Search results for: infectious bronchitis virus

Authors: Andi Aliah Hidayani, Asmi Citra Malina A. R. Tassakka, Andi Parenrengi

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Vanname Shrimp is one of the high yielding varieties that are more resistant to virus attacks. However, now this shrimp more death due to virus attack such as white spot disease caused by white spot syndrome virus (WSSV). Various efforts have done to prevent the disease, like immunostimulatory, probiotics, and vaccine. White spot syndrome virus (WSSV) envelope protein VP19 gene is important because of its involvement in the system infection of shrimp. This study aimed to isolate and characterize an envelope protein VP19 – encoding gene of WSSV using WSSV infected Vanname Shrimp sample from some areas in South Sulawesi (Pangkep, Barru and Pinrang). The genomic of DNA were isolated from shrimp muscle using DTAB-CTAB method. Isolation of gene encoding envelope protein VP19 WSSV ws successfully performed with the results of the length of DNA fragment was 387 bp. The results of homology analysis using BLASTn homology suggested that these isolates genes from Barru, Pangkep and Pinrang have closest relationship with isolates from Mexican.

Keywords: vanname, shrimp, WSSV, viral protein 19

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Authors: Magno Enrique Mendoza Meza

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This paper deals with the comparison of three control techniques: the hysteresis on-off control (HyOOC), the hybrid on-off control (HOOC) and the simple Structured Treatment Interruptions (sSTI). These techniques are applied to the mathematical model developed by Kirschner and Webb. To compare these techniques we use a cost functional that minimize the wild-type virus population and the mutant virus population, but the main objective is to minimize the systemic cost of treatment and maximize levels of healthy CD4+ T cells. HyOOC, HOOC, and sSTI are applied to the drug therapies using a reverse transcriptase and protease inhibitors; simulations show that these controls maintain the uninfected cells in a small, bounded neighborhood of a pre-specified level. The controller HyOOC and HOOC are designed by appropriate choice of virtual equilibrium points.

Keywords: virus dynamics, on-off control, hysteresis, multi-drug therapies

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Authors: Getachew Adugna

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Background: Chronic energy malnutrition and human immune deficiency virus among pregnant women are highly prevalent in Sub-Saharan Africa, and they are interrelated in a vicious cycle. However, the prevalence of chronic energy malnutrition and its determinant factors among human immune deficiency virus-positive pregnant women is not well studied in Ethiopia and Addis Ababa in particular. Objective: To determine the prevalence & associated factors of chronic energy malnutrition among human immune deficiency virus-positive pregnant women in health centres of Addis Ababa Ethiopia. Methods: An institution-based cross-sectional study was conducted and a systematic random sampling technique was used to select study subjects. A total of 253 study subjects were enrolled in the study—a structured and pre-tested questionnaire collected sociodemographic, maternal health-related, and nutritional-related variables. MUAC measurements were taken and medical charts were reviewed. Bi-variable and multi-variable logistic regression analyses were used to assess the effect of different factors on chronic energy malnutrition. Result: The overall prevalence of chronic energy malnutrition was 32.0%. It was significantly associated with dietary counselling (AOR: 0.062; 95%CI: 0.007, 0.549), CD4 level (AOR: 0.219; 95%CI: 0.025, 1.908), and clinical stage (AOR: 0.127; 95%CI: 0.053, 0.305). Conclusions: The prevalence of chronic energy malnutrition among Human Immune deficiency virus-infected pregnant women in Addis Ababa was high and Nutritional Intervention should be an integral part of the HIV care program.

Keywords: chronic energy malnutrition, HIV, MUAC, Addis Ababa

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Authors: Sajjad Jafari, Reza Akbari

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Background and Aim: Chrysin, a flavonoid compound found in medicinal plants, honey, and propolis, has potential biological activities that make it an important perspective in the treatment of infectious and cancer diseases. The aim of this review study is to evaluate the biological activities of chrysin in the treatment of infectious and cancer diseases. Material and Methods: The present study is a review study that searched reputable scientific databases such as PubMed, Google Scholar, Scopus, and Web of Science from 2000 to 2023 using keywords such as antimicrobial, antifungal, chrysin, anticancer, antioxidants, and infectious diseases. The researchers examined 25 articles to determine the biological activities of chrysin. Results: Chrysin has high inhibitory or lethal activities on gram-positive and gram-negative bacteria, including Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, and Enterococcus faeces. It also has anti-biofilm effects and antifungal effects on strains such as Aspergillus niger and Candida albicans. Chrysin also has anticancer effects on various cancers, including colorectal cancer, pancreatic cancer, breast cancer, and MCF-7 cancer, which have been confirmed in vitro and in vivo. Conclusion: Chrysin has the potential as an important therapeutic option in the treatment of infectious and cancer diseases. Its high antimicrobial and anticancer activities, combined with its low toxicity in nanoparticle form, make it a promising candidate for further clinical trials. The production of anti-microbial and anti-cancer drugs from natural substances, such as chrysin, is a valuable contribution to the field of medicine.

Keywords: chrysin, antimicrobial, anticancer, infectious diseases

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Authors: Alhaji Modu Bukar, Faez Firdaus Abdullah Jesse, Che Azurahanim Che Abdullah, Mustapha M. Noordin, Mohd-Lila Mohd Azmia

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Orf virus (ORFV) is the causative agent of a proliferative skin lesion known as contagious ecthyma in sheep and goats. Currently used live attenuated vaccines against ORFV infection have been reported to cause severe outbreaks in vaccinated animals. In this study, we investigated the immunogenicity of the B2L and F1L proteins of the virus, which are thought to elicit a protective immune response The 6-week-old 50 female mice were divided into 8 groups: seven experimental groups and one control group. Each animal in the experimental group received an initial immunisation with the nanoparticles or proteins coated with the nanoparticles, followed by two booster immunizations with the same products 14 days apart. Ten days after the last booster inoculation, the mice were either humanely killed or lethally challenged with UPM /HSN-2-ORFV at a dose of 106 TCID50/mL in a volume of 50 μl. The spleen was examined for histopathological changes and quantification of T cells by flow cytometry. On the other hand, the degree of protection of mice from the lethal virus was evaluated by lesion size, weight loss, and histopathological examination of skin and liver. The results showed that mice immunised with rB2L alone, rB2L-Al₂O₃-NPs, rB2L/rF1L, and rB2L/rF1L-Al₂O₃-NPs elicited statistically higher levels of anti-rB2L and/or rF1L-specific IgA/IgG and CD4/CD8 cell immune responses than mice in the control groups (p < 0.01). The vaccine candidate did not exhibit severe skin damage after monitoring histopathology, morbidity, and mortality. Overall, the results suggest that recombinant rB2L and rF1L antigens may be useful universal vaccine candidates against ORFV infections.

Keywords: orf virus, antigen nanoparticles, virus, nanoparticles

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Authors: Muhammad Yousaf Ali, Shahid Mansoor, Javeria Qazi, Imran Amin, Musarrat Shaheen

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Cotton leaf curl disease (CLCuD) is the major threat to the cotton crop and is transmitted by whitefly (Bemisia tabaci). Since multiple begomoviruses and associated satellites are involved in CLCuD, approaches based on the concept of broad-spectrum resistance are essential for effective disease control. Gro-El and G5 are two proteins from whitefly endosymbiont and M13 bacteriophage origin, respectively. Gro-El encapsulates the virus particle when it enters the whitefly and protects the virus from the immune system of the whitefly as well as prevents viral expression in it. This characteristic of Gro-El can be exploited to get resistance against viruses if expressed in plants. G5 is a single-stranded DNA binding protein, expression of which in transgenic plants will stop viral expression on its binding with ssDNA. The use of tissue-specific promoters is more efficient than constitutive promoters. Transgenics of Nicotiana benthamiana for Gro-El under constitutive promoter and Gro-El under phloem specific promoter were made. In comparison to non-transgenic plants, transgenic plants with Gro-El under NSP promoter showed promising results when challenged against cotton leaf curl Multan virus (CLCuMuV) along with cotton leaf curl Multan beta satellite (CLCuMB), cotton leaf curl Khokhran virus (CLCuKoV) along with cotton leaf curl Multan beta satellite (CLCuMB) and Pedilenthus leaf curl virus (PedLCV) along with Tobacco leaf curl beta satellite (TbLCB).

Keywords: cotton leaf curl disease, whitefly, endosymbionts, transgenic, resistance

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Authors: Abdolreza Esmaeilzadeh, Maryam Erfanmanesh, Sousan Ghasemi, Farzaneh Mohammadi

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Background: Hepatitis C Virus (HCV), a public health problem, is an enveloped, single-stranded RNA virus and a member of the Hepacivirus genus of the Flaviviridae family. Liver cancer, cirrhosis, and end-stage liver are the outcomes of chronic infection with HCV. HCV isolates show significant heterogeneity in genetics around the world. Therefore, determining HCV genotypes is a vital step in determining prognosis and planning therapeutic strategies. Materials and Methods: Serum samples of 136 patients were collected and analyzed for anti-HCV antibodies using ELISA (The enzyme-linked immunosorbent assay) method. Then, positive samples were exposed to RT-PCR, which was performed under standard condition. Afterwards, they investigated for genotyping using allele-specific PCR (AS-PCR), and HCV genotype 2.0 line probe assay (LiPA). Results: Samples indicated 216 bp bands on 2% agarose gel. Analyses of the results demonstrated that the most dominant subtype was 3a with frequency of 38.26% in Zanjan Province followed by subtypes of 1b, 1a, 2, and 4 with frequencies of 25.73%, 22.05%, 5.14%, and 4.41%, respectively. The frequency of unknown HCV genotypes was 4.41%. Conclusions: According to the results, it was found that HCV high prevalent genotype in Zanjan is subtype 3a. Analysis of the results provides identification of certain HCV genotypes, and these valuable findings could affect the type and duration of the treatment.

Keywords: anti-HCV antibody, Hepatitis C Virus (HCV), genotype, RT-PCR, AS-PCR

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Authors: Samaneh Farokhirad, Fatemeh Ahmadpoor

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Biological membranes are constantly in contact with various filamentous soft nanostructures that either reside on their surface or are being transported between the cell and its environment. In particular, viral infections are determined by the interaction of viruses (such as filovirus) with cell membranes, membrane protein organization (such as cytoskeletal proteins and actin filament bundles) has been proposed to influence the mechanical properties of lipid membranes, and the adhesion of filamentous nanoparticles influence their delivery yield into target cells or tissues. The goal of this research is to integrate the rapidly increasing but still fragmented experimental observations on the adhesion and self-assembly of nanofilaments (including filoviruses, actin filaments, as well as natural and synthetic nanofilaments) on cell membranes into a general, rigorous, and unified knowledge framework. The global outbreak of the coronavirus disease in 2020, which has persisted for over three years, highlights the crucial role that nanofilamentbased delivery systems play in human health. This work will unravel the role of a unique property of all cell membranes, namely flexoelectricity, and the significance of nanofilaments’ flexibility in the adhesion and self-assembly of nanofilaments on cell membranes. This will be achieved utilizing a set of continuum mechanics, statistical mechanics, and molecular dynamics and Monte Carlo simulations. The findings will help address the societal needs to understand biophysical principles that govern the attachment of filoviruses and flexible nanofilaments onto the living cells and provide guidance on the development of nanofilament-based vaccines for a range of diseases, including infectious diseases and cancer.

Keywords: virus nanofilaments, cell mechanics, computational biophysics, statistical mechanics

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Authors: Hossein Rassi, Marjan Moradi Fard, Samaneh Niko

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Human papillomavirus type 16 and 18 are closely associated with the development of human cervical carcinoma, which is one of the most common causes of cancer death in women worldwide. At present, HPV type 18 accounts for about 34 % of all HPV infections in Iran and the most promising vaccine against HPV infection is based on the L1 major capsid protein. The L1 protein of HPV18 has the capacity to self-assemble into capsomers or virus-like particles (VLPs) that are non-infectious, highly immunogenic and allowing their use in vaccine production. The methylotrophic yeast Pichia pastoris is an efficient and inexpensive expression system used to produce high levels of heterologous proteins. In this study we expressed HPV18 L1 VLPs in P. pastoris. The gene encoding the major capsid protein L1 of the high-risk HPV type 18 was isolated from Iranian patient by PCR and inserted into pTG19-T vector to obtain the recombinant expression vector pTG19-HPV18-L1. Then, the pTG19-HPV18-L1 was transformed into E. coli strain DH5α and the recombinant protein HPV18 L1 was expressed under IPTG induction in soluble form. The HPV18 L1 gene was excised from recombinant plasmid with XhoI and EcoRI enzymes and ligated into the yeast expression vector pPICZα linearized with the same enzymes, and transformed into P. pastoris. Induction and expression of HPV18 L1 protein was demonstrated by BMGY/BMMY and RT PCR. The parameters for induced cultivation for strain in P. pastoris KM71 with HPV16L1 were investigated in shaking flask cultures. After induced cultivation BMMY (pH 7.0) medium supplemented with methanol to a final concentration of 1.0% every 24 h at 37 degrees C for 96 h, the recombinant produced 78.6 mg/L of L1 protein. This work offers the possibility for the production of prophylactic vaccine for cervical carcinoma by P. pastoris for HPV-18 L1 gene. The VLP-based HPV vaccines can prevent persistent HPV18 infections and cervical cancer in Iran. The HPV-18 L1 gene was expressed successfully in E.coli, which provides necessary basis for preparing HPV-18 L1 vaccine in human. Also, HPV type 6 L1 proteins expressed in Pichia pastoris will facilitate the HPV vaccine development and structure-function study.

Keywords: Pichia pastoris, L1 virus-like particles, human papillomavirus type 18, biotechnology

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Authors: Praskovya Britskaya, Fatima Shaizadina, Alua Omarova, Nessipkul Alysheva

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Planned preventive vaccination, which is carried out in the Republic of Kazakhstan, promoted permanent decrease in the incidence of measles and viral hepatitis B. In the structure of VHB patients prevail people of young, working age. Monitoring of infectious incidence, monitoring of coverage of immunization of the population, random serological control over the immunity enable well-timed identification of distribution of the activator, effectiveness of the taken measures and forecasting. The serological blood analysis was conducted in indicator groups of the population of Central Kazakhstan for the purpose of identification of antibody titre for vaccine preventable infections (measles, viral hepatitis B). Measles antibodies were defined by method of enzyme-linked assay (ELA) with test-systems "VektoKor" – Ig G ('Vektor-Best' JSC). Antibodies for HBs-antigen of hepatitis B virus in blood serum was identified by method of enzyme-linked assay (ELA) with VektoHBsAg test systems – antibodies ('Vektor-Best' JSC). The result of the analysis is positive, the concentration of IgG to measles virus in the studied sample is equal to 0.18 IU/ml or more. Protective level of concentration of anti-HBsAg makes 10 mIU/ml. The results of the study of postvaccinal measles immunity showed that the share of seropositive people made 87.7% of total number of surveyed. The level of postvaccinal immunity to measles in age groups differs. So, among people older than 56 the percentage of seropositive made 95.2%. Among people aged 15-25 were registered 87.0% seropositive, at the age of 36-45 – 86.6%. In age groups of 25-35 and 36-45 the share of seropositive people was approximately at the same level – 88.5% and 88.8% respectively. The share of people seronegative to a measles virus made 12.3%. The biggest share of seronegative people was found among people aged 36-45 – 13.4% and 15-25 – 13.0%. The analysis of results of the examined people for the existence of postvaccinal immunity to viral hepatitis B showed that from all surveyed only 33.5% have the protective level of concentration of anti-HBsAg of 10 mIU/ml and more. The biggest share of people protected from VHB virus is observed in the age group of 36-45 and makes 60%. In the indicator group – above 56 – seropositive people made 4.8%. The high percentage of seronegative people has been observed in all studied age groups from 40.0% to 95.2%. The group of people which is least protected from getting VHB is people above 56 (95.2%). The probability to get VHB is also high among young people aged 25-35, the percentage of seronegative people made 80%. Thus, the results of the conducted research testify to the need for carrying out serological monitoring of postvaccinal immunity for the purpose of operational assessment of the epidemiological situation, early identification of its changes and prediction of the approaching danger.

Keywords: antibodies, blood serum, immunity, immunoglobulin

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Authors: Asma Alhuqail, Nagwa Aref

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Programmed cell death resembles a real nature active defense in Datura metel against TMV after three days of virus infection. Physiological plant response was assessed for asymptomatic healthy and symptomatic infected detached leaves. The results indicated H2O2 and Chlorophyll-a as the most potential parameters. Chlorophyll-a was considered the only significant predictor variant for the H2O2 dependent variant with a P value of 0.001 and R-square of 0.900. The plant immune response was measured within three days of virus infection using the cutoff value of H2O2 (61.095 lmol/100 mg) and (63.201 units) for the tail moment in the Comet Assay. Their percentage changes were 255.12% and 522.40% respectively which reflects the stress of virus infection in the plant. Moreover, H2O2 showed 100% specificity and sensitivity in the symptomatic infected group using the receiver-operating characteristic (ROC). All tested parameters in the symptomatic infected group had significant correlations with twenty-five positive and thirty-one negative correlations where the P value was <0.05 and 0.01. Chlorophyll-a parameter had a crucial role of highly significant correlation between total protein and salicylic acid. Contrarily, this correlation with tail moment unit was (r = _0.930, P <0.01) where the P value was < 0.01. The strongest significant negative correlation was between Chlorophyll-a and H2O2 at P < 0.01, while moderate negative significant correlation was seen for Chlorophyll-b where the P value < 0.05. The present study discloses the secret of the three days of rapid transient production of activated oxygen species (AOS) that was enough for having potential quantitative physiological parameters for defensive plant response toward the virus.

Keywords: programmed cell death, plant–adaptive immune response, hydrogen peroxide (H2O2), physiological parameters

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Authors: Asrat M.Belachew, Tiago Pereira, Institute of Mathematics, Computer Sciences, Avenida Trabalhador São Carlense, 400, São Carlos, 13566-590, Brazil

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Infectious diseases are among the most prominent threats to human beings. They cause morbidity and mortality to an individual and collapse the social, economic, and political systems of the whole world collectively. Mathematical models are fundamental tools and provide a comprehensive understanding of how infectious diseases spread and designing the control strategy to mitigate infectious diseases from the host population. Modeling the spread of infectious diseases using a compartmental model of inhomogeneous populations is good in terms of complexity. However, in the real world, there is a situation that accounts for heterogeneity, such as ages, locations, and contact patterns of the population which are ignored in a homogeneous setting. In this work, we study how classical an SEIR infectious disease spreading of the compartmental model can be extended by incorporating the mobility of population between heterogeneous cities during an outbreak of infectious disease. We have formulated an SEIR multi-cities epidemic spreading model using a system of 4k ordinary differential equations to describe the disease transmission dynamics in k-cities during the day and night. We have shownthat the model is epidemiologically (i.e., variables have biological interpretation) and mathematically (i.e., a unique bounded solution exists all the time) well-posed. We constructed the next-generation matrix (NGM) for the model and calculated the basic reproduction number R0for SEIR-epidemic spreading model with cities mobility. R0of the disease depends on the spectral radius mobility operator, and it is a threshold between asymptotic stability of the disease-free equilibrium and disease persistence. Using the eigenvalue perturbation theorem, we showed that sending a fraction of the population between cities decreases the reproduction number of diseases in interconnected cities. As a result, disease transmissiondecreases in the population.

Keywords: SEIR-model, mathematical model, city mobility, epidemic spreading

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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: Esther N. Maina, Anna Skowronska, Sridhar Chaganti, Malcolm A. Taylor, Paul G. Murray, Tatjana Stankovic

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Epstein-Barr virus (EBV) has been implicated in the pathogenesis of human tumours of B-cell origin. The demonstration that a proportion of Hodgkin lymphomas and all Burkitt’s lymphomas harbour EBV suggests that the virus contributes to the development of these malignancies. However, the mechanisms of lymphomagenesis remain largely unknown. To determine whether EBV causes DNA damage and alters DNA damage response in cells of EBV-driven lymphoma origin, Germinal Centre (GC) B cells were infected with EBV and DNA damage responses to gamma ionising radiation (IR) assessed at early time points (12hr – 72hr) after infection and prior to establishment of lymphoblastoid (LCL) cell lines. In the presence of EBV, we observed induction of spontaneous DNA DSBs and downregulation of ATM-dependent phosphorylation in response to IR. This downregulation coincided with reduced ability of infected cells to repair IR-induced DNA double-strand breaks, as measured by the kinetics of gamma H2AX, a marker of double-strand breaks, and by the tail moment of the comet assay. Furthermore, we found that alteration of DNA damage responses coincided with the expression of LMP-1 protein. The presence of the EBV virus did not affect the localization of the ATM-dependent DNA repair proteins to sites of damage but instead lead to an increased expression of PP5, a phosphatase that regulates ATM function. The impact of the virus on DNA repair was most prominent 24h after infection, suggesting that this time point is crucial for the viral establishment in B cells. Our results suggest that during an early infection EBV virus dampens crucial cellular responses to DNA double-strand breaks which facilitate successful viral infection, but at the same time might provide the mechanism for tumor development.

Keywords: EBV, ATM, DNA damage, germinal center cells

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Authors: Shinta Stri Ayuda Nur Setyaningsih

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Anterior uveitis is often triggered by viral infections such as herpes simplex virus (HSV) and cytomegalovirus (CMV). This study aims to provide an overview of the demographic and clinical characteristics of patients with anterior uveitis caused by CMV and HSV infection at PMN Cicendo Eye Hospital Bandung. This study used a retrospective observational method. Data were collected from the medical records of patients who visited the PMN Infection and Immunology Polyclinic at Cicendo Eye Hospital between February and July 2023. The results showed that anterior uveitis associated with HSV and CMV viruses often occurs in the elderly and more in women. The most common clinical symptoms are red eyes and decreased visual acuity, with a gradual onset of symptoms. Complications that often arise are cataracts and glaucoma. This study provides a deeper understanding of anterior uveitis caused by infection with HSV and CMV viruses.

Keywords: uveitis anterior, cytomegavirus, herpes simplex virus type I ELISA

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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

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Authors: Shuofeng Yuan, Bojian Zheng

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Assembly of the heterotrimeric polymerase complex of influenza virus from the individual subunits PB1, PA, and PB2 is a prerequisite for viral replication, in which the interaction between the N-terminal of PB1 (PB1N) and the C terminal of PA (PAC) may be a desired target for antiviral development. In this study, we first compared the feasibility of high throughput screening by enzyme-linked immunosorbent assay (ELISA) and fluorescence polarization (FP) assay. Among the two, ELISA was demonstrated to own broader dynamic range so that it was used for screening inhibitors, which blocked PA and PB1 interaction. Several binding inhibitors of PAC-PB1N were identified and subsequently tested for the antiviral efficacy. Apparently, 3-(2-chlorophenyl)-6-ethyl-7-methyl[1,2,4]triazolo[4,3-a]pyrimidin-5-ol, designated ANA-1, was found to be a strong inhibitor of PAC-PB1N interaction and act as a potent antiviral agent against the infections of multiple subtypes of influenza A virus, including H1N1, H3N2, H5N1, H7N7, H7N9 and H9N2 subtypes, in cell cultures. Intranasal administration of ANA-1 protected mice from lethal challenge and reduced lung viral loads in H1N1 virus infected BALB/c mice. Docking analyses predicted that ANA-1 bound to an allosteric site of PAC, which would cause conformational changes thereby disrupting the PAC-PB1N interaction. Overall, our study has identified a novel compound with potential to be developed as an anti-influenza drug.

Keywords: influenza, antiviral, viral polymerase, compounds

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Authors: Amal Raqib Shameran, Ghanim Aboud Al-Mola

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Respiratory syncytial virus (hRSV) is the major pathogens of respiratory tract infections (RTI) among infants and children in the world. They are classified in family Paramyxoviridae and sub-family Pneumovirinae. The current work aimed to detect the role of RSV in the lower respiratory tract infection (LRTI) in Hilla, Iraq. The samples were collected from 50 children who were admitted to hospital suffering from lower respiratory tract infections (LRTI). 50 nasal and pharyngeal swabs were taken from patients at the period from January 2010 till April 2011, hospitalized in Hilla Maternity and Children Hospital. The results showed that the proportion of children infected with hRSV accounted for 24% 12/50 with lower respiratory tract infections (LRTI) when they tested by polymerase chain reaction (RT-PCR).

Keywords: respiratory syncytial virus, respiratory tract infections, infants, polymerase chain reaction (PCR)

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Authors: Shittu Bisi Agnes

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Ebola virus Diseases outbreak in Nigeria caused a lot of concerns considering the mode of transmission and no known cure discovered. Therefore a lot of safety measures were taken which eventually led to the eradication of the virus in Nigeria. This therefore attempted to determine the various safety measures, how socio-economic characteristic of the people affected adherence to safety measures. And provide reasonable recommendations for total eradication of the virus, future outbreak and general environmental safety Data were collected with the aid of well structured questionnaires and administered 180 randomly selected of the state and oral interview was also utilize. Data collected were analysed using both descriptive tools and inferential statistics vis-a-vis regression analysis. Finding showed that 70.5% was strongly adhere to almost all the measures, 15.2% was fairly advent, 3% was poorly observing the selected measures while 1.3% was in different. 65% of the respondents was strongly aware of the advent of ebola virus diseases, 20% was fairly in awareness, 8.5% was poorly in awareness while 6.55% was in aware of any disease outbreak. Safety measures put forwards were; hand washing, use of hand sanitize-rs, no shaking of hands non-consumption of wildlife games(Bush Meat) and general health and environmental safety measures. It was recommended that policy instrument to increase peoples income will accelerate eradication of diseases as this will enable households to pay for monetary safety measures, health and environmental education, in form of talk shop, workshop, lectures could be organised at the political ward levels, schools, market women, religious bodies functional unions and mass media.

Keywords: ebola diseases, pay, safety, outbreak

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Authors: Retno A. Vinarti, Lucy M. Hederman

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This research describes a knowledge model for a system which give personalized alert to users about infectious disease risks in the context of weather, location and time. The knowledge model is based on established epidemiological concepts augmented by information gleaned from infection-related data repositories. The existing disease risk prediction research has more focuses on utilizing raw historical data and yield seasonal patterns of infectious disease risk emergence. This research incorporates both data and epidemiological concepts gathered from Atlas of Human Infectious Disease (AHID) and Centre of Disease Control (CDC) as basic reasoning of infectious disease risk prediction. Using CommonKADS methodology, the disease risk prediction task is an assignment synthetic task, starting from knowledge identification through specification, refinement to implementation. First, knowledge is gathered from AHID primarily from the epidemiology and risk group chapters for each infectious disease. The result of this stage is five major elements (Person, Infectious Disease, Weather, Location and Time) and their properties. At the knowledge specification stage, the initial tree model of each element and detailed relationships are produced. This research also includes a validation step as part of knowledge refinement: on the basis that the best model is formed using the most common features, Frequency-based Selection (FBS) is applied. The portion of the Infectious Disease risk model relating to Person comes out strongest, with Location next, and Weather weaker. For Person attribute, Age is the strongest, Activity and Habits are moderate, and Blood type is weakest. At the Location attribute, General category (e.g. continents, region, country, and island) results much stronger than Specific category (i.e. terrain feature). For Weather attribute, Less Precise category (i.e. season) comes out stronger than Precise category (i.e. exact temperature or humidity interval). However, given that some infectious diseases are significantly more serious than others, a frequency based metric may not be appropriate. Future work will incorporate epidemiological measurements of disease seriousness (e.g. odds ratio, hazard ratio and fatality rate) into the validation metrics. This research is limited to modelling existing knowledge about epidemiology and chain of infection concepts. Further step, verification in knowledge refinement stage, might cause some minor changes on the shape of tree.

Keywords: epidemiology, knowledge modelling, infectious disease, prediction, risk

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Authors: Devender Mandala, Paul Watts

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Cis-Nucleosides mainly lamivudine (3TC) and emtricitabine (FTC) are an important tool in the treatment of Human immune deficiency virus (HIV), Hepatitis B virus (HBV) and Human T-Lymotropoic virus (HTLV). Lamivudine and emtricitabine are potent nucleoside analog reverse transcriptase inhibitors (nRTI). These two drugs are synthesized by a four-stage process from the starting materials: menthyl glyoxylate hydrate and 1,4-dithane-2,5-diol to produce the 5-hydroxy oxathiolane which upon acetylation with acetic anhydride to yield 5-acetoxy oxathiolane. Then glycosylation of this acetyl product with silyl protected nucleoside to produce the intermediate. The reduction of this intermediates can provide the final targets. Although there are several different methods reported for the synthesis of lamivudine and emtricitabine as a single enantiomer, we required an efficient route, which was suitable for large-scale synthesis to support the development of these compounds. In this process, we successfully prepared the intermediates of lamivudine and emtricitabine without using any solvents and catalyst, thus promoting the green synthesis. All the synthesized compound were confirmed by TLC, GC, Mass, NMR and 13C NMR spectroscopy.

Keywords: emtricitabine, green synthesis, lamivudine, nucleoside

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Authors: Lilia M. Fernando, Matthew K. Vasher, Evangelyn C. Alocilja

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This paper describes the development of a DNA-based nanobiosensor to detect the dengue virus in mosquito using electrically active magnetic (EAM) nanoparticles as the concentrator and electrochemical transducer. The biosensor detection encompasses two sets of oligonucleotide probes that are specific to the dengue virus: the detector probe labeled with the EAM nanoparticles and the biotinylated capture probe. The DNA targets are double hybridized to the detector and the capture probes and concentrated from nonspecific DNA fragments by applying a magnetic field. Subsequently, the DNA sandwiched targets (EAM-detector probe–DNA target–capture probe-biotin) are captured on streptavidin modified screen printed carbon electrodes through the biotinylated capture probes. Detection is achieved electrochemically by measuring the oxidation–reduction signal of the EAM nanoparticles. Results indicate that the biosensor is able to detect the redox signal of the EAM nanoparticles at dengue DNA concentrations as low as 10 ng/ul.

Keywords: dengue, magnetic nanoparticles, mosquito, nanobiosensor

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Authors: Nino G. Vepkhvadze, Tea Enukidze

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Every year the number of countries around the world face the risk of the spread of infectious diseases that bring significant ecological and social-economic damage. Hence, the importance of food product safety is emphasized that is the issue of interest for many countries. To solve them, it’s necessary to conduct preventive measures against the diseases, have accurate diagnostic results, leadership, and management. The Peste des petits ruminants (PPR) disease is caused by a morbillivirus closely related to the rinderpest virus. PPR is a transboundary disease as it emerges and evolves, considered as one of the top most damaging animal diseases. The disease imposed a serious threat to sheep-breeding when the farms of sheep, goats are significantly growing within the country. In January 2016, PPR was detected in Georgia. Up to present the origin of the virus, the age relationship of affected ruminants and the distribution of PPRV in Georgia remains unclear. Due to the nature of PPR, and breeding practices in the country, reemerging of the disease in Georgia is highly likely. The purpose of the studies is to provide laboratories with efficient tools allowing the early detection of PPR emergence and re-emergences. This study is being accomplished under the Biological Threat Reduction Program project with the support of the Defense Threat Reduction Agency (DTRA). The purpose of the studies is to investigate the samples and identify areas at high risk of the disease. Georgia has a high density of small ruminant herds bred as free-ranging, close to international borders. Kakheti region, Eastern Georgia, will be considered as area of high priority for PPR surveillance. For this reason, in 2019, in Kakheti region investigated n=484 sheep and goat serum and blood samples from the same animals, utilized serology and molecular biology methods. All samples were negative by RT-PCR, and n=6 sheep samples were seropositive by ELISA-Ab. Future efforts will be concentrated in areas where the risk of PPR might be high such as international bordering regions of Georgia. For diagnostics, it is important to integrate the PPRV knowledge with epidemiological data. Based on these diagnostics, the relevant agencies will be able to control the disease surveillance.

Keywords: animal disease, especially dangerous pathogen, laboratory diagnostics, virus

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Authors: Isomar Lima da Silva, Aristeu Jonatas Leite de Oliveira, Roberto Maia Augusto

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Biological isolation capsules are equipment commonly used in the control and prevention of infectious diseases in the hospital environment. This type of equipment, combined with pre-established medical protocols, contributes significantly to the containment of highly transmissible pathogens such as COVID-19. Due to its hermetic isolation, it allows more excellent patient safety, protecting companions and the health team. In this context, this work presents the development, testing, and validation of a medical capsule to treat patients affected by COVID-19. To this end, requirements such as low cost and easy handling were considered to meet the demand of people infected with the virus in remote locations in the Amazon region and/or where there are no ICU beds and mechanical ventilators for orotracheal intubation. Conceived and developed in a partnership between SAMEL Planos de Saúde and Instituto Conecthus, the device entitled "Vanessa Capsule" was designed to be used together with the NIV protocol (non-invasive ventilation), has an automatic exhaust system and filters performing the CO2 exchange, in addition to having BiPaps ventilatory support equipment (mechanical fans) in the Cabin Kit. The results show that the degree of effectiveness in protecting against infection by aerosols, with the protection cabin, is satisfactory, implying the consideration of the Vanessa capsule as an auxiliary method to be evaluated by the health team. It should also be noted that the medical observation of the evaluated patients found that the treatment against the COVID-19 virus started earlier with non-invasive mechanical ventilation reduces the patient's suffering and contributes positively to their recovery, in association with isolation through the Vanessa capsule.

Keywords: COVID-19, mechanical ventilators, medical capsule, non-invasive ventilation

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Authors: D. Leibman, D. Wolf, A. Gal-On

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Viral diseases of tomato crops result in heavy yield losses and may even jeopardize the production of these crops. Classical tomato breeding for disease resistance against Tomato yellow leaf curl virus (TYLCV), leads to partial resistance associated with a number of recessive genes. To author’s best knowledge Pepino mosaic virus (PepMV) genetic resistance is not yet available. The generation of viral resistance by means of genetic engineering was reported and implemented for many crops, including tomato. Transgenic resistance against viruses is based, in most cases, on Post Transcriptional Gene Silencing (PTGS), an endogenous mechanism which destroys the virus genome. In this work, we developed immunity against PepMV and TYLCV in a tomato based on a PTGS mechanism. Tomato plants were transformed with a hairpin-construct-expressed transgene-derived double-strand-RNA (tr-dsRNA). In the case of PepMV, the binary construct harbored three consecutive fragments of the replicase gene from three different PepMV strains (Italian, Spanish and American), to provide resistance against a range of virus strains. In the case of TYLCV, the binary vector included three consecutive fragments of the IR, V2 and C2 viral genes constructed in a hairpin configuration. Selected transgenic lines (T0) showed a high accumulation of transgene siRNA of 21-24 bases, and T1 transgenic lines showed complete immunity to PepMV and TYLCV. Graft inoculation displayed immunity of the transgenic scion against PepMV and TYLCV. The study presents the engineering of resistance in tomato against two serious diseases, which will help in the production of high-quality tomato. However, unfortunately, these resistant plants have not been implemented due to public ignorance and opposition against breeding by genetic engineering.

Keywords: PepMV, PTGS, TYLCV, tr-dsRNA

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Authors: Shuafeng Yuan, Bojian Zheng

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The RNA-dependent RNA polymerase of influenza a virus comprises conserved and independently folded subdomains with defined functionalities. The N-terminal domain of the PA subunit (PAN) harbors the endonuclease function so that it can serve as a desired target for drug discovery. To identify a class of anti-influenza inhibitors that impedes PAN endonuclease activity, a screening approach that integrated the fluorescence resonance energy transfer based endonuclease inhibitor assay with the DNA gel-based endonuclease inhibitor assay was conducted, followed by the evaluation of antiviral efficacies and potential cytotoxicity of the primary hits in vitro and in vivo. A small-molecule compound ANA-0 was identified as a potent inhibitor against the replication of multiple subtypes of influenza A virus, including H1N1, H3N2, H5N1, H7N7, H7N9 and H9N2, in cell cultures. Combinational treatment of zanamivir and ANA-0 exerted synergistic anti-influenza effect in vitro. Intranasal administration of ANA-0 protected mice from lethal challenge and reduced lung viral loads in H1N1 virus infected BALB/c mice. Docking analyses predicted ANA-0 bound the endonuclease cavity of PAN by interacting with the metal-binding and catalytic residues. In summary, ANA-0 shows potential to be developed to novel anti-influenza agents.

Keywords: anti-influenza, novel compound, inhibition of endonuclease, PA

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Authors: Shittu Bisi Agnes

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Studies on households’ willingness to pay for environmental and general health safety in the advent of Ebola virus Diseases in Nigeria was carried out. This is aimed at revealing the means by which the virus was eventually eradicated in Nigeria as widely claimed in the media. This study therefore attempted to determine the environmental and general health condition in the State Of Osun, how socio-economic characteristics of the people affected willingness to pay. And also provide platform for the reduction of environmental and general health problems. Data were collected with the aid of well-structured questionnaire and administer 150 randomly selected people of study area, and oral interview was also utilized. Data collected were analyzed using both descriptive tools and inferential statistics vis-a-viz regression analysis. Findings showed 92.5% of respondents was aware of ebola virus diseases outbreak in Nigeria, 8.5% was unaware of any disease outbreak. And 65.7% of respondents was strongly willing to pay for environmental and general health safety 27.1% was fairly willing, 5.7% was indifferent and 1.7% was unwilling to pay. 5% rated the level of environmental and general health condition in the area has been good, 53.6% rated theirs has been fair, 33.6% as been poor. The average willingness to pay per household per month were #500.00, #250.00, #150.00 and #100.00 respectively for the four categories. It was recommended that policy instruments to increase peoples' income will accelerate eradication of environmental and general health problems, environmental health education in form of talk shop, workshop, lectures and seminars could be organized at the political ward levels, churches, mosque, and at schools. Environmental and general health safety related information could be disseminated through mass media, market women, and functional unions.

Keywords: ebola virus diseases (EVD), socio-economic, safety, pay, Osun

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Authors: Seyedeh Nasim Mirbahari, Taha Azad, Mehdi Totonchi

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Oncolytic viruses, which only replicate in tumor cells, are being extensively studied for their use in cancer therapy. A particular virus known as the vaccinia virus, a member of the poxvirus family, has demonstrated oncolytic abilities glioma. Treating Glioma with traditional methods such as chemotherapy and radiotherapy is quite challenging. Even though oncolytic viruses have shown immense potential in cancer treatment, their effectiveness in glioblastoma treatment is still low. Therefore, there is a need to improve and optimize immunotherapies for better results. In this study, we have designed oncoVV-TT, which can more effectively target tumor cells while minimizing replication in normal cells by replacing the thymidine kinase gene with a luc-p2a-GFP gene expression cassette. Human glioblastoma cell line U251 MG, rat glioblastoma cell line C6, and non-tumor cell line HFF were plated at 105 cells in a 12-well plates in 2 mL of DMEM-F2 medium with 10% FBS added to each well. Then incubated at 37°C. After 16 hours, the cells were treated with oncoVV-TT at an MOI of 0.01, 0.1 and left in the incubator for a further 24, 48, 72 and 96 hours. Viral replication assay, fluorescence imaging and viability tests, including trypan blue and crystal violet, were conducted to evaluate the cytotoxic effect of oncoVV-TT. The finding shows that oncoVV-TT had significantly higher cytotoxic activity and proliferation rates in tumor cells in a dose and time-dependent manner, with the strongest effect observed in U251 MG. To conclude, oncoVV-TT has the potential to be a promising oncolytic virus for cancer treatment, with a more cytotoxic effect in human glioblastoma cells versus rat glioma cells. To assess the effectiveness of vaccinia virus-mediated viral therapy, we have tested U251mg and C6 tumor cell lines taken from human and rat gliomas, respectively. The study evaluated oncoVV-TT's ability to replicate and lyse cells and analyzed the survival rates of the tested cell lines when treated with different doses of oncoVV-TT. Additionally, we compared the sensitivity of human and mouse glioma cell lines to the oncolytic vaccinia virus. All experiments regarding viruses were conducted under biosafety level 2. We engineered a Vaccinia-based oncolytic virus called oncoVV-TT to replicate specifically in tumor cells. To propagate the oncoVV-TT virus, HeLa cells (5 × 104/well) were plated in 24-well plates and incubated overnight to attach to the bottom of the wells. Subsequently, 10 MOI virus was added. After 48 h, cells were harvested by scraping, and viruses were collected by 3 sequential freezing and thawing cycles followed by removal of cell debris by centrifugation (1500 rpm, 5 min). The supernatant was stored at −80 ◦C for the following experiments. To measure the replication of the virus in Hela, cells (5 × 104/well) were plated in 24-well plates and incubated overnight to attach to the bottom of the wells. Subsequently, 5 MOI virus or equal dilution of PBS was added. At the treatment time of 0 h, 24 h, 48 h, 72 h and 96 h, the viral titers were determined under the fluorescence microscope (BZ-X700; Keyence, Osaka, Japan). Fluorescence intensity was quantified using the imagej software according to the manufacturer’s protocol. For the isolation of single-virus clones, HeLa cells seeded in six-well plates (5×105 cells/well). After 24 h (100% confluent), the cells were infected with a 10-fold dilution series of TianTan green fluorescent protein (GFP)virus and incubated for 4 h. To examine the cytotoxic effect of oncoVV-TT virus ofn U251mg and C6 cell, trypan blue and crystal violet assay was used.

Keywords: oncolytic virus, immune therapy, glioma, vaccinia virus

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Authors: Jacob Medupe

Abstract:

Life as we know it has changed since the global outbreak of Coronavirus Disease 2019 (COVID-19) and business as usual will not continue. The human impact of the COVID-19 crisis is already immeasurable. Moreover, COVID-19 has already negatively impacted economies, livelihoods and disrupted food systems around the world. The disruptive nature of the Corona virus has affected every sphere of life including the culture and teaching and learning. Right now the majority of education research is based around classroom management techniques that are no longer necessary with digital delivery. Instead there is a great need for new data about how to make the best use of the one-on-one attention that is now becoming possible (Diamandis & Kotler, 2014). The COVID-19 pandemic has necessitated an environment where the South African learners are focused to adhere to social distancing in order to minimise the wild spread of the Corona virus. This arrangement forces the student to utilise the online classroom technologies to continue with the lessons. The historical reality is that the country has not made much strides on the closing of the digital divide and this is particularly a common status quo in the deep rural areas. This will prove to be a toll order for most of the learners affected by the Corona Virus to be able to have a seamless access to the online learning facilities. The paper will seek to look deeply into this reality and how the Corona virus has brought us to the reality that South Africa remains a deeply unequal society in every sphere of life. The study will also explore the state of readiness for education system around the online classroom environment.

Keywords: virtual learning, virtual classroom, COVID-19, Corona virus, internet connectivity, blended learning, online learning, distance education, e-learning, self-regulated Learning, pedagogy, digital literacy

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Authors: Olga Smilevska Spasova, Katerina Boshkovska, Gorica Popova, Mirjana Popovska

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Introduction: Pneumonia is an infection of the pulmonary parenchyma. HPIV 3 is one of four viruses that is a member of the Paramyxoviridae family designated types 1-4 that have a nonsegmented, single-stranded RNA genome with a lipid-containing envelope. They are spread from the respiratory tract by aerosolized secretions or by direct contact with secretions. Type 3 is endemic and can cause serious illness in immunocompromised patients. Illness caused by parainfluenza occurs shortly after inoculation with the virus. The level of immunoglobulin A antibody in serum is the best predictor of susceptibility to infection. Streptococcus pneumonia or pneumococcus is a Gram-positive, spherical bacteria, usually found in pairs and it is a member of the genus Streptococcus. Streptococcus pneumonia resides asymptomatically in healthy carriers typically colonizing the respiratory tract, sinuses, and nasal cavity. In individuals with weaker immune systems like young infants, pneumococcal bacterium is the most common cause of community-acquired pneumonia in the world. Case Report: The aim is to present a case of lower respiratory tract infection in an infant caused by parainfluenza virus 3, S. pneumonia and undifferentiated gram-negative bacteria that was successfully treated. The infant is with a history of recurrent episodes of wheezing in the past 3mounts.Infant of 10months presents 2weeks before admittance with high fever, runny nose, and cough. The primary pediatrician prescribed oral cefpodoxime for 10days and inhaled salbutamol. Two days before admittance in hospital the infant with high fever, cough, and difficulty breathing. At admittance, infant is pale, anxious with rapid respirations, cough, wheezing and tachycardia. On auscultation: vesicular breathing sounds with high pitched wheezing and on the right coarse crackles. Investigations: Blood analysis: RBC: 4, 7 x1012L, WBC: 8,3x109L: Neut: 42.73% Lym: 41.57%, Hgb: 9.38 g/dl MCV: 62.7fl, MCH: 20.0pg MCHC: 31.8 g/dl RDW: 18.7% Plt-307.9 x109LCRP: 2,5mg/l, serum iron-7.92umol/l, O2sat-97% on blood gas analysis, puls-125/min.X-ray of chest with hyperinflationand right pericardial consolidation. Microbiological analysis of sputum sample is positive for undifferentiated gram-negative bacteria (colonizer)–resistant to cefotaxime, ampicillin, cefoxitin, sulfamet.+trimetoprim and sensitive to amikacin, gentamicin, and ciprofloxacin. Molecular multiplex RT-PCR for 19 viruses and multiplex PCR for 7 bacteria test for respiratory pathogens positive for Parainfluenza virus 3(Ct=22.73), Streptococcus pneumonia (Ct=26.75).IED: IgG-9.31g/l, IgA-0.351g/l, IgM-0.86g/l. Therapy: Treatment was started with inhaled salbutamol, intravenous antibiotic cefotaxime as well as systemic corticosteroids. On day 7 because of slow clinical resolution of chest auscultation findings and an etiologic clue with a positive sputum sample for resistant undifferentiated gram negative bacteria, a second intravenous antibiotic was administered amikacin. The infant is discharged on day 14 with resolution of clinical findings. Conclusion: Mixed co-infections with respiratory viruses and bacteria in immunocompromised infants are likely to lead to a more severe form of community acquired pneumonia that will need hospitalization.

Keywords: HPIV- 3, infant, pneumonia, S. pneumonia, x-ray chest

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