Strongyloides-Specific IgE Phage cDNA Clones and Development of a Novel ELISA for Strongyloidiasis.
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ABSTRACT:
SUBMITTER: Ahmad H
PROVIDER: S-EPMC8228214 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BACKGROUND: Salmonid alphavirus (SAV) is a widespread pathogen in European aquaculture of salmonid fish. Distinct viral subtypes have been suggested based on sequence comparisons and some of these have different geographical distributions. In Norway, only SAV subtype 3 have so far been identified. Little is known about viral mechanisms important for pathogenesis and transmission. Tools for detailed exploration of SAV genomes are therefore needed. RESULTS: Infectious cDNA clones in which a genome of subtype 3 SAV is under the control of a CMV promoter were constructed. The clones were designed to express proteins that are putatively identical to those previously reported for the SAVH20/03 strain. A polyclonal antiserum was raised against a part of the E2 glycoprotein in order to detect expression of the subgenomic open reading frame (ORF) encoding structural viral proteins. Transfection of the cDNA clone revealed the expression of the E2 protein by IFAT, and in serial passages of the supernatant the presence of infectious recombinant virus was confirmed through RT-PCR, IFAT and the development of a cytopathic effect similar to that seen during infection with wild type SAV. Confirmation that the recovered virus originated from the infectious plasmid was done by sequence identification of an introduced genetic tag. The recombinant virus was infectious also when an additional ORF encoding an EGFP reporter gene under the control of a second subgenomic alphavirus promoter was added. Finally, we used the system to study the effect of selected point mutations on infectivity in Chinook salmon embryo cells. While introduced mutations in nsP2197, nsP3263 and nsP3323 severely reduced infectivity, a serine to proline mutation in E2206 appeared to enhance the virus titer production. CONCLUSION: We have constructed infectious clones for SAV based on a subtype 3 genome. The clones may serve as a platform for further functional studies.
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Project description:BackgroundStrongyloides stercoralis infects human hosts mainly through skin contact with contaminated soil. The result is strongyloidiasis, a parasitic disease, with a unique cycle of auto-infection causing a variety of symptoms and signs, with possible fatality from hyper-infection. Australian Indigenous community members, often living in rural and remote settings, are exposed to and infected with S. stercoralis. The aim of this review is to determine barriers to control of strongyloidiasis. The purpose is to contribute to the development of initiatives for prevention, early detection and effective treatment of strongyloidiasis.Methodology/principle findingsSystematic search reviewing research published 2012 and earlier was conducted. Research articles discussing aspects of strongyloidiasis, context of infection and overall health in Indigenous Australians were reviewed. Based on the PRISMA statement, the systematic search of health databases, Academic Search Premier, Informit, Medline, PubMed, AMED, CINAHL, Health Source Nursing and Academic was conducted. Key search terms included strongyloidiasis, Indigenous, Australia, health, and community. 340 articles were retrieved with 16 original research articles published between 1969 and 2006 meeting criteria. Review found barriers to control defined across three key themes, (1) health status, (2) socioeconomic status, and (3) health care literacy and procedures.Conclusions/significanceThis study identifies five points of intervention: (1) develop reporting protocols between health care system and communities; (2) test all Indigenous Australian patients, immunocompromised patients and those exposed to areas with S. stercoralis; (3) health professionals require detailed information on strongyloidiasis and potential for exposure to Indigenous Australian people; (4) to establish testing and treatment initiatives within communities; and (5) to measure and report prevalence rates specific to communities and to act with initiatives based on these results. By defining barriers to control of strongyloidiasis in Australian Indigenous people, improved outcomes of prevention, treatment of strongyloidiasis and increased health overall are attainable.
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Project description:Critical to managing the spread of COVID-19 is the ability to diagnose infection and define the acquired immune response across the population. While genomic tests for the novel Several Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) detect the presence of viral RNA for a limited time frame, when the virus is shed in the upper respiratory tract, tests able to define exposure and infection beyond this short window of detectable viral replication are urgently needed. Following infection, antibodies are generated within days, providing a durable read-out and archive of exposure and infection. Several antibody tests have emerged to diagnose SARS-CoV-2. Here we report on a qualified quantitative ELISA assay that displays all the necessary characteristics for high-throughput sample analysis. Collectively, this test offers a quantitative opportunity to define both exposure and levels of immunity to SARS-CoV-2.
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Project description:Reverse genetics systems represent an important tool for studying the molecular and functional processes of viral infection. Citrus leprosis virus C (CiLV-C) (genus Cilevirus, family Kitaviridae) is the main pathogen responsible for the citrus leprosis (CL) disease in Latin America, one of the most economically important diseases of the citrus industry. Molecular studies of this pathosystem are limited due to the lack of infectious clones. Here, we report the construction and validation of a CiLV-C infectious cDNA clone based on an agroinfection system. The two viral RNA segments (RNA1 and RNA2) were assembled into two binary vectors (pJL89 and pLXAS). Agroinfiltrated Nicotiana benthamiana plants showed a response similar to that observed in the natural infection process with the formation of localized lesions restricted to the inoculated leaves. The virus recovered from the plant tissue infected with the infectious clones can be mechanically transmitted between N. benthamiana plants. Detection of CiLV-C subgenomic RNAs (sgRNAs) from agroinfiltrated and mechanically inoculated leaves further confirmed the infectivity of the clones. Finally, partial particle-purification preparations or sections of CiLV-C-infected tissue followed by transmission electron microscopy (TEM) analysis showed the formation of CiLV-C virions rescued by the infectious clone. The CiLV-C reverse genetic system now provides a powerful molecular tool to unravel the peculiarities of the CL pathosystem.
| S-EPMC10682248 | biostudies-literature
Project description:Strongyloidiasis is caused by the nematode Strongyloides stercoralis which has the unique ability to reproduce and complete its entire life cycle within the human host through its autoinfection cycle. Diagnosis of this infection is important because of its potential to cause fatal hyperinfection syndrome or disseminated infections in those with defective cellular immunity. Parasitological methods based on faecal microscopy and culture often fail to detect low-intensity infections. A multiplex polymerase chain reaction (PCR) assay was developed for the detection of S. stercoralis, Ascaris lumbricoides, and Enterobius vermicularis by designing primers specific for the ITS1 region of ribosomal DNA of S. stercoralis and A. lumbricoides and 18S region of rRNA of E. vermicularis. A 61-year-old patient presented with chronic gastrointestinal and respiratory symptoms and weight loss with a stool microscopy positive for helminth larvae. Stool cultures with the Harada-Mori technique yielded L3 larvae which were identified as S. stercoralis based on morphology. The multiplex PCR performed on DNA extracted from stool elicited the expected band at 129 bp on gel electrophoresis of the PCR yield providing molecular evidence of intestinal strongyloidiasis. The patient's gastrointestinal symptoms improved with a six-day course of albendazole (400 mg twice daily). Negative posttreatment stool microscopy, culture, and PCR confirmed successful clearance of infection. Molecular-based PCR assay is a promising tool to diagnose and assess the therapeutic efficacy of anthelmintics in intestinal helminthiases such as strongyloidiasis.
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Project description:A large collection of full-length cDNAs is essential for the correct annotation of genomic sequences and for the functional analysis of genes and their products. We obtained a total of 39,936 soybean cDNA clones (GMFL01 and GMFL02 clone sets) in a full-length-enriched cDNA library which was constructed from soybean plants that were grown under various developmental and environmental conditions. Sequencing from 5' and 3' ends of the clones generated 68 661 expressed sequence tags (ESTs). The EST sequences were clustered into 22,674 scaffolds involving 2580 full-length sequences. In addition, we sequenced 4712 full-length cDNAs. After removing overlaps, we obtained 6570 new full-length sequences of soybean cDNAs so far. Our data indicated that 87.7% of the soybean cDNA clones contain complete coding sequences in addition to 5'- and 3'-untranslated regions. All of the obtained data confirmed that our collection of soybean full-length cDNAs covers a wide variety of genes. Comparative analysis between the derived sequences from soybean and Arabidopsis, rice or other legumes data revealed that some specific genes were involved in our collection and a large part of them could be annotated to unknown functions. A large set of soybean full-length cDNA clones reported in this study will serve as a useful resource for gene discovery from soybean and will also aid a precise annotation of the soybean genome.
| S-EPMC2608845 | biostudies-literature
Project description:Strongyloidiasis is a much-neglected soil born helminthiasis caused by the nematode Strongyloides stercoralis. Human derived S. stercoralis can be maintained in dogs in the laboratory and this parasite has been reported to also occur in dogs in the wild. Some authors have considered strongyloidiasis a zoonotic disease while others have argued that the two hosts carry host specialized populations of S. stercoralis and that dogs play a minor role, if any, as a reservoir for zoonotic S. stercoralis infections of humans. We isolated S. stercoralis from humans and their dogs in rural villages in northern Cambodia, a region with a high incidence of strongyloidiasis, and compared the worms derived from these two host species using nuclear and mitochondrial DNA sequence polymorphisms. We found that in dogs there exist two populations of S. stercoralis, which are clearly separated from each other genetically based on the nuclear 18S rDNA, the mitochondrial cox1 locus and whole genome sequence. One population, to which the majority of the worms belong, appears to be restricted to dogs. The other population is indistinguishable from the population of S. stercoralis isolated from humans. Consistent with earlier studies, we found multiple sequence variants of the hypervariable region I of the 18 S rDNA in S. stercoralis from humans. However, comparison of mitochondrial sequences and whole genome analysis suggest that these different 18S variants do not represent multiple genetically isolated subpopulations among the worms isolated from humans. We also investigated the mode of reproduction of the free-living generations of laboratory and wild isolates of S. stercoralis. Contrary to earlier literature on S. stercoralis but similar to other species of Strongyloides, we found clear evidence of sexual reproduction. Overall, our results show that dogs carry two populations, possibly different species of Strongyloides. One population appears to be dog specific but the other one is shared with humans. This argues for the strong potential of dogs as reservoirs for zoonotic transmission of S. stercoralis to humans and suggests that in order to reduce the exposure of humans to infective S. stercoralis larvae, dogs should be treated for the infection along with their owners.
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Project description:The Mammalian Gene Collection (MGC) consortium (http://mgc.nci.nih.gov) seeks to establish publicly available collections of full-ORF cDNAs for several organisms of significance to biomedical research, including human. To date over 15,200 human cDNA clones containing full-length open reading frames (ORFs) have been identified via systematic expressed sequence tag (EST) analysis of a diverse set of cDNA libraries; however, further systematic EST analysis is no longer an efficient method for identifying new cDNAs. As part of our involvement in the MGC program, we have developed a scalable method for targeted recovery of cDNA clones to facilitate recovery of genes absent from the MGC collection. First, cDNA is synthesized from various RNAs, followed by polymerase chain reaction (PCR) amplification of transcripts in 96-well plates using gene-specific primer pairs flanking the ORFs. Amplicons are cloned into a sequencing vector, and full-length sequences are obtained. Sequences are processed and assembled using Phred and Phrap, and analyzed using Consed and a number of bioinformatics methods we have developed. Sequences are compared with the Reference Sequence (RefSeq) database, and validation of sequence discrepancies is attempted using other sequence databases including dbEST and dbSNP. Clones with identical sequence to RefSeq or containing only validated changes will become part of the MGC human gene collection. Clones containing novel splice variants or polymorphisms have also been identified. Our approach to clone recovery, applied at large scale, has the potential to recover many and possibly most of the genes absent from the MGC collection.
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Project description:In reverse genetic experiments we have isolated recombinant mumps viruses (rMuV) that carry large numbers of mutations clustered in small parts of their genome, which are not caused by biased hyper-mutation. In two separate experiments we obtained such recombinant viruses: one virus had 11 mutations in the V/P region of the genome; the other, which also contained an extra transcription unit encoding green fluorescent protein (EGFP), had 32 mutations in the N gene. These specific sets of mutations have not been observed in naturally occurring MuV isolates. Unusually, the vast majority of the mutations (48/51) were synonymous. On passage in Vero cells and human B-LCL cells, a B lymphocyte-like cell line, these mutations appear stable as no reversion occurred to the original consensus sequence, although mutations in other parts of the genome occurred and changed in frequency during passage. Defective interfering RNAs accumulate in passage in Vero cells but not in B-LCL cells. Interestingly, in all passaged samples the level of variation in the EGFP gene is the same as in the viral genes, though it is unlikely that this gene is under any functionality constraint. What mechanism gave rise to these viruses with clustered mutations and their stability remains an open question, which is likely of interest to a wider field than mumps reverse genetics.
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Project description:The recently emerged highly virulent variants of porcine epidemic and diarrhea virus (PEDV) remain a huge threat to the worldwide swine industry. Here, we describe the development of a bacterial artificial chromosome (BAC) reverse genetics system for PEDV based on two recent Chinese field isolates, namely CHM2013 and BJ2011C. Phylogenetically, CHM2013 is closely related to the vaccine strain SM98 whereas the isolate BJ2011C belongs to the GIIb group, a cluster that contains many recent pandemic strains. The full-length cDNA clones of the two isolates were constructed into BAC under the control of CMV promoter. The rescued viruses rBJ2011C and rCHM2013 were found to replicate at the kinetics similar to their respective parental viruses in cell culture. When tested in the 2-day-old pig model, rBJ2011C caused severe diarrhea of piglets with extensive damages to the intestinal epithelium, leading to 100% fatality within 48 hours. In contrast, the rCHM2013-inoculated piglets all survived with only very minor tissue damage observed. Thus, we have successfully established a convenient platform for PEDV genome manipulation. This study also represents the first description of a DNA-launched reverse genetics system for the highly virulent PEDV.
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