Viral Agents Research Articles

B-147 Evaluation of Laboratory Database: Is there a Causal Relationship between COVID-19 and Type 1 Diabetes

Abstract Background Type 1 diabetes mellitus (T1DM) constitutes about 5% of all diagnosed cases of diabetes. The global incidence is increasing over the past few years. To date, the pathophysiology of the disease is not fully understood. with descriptions of activation of the immune system by some viral agents. SARS-CoV-2 has been suggested as a potential inducer of new-onset type 1 diabetes mellitus (T1DM). DASA has one of the largestData and Analytics areas in Diagnostic Medicine. The use of laboratory data can represent an important tool in broader knowledge of this relationship. This study aimed to evaluate the frequency of new-onset T1DM in the years 2019, 2020 and 2021. Methods This study was done through an observational, retrospective big data laboratory approach with a network that extends over a large part of Brazil to analyze whether there was an increase in T1DM cases. Individuals aged 18 years or younger were evaluated from January 2019 to December 2021. The subjects were characterized with possible new-onset T1DM through the presence of glycated hemoglobin equal to or greater than 6.5% in the presence of at least one disease-related antibody. The antibodies evaluated were: anti-GAD, anti-islet, anti-insulin and anti-zinc transporter type 8. For data evaluation, the means and standard deviations and the chi-square test were used. The results of patients who underwent RT PCR COVID19 were evaluated. Results A total of 431 060 subjects (<18 years) were evaluated, with 229 201 female (53.17%). Table 1 shows the distribution of data over the years evaluated, the number of patients with glycated levels greater than 6.5% and the number of probable new cases of DM1 from the positivity of at least 1 antibody. In the years evaluated, there was no statistical difference in the incidence of the disease between the sexes (2019 - 51.2%; P:0.92, 2020 - 50.3%; P:0.99 and 2021 - 48.9%; P: 0.67). The mean age with standard deviation in the years 2019, 2020 and 2021, respectively, was: 12.4% ± 4.4; 12.6 ± 4.3 and 12.5% ± 4.3. The mean values of glycated hemoglobin of patients with a value ≥6.5% with standard deviation in the years 2019, 2020 and 2021, respectively, were: 9.3% ± 2.1; 9.2 ± 2.5 and 9.3% ± 3.4. When comparing the incidence of the disease between the years, there was a significant increase in cases when comparing the years 2021 with 2020 and 2019 (P: 0.01 and P < 0.01, respectively). Of the total number of patients in 2020, only 5 underwent RT PCR COVID 19, with no positive result, and in 2021 a total of 32, with 7 positive, corresponding to 21.8% of cases. Conclusion The present study, using a large database, seems to demonstrate an increase in the incidence of DM1 among children diagnosed with diabetes. Prospective studies that evaluate the possible relationship of the disease with the SARS-CoV-2 virus may show this agent as a trigger of disease-related automunity.

A-304 A RT-PCR Assay for Detection and Quantification of Hepatitis D Virus (HDV)

Abstract Background Hepatitis D virus (HDV), also called Delta Hepatitis, is associated with the presence of the Hepatitis B virus (HBV) and is responsible for the infection and inflammation of liver cells. It is estimated that 15–20 million people worldwide are affected by HDV. In Brazil, Hepatitis D has high prevalence rates in the Amazon Basin. HDV is an enveloped virus with a 36 nm average diameter. The HDV virion involves a negative-sense, single-stranded circular RNA molecule associated with an inner-core delta antigen surrounded by envelope glycoproteins. HDV-infected individuals may not show signs or symptoms of the disease. When present, the most common are: tiredness, dizziness, nausea and/or vomiting, fever, abdominal pain, and yellow skin/eyes. Chronic Hepatitis D is considered the most severe form of chronic viral hepatitis, with more rapid progression to cirrhosis and an increased risk for decompensation, hepatocellular carcinoma (HCC), and death. Molecularly, determining the concentration of virus circulating in the blood of HDV patients is extremely important for diagnosis. The Quantitative Polymerase Chain Reaction (qPCR) is a molecular technique capable of detecting the viral genome with high sensitivity and specificity, ensuring the safety of the result. Therefore, this study aims to describe the analytical validation of a one-step RT-qPCR assay for HDV detection. Methods Assay performance was evaluated using commercial quantified positive control to HDV (1000, 100, 10, 1, and 0.5 IU/µL), and the parameters of analysis included: standardization assays, efficiency, and detection limit. For specificity evaluation, positive samples for Epstein-Barr Virus (EBV), Herpes Simplex Virus (HSV), Cytomegalovirus (CMV), Hepatitis B Virus (HBV), and Hepatitis C Virus (HCV) were used. RNA isolation was performed with the MagNA Pure 24 Total NA Isolation Kit at the automated platform MagNA Pure 24 System (Roche Diagnostics, Basel, Switzerland), according to the manufacturer's instructions. Altona RealStar HDV RT-PCR Kit 1.0 (Altona Diagnostics, Hamburg, Germany) was used to directly amplify RNA samples on the qPCR instrument, according to the manufacturer's instructions. Results The standardization assays indicated good qPCR amplification curves and high fluorescence levels between the controls evaluated. The assay demonstrated an average efficiency of 95%, with a coefficient of determination (r2) of 0.99. The detection limit of the test was 1 IU/µL for the HDV target (95% confidence interval). No cross-reactions with closely related viruses were observed (15 HDV-negative human plasma samples), indicating 100% specificity. The experiments performed to evaluate the precision demonstrated optimal repeatability and reproducibility. Conclusion The HDV diagnostic is required to ensure sensitive, specific and accurate identification of the viral agent and thus prompt surveillance actions such as epidemiological monitoring and control measures of infection. The RT-qPCR assay described here provides a reliable, highly specific, and sensitive method for the detection and quantification of HDV. Nevertheless, the assay needs to be evaluated in conjunction with a clinical and epidemiological context.

A-301 Analytical and Clinical Performance of the Molecular Method for Monkeypox Virus Detection

Abstract Background Monkeypox (MPX) is a zoonose caused by the Monkeypox virus (MPXV) belonging to the Orthopoxvirus genus of the Poxviridae family. Currently, MPXV is responsible for more than 84 000 infections worldwide, resulting in major social impacts. Although the reservoir is unknown, the main hosts are small rodents, typical of the tropical forests of West and Central Africa. The transmission occurs by interactions of infected animal-to-person (direct contact with the blood, bodily fluids, or cutaneous/mucosal lesions) and person-to-person (close contact with respiratory secretions, skin lesions, or recently contaminated objects). After infection, there is an incubation period of approximately 1–2 weeks (asymptomatic and non-transmissible), followed by the development of skin rashes over the body, lymphadenopathy, and systemic symptoms (fever, malaise, headache, myalgia, and fatigue). The rapid and accurate diagnosis of MPX is extremely important for combating the virus and also for epidemiological tracking. In this sense, molecular methods have been shown essential for the accurate detection and characterization of MPXV. Therefore, this study aims to describe the analytical and clinical validation of real-time polymerase chain reaction (qPCR) assay for MPXV detection. Methods The qPCR assay used in this validation was based on recommendations published by the Center for Disease Control and Prevention (CDC). The primer/probe sequence was commercially acquired and included the analysis of a MPXV-specific genomic region (CADT ID APNKYAD; Thermo Fisher, USA). Assay performance was evaluated using inactivated viral isolates and positive samples with known MPXV positive results. The analysis parameters included: (i) assay verification (performance evaluation of probes and reagents); (ii) Analytical sensitivity; (iii) Limit of detection (LOD); (iv) Analytical specificity (interference study with Herpes simplex virus and Varicella-Zoster virus); and (v) clinical validation (10 positive skin rashes samples for the MPXV). Results The assay demonstrated good performance visualized by the qPCR amplification curves (low cycle threshold (Ct) value and high fluorescence level) with an efficiency >92%. Analytical sensitivity and LOD tests were 100% and 50 copies/µL, respectively, considering a 95% confidence interval. The regression equations obtained show satisfactory reaction conditions with a positive correlation between the variables (coefficient of determination (r2) = 0.99). The assays indicated no cross-reactions with closely related viruses, demonstrating 100% of specificity. Finally, clinical validation showed 100% agreement with the expected results for all samples evaluated. Conclusion qPCR can play an important role in the rapid and accurate diagnosis of several viruses. The analytical and clinical validation provided data indicating high sensitivity and specificity for MPX detection. This study allowed the implementation of a sensitive, specific, and accurate molecular test for viral agent identification and allow prompt surveillance action by health such as epidemiological monitoring and control measures of these infections.

CHARACTERISTICS OF ROOT CANAL MICROBIOTA IN ACUTE PULPITIS OF TEMPORARY TEETH

Aims. To determine the representatives of the microbiota of the root canals seeded in acute forms of pulpitis of temporary teeth among children of the Transcarpathian region, taking into account the endemic features of the Transcarpathian region and its influence on the course of the carious process.
 Materials and methods. Collection of biomaterial was carried out using transport tubes with Amies transport medium. The biomaterial was delivered to the laboratory within 2-4 hours after selection. In order to evaluate the microbiota of the root canals, cultivation of biomaterial was carried out on differential diagnostic nutrient media. The material was sown by the method of sector sowing according to Gold: Saburo for the cultivation of microscopic mushrooms; blood agar (MPA + 5% blood) Streptococcus and Neisseria bacteria; bacteria of the family Enterobacteriaceae Endo and Levin; yolk-salt agar with mannitol bacteria of the genus Staphylococcus; enterococcus agar for isolation of enterococci (Farmaktiv, Ukraine). Additionally, the biomaterial was sown on a chromogenic medium (bioMerieux, France). Microorganisms were identified by cultural, morphological and biochemical characteristics using API-test systems (bioMerieux). The results of the quantitative and qualitative evaluation of the microbiota of the oral cavity were expressed in CFU/ml.
 Results and discussion. Today, in the daily practice of a dentist, among issues of diagnosis, prevention and treatment of dental diseases, the study of the microbiota of the oral cavity, which is a collection of different taxonomic groups of microbes that inhabit the oral cavity and enter into biochemical, immunological and other relationships with macroorganism and each other.
 At the same time, the microbial landscape of the oral cavity is represented by two types of microbiota: obligate, that is, one that is constantly present in the oral cavity and is mainly saprophytic, and provides metabolic processes and the function of protecting the body from virulent infectious agents, and facultative, which is represented opportunistic microorganisms, which, when immune protection is reduced, acquire aggressive properties and contribute to the development of diseases [1].
 Conclusion. Opportunistic bacteria play a significant role in the development of inflammatory diseases. Considering the fact that pulpitis is an inflammatory reaction caused by the activity of opportunistic microflora, the study of the characteristics of the microbiota of root canals is relevant, which will help to improve the selection of antiseptic agents for medicinal treatment of root canals in order to obtain positive delayed treatment results.

Tokat İli Asma Alanında Bazı Asma Virüslerinin Moleküler Olarak Belirlenmesi ve Grapevine Virus A'nın Filogenetik Analizi

Grapevine (Vitis spp.) is one of the major fruit crop with high socioeconomic importance for Turkey. In vineyards, many harmful organism, especially virus infections, weaken the plant and lead to decreases in yield and quality, so it takes the lead in quarantine and certification. This study was carried out to determine some viral agents that cause yield loss in vines produced in Tokat, where viticulture is very important. Samples were collected from young leaves and one-year-old shoots of grapevines showing virus symptoms from some vineyard areas in Tokat Center and its districts. Collected 189 grapevine samples were subjected to the RT-PCR test, which is a molecular method using virus-specific primers, to detect the presence of Grapevine pinot gris virus (GPGV), Grapevine virus A (GVA), Strawberry latent ringspot virus (SLRSV). Out of a total of 189 plant samples, 80 (42.32%) of GVA, 3 (1.58%) of GPGV were detected and SLRSV (0%) was not detected. More than one virus was found in 2 (1.05%) of 189 tested samples. It was determined that the most common virus was GVA, the least detected virus was GPGV in plant samples collected from Tokat Center and its districts. Bidirectional sequence analysis of RT-PCR products of GVA-infected isolates were performed and phylogenetic analyzes were done by comparing them with reference isolates after they were aligned with the MEGAX computer program. Based on phylogenetic analysis studies, GVA showed differential branching with isolates registered in GenBank and isolates obtained in the study. GVA-infected isolates showed similarity with reference isolates at rates of 92-94%. In this study, molecular analysis of Turkish GVA isolates was performed. This molecular information is important as it will shed light on future studies.

Імунозапальні зміни важкого перебігу COVID-19 у вагітних

Pregnant women are the most sensitive contingent to the effects of the virus and this is due to adaptive physiological changes in the woman’s bronchopulmonary system and features of the immune system. “Physiological tolerance” is formed during pregnancy and this is accompanied by a decrease in the level of T-helpers and natural killers in pregnant women and leads to a decrease in the resistance of the woman’s body to the action of altering viral and bacterial agents. Purpose - to reveal the peculiarities and clarify the nature of clinical and instrumental changes in pregnant women with a severe course of COVID-19 under dynamic observation. Materials and methods. The study included 40 pregnant women with coronavirus disease with a gestational age of 22 to 41 weeks, who formed the main group. The control group consisted of 40 women whose pregnancies were not complicated by coronavirus disease. The course of the disease and the influence of the coronavirus infection on the course of pregnancy were evaluated according to the results of: general - clinical examination, laboratory examination (general blood test, biochemical blood test, coagulogram, C-reactive protein, D-dimer, procalcitonin and interleukin 6), instrumental examination (cardiotocographic research, pulse oximetry). Results. Peculiarities of changes in clinical and laboratory indicators in pregnant women were studied depending on the stage of development of the coronavirus disease. During the study, it was established: a significant increase in the level of indicators in accordance with the control group, namely an increase in leukocytes by 38.2% to 10.07±1.59×109/l (р<0.05), an increase in thrombocytes by 35% to 314.36±49.70 (р<0.05), an increase in monocytes by 22.5% to 7.08±1.12 and a decrease in lymphocytes to 13.46±2.13, which is 51.2% lower than the indicators of the control group. The level of C-reactive protein in pregnant women with COVID-19 is probably higher than in all groups of examined patients. The level of D-dimer exceeded the indicators of the control group in patients of all groups with the maximum values of indicators in patients at the time of admission to department of anaesthesiology and intense therapy (DAIT) and was 4867.42 ng FEU/ml, which was 6.3 times higher than the level of the control group (p<0.05). It should be noted that the amount of interleukin-6 in patients of all studied groups was statistically higher than that of the control group. Conclusions. Immune-inflammatory blood changes in pregnant women occur in the case of a severe course of COVID-19 and are accompanied by an increase in the level of interleukin-6 at all stages of the severe course of the disease. СRP is a marker of the severe course of the coronavirus disease in pregnant women. The identified changes are accompanied by coagulopathy and hypercoagulation with increased D-dimer content in patients with a severe course of the disease. The research was carried out in accordance with the principles of the Helsinki Declaration. The study protocol was approved by the Local Ethics Committee of all participating institutions. The informed consent of the patient was obtained for conducting the studies. No conflict of interests was declared by the authors.

The role of DC-SIGN as a trans-receptor in infection by MERS-CoV.

DC-SIGN is a C-type lectin expressed in myeloid cells such as immature dendritic cells and macrophages. Through glycan recognition in viral envelope glycoproteins, DC-SIGN has been shown to act as a receptor for a number of viral agents such as HIV, Ebola virus, SARS-CoV, and SARS-CoV-2. Using a system of Vesicular Stomatitis Virus pseudotyped with MERS-CoV spike protein, here, we show that DC-SIGN is partially responsible for MERS-CoV infection of dendritic cells and that DC-SIGN efficiently mediates trans-infection of MERS-CoV from dendritic cells to susceptible cells, indicating a potential role of DC-SIGN in MERS-CoV dissemination and pathogenesis.

Stimuli-Responsive Hydrogel Microcapsules Harnessing the COVID-19 Immune Response for Cancer Therapeutics.

The combination of gene therapy and immunotherapy concepts, along recent advances in DNA nanotechnology, have the potential to provide important tools for cancer therapies. We present the development of stimuli-responsive microcapsules, loaded with a viral immunogenetic agent, harnessing the immune response against the Coronavirus Disease 2019, COVID-19, to selectively attack liver cancer cells (hepatoma) or recognize breast cancer or hepatoma, by expression of green fluorescence protein, GFP. The pH-responsive microcapsules, modified with DNA-tetrahedra nanostructures, increased hepatoma permeation by 50%. Incorporation of a GFP-encoding lentivirus vector inside the tumor-targeting pH-stimulated miRNA-triggered and Alpha-fetoprotein-dictated microcapsules enables the demonstration of neoplasm selectivity, with approximately 5,000-, 8,000- and 50,000-fold more expression in the cancerous cells, respectively. The incorporation of the SARS-CoV-2 spike protein in the gene vector promotes specific recognition of the immune-evading hepatoma by the COVID-19-analogous immune response, which leads to cytotoxic and inflammatory activity, mediated by serum components taken from vaccinated or recovered COVID-19 patients, resulting in effective elimination of the hepatoma (>85% yield).

Stimuli‐Responsive Hydrogel Microcapsules Harnessing the COVID‐19 Immune Response for Cancer Therapeutics

AbstractThe combination of gene therapy and immunotherapy concepts, along recent advances in DNA nanotechnology, have the potential to provide important tools for cancer therapies. We present the development of stimuli‐responsive microcapsules, loaded with a viral immunogenetic agent, harnessing the immune response against the Coronavirus Disease 2019, COVID‐19, to selectively attack liver cancer cells (hepatoma) or recognize breast cancer or hepatoma, by expression of green fluorescence protein, GFP. The pH‐responsive microcapsules, modified with DNA‐tetrahedra nanostructures, increased hepatoma permeation by 50 %. Incorporation of a GFP‐encoding lentivirus vector inside the tumor‐targeting pH‐stimulated miRNA‐triggered and Alpha‐fetoprotein‐dictated microcapsules enables the demonstration of neoplasm selectivity, with approximately 5,000‐, 8,000‐ and 50,000‐fold more expression in the cancerous cells, respectively. The incorporation of the SARS‐CoV‐2 spike protein in the gene vector promotes specific recognition of the immune‐evading hepatoma by the COVID‐19‐analogous immune response, which leads to cytotoxic and inflammatory activity, mediated by serum components taken from vaccinated or recovered COVID‐19 patients, resulting in effective elimination of the hepatoma (>85 % yield).

Impact of bronchoalveolar lavage on the management of immunocompromised hosts

BackgroundRespiratory infections are an important cause of morbidity and mortality in immunocompromised individuals. Fiberoptic bronchoscopy with bronchoalveolar lavage (BAL) is an important tool to detect infectious agents in immunocompromised patients with low respiratory tract infections (LRTI). Research QuestionBAL changes the management of immunocompromised patients with suspected LRTI. Study Design and MethodsImmunocompromised patients with a suspicion of LRTI underwent diagnostic BAL. The primary composite outcome consisted of pre-defined modifications in the management of the immunocompromised patients following BAL. We quantified the impact of bronchoscopy up to 30 days after the procedure. ResultsA total of 2666 visits from 1301 patients were included in the study and immunosuppression was classified as haematological (n = 1040; 544 patients), solid organ transplantation (n = 666; 107 patients) and other causes (n = 960; 650 patients). BAL led to a change in management in 52.36% (n = 1396) of all cases. This percentage, as well as the 30-day mortality differed significantly amongst the three groups. Age, C-reactive protein and aetiology of infection determined significantly the risk of 30-day mortality in all patients. In 1.89% (n = 50) of all cases, a combination of 2 respiratory viral agents was identified and 24.23% (n = 646) were diagnosed with a single respiratory viral agent. InterpretationBAL leads to changes in management in the majority of immunosuppressed patients. There is a high prevalence of multimicrobial infections and respiratory viral infections in immunocompromised patients with respiratory symptoms. Individual virus infection is associated with diverse risk of a negative outcome.

Designing a novel multi-epitopes pan-vaccine against SARS-CoV-2 and seasonal influenza: in silico and immunoinformatics approach

The merger of COVID-19 and seasonal influenza infections is considered a potentially serious threat to public health. These two viral agents can cause extensive and severe lower and upper respiratory tract infections with lung damage with host factors. Today, the development of vaccination has been shown to reduce the risk of hospitalization and mortality from the COVID-19 virus and influenza epidemics. Therefore, this study contributes to an immunoinformatics approach to producing a vaccine that can elicit strong and specific immune responses against COVID-19 and influenza A and B viruses. The NCBI, GISAID, and Uniprot databases were used to retrieve sequences. Linear B cell, Cytotoxic T lymphocyte, and Helper T lymphocyte epitopes were predicted using the online servers. Population coverage of MHC I epitopes worldwide for SARS-CoV-2, Influenza virus H3N2, H3N2, and Yamagata/Victoria were 99.93%, 68.67%, 68.38%, and 85.45%, respectively. Candidate epitopes were linked by GGGGS, GPGPG, and KK linkers. Different epitopes were permutated several times to form different peptides and then screened for antigenicity, allergenicity, and toxicity. The vaccine construct was analyzed for physicochemical properties, conformational B-cell epitopes, interaction with Toll-like receptors, and IFN-gamma-induced. Immune stimulation response of final construct was evaluated using C-IMMSIM. Eventually, the final construct sequence was codon-optimized for Escherichia coli K12 and Homo sapiens to design a multi-epitope vaccine and mRNA vaccine. In conclusion, due to the variable nature of SARS-CoV-2 and influenza proteins, the design of a multi-epitope vaccine can protect against all their standard variants, but laboratory validation is required. Communicated by Ramaswamy H. Sarma

The utility of a Bayesian predictive model to forecast neuroinvasive West Nile virus disease in the United States of America, 2022.

Arboviruses (arthropod-borne-viruses) are an emerging global health threat that are rapidly spreading as climate change, international business transport, and landscape fragmentation impact local ecologies. Since its initial detection in 1999, West Nile virus has shifted from being a novel to an established arbovirus in the United States of America. Subsequently, more than 25,000 cases of West Nile neuro-invasive disease have been diagnosed, cementing West Nile virus as an arbovirus of public health importance. Given its novelty in the United States of America, high-risk ecologies are largely underdefined making targeted population-level public health interventions challenging. Using the Centers for Disease Control and Prevention ArboNET neuroinvasive West Nile virus data from 2000-2021, this study aimed to predict neuroinvasive West Nile virus human cases at the county level for the contiguous USA using a spatio-temporal Bayesian negative binomial regression model. The model includes environmental, climatic, and demographic factors, as well as the distribution of host species. An integrated nested Laplace approximation approach was used to fit our model. To assess model prediction accuracy, annual counts were withheld, forecasted, and compared to observed values. The validated models were then fit to the entire dataset for 2022 predictions. This proof-of-concept mathematical, geospatial modelling approach has proven utility for national health agencies seeking to allocate funding and other resources for local vector control agencies tackling West Nile virus and other notifiable arboviral agents.

Agave attenuata and Agave amica, new hosts of Tuberose mild mosaic virus in Mexico.

Agave attenuata is a Mexican wild plant originally from highlands in the central and occidental mountains of Mexico. This species, known as "swan´s neck agave", is used only as an ornamental plant in public and private gardens. No virus had previously been reported from A. attenuata before this study. In a survey conducted in a commercial greenhouse in Cuautla, Morelos, in 2018, several plants were observed with symptoms of green mosaic and streaks, consistent with a putative viral infection. Sap inoculation from symptomatic A. attenuata plants to herbaceous indicator plants (Nicotiana benthamiana and N. tabacum) failed to produce symptoms in the mechanically inoculated plants. ELISA specific test to CMV, TEV, AMV, TMV and Potyvirus Group (Agdia, Inc.), was positive only for the last one (Chen and Chang, 1998). To determine the identity of the potyvirus involved, total nucleic acid extracts from 100 mg of symptomatic leaves (Trizol reagent; Gibco BRL Life Technologies, England) were used as template in RT-PCR with genus-specific potyvirus primers POT1-POT2, which targeted the variable 5´ terminal half of the coat protein gene of potyviruses (Colinet et al. 1998). The expected 900 bp amplicon was consistently detected in 10 symptomatic A. attenuata plants whereas no PCR products were obtained from 15 asymptomatic A. attenuata plants collected from the "Agaves de México" section at the 'Botanic Garden' of the Instituto de Biología de la UNAM, México. The amplicons were sequenced by the Sanger´s method and the obtained nucleotide (nt) sequences (Acc. No KY190217.1; OP964597-598) and their derived amino acid (aa) sequences were 94.68% to 95.80% similar to an isolate of Tuberose mild mosaic virus (TuMMV; Potyvirus; (Acc. No ON116187.1) characterized from Agave amica in India (Raj et al. 2009). Interestingly, A. amica (formerly Poliantes tuberose) is also a wild Mexican plant that is geographically distributed in the central and south regions of Mexico and is currently being commercially cultivated as an ornamental plant. Plants of A. amica (n=10) showing yellow mild streak were collected from commercial greenhouse and tested positive for TuMMV by RT-PCR and Sanger sequencing (No Acc. OP964599-601 levels) described above. The derived TuMMV sequences from A. attenuata and A. amica were 99-100% similar to each other at the nt/aa level. To exclude the involvement of additional viral agents in the disease, high-throughput sequencing analysis was performed separately for each species of Agave on total RNA extracts from a composite sample of symptomatic leaf tissues using Illumina´s Next Seq 500 platform. Analysis of the obtained 13,260,700 reads (each 75 nt) by the Trinity software, with a total number of sequences of 22,793, resulted in the identification of a single viral contig of 9500 nt for A. attenuata (Acc. No OP964595). Similarly, for A. amica, 27,262,248 reads were obtained, with a total number of sequences of 23,269, resulting in the identification of a single viral contig of 8500 nt (ACC. No OP964602). These contigs showed an identity percentage of 96%/88% and 98%/96% for nucleotides and amino acids, respectively, compared to an isolate of TuMMV from India (Acc. OM293939). Mexico is a center of origin for numerous species of genus Agave which have high economic, social, and ecological impact. TuMMV could be a threat to these plants and potentially to other unknown susceptible crops. To our knowledge, this is the first report of TuMMV in A. attenuata and A. amica in Mexico. REFERENCE Chen, C. C., and Chang, C. A. 1998. Characterization of a potyvirus causing mild mosaic on tuberose. Plant Dis. 82:45-49. Colinet, D., Nguyen, M., Kummert, J., Lepoivre, P., and Xia, F. Z. 1998. Differentiation among potyviruses infecting sweet potato based on genus- and virus-specific reverse transcription polymerase chain reaction. Plant Dis. 82:223-229. Raj, S.K., Snehi, S.K., Kumar, S., Ram, T. and Goel, A.K. 2009. First report of Tuberose mild mosaic potyvirus from tuberose (Polianthes tuberosa L.) in India. Australasian Plant Dis. Notes 4, 93-95.

Development and Validation of a Panel of One-Step Four-Plex qPCR/RT-qPCR Assays for Simultaneous Detection of SARS-CoV-2 and Other Pathogens Associated with Canine Infectious Respiratory Disease Complex.

Canine infectious respiratory disease complex (CIRDC) is the primary cause of respiratory disease in the canine population and is caused by a wide array of viruses and bacterial pathogens with coinfections being common. Since its recognition in late 2019, Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) has been reported to cause respiratory disease in dogs. Therefore, the rapid detection and differentiation of SARS-CoV-2 from other common viral and bacterial agents is critical from a public health standpoint. Here, we developed and validated a panel of four one-step multiplex qPCR/RT-qPCR assays for the detection and identification of twelve pathogens associated with CIRDC (canine adenovirus-2, canine distemper virus, canine herpesvirus-1, canine influenza A virus, canine parainfluenza virus, canine pneumovirus, canine respiratory coronavirus, SARS-CoV-2, Bordetella bronchiseptica, Streptococcus equi subsp. zooepidemicus, Mycoplasma cynos, and M. canis), as well as the identification of three main CIV subtypes (i.e., H3N2, H3N8, and H1N1). All developed assays demonstrated high specificity and analytical sensitivity. This panel was used to test clinical specimens (n = 76) from CIRDC-suspected dogs. M. canis, M. cynos, and CRCoV were the most frequently identified pathogens (30.3%, 25.0%, and 19.7% of samples, respectively). The newly emerging pathogens CPnV and SARS-CoV-2 were detected in 5.3% of samples and coinfections were identified in 30.3%. This new multiplex qPCR/RT-qPCR panel is the most comprehensive panel developed thus far for identifying CIRDC pathogens, along with SARS-CoV-2.

Generation and Characterisation of Monoclonal Antibodies against Nairobi Sheep Disease Virus Nucleoprotein.

Nairobi sheep disease (NSD), caused by the viral agent NSD virus (NSDV), is a haemorrhagic fever disease affecting and inducing high mortality in sheep and goat populations. NSDV belongs to the genus Orthonairovirus of the Nairoviridae family from the order Bunyavirales. Other viruses circulating in livestock such as Crimean-Congo haemorrhagic fever virus (CCHFV) and Dugbe virus (DUGV) are members of the same genus and are reported to share antigenic features. There are very few available materials to study NSDV infection both in vitro and in vivo. In the present work, we characterised two monoclonal antibodies generated in mice that recognise NSDV specifically but not CCHFV or DUGV, along with a potential use to define virus-infected cells, using flow cytometry. We believe this tool can be useful for research, but also NSDV diagnostics, especially through immunological staining.

Clinical presentations and microbiological analysis of cerebrospinal fluid samples in cases of suspected bacterial meningitis patients attending a 1600 bedded teaching hospital from 2019 to 2022: A retrospective study.

Meningitis can be attributed to bacterial, fungal, or viral agents. In this study, we demonstrate the common bacterial agents causing meningitis along with their antibiotics susceptibility pattern in patients of all age groups. This retrospective, observational study was carried out in the Department of Microbiology with cerebrospinal fluid (CSF) samples collected from November 2019 to May 2022. We collected 1986 nonrepeat CSF samples from clinically suspected patients of bacterial meningitis, and clinical information about the patients was extracted from the hospital information system. Out of the 1986 CSF samples included in our study, 150 (7.55%) were found to be positive on bacterial culture. Most of our patients were in the age group of 0-20 years. Common clinical manifestations observed in our patients were: high-grade fever, 87 patients (58%); severe headache, 126 patients (84%); neck rigidity, 47 patients (31.3%); altered mental status, 76 patients (50.7%) and photophobia, 83 patients (55.3%). The most commonly isolated bacteria was Acinetobacter species (30%). The mean length of hospitalization (37.76 ± 25.30), the mean total cell count, high levels of protein (mg/dl) and low levels of glucose (mg/dl) of CSF were statistically significant in meningitis caused by multidrug-resistant bacteria. We recognized the spectrum of pathogens causing meningitis at our center along with the antibiotic resistance pattern to guide and facilitate early treatment by primary health care professionals and family medicine practitioners.

Association of different microbes and pathogenic factors in cases of infectious bovine keratoconjunctivitis in cattle from Eastern Kazakhstan.

Infectious bovine keratoconjunctivitis (IBK) causes a significant economic loss to cattle industries in many countries, including Kazakhstan. Although Moraxella bovis is recognized as an etiologic agent of IBK, other bacterial and viral agents have been suspected to play a role in the pathogenesis of this disease. This study aimed to evaluate samples collected from the eyes of IBK-affected cattle in Eastern Kazakhstan at different stages of IBK for the presence of Mor. bovis, Moraxella bovoculi, Mycoplasma bovis, Mycoplasma bovoculi, and Bovine Herpes Virus Type 1 (BHV-1) and to characterize Mor. bovoculi pilA gene sequence diversity from Mor. bovoculi positive samples. Individual ocular swabs (n = 168) were collected from cattle that had clinical signs of IBK during the summer of 2022 on farms in the Abay region of Kazakhstan. Eye lesion scores (1, 2, and 3) were assigned depending on the degree of ocular damage. Infectious bovine keratoconjunctivitis-associated organisms were detected using a multiplex real-time polymerase chain reaction assay. The Mor. bovoculi pilA gene was sequenced from Mor. bovoculi positive samples. Mycoplasma bovis and BHV-1 were not detected in any of the collected samples. Mycoplasma bovoculi was identified in the majority of samples overall, usually in mixed infection with Moraxella spp. Moraxella bovoculi was detected in 76.2% of animals and predominated in animals with eye lesion scores 2 and 3. Mycoplasma bovoculi was detected only in association with Mor. bovis and/or Mor. bovoculi in animals with eye lesion scores 2 and 3. Moraxella bovis was found in 57.7% of animals and was always identified in association with another organism. Sequencing of the pilA gene in 96 samples from Mor. bovoculi positive samples identified five PilA groups. The majority belonged to PilA group A. However, three new PilA groups were identified and designated PilA groups N, O, and P. The results indicate a high prevalence of Myc. bovoculi and Mor. bovoculi in eyes of cattle with IBK on livestock farms in Eastern Kazakhstan. Additional novel Mor. bovoculi PilA groups were identified.

An innovative 4.0 system to prevent the spreading of the diseases which threaten the globalized world

PurposeThe purpose of this research is to provide an effective contribution to contrast the spread of Covid19. Therefore, the authors aimed to model a new strategy (technologies and processes), using the principles made available by Industry 4.0.MethodThe strategy consists in an IoT thermoscanner (developed by the authors, strategically placed throughout the settlement), and an innovative method of disinfection (achieved by redesigning the sanitization processes, using UV-C rays and gaseous Ozone produced by IoT machines, again conceptualized and developed by the authors, being capable of reproducing the Chapman Cycle and its associated benefits). This method was discussed in the article "Sanitizing of Confined Spaces Using Gaseous Ozone Produced by 4.0 Machines," which was presented at the WCE 2021 IAENG Congress (Best Paper Award of the 2021 International Conference of Systems Biology and Bioengineering).ResultThe results consist in: 1. an absolute disinfection system based on a reversible cycle Oxygen-Ozone-Oxygen, with quick re-habitability of the treated rooms, at a minimum treatment costs, without expensive and harmful chemicals or moist water vapor (incompatible by nature with paper and electronics); 2. a 4.0 device for quick detection of fever; 3. clear processes for disease spread prevention.ConclusionThe target contribution was widely achieved, providing machinery, processes and procedures. The authors aim now to extend the solution proposed to any other type virus, bacteria, or pathogen agent introduced by subjects who, despite being unaware of acting as vectors, develop infection along their stay in hotels, offices or any other public place.

In silico Study of Alkaloid Compounds with Computational Approach for Selection of Drug Leads for COVID-19

Background: The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the virulent source of COVID-19 disease. As a result of the rapid transmission of the viral agent and deficiency of specific drugs against the virus, a worldwide pandemic ensued with a terrifying death toll. Thus there is tremendous urgency to discover substances for the development of specific COVID-19 drugs. With increasing public interest in natural products, this study aims to discover alkaloid compounds capable of inhibiting SARS-CoV-2 with the assistance of bioinformatics. Methods: In this work, 298 alkaloids with reported antiviral properties were identified, and their biological activities were validated with QSAR analysis using the Pass Online server until only 7 alkaloids remained. Molecular docking studies for these 7 alkaloids onto SARS-CoV-2 3CLpro, a protein involved in viral replication, were carried out with AutoDock Vina, followed by in silico visualization of the protein-alkaloid interaction with Ligplot+ program and prediction of ADME-Tox properties of the alkaloids using Toxtree program and SwissADME online server. Results: Fangchinoline, phenanthroindolizidine, and polyalthenol are predicted to have strong binding affinity with SARS-CoV-2 3CLpro. Visualization of the molecular interactions between the ligand and protein target, however, showed that homonojirimycin formed the most hydrogen bonds with the protein binding site. Most of the alkaloids have little to no violation of Lipinski’s Rule of 5, easy to moderate synthetic accessibility, and good pharmacokinetic properties. Conclusion: Fangchinoline, phenanthroindolizidine, and polyalthenol exhibited high binding affinity values to SARS-CoV-2 3CLpro, with polyalthenol predicted to possess the strongest binding interactions to the active site of the protein. Polyalthenol and phenanthroindolizidine confer the most versatility in terms of bioavailability, however, supplementary observation of phenanthroindolizidine for the prospect of mutagenicity is required before it can be recommended for further drug development

Environmental Health and Safety Offers a Biosafety Risk Assessment for a Theoretical Model of a Gene Therapy Process Transfer from Research and Development to Large-Scale Manufacturing.

This article provides a strategy by which a manufacturing process with a Biosafety Level 2 (BL2) designation can be downgraded to Biosafety Level 1 (BL1). The principles of the downgrading process are based on the robust contamination controls in clinical and commercial manufacturing, which typically are not part of Research and Development processes. These strict requirements along with the application of current Good Manufacturing Practice (cGMP) principles provide a framework by which processes can be suitably managed and controlled to mitigate biohazard risk, specifically for cell lines that may be contaminated with human pathogenic viral agents. We demonstrate how a risk assessment guide was used to define the risk profile of a theoretical process with a human cell line intended for clinical/commercial application. Based on the risk assessment, key BL2 elements were identified as suitable for downgrading, including facility containment controls, emergency spill response plans, and storage and shipping requirements. For various reasons, some aspects of the systems were deemed unsuitable for downgrading due to the severity of the control risk and, therefore, remained at BL2. We have used an established risk assessment guide to show how cGMP compliments and augments biosafety containment. We provide justification for downgrading from BL2 to BL1 for clinical and commercial cell and gene therapy manufacturing with human cell lines.