Dengue NS1 ELISA can be used for preliminary viral detection in mosquitoes as more than 90% of the samples were found positive compared to RT-PCR and viral load estimation.

Keywords: co-infection, dengue, reproductive fitness, viral quantification

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Authors: Houda Haddad, Nolwen Jouvenet, Maxime Chazal, Frédéric Tangy, Amira Zairi

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Zika virus represents the primary cause of infection during pregnancy and can lead to various neurological disorders, such as microcephaly and Guillain-Barré syndrome, affecting both children and adults. This infection is also associated with urological and nephrological problems. So far, evidence of mosquito-borne Zika virus infection has been reported in a total of 89 countries and territories. However, surveillance efforts primarily concentrate on outbreaks that this virus can cause, yet the measures implemented are typically limited. Currently, there are no specific treatments or vaccines designed for the prevention or treatment of Zika virus infection or its associated disease. The development of effective therapeutic agents presents an urgent need. Importantly, an alternative for advancing the discovery of molecules could be dermaseptins, a family of antimicrobial peptides known for their potential antiviral properties. In this study, we carried out the synthesis of dermaseptins and their analogs and subsequently assessed the bioactivity tests against Zika virus (ZIKV PF13) of dermaseptins B2 and S4 and their derivatives. The cytotoxicity of these peptides was investigated on the HMC3 cell line and HeLa cells by CellTiter-Glo® Luminescent Cell Viability Assay. Thereafter, we evaluated the antiviral activity caused by the action of our dermaseptins on the viral envelope using the Fluorescence Activated Cell Sorting (FACS). The cytotoxicity of our molecules was concentration-dependent at microgram concentrations except for dermaseptin B2 and its derivative, which present no toxicity against HeLa and HMC3 cell lines. It was observed that all tested analogs from the S4 family exhibited antiviral activity with low concentrations ranging from 3 to 12.5 μg/mL, unlike the native B2 and its derivative, which increased the infectivity. Pre-incubating of dermaseptins with ZIKV PF13 before infection revealed that these derivatives inhibit the initial stages of virus infection. In summary, these results suggest that dermaseptins could serve as lead structures for the development of potent antiviral agents against Zika virus infections.

Keywords: dermaseptin B2, dermaseptin S4, analogs, zika virus, neurological infections, antiviral activity

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Authors: Mahboobeh Mohadeszadeh, Majid Saghi

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Polyoxometalates (POMs) are important inorganic compounds that have been considered specifically in recent years due to abundant attributes and applications. Those POMs that have one central tetrahedral atom called keggin. The binding Amino-acid groups to keggin structure give the antivirus effect to these compounds. A new organic-inorganic hybrid structure, with formula (Gly)2H4SiW12O40 was synthesized. Investigation on Anti-viral effect of this compound showed the (Gly)2H4SiW12O40 prevents infection of Tobacco Mosaic Virus (TMV) on the Nicotianatabacum plants.

Keywords: Polyoxometalate, Keggin, Organic-inorganic salt, TMV

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Authors: Mahboobeh Mohadeszadeh, Majid Saghi

Abstract:

Polyoxometalates (POMs) are important inorganic compounds that have been considered specifically in recent years due to abundant attributes and applications. Those POMs that have one central tetrahedral atom called keggin. The binding Amino-acid groups to keggin structure give the antivirus effect to these compounds. A new organic-inorganic hybrid structure, with formula (Gly)2H4SiW12O40 was synthesized. Investigation on Anti-viral effect of this compound showed the (Gly)2H4SiW12O40 prevents infection of Tobacco Mosaic Virus (TMV) on the Nicotianatabacum plants.

Keywords: polyoxometalate, keggin, organic-inorganic salt, TMV

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Authors: Maia Kukhaleishvili, Ekaterine Bulauri, Iveta Megrelishvili, Tamar Shamatava, Tamar Chipashvili

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Plant viruses can cause loss of yield and quality in a lot of important crops. Symptoms of pathogens are variable depending on the cultivars and virus strain. Selection of resistant potato varieties would reduce the risk of virus transmission and significant economic impact. Other way to avoid reduced harvest yields is regular potato seed production sampling and testing for viral infection. The aim of this study was to determine the occurrence and distribution of viral diseases according potato cultivars for further selection of virus-free material in Georgia. During the summer 2015- 2016, 5 potato cultivars (Sante, Laura, Jelly, Red Sonia, Anushka) at 5 different farms located in Akhalkalaki were tested for 6 different potato viruses: Potato virus A (PVA), Potato virus M (PVM), Potato virus S (PVS), Potato virus X (PVX), Potato virus Y (PVY) and potato leaf roll virus (PLRV). A serological method, Double Antibody Sandwich-Enzyme linked Immunosorbent Assay (DASELISA) was used at the laboratory to analyze the results. The result showed that PVY (21.4%) and PLRV (19.7%) virus presence in collected samples was relatively high compared to others. Researched potato cultivars except Jelly and Laura were infected by PVY with different concentrations. PLRV was found only in three potato cultivars (Sante, Jelly, Red Sonia) and PVM virus (3.12%) was characterized with low prevalence. PVX, PVA and PVS virus infection was not reported. It would be noted that 7.9% of samples were containing PVY/PLRV mix infection. Based on the results it can be concluded that PVY and PLRV infections are dominant in all research cultivars. Therefore significant yield losses are expected. Systematic, long-term control of potato viral infection, especially seed-potatoes, must be regarded as the most important factor to increase seed productivity.

Keywords: virus, potato, infection, diseases

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Authors: Sergio Alejandro Cuevas, Catherine Etchebest, Fernando Luis Barroso Da Silva

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The flavivirus genus has several organisms responsible for generating various diseases in humans. Special in Brazil, Zika (ZIKV), Dengue (DENV) and Yellow Fever (YFV) viruses have raised great health concerns due to the high number of cases affecting the area during the last years. Diagnostic is still a difficult issue since the clinical symptoms are highly similar. The understanding of their common structural/dynamical and biomolecular interactions features and differences might suggest alternative strategies towards differential serological diagnostics and therapeutic intervention. Due to their immunogenicity, the primary focus of this study was on the ZIKV, DENV and YFV non-structural proteins 1 (NS1) protein. By means of computational studies, we calculated the main physical chemical properties of this protein from different strains that are directly responsible for the biomolecular interactions and, therefore, can be related to the differential infectivity of the strains. We also mapped the electrostatic differences at both the sequence and structural levels for the strains from Uganda to Brazil that could suggest possible molecular mechanisms for the increase of the virulence of ZIKV. It is interesting to note that despite the small changes in the protein sequence due to the high sequence identity among the studied strains, the electrostatic properties are strongly impacted by the pH which also impact on their biomolecular interactions with partners and, consequently, the molecular viral biology. African and Asian strains are distinguishable. Exploring the interfaces used by NS1 to self-associate in different oligomeric states, and to interact with membranes and the antibody, we could map the strategy used by the ZIKV during its evolutionary process. This indicates possible molecular mechanisms that can explain the different immunological response. By the comparison with the known antibody structure available for the West Nile virus, we demonstrated that the antibody would have difficulties to neutralize the NS1 from the Brazilian strain. The present study also opens up perspectives to computationally design high specificity antibodies.

Keywords: zika, biomolecular interactions, electrostatic interactions, molecular mechanisms

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Authors: Mahboobeh Mohadeszadeh, Majid Saghi

Abstract:

Polyoxometalates (POMs) are important inorganic compounds that have been considered specifically in recent years due to abundant attributes and applications. Those POMs that have one central tetrahedral atom called keggin. The binding Amino-acid groups to keggin structure give the antivirus effect to these compounds. A new organic-inorganic hybrid structure, with formula (Gly)2H4SiW12O40, was synthesized. Investigation on the anti-viral effect of this compound showed the (Gly)2H4SiW12O40 prevents infection of Tobacco Mosaic Virus (TMV) on the Nicotianatabacum plants.

Keywords: polyoxometalate, keggin, organic-inorganic salt, TMV

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Authors: Hueiwang Anna Jeng, Norou Diawara, Nancy Welch, Cynthia Jackson, Rekha Singh, Kyle Curtis, Raul Gonzalez, David Jurgens, Sasanka Adikari

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Modeling effort is needed to predict the COVID-19 trends for developing management strategies and adaptation measures. The objective of this study was to assess whether SARS-CoV-2 viral load in wastewater could serve as a predictor for forecasting COVID-19 cases, hospitalization cases, and death cases using copula-based time series modeling. SARS-CoV-2 RNA load in raw wastewater in Chesapeake VA was measured using the RT-qPCR method. Gaussian copula time series marginal regression model, incorporating an autoregressive moving average model and the copula function, served as a forecasting model. COVID-19 cases were correlated with wastewater viral load, hospitalization cases, and death cases. The forecasted trend of COVID-19 cases closely paralleled one of the reported cases, with over 90% of the forecasted COVID-19 cases falling within the 99% confidence interval of the reported cases. Wastewater SARS-CoV-2 viral load could serve as a predictor for COVID-19 cases and hospitalization cases.

Keywords: COVID-19, modeling, time series, copula function

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Authors: Daniel Todorov, Anton Hinkov, Petya Angelova, Kalina Shishkova, Venelin Tsvetkov, Stoyan Shishkov

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Human herpes viruses (HHV) are ubiquitous pathogens with a pandemic spread across the globe. HHV type 1 is the main causative agent of cold sores and fever blisters around the mouth and on the face, whereas HHV type 2 is generally responsible for genital herpes outbreaks. The treatment of both viruses is more or less successful with antivirals from the nucleoside analogues group. Their wide application increasingly leads to the emergence of resistant mutants In the past, medicinal plants have been used to treat a number of infectious and non-infectious diseases. Their diversity and ability to produce the vast variety of secondary metabolites according to the characteristics of the environment give them the potential to help us in our warfare with viral infections. The variable chemical characteristics and complex composition is an advantage in the treatment of herpes since the emergence of resistant mutants is significantly complicated. The screening process is difficult due to the lack of standardization. That is why it is especially important to follow the mechanism of antiviral action of plants. On the one hand, it may be expected to interact with its compounds, resulting in enhanced antiviral effects, and the most appropriate environmental conditions can be chosen to maximize the amount of active secondary metabolites. During our study, we followed the activity of various plant extracts on the viral replication cycle as well as their effect on the extracellular virion. We obtained our results following the logical sequence of the experimental settings - determining the cytotoxicity of the extracts, evaluating the overall effect on viral replication and extracellular virion.During our research, we have screened a variety of plant extracts for their antiviral activity against both virus replication and the virion itself. We investigated the effect of the extracts on the individual stages of the viral replication cycle - viral adsorption, penetration and the effect on replication depending on the time of addition. If there are positive results in the later experiments, we had studied the activity over viral adsorption, penetration and the effect of replication according to the time of addition. Our results indicate that some of the extracts from the Lamium album have several targets. The first stages of the viral life cycle are most affected. Several of our active antiviral agents have shown an effect on extracellular virion and adsorption and penetration processes. Our research over the last decade has shown several curative antiviral plants - some of which are from the Lamiacea family. The rich set of active ingredients of the plants in this family makes them a good source of antiviral preparation.

Keywords: human herpes virus, antiviral activity, Lamium album, Nepeta nuda

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Authors: Rachel Matar, Maxime Merheb

Abstract:

Chemical drugs have been used for many centuries as the only way to cure diseases until the novel gene therapy has been created in 1960. Gene therapy is based on the insertion, correction, or inactivation of genes to treat people with genetic illness (1). Gene therapy has made wonders in Parkison’s, Alzheimer and multiple sclerosis. In addition to great promises in the healing of deadly diseases like many types of cancer and autoimmune diseases (2). This method implies the use of recombinant DNA technology with the help of different viral and non-viral vectors (3). It is nowadays used in somatic cells as well as embryos and gametes. Beside all the benefits of gene therapy, this technique is deemed by some opponents as an ethically unacceptable treatment as it implies playing with the genes of living organisms.

Keywords: gene therapy, genetic disease, cancer, multiple sclerosis

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Authors: Uraib Sharaha, Guy Beck, Joseph Kapelushnik, Adam H. Agbaria, Itshak Lapidot, Shaul Mordechai, Ahmad Salman, Mahmoud Huleihel

Abstract:

Infectious diseases cause a significant burden on the public health and the economic stability of societies all over the world for several centuries. A reliable detection of the causative agent of infection is not possible based on clinical features, since some of these infections have similar symptoms, including fever, sneezing, inflammation, vomiting, diarrhea, and fatigue. Moreover, physicians usually encounter difficulties in distinguishing between viral and bacterial infections based on symptoms. Therefore, there is an ongoing need for sensitive, specific, and rapid methods for identification of the etiology of the infection. This intricate issue perplex doctors and researchers since it has serious repercussions. In this study, we evaluated the potential of the mid-infrared spectroscopic method for rapid and reliable identification of bacterial and viral infections based on simple peripheral blood samples. Fourier transform infrared (FTIR) spectroscopy is considered a successful diagnostic method in the biological and medical fields. Many studies confirmed the great potential of the combination of FTIR spectroscopy and machine learning as a powerful diagnostic tool in medicine since it is a very sensitive method, which can detect and monitor the molecular and biochemical changes in biological samples. We believed that this method would play a major role in improving the health situation, raising the level of health in the community, and reducing the economic burdens in the health sector resulting from the indiscriminate use of antibiotics. We collected peripheral blood samples from young 364 patients, of which 93 were controls, 126 had bacterial infections, and 145 had viral infections, with ages lower than18 years old, limited to those who were diagnosed with fever-producing illness. Our preliminary results showed that it is possible to determine the infectious agent with high success rates of 82% for sensitivity and 80% for specificity, based on the WBC data.

Keywords: infectious diseases, (FTIR) spectroscopy, viral infections, bacterial infections.

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Authors: Sila Akhan, Muge Toygar, Murat Sayan, Simge Fidan

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Chronic viral hepatitis B, C, and D can cause hepatocellular carcinoma (HCC), cirrhosis and death. The proper treatment reduce the risk of development of HCC importantly, but not to zero point. Materials and Methods: We analysed retrospectively our chronic viral hepatitis B, C and D patients who attended to our Infectious Diseases policlinic between 2004-2018. From 589 biopsy-proven chronic hepatitis patients 3 have hepatocellular carcinoma on our follow up. First case is 74 years old patient. His HCV infection diagnosis was made 8 years ago. First treatment was pegylated interferon plus ribavirin only 28 weeks, because of HCV RNA breakthrough under treatment. In 2013 he was retreated with telaprevir, pegylated interferon plus ribavirin 24 weeks. But at the end of the therapy HCV RNA was found 1.290.000 IU/mL. He has abdominal ultrasonography (US) controls and alpha-fetoprotein (AFP) at 6 months intervals. All seemed normal until 2015 then he has an abdominal magnetic resonance imaging (MRI) and found HCC by chance. His treatment began in Oncology Clinic after verified with biopsy of HCC. And then sofosbuvir/ledipasvir was given to him for HCV 24 weeks. Sustained virologic response (SVR) was obtained. He is on cure for HCV infection and under control of Oncology for HCC. Second patient is 36 years old man. He knows his HBV infection since 2008. HBsAg and HBeAg positive; HDV RNA negative. Liver biopsy revealed grade:4, stage 3-4 according modified Knodell scoring system. In 2010 tenofovir treatment was began. His abdominal US and AFP were normal. His controls took place at 6 months intervals and HBV DNA negative, US, and AFP were normal until 2016 continuously. AFP found 37 above the normal range and then HCC was found in MRI. Third patient is 57 years old man. As hepatitis B infection was first diagnosed; he has cirrhosis and was began tenofovir as treatment. In short time he has HCC despite normal AFP values. Conclusion: In Mediterranian countries including Turkey naturally occurring pre-S/S variants are more than 75% of all chronic hepatitis B patients. This variants may contribute to the development of progressive liver damage and hepatocarcinogenesis. HCV-induced development of HCC is a gradual process and is affected by the duration of disease and viral genotype. All the chronic viral hepatitis patients should be followed up in 6 months intervals not only with US and AFP for HCC. Despite they have proper treatment there is always the risk development of HCC. Chronic hepatitis patients cannot be dropped from follow up even treated well.

Keywords: HCC, HCV, HBV, DAA

Procedia PDF Downloads 118

Authors: Dalia. G. Aseel, E. E. Hafez, S. M. Hammad

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Viral RNAs of both potato leaf roll virus (PLRV) and potato virus Y (PVY) were extracted from infected potato leaves collected from different Egyptian regions. Differential Display Polymerase Chain Reaction (DD-PCR) using (Endogluconase, β-1,3-glucanases, Chitinase, Peroxidase and Polyphenol oxidase) primers (forward strand) for was performed. The obtained data revealed different banding patterns depending on the viral type and the region of infection. Regarding PLRV, a 58 up regulated and 19 down regulated genes were detected, while, 31 up regulated and 14 down regulated genes were observed in case of PVY. Based on the nucleotide sequencing, variable phylogenetic relationships were reported for the three sequenced genes coding for: Induced stolen tip protein, Disease resistance RPP-like protein and non-specific lipid-transfer protein. In a complementary approach, using the quantitative Real-time PCR, the expressions of PRs genes understudy were estimated in the infected leaves by PLRV and PVY of three potato cultivars (Spunta, Diamont and Cara). The infection with both viruses inhibited the expressions of the five PRs genes. On the contrary, infected leaves by PLRV or PVY elevated the expression of some defense genes. This interaction also may be enhanced and/or inhibited the expression of some genes responsible for the plant defense mechanisms.

Keywords: PLRV, PVY, PR genes, DD-PCR, qRT-PCR, sequencing

Procedia PDF Downloads 315

Authors: Saleh Alkarri, Melinda Frame, Dimple Sharma, John Cairney, Lee Maddan, Jin H. Kim, Jonathan O. Rayner, Teresa M. Bergholz, Muhammad Rabnawaz

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Reported herein is an investigation of anti-bacterial and anti-virus properties, mode of action of Mg(OH)₂ and copper-infused Mg(OH)₂ nanoplatelets (NPs) on melt-compounded and thermally embossed polypropylene (PP) surfaces. The anti-viral activity for the NPs was studied in aqueous liquid suspensions against SARS-CoV-2, and the mode of action was investigated on neat NPs and PP samples that were thermally embossed with NPs. Anti-bacterial studies for melt-compounded NPs in PP confirmed approximately 1 log reduction of E. coli populations in 24 h, while for thermally embossed NPs, an 8 log reduction of E. coli populations was observed. In addition, the NPs exhibit anti-viral activity against SARS-CoV-2. Fluorescence microscopy revealed that reactive oxygen species (ROS) is the main mode of action through which Mg(OH)₂ and Cu-Infused Mg(OH)₂act against microbes. Plastics with anti-microbial surfaces from where biocides are non-leachable are highly desirable. This work provides a general fabrication strategy for developing anti-microbial plastic surfaces.

Keywords: anti-microbial activity, E. coli K-12 MG1655, anti-viral activity, SARS-CoV-2, copper-infused magnesium hydroxide, non-leachable, ROS, compounding, surface embossing, dyes

Procedia PDF Downloads 49

Authors: Charles Bijjah Nkhata, Memory Nekati Mvula, Milton Masautso Kalongonda, Martha Masamba, Isaac Thom Shawa

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Viral Hepatitis is a serious public health concern globally with deaths estimated at 1.4 million annually due to liver fibrosis, cirrhosis, and hepatocellular carcinoma. Hepatitis B and C are the most common viruses that cause liver damage. However, the majority of infected individuals are unaware of their serostatus. Viral Hepatitis has contributed to maternal and neonatal morbidity and mortality. There is no updated data on the Epidemiology of hepatitis B and C among pregnant mothers in Malawi. To assess the epidemiology of Hepatitis B and C viruses among pregnant women at Queen Elizabeth Central Hospital. Specific Objectives • To determine sero-prevalence of HBsAg and Anti-HCV in pregnant women at QECH. • To investigate risk factors associated with HBV and HCV infection in pregnant women. • To determine the distribution of HBsAg and Anti-HCV infection among pregnant women of different age group. A descriptive cross-sectional study was conducted among pregnant women at QECH in last quarter of 2021. Of the 114 pregnant women, 96 participants were consented and enrolled using a convenient sampling technique. 12 participants were dropped due to various reasons; therefore 84 completed the study. A semi-structured questionnaire was used to collect socio-demographic and behavior characteristics to assess the risk of exposure. Serum was processed from venous blood samples and tested for HBsAg and Anti-HCV markers utilizing Rapid screening assays for screening and Enzyme Linked Immunosorbent Assay for confirmatory. A total of 84 pregnant consenting pregnant women participated in the study, with 1.2% (n=1/84) testing positive for HBsAg and nobody had detectable anti-HCV antibodies. There was no significant link between HBV and HCV in any of the socio-demographic data or putative risk variables. The findings indicate a viral hepatitis prevalence lower than the set range by the WHO. This suggests that HBV and HCV are rare in pregnant women at QECH. Nevertheless, accessible screening for all pregnant women should be provided. The prevention of MTCT is key for reduction and prevention of the global burden of chronic viral Hepatitis.

Keywords: viral hepatitis, hepatitis B, hepatitis C, pregnancy, malawi, liver disease, mother to child transmission

Procedia PDF Downloads 147

Authors: Shayli Varasteh Moradi, Wayne A. Johnston, Dejan Gagoski, Kirill Alexandrov

Abstract:

The demand for a rapid and more efficient technique to identify protein-protein interaction particularly in the areas of therapeutics and diagnostics development is growing. The method described here is a rapid in vitro protein-protein interaction analysis approach based on AlphaLISA technology combined with Leishmania tarentolae cell-free protein production (LTE) system. Cell-free protein synthesis allows the rapid production of recombinant proteins in a multiplexed format. Among available in vitro expression systems, LTE offers several advantages over other eukaryotic cell-free systems. It is based on a fast growing fermentable organism that is inexpensive in cultivation and lysate production. High integrity of proteins produced in this system and the ability to co-express multiple proteins makes it a desirable method for screening protein interactions. Following the translation of protein pairs in LTE system, the physical interaction between proteins of interests is analysed by AlphaLISA assay. The assay is performed using unpurified in vitro translation reaction and therefore can be readily multiplexed. This approach can be used in various research applications such as epitope mapping, antigen-antibody analysis and protein interaction network mapping. The intra-viral protein interaction network of Zika virus was studied using the developed technique. The viral proteins were co-expressed pair-wise in LTE and all possible interactions among viral proteins were tested using AlphaLISA. The assay resulted to the identification of 54 intra-viral protein-protein interactions from which 19 binary interactions were found to be novel. The presented technique provides a powerful tool for rapid analysis of protein-protein interaction with high sensitivity and throughput.

Keywords: AlphaLISA technology, cell-free protein expression, epitope mapping, Leishmania tarentolae, protein-protein interaction

Procedia PDF Downloads 216

Authors: Sofia Papadimitriou

Abstract:

INTRODUCTION: The differential diagnosis between acute viral and bacterial infections is an important cost-effectiveness parameter at the stage of the treatment process in order to achieve the maximum benefits in therapeutic intervention by combining the minimum cost to ensure the proper use of antibiotics.The discovery of sensitive and robust molecular diagnostic tests in response to the role of the host in infections has enhanced the accurate diagnosis and differentiation of infections. METHOD: The study used a sample of six independent blood samples (total=756) which are associated with human proteins-proteins, each of which at the transcription stage expresses a different response in the host network between viral and bacterial infections.Τhe individual blood samples are subjected to a sequence of computer filters that identify a gene panel corresponding to an autonomous diagnostic score. The data set and the correspondence of the gene panel to the diagnostic patents a new Bangalore -Viral Bacterial (BL-VB). FINDING: We use a biomarker based on the blood of 10 genes(Panel-VB) that are an important prognostic value for the detection of viruses from bacterial infections with a weighted average AUROC of 0.97(95% CL:0.96-0.99) in eleven independent samples (sets n=898). We discovered a base with a patient score (VB 10 ) according to the table, which is a significant diagnostic value with a weighted average of AUROC 0.94(95% CL: 0.91-0.98) in 2996 patient samples from 56 public sets of data from 19 different countries. We also studied VB 10 in a new cohort of South India (BL-VB,n=56) and found 97% accuracy in confirmed cases of viral and bacterial infections. We found that VB 10 (a)accurately identifies the type of infection even in unspecified cases negative to the culture (b) shows its clinical condition recovery and (c) applies to all age groups, covering a wide range of acute bacterial and viral infectious, including non-specific pathogens. We applied our VB 10 rating to publicly available COVID 19 data and found that our rating diagnosed viral infection in patient samples. RESULTS: Τhe results of the study showed the diagnostic power of the biomarker VB 10 as a diagnostic test for the accurate diagnosis of acute infections in recovery conditions. We look forward to helping you make clinical decisions about prescribing antibiotics and integrating them into your policies management of antibiotic stewardship efforts. CONCLUSIONS: Overall, we are developing a new property of the RNA-based biomarker and a new blood test to differentiate between viral and bacterial infections to assist a physician in designing the optimal treatment regimen to contribute to the proper use of antibiotics and reduce the burden on antimicrobial resistance, AMR.

Keywords: acute infections, antimicrobial resistance, biomarker, blood transcriptome, systems biology, classifier diagnostic score

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Authors: Reshu Saxena, R. K. Tripathi

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HIV-1 transmission and spread involves significant host-virus interaction. Potential targets for prevention of HIV-1 lies at the site of mucosal barriers. Thus a better understanding of how HIV-1 infects target cells at such sites and lead their invasion is required, with prime focus on the host determinants regulating HIV-1 spread. HIV-1 Nef is important for viral infectivity and pathogenicity. It promotes HIV-1 replication, facilitating immune evasion by interacting with various host factors and altering cellular pathways via multiple protein-protein interactions. In this study nef was sequenced from HIV-1 patients, and showed specific mutations revealing sequence variability in nef. To explore the difference in Nef functionality based on sequence variability we have studied the effects of HIV-1 Nef in human SupT1 T cell line and (THP-1) monocyte-macrophage cell lines through proteomics approach. 2D-Gel Electrophoresis in control and Nef-transfected SupT1 cells demonstrated several differentially expressed proteins with significant modulation of alpha-enolase. Through further studies, effects of Nef on alpha-enolase regulation were found to be cell lineage-specific, being stimulatory in macrophages/monocytes, inhibitory in T cells and without effect in HEK-293 cells. Cell migration and invasion studies were employed to determine biological function affected by Nef mediated regulation of alpha-enolase. Cell invasion was enhanced in THP-1 cells but was inhibited in SupT1 cells by wildtype nef. In addition, the modulation of enolase and cell invasion remained unaffected by a unique nef variant. These results indicated that regulation of alpha-enolase expression and invasive property of host cells by Nef is sequence specific, suggesting involvement of a particular motif of Nef. To precisely determine this site, we designed a heptapeptide including the suggested alpha-enolase regulating sequence of nef and a nef mutant with deletion of this site. Macrophages/monocytes being the major cells affected by HIV-1 at mucosal barriers, were particularly investigated by the nef mutant and peptide. Both the nef mutant and heptapeptide led to inhibition of enhanced enolase expression and increased invasiveness in THP-1 cells. Together, these findings suggest a possible mechanism of host invasion by HIV-1 through Nef mediated regulation of alpha-enolase and identifies a potential therapeutic target for HIV-1 entry at mucosal barriers.

Keywords: HIV-1 Nef, nef variants, host-virus interaction, tissue invasion

Procedia PDF Downloads 387

Authors: Ren-Jye Lin, Li-Hsiung Lin, Bing-Cheng Liu, Ching-Len Liao

Abstract:

The human zinc finger protein 36-like protein family, containing zinc finger protein 36-like 1 (ZFP36L1) and zinc finger protein 36-like 2 (ZFP36L2), belongs to CCCH-type zinc-finger protein identified as an RNA-binding protein that participates in controlling posttranscriptional regulation via RNA decay pathways. Recently, we demonstrated that human ZFP36L1 showed potent antiviral activity against flavivirus Infection by both 5´-3´ XRN1 and 3´-5´RNA-exosome RNA decay pathways (Journal of Virology 2022 Jan 12;96(1): e0166521). However, another zinc finger protein 36-like protein member, ZFP36L2, in the host defense response against flaviviruses has yet to be addressed. Here, we also demonstrate that ZFP36L2 functions as a host innate defender against flaviviruses, including Japanese encephalitis virus (JEV) and dengue virus (DENV). Overexpression of ZFP36L2 reduced JEV and DENV infection, and ZFP36L2 knockdown significantly promoted viral replication. Distinct from the antiviral mechanism of ZFP36L1, ZFP36L2 inhibits flavivirus infection by only a 5´-3´ XRN1-mediated RNA decay pathway but not the 3´-5´RNA-exosome RNA decay pathway. Human ZFP36L1 and ZFP36L2 can restrict flavivirus replication by directly binding and destabilizing viral RNA. Thus, for the first time, human zinc finger protein 36-like family members, ZFP36L1 and ZFP36L2, are identified as host antiviral factors that can bind and degrade flavivirus viral RNA by diverse antiviral mechanisms.

Keywords: ZFP36L1, ZFP36L2, 5'-3' exonuclease XRN1, antiviral mechansim

Procedia PDF Downloads 55

Authors: Mohamad Ammar Ayass, Natalya Griko, Victor Pashkov, Wanying Cao, Kevin Zhu, Jin Zhang, Lina Abi Mosleh

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Respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent for coronavirus disease 2019 (COVID-19). Early evidence pointed at the angiotensin-converting enzyme 2 (ACE-2) expressed on the epithelial cells of the lung as the main entry point of SARS-CoV-2 into the cells. The viral entry is mediated by the binding of the Receptor Binding Domain (RBD) of the spike protein that is expressed on the surface of the virus to the ACE-2 receptor. As the number of SARS-CoV-2 variants continues to increase, mutations arising in the RBD of SARS-CoV-2 may lead to the ineffectiveness of RBD targeted neutralizing antibodies. To address this limitation, the objective of this study is to develop a combination of aptamers that target different regions of the RBD, preventing the binding of the spike protein to ACE-2 receptor and subsequent viral entry and replication. A safe and innovative biomedical tool was developed to inhibit viral infection and reduce the harms of COVID-19. In the present study, DNA aptamers were developed against a recombinant trimer S protein using the Systematic Evolution of Ligands by Exponential enrichment (SELEX). Negative selection was introduced at round number 7 to select for aptamers that bind specifically to the RBD domain. A series of 9 aptamers (ADI2010, ADI2011, ADI201L, ADI203L, ADI205L, ADIR68, ADIR74, ADIR80, ADIR83) were selected and characterized with high binding affinity and specificity to the RBD of the spike protein. Aptamers (ADI25, ADI2009, ADI203L) were able to bind and pull down endogenous spike protein expressed on the surface of SARS-CoV-2 virus in COVID-19 positive patient samples and determined by liquid chromatography- tandem mass spectrometry analysis (LC-MS/MS). LC-MS/MS data confirmed that aptamers can bind to the RBD of the spike protein. Furthermore, results indicated that the combination of the 9 best aptamers inhibited the binding of the purified trimer spike protein to the ACE-2 receptor found on the surface of Vero E6 cells. In the same experiment, the combined aptamers displayed a better neutralizing effect than antibodies. The data suggests that the selected aptamers could be used in therapy to neutralize the effect of the SARS-CoV-2 virus by inhibiting the interaction between the RBD and ACE-2 receptor, preventing viral entry into target cells and therefore blocking viral replication.

Keywords: aptamer, ACE-2 receptor, binding inhibitor, COVID-19, spike protein, SARS-CoV-2, treatment

Procedia PDF Downloads 171