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The emergence of SARS-CoV-2 variants has posed a challenge to disease control efforts worldwide. This study explored the genomic diversity and phylogenetic relationship of SARS-CoV-2 variants reported in Pakistan. Our objective was to understand the transmission dynamics of different lineages within the country. We retrieved and analyzed spike protein sequences from Pakistan and compared them with reference sequences reported worldwide. Our analysis revealed the clustering of Pakistan-origin isolates in nine different clades representing different regions worldwide, suggesting the transmission of multiple lineages within the country. We found 96 PANGO lineages of SARS-CoV-2 in Pakistan, and 64 of these corresponded to 4 WHO-designated variants: Alpha, Beta, Delta, and Omicron. The most dominant variants in Pakistan were Alpha (B.1.1.7), Beta (B.1.351), Delta (B.1.617.2, AY.108), and Omicron (BA.2.75, BA.5.2), and the N-terminal domain and receptor binding regions were the most hypervariable regions of the spike gene. Compared to the reference strain, characteristic substitutions were found in dominant variants. Our findings emphasize the importance of continuously monitoring and assessing nucleotide and residue substitutions over time to understand virus evolutionary trends better and devise effective disease control interventions.

Campylobacter jejuni is a major cause of gastroenteritis in humans. It has been reported that the pathogenesis of C. jejuni is closely related to the formation, adhesion, and invasion of flagella toxin in host epithelial cells. A putative transcriptional regulator, known as cj0440c, is thought to be involved in the regulation of flagellar synthesis. However, confirmation of this hypothesis requires deep insight into the regulation mechanism of cj0440c and its possible relationship with different antibiotics. Therefore, the study explained here was designed to determine the relationship and function (phenotypically and genotypically) of cj0440c in the flagellar synthesis of C. jejuni NCTC11168. The study determined the mode of expression of cj0440c and flagella-related genes under exposure to various drugs. To verify the involvement of cj0440c protein in the metabolic pathway of thiamine, an enzymatic hydrolysis experiment was performed and analyzed through the application of mass spectrometry. The overexpression vector of C. jejuni NCTC11168 was also constructed to find out whether or not target genes were regulated by cj0440c. The findings of the study showed that cj0440c and other flagella-related genes were expressed differentially under the influence of various antibiotics including erythromycin, tylosin, azithromycin, gentamicin, etimicin, enrofloxacin, gatifloxacin, tetracycline, and tigecycline. The analysis showed that the cj0440c protein did not catalyze the degradation of thiamine. In conclusion, the study aids in the understanding of the inter-relationship between the regulatory mechanism of flagella genes and the thiamine metabolic pathway.

The concepts of service-oriented computing (SOC) have previously been used in the embedded systems domain, mostly at the device level in ad hoc manners to achieve advantages of device integration. However, SOC has not been used for the development of embedded software systems (ESS), i.e., software controlling the embedded devices. To fill this gap, a process model is proposed in this article that allows the systematic development of ESS based on SOC concepts. The proposed process consists of analysis and design phases of embedded software development. The analysis phase is concerned with the collection of system information and preparation for system design. Based on this, the service-based software architecture is developed in the design phase. The effectiveness of the proposed process model is demonstrated through its application in the Smart Home case study.

Iodine complexes have known antimicrobial properties along with reported in-vitro antiviral activity for several viruses. Renessans is one such product with iodine complexes and ascorbic acid. The present study was designed to determine its efficacy for SARS-CoV-2 in Rhesus macaque. Rhesus macaque were assigned to: A) prophylactic group (n = 3), (B) treatment group (n = 3), (C) infection control group (n = 4), and (D) negative control group (n = 4). Groups A, B, and C were challenged with 2 × 106 TCID of SARS-CoV-2. The prophylactic group (A) was administered Renessans from 5 days before infection till 8 days postinfection (DPI). The treatment group (B) was administered Renessans from 3 till 8 DPI. Group C was administered water-insoluble fractions only. Nasal swabs from all monkeys of groups A, B, and C remained positive for SARS-CoV-2 till 2 and 7 DPI, while the swabs became negative for groups A and B at 14 DPI. Likewise, fecal matter of monkeys in group A returned negative results during the experiment, while that of group B had significantly decreased viral load (101.5 genome copies/mL) compared to group C (103 genome copies/mL). Hence, it is concluded that Renessans has in-vivo SARS-CoV-2 activity and may result in early clearance of SARS-CoV-2.

Since the emergence of COVID-19 pandemic in China in late 2019, scientists are striving hard to explore non-toxic, viable anti-SARS-CoV-2 compounds or medicines. We determined In vitro anti-SARS-CoV-2 activity of oral formulations (syrup and capsule)of an Iodine-complex (Renessans). First, cell cytotoxicity of Renessans on the Vero cells was determined using MTT assay. Afterwards, the antiviral activity of Renessans was determined using viral inhibition assays and TCID50. For this, nontoxic concentrations of the Renessans were used. The results showed that Renessans is nontoxic to the cells up to 50 µg/mL. At 1.5 µg/mL concentration, SARS-CoV-2 production was significantly reduced to 101.43 TCID50 and 101.58 TCID50 for the syrup and capsule, respectively, as compare to virus infected control cells 106.08 TCID50 and we found the dose dependent inhibition of virus replication in the presence of Renessans. Renessans inhibited SARS-CoV-2 with an EC50 value of 0.425 µg/mL and 0.505 µg/mL for syrup and capsule, respectively. Furthermore, there was no virus detected at concentration of 50 µg/mL of Renessans. This study indicates that Renessans, containing iodine, have potential activity against SARS-CoV-2 which needs to be further investigated in human clinical trials.

Iodine complexes have known antimicrobial properties along with reported

Coronaviruses (CoVs) infect a wide range of domestic and wild mammals. These viruses have a potential and tendency to cross-species barriers and infect humans. Novel human coronavirus 2019-nCoV (hCoV-19) emerged from Wuhan, China, and has caused a global pandemic. Genomic features of SARS-CoV-2 may attribute inter-species transmission and adaptation to a novel host, and therefore is imperative to explicate the evolutionary dynamics of the viral genome and its propensity for differential host selection. We conducted an in silico analysis of all the coding gene sequences of SARS-CoV-2 strains (n = 39) originating from a range of non-human mammalian species, including pangolin, bat, dog, cat, tiger, mink, mouse, and the environmental samples such as wastewater, air and surface samples from the door handle and seafood market. Compared to the reference SARS-CoV-2 strain (MN908947; Wuhan-Hu-1), phylogenetic and comparative residue analysis revealed the circulation of three variants, including hCoV-19 virus from humans and two hCoV-19-related precursors from bats and pangolins. A lack of obvious differences as well as a maximum genetic homology among dog-, cat-, tiger-, mink-, mouse-, bat- and pangolin-derived SARS-CoV-2 sequences suggested a likely evolution of these strains from a common ancestor. Several residue substitutions were observed in the receptor-binding domain (RBD) of the spike protein, concluding a promiscuous nature of the virus for host species where genomic alternations may be required for the adaptation to novel host/s. However, such speculation needs in vitro investigations to unleash the influence of substitutions towards species-jump and disease pathogenesis. Electronic supplementary material The online version of this article (10.1007/s11033-020-05879-5) contains supplementary material, which is available to authorized users.

Repeated cases of low pathogenic influenza A/H9N2 virus (IAV/H9N2) have been reported in commercial chickens since its emergence in 1998 in Pakistan. However, recently increased mortality and severe respiratory complications under field conditions have been noticed, suggesting concomitant influenza infections with respiratory viral and/or bacterial pathogens. Therefore, the present study aimed to investigate the presence of IAV/H9N2 coinfecting with multiple viral and bacterial pathogens in broiler chicken flocks. We surveyed 60 broiler flocks with respiratory signs from March through July 2019 in Punjab, Pakistan. Suspected flocks were screened for the presence of IAV using a lateral-flow device. Tracheal, cloacal, and bone marrow samples were collected and further tested for seven viral agents (chicken anemia; Newcastle disease; infectious bronchitis; infectious laryngeotracheitis [ILT]; and IAV subtypes H9, H7, and H5) and three bacterial agents (

Vaccines are one of the efficient means available so far for preventing and controlling the infection rate of COVID-19. Several researchers have focused on the whole virus’s (SARS-CoV-2) inactivated vaccines which are economically efficient to produce. In Pakistan, multiple variants of SARS-CoV-2 have been reported since the start of the pandemic in February 2020. Due to the continuous evolution of the virus and economic recessions, the present study was designed to develop an indigenous inactivated SARS-CoV-2 vaccine that might help not only to prevent the COVID-19 in Pakistan, it will also save the country’s economic resources. The SARS-CoV-2 were isolated and characterized using the Vero-E6 cell culture system. The seed selection was carried out using cross-neutralization assay and phylogenetic analysis. The selected isolate of SARS-CoV-2 (hCoV-19/Pakistan/UHSPK3-UVAS268/2021) was inactivated using beta-propiolactone followed by vaccine formulation using Alum adjuvant, keeping the S protein concentration as 5 μg/dose. The vaccine efficacy was evaluated by in vivo immunogenicity testing in laboratory animals and in in vitro microneutralization test. The phylogenetic analysis revealed that all the SARS-CoV-2 isolates reported from Pakistan nested into different clades, representing multiple introductions of the virus into Pakistan. The antisera raised against various isolates from different waves in Pakistan showed a varied level of neutralization titers. However, the antisera produced against a variant (hCoV-19/Pakistan/UHSPK3-UVAS268/2021; fourth wave) efficiently neutralized (1:64–1:512) all the tested SARS-CoV-2 isolates. The inactivated whole virus vaccine of SARS-CoV-2 was safe and it also elicited a protective immune response in rabbits and rhesus macaques on the 35th-day post-vaccination. The activity of neutralizing antibodies of vaccinated animals was found at 1:256–1:1024 at 35 days post-vaccination, indicating the effectiveness of the double-dose regime of the indigenous SARS-CoV-2 vaccine.

Recently, research on vehicular ad hoc networks (VANETs) has gained momentum all over the world. These emerging networks promise to make our driving experience more efficient, safer, comfortable, and enjoyable. VANETs have the potential to support a wide range of interesting applications. One such promising application area is the discovery of roadside services on the highways. Drivers need an efficient way to find these services during their journeys. However, due to the unique features of VANETs, the design and implementation of such applications is a challenging task. In this paper, we propose an application layer beaconing-based roadside services discovery protocol (RSDP). The RSDP is a lightweight, beacon-based protocol, which uses store-carry-and-response technique and opportunistic vehicle-to-vehicle communication. Our solution is efficient for business owners, as the vehicles move from one place to another to carry service advertisements and opportunistically forward them to other vehicles upon request. Our system architecture is fully based on the VANET standard protocols stack. We performed extensive simulations to evaluate the performance of RSDP in a multilane highway scenario. The results demonstrate the effectiveness of our solution in terms of services discovery.

Renessans is an iodine complex which has proven in vitro antiviral activity including Anti-SARS-CoV-2 activity. The present study was designed to determine its efficacy against SARS-CoV-2 in monkeys (Rhesus macaque). A total of 14 monkeys were divided into four groups: A) Prophylactic group (n=03), (B) Treatment group (n=03), (C) infection control group (n=04) and (D) negative control group (n=04) and were housed in BSL-3 Animal facility while group D was housed at another animal house. Group A was administered with Renessans @ 2.85 mg/7 kg from 5 days prior to the infection to 08 days post infections (DPI). Group B was administered with Renessans from 03-08 DPI @ 2.85 mg/7 kg. Group C was administered with WIF only. The infection @ 2 × 106 TCID of SARS-CoV-2 was given to all group monkeys through intranasal and oral route under anesthesia. Nasal swab samples (at different times) and fecal matter on daily basis were collected for the detection of SARS-CoV-2 through real-time quantitative PCR. Three monkeys (one from each of group A, B and C) were euthanized at 07 DPI to determine the gross pathological lesions and SARS-CoV-2 detection from internal tissues. Nasal swabs from all the monkeys from group A, B and C were positive for SARS-CoV-2 at 02 and 07 DPI (Day 05 of treatment). At 14 DPI, all (100%) nasal swabs from group A were negative for SARS-CoV-2 while 50% and 100% were positive from group B and C, respectively. At 21 DPI, monkeys from group B were negative and all in group C were still positive for SARS-CoV-2. Similarly, fecal matter of monkeys in group A and B was returned negative in significantly lesser time as compared to monkeys from infection control group. Based on these research findings it is concluded that the Renessans has in-vivo SARS-CoV-2 activity and may result in early clearance of SARS-CoV-2. Therefore, a clinical trial of the drug in COVID-19 patients may reveal its anti-COVID-19 potential.

The visual exploration of retinal blood vessels assists ophthalmologists in the diagnoses of different abnormalities of the eyes such as diabetic retinopathy, glaucoma, cardiovascular ailment, high blood pressure, arteriosclerosis, and age-related macular degeneration. The manual inspection of retinal vasculature is an extremely challenging and tedious task for medical experts due to the complex structure of an eye, tiny blood vessels, and variation in vessels width. Several automatic retinal vessels extraction techniques have been proposed in contemporary literature, which assist ophthalmologists in the timely identification of an eye disorders. However, due to the fast evolution of such techniques, a comprehensive survey is needed. This survey presents a comprehensive review of such techniques, strategies, and algorithms presented to date. The techniques are classified into logical groups based on the underlying methodology employed for retinal vessel extraction. The performance of existing techniques is reported on the publicly accessible datasets in term of various performance measures, providing a valuable comparison among the techniques. Thus, this survey presents a valuable resource for researchers working toward automatic extraction of retinal vessels.

The crowd phenomenon frequently occurs in dense urban living environments. Crowd counting or estimation helps to develop management strategies such as designing safe public places and evacuation plan for emergencies. These strategies are different depending upon the type of event such as political and public demonstrations, sports, and religious events. However, estimating the number of people in crowds at closed or open environments is quite challenging because of the dynamics involved in the process. In addition, crowd estimation itself poses challenges due to randomness in crowd behavior, motion, and an area’s geometric specifications. Crowd behavior as well as the area parameters is studied before suggesting any possible technological solution for managing a crowd. This article presents a theoretical understanding of the major crowd size estimation approaches that cannot be achieved through the study of existing survey papers in this area, because the existing survey papers focus on particular technologies/specific areas with no or brief description of the involved steps. Besides, this article also highlights the strength and weakness of crowd size estimation solutions and their possible applications. It is, therefore, believed that the provided information would assist in developing an intelligent system for crowd management.

The increasing use of software in Embedded Real-Time Systems (ERTS) has allowed the application of software engineering methods and techniques in their development. In this regard, Service-Oriented Computing (SOC) has previously been used to reduce the development complexity of ERTS. However, the concepts of SOC are mostly used at the implementation level without any focus on system design. To fill this gap, the author has previously developed a meta-model for service-oriented development of ERTS. Although the proposed meta-model allows the high-level modeling of ERTS in terms of service-oriented concepts, it covers only the structural requirements. Therefore, the meta-model is further extended in this paper to include the behavior modeling of the system. The effectiveness of the proposed mechanism is demonstrated with the help of Traffic Light Controller and Arbiter case studies. Following the model-based system engineering process, the high-level models can be transformed into the low-level models.

The development complexity of Distributed Embedded Real-Time Systems (DERTS) can be reduced by the use of Service-Oriented Computing (SOC). However, the existing modeling methods allow modeling of either DERTS or SOC concepts and there is a lack of meta-model for Service-Oriented development of DERTS. This paper proposes a service-based meta-model for DERTS, along with the constraints of the elements of the meta-model. A Smart Home case study was designed to validate the meta-model. This meta-model could not only be beneficial for Service-Oriented development of DERTS, but can also be used at the Platform Independent Model (PIM) level of MDD.

The benefits of Domain Specific Modeling Languages (DSML), for modeling and design of cyber physical systems, have been acknowledged in previous years. In contrast to general purpose modeling languages, such as Unified Modeling Language, DSML facilitates the modeling of domain specific concepts. The objective of this work is to develop a simple graphical DSML for cyber physical systems, which allow the unified modeling of the structural and behavioral aspects of a system in a single model, and provide model transformation and design verification support in future. The proposed DSML was defined in terms of its abstract and concrete syntax. The applicability of the proposed DSML was demonstrated by its application in two case studies: Traffic Signal and Arbiter case studies. The results showed that the proposed DSML produce simple and unified models with possible model transformation and verification support.

A smart space is an increasingly important application of Internet of Things (IoT), which is being developed throughout the world for different purposes ranging from home automation to smart grids. However, a common approach for requirement capture and specification at an abstract level is needed in smart space development that is independent of the continuously changing technology. To provide a systematic process, the standard software engineering practices can be used in the smart space development, and hence take advantages of. In this context, this paper contributes in the field of requirement engineering for smart spaces by introducing the requirement specification technique based on software engineering Use Case concept. The conventional Use Case structure and the way it is described are modified to suit the requirements of smart spaces. The case of a hypothetical smart space development is considered, where the proposed requirement specification technique is applied and a Use Case repository is developed that can be used as a canonical resource for other researchers to draw upon. The implementation provides profound descriptions on how to use the proposed requirement specification technique during the development of smart spaces and in other application areas of IoT. This work can be used as a first step towards defining the smart space development framework, of which the proposed technique would be a key element.

The composite service design modeling is an essential process of the service-oriented software development life cycle, where the candidate services, composite services, operations and their dependencies are required to be identified and specified before their design. However, a systematic service-oriented design modeling method for composite services is still in its infancy as most of the existing approaches provide the modeling of atomic services only. For these reasons, a new method (ComSDM) is proposed in this work for modeling the concept of service-oriented design to increase the reusability and decrease the complexity of system while keeping the service composition considerations in mind. Furthermore, the ComSDM method provides the mathematical representation of the components of service-oriented design using the graph-based theoryto facilitate the design quality measurement. To demonstrate that the ComSDM method is also suitable for composite service design modeling of distributed embedded real-time systems along with enterprise software development, it is implemented in the case study of a smart home. The results of the case study not only check the applicability of ComSDM, but can also be used to validate the complexity and reusability of ComSDM. This also guides the future research towards the design quality measurement such as using the ComSDM method to measure the quality of composite service design in service-oriented software system.

The Worst-Case Execution Time WCET of real-time systems is mainly influenced by the program design, its execution environment and the input data. To cover the last factor in the context of WCET estimation, the objective of this work is to generate the test-data that maximize the execution times of the parallel real-time systems. In this paper, a test-data generation technique is proposed that uses Genetic Algorithms to automatically generate the input data, to be used for testing of parallel real-time systems. The proposed technique was applied to a parallel embedded application --- Stringsearch. The result was an analysis that took as input the parallel program and generated the test-data that cause maximal execution times. The generated test-data showed improvements by exercising long execution times in comparison to randomly generated input data.

The service composition process depends on effective service discovery, matching, and selection. Although, service discovery and matching has been thoroughly researched for software services, an effective service discovery and matching mechanism is needed for services representing device functionality. In this chapter, an ontology-based service description method and service discovery and matching mechanism is proposed for Distributed Embedded Real-Time Systems (DERTS). For service description, the existing ontology specification OWL-S is modified to make it suitable for DERTS. The proposed mechanism was applied to a scenario of Smart Home case study for testing. The proposed service description method and discovery and matching mechanism set foundations can be used for the dynamic service composition in DERTS and in any ubiquitous environment.

The successful implementation of Service-Oriented development of Distributed Embedded Real-time Systems (DERTS) in the recent years has proved the importance of Service-Orientation over previous paradigms. However, efforts are still needed on systematic Service-Oriented design of DERTS, especially on the modelling of DERTS. The UML standard profile MARTE can be used for modelling of embedded real-time system while UML standard profile SoaML can be used to represent Service-Oriented concepts. Therefore, a thorough investigation of both standard profiles is needed to determine which parts of these profiles are useful for Service-Oriented DERTS development. This paper investigates these profiles in order to highlight the portions of the two profiles and provides some suggestions that can be utilized for Service-Oriented modelling of DERTS. The results of this investigation would be useful in developing a Service-Oriented based meta-model for DERTS.

The increasing use and reliance on Distributed Embedded Real-Time Systems (DERTS) demand the enhancement of their capabilities. This along with the unique characteristics of DERTS, such as resource constraints, make the design and development more complex. State-of-the-art software development methods like Service-Oriented Computing (SOC) and Model-Driven Development can be used to cope with these complexities. However, both of the methods require the system to be designed at a higher level of abstraction and the details are provided at lower levels. Such design models for DERTS with different levels of abstractions are still currently unavailable. This paper presents four basic design models representing different levels of abstraction, to be used in Service-Oriented DERTS design. The proposed models were applied in the Home Automation domain for verification purposes. This level-by-level modelling does not only simplify the design process, but can also be used as part of a Service-Oriented modelling framework for DERTS. Furthermore, these design models can support service composition during Service-Oriented DERTS development. The service composition modelling would be beneficial for CASE tools development in supporting designers working at different levels of abstraction.

Analysis and design are the key phases in any software development life cycle. Service identification is one of the most important steps performed in the analysis phase of service-oriented software development life cycle. Service-orientation has been applied successfully in the domain of distributed embedded real-time system because of the advantages it provides such as reusability and interoperability. However, a systematic service-oriented development methodology for distributed embedded real-time system is still missing. A step-by-step guideline for service identification is presented in this study as part of an attempt to define the analysis phase of service-oriented software development life cycle for distributed embedded real-time system. The Smart Home case study is applied to verify the guidelines. This study provides profound descriptions on how to identify services for distributed embedded real-time system. Moreover, this work can be used as a first step towards a systematic service-oriented software development life cycle for distributed embedded real-time systems.