ViDiT–CACTUS: an inexpensive and versatile library preparation and sequence analysis method for virus discovery and other microbiology applications

2018 ◽  
Vol 64 (10) ◽  
pp. 761-773 ◽  
Author(s):  
Joost T.P. Verhoeven ◽  
Marta Canuti ◽  
Hannah J. Munro ◽  
Suzanne C. Dufour ◽  
Andrew S. Lang
Keyword(s):  
Influenza A ◽  
High Throughput Sequencing ◽  
Low Cost ◽  
Quality Data ◽  
Library Preparation ◽  
Virus Discovery ◽  
Influenza A Viruses ◽  
Wide Range ◽  
Functional Profiling ◽  
Biology Laboratory

High-throughput sequencing (HTS) technologies are becoming increasingly important within microbiology research, but aspects of library preparation, such as high cost per sample or strict input requirements, make HTS difficult to implement in some niche applications and for research groups on a budget. To answer these necessities, we developed ViDiT, a customizable, PCR-based, extremely low-cost (less than US$5 per sample), and versatile library preparation method, and CACTUS, an analysis pipeline designed to rely on cloud computing power to generate high-quality data from ViDiT-based experiments without the need of expensive servers. We demonstrate here the versatility and utility of these methods within three fields of microbiology: virus discovery, amplicon-based viral genome sequencing, and microbiome profiling. ViDiT–CACTUS allowed the identification of viral fragments from 25 different viral families from 36 oropharyngeal–cloacal swabs collected from wild birds, the sequencing of three almost complete genomes of avian influenza A viruses (>90% coverage), and the characterization and functional profiling of the complete microbial diversity (bacteria, archaea, viruses) within a deep-sea carnivorous sponge. ViDiT–CACTUS demonstrated its validity in a wide range of microbiology applications, and its simplicity and modularity make it easily implementable in any molecular biology laboratory, towards various research goals.

scholarly journals Eco-Epidemiological Evidence of the Transmission of Avian and Human Influenza A Viruses in Wild Pigs in Campeche, Mexico

Viruses ◽  
2020 ◽  
Vol 12 (5) ◽  
pp. 528
Author(s):  
Brenda Aline Maya-Badillo ◽  
Rafael Ojeda-Flores ◽  
Andrea Chaves ◽  
Saul Reveles-Félix ◽  
Guillermo Orta-Pineda ◽  
...  
Keyword(s):  
Influenza A ◽  
Influenza Viruses ◽  
Influenza A Viruses ◽  
Serum Samples ◽  
Fragmented Habitats ◽  
Wild Pigs ◽  
Wide Range ◽  
Influenza Transmission ◽  
Human Viruses ◽  
Positive Results

Influenza, a zoonosis caused by various influenza A virus subtypes, affects a wide range of species, including humans. Pig cells express both sialyl-α-2,3-Gal and sialyl-α-2,6-Gal receptors, which make them susceptible to infection by avian and human viruses, respectively. To date, it is not known whether wild pigs in Mexico are affected by influenza virus subtypes, nor whether this would make them a potential risk of influenza transmission to humans. In this work, 61 hogs from two municipalities in Campeche, Mexico, were sampled. Hemagglutination inhibition assays were performed in 61 serum samples, and positive results were found for human H1N1 (11.47%), swine H1N1 (8.19%), and avian H5N2 (1.63%) virus variants. qRT-PCR assays were performed on the nasal swab, tracheal, and lung samples, and 19.67% of all hogs were positive to these assays. An avian H5N2 virus, first reported in 1994, was identified by sequencing. Our results demonstrate that wild pigs are participating in the exposure, transmission, maintenance, and possible diversification of influenza viruses in fragmented habitats, highlighting the synanthropic behavior of this species, which has been poorly studied in Mexico.

scholarly journals Epidemiology of Severe Acute Respiratory Infection (SARI) Cases at a sentinel site in Egypt, 2013–15

2019 ◽  
Vol 42 (3) ◽  
pp. 525-533 ◽  
Author(s):  
Mohamed M Elhakim ◽  
Sahar K Kandil ◽  
Khaled M Abd Elaziz ◽  
Wagida A Anwar
Keyword(s):  
Respiratory Infection ◽  
Acute Respiratory Infection ◽  
Influenza A ◽  
Case Definition ◽  
Quality Data ◽  
Influenza B ◽  
Sentinel Site ◽  
Influenza A Viruses ◽  
High Quality Data ◽  
Severe Acute Respiratory Infection

Abstract Background Sentinel surveillance for severe acute respiratory infection (SARI) in Egypt began in 2006 and occurs at eight sites. Avian influenza is endemic, and human cases of influenza A (H5N1) have been reported annually since 2006. This study aimed to describe the epidemiology of SARI at a major sentinel site in the country. Methods Data included in the study were collected from a major SARI sentinel site in Egypt during three consecutive years (2013–15). Results A total of 1254 SARI patients conforming to the WHO case definition were admitted to the sentinel site, representing 5.6% of admitted patients for all causes and 36.6% of acute respiratory infection patients. A total of 99.7% of the patients were tested, and 21.04% tested positive; 48.7% of cases involved influenza A viruses, while 25% involved influenza B. The predominant age group was under 5 years of age, accounting for 443 cases. The seasonality of the influenza data conformed to the Northern Hemisphere pattern. Conclusions The present study’s results show that SARI leads to substantial morbidity in Egypt. There is a great need for high-quality data from the SARI surveillance system in Egypt, especially with endemic respiratory threats such as influenza A (H5N1) in Egypt.

scholarly journals Analysis of Coinfections with A/H1N1 Strain Variants among Pigs in Poland by Multitemperature Single-Strand Conformational Polymorphism

2015 ◽  
Vol 2015 ◽  
pp. 1-9 ◽  
Author(s):  
Krzysztof Lepek ◽  
Beata Pajak ◽  
Lukasz Rabalski ◽  
Kinga Urbaniak ◽  
Krzysztof Kucharczyk ◽  
...  
Keyword(s):  
Influenza A ◽  
Cost Effective ◽  
Single Strand ◽  
Surveillance Systems ◽  
Monitoring And Control ◽  
Influenza A Viruses ◽  
Single Strand Conformational Polymorphism ◽  
Conformational Polymorphism ◽  
Wide Range ◽  
Different Strains

Monitoring and control of infections are key parts of surveillance systems and epidemiological risk prevention. In the case of influenza A viruses (IAVs), which show high variability, a wide range of hosts, and a potential of reassortment between different strains, it is essential to study not only people, but also animals living in the immediate surroundings. If understated, the animals might become a source of newly formed infectious strains with a pandemic potential. Special attention should be focused on pigs, because of the receptors specific for virus strains originating from different species, localized in their respiratory tract. Pigs are prone to mixed infections and may constitute a reservoir of potentially dangerous IAV strains resulting from genetic reassortment. It has been reported that a quadruple reassortant, A(H1N1)pdm09, can be easily transmitted from humans to pigs and serve as a donor of genetic segments for new strains capable of infecting humans. Therefore, it is highly desirable to develop a simple, cost-effective, and rapid method for evaluation of IAV genetic variability. We describe a method based on multitemperature single-strand conformational polymorphism (MSSCP), using a fragment of the hemagglutinin (HA) gene, for detection of coinfections and differentiation of genetic variants of the virus, difficult to identify by conventional diagnostic.

Influenza viruses: transmission between species

1980 ◽  
Vol 288 (1029) ◽  
pp. 439-447 ◽  
Keyword(s):  
Influenza A ◽  
Direct Evidence ◽  
Swine Influenza ◽  
Indirect Evidence ◽  
Preliminary Evidence ◽  
Influenza Viruses ◽  
Surface Proteins ◽  
Aquatic Birds ◽  
Influenza A Viruses ◽  
Wide Range

The only direct evidence for transmission of influenza viruses between species comes from studies on swine influenza viruses. Antigenically and genetically identical Hsw1N1 influenza viruses were isolated from pigs and man on the same farm in Wisconsin, U.S.A. The isolation of H3N2 influenza viruses from a wide range of lower animals and birds suggests that influenza viruses of man can spread to the lower orders. Under some conditions the H3N2 viruses can persist for a number of years in some species. The isolation, from aquatic birds, of a large number of influenza A viruses that possess surface proteins antigenically similar to the viruses isolated from man, pigs and horses provides indirect evidence for inter-species transmission. There is now a considerable body of evidence which suggests that influenza viruses of lower animals and birds may play a role in the origin of some of the pandemic strains of influenza A viruses. There is no direct evidence that the influenza viruses in aquatic birds are transmitted to man, but they may serve as a genetic pool from which some genes may be introduced into humans by recombination. Preliminary evidence suggests that the molecular basis of host range and virulence may be related to the RNA segments coding for one of the polymerase proteins (P3) and for the nucleoprotein (NP).

scholarly journals Avian Influenza A Virus Pandemic Preparedness and Vaccine Development

Vaccines ◽  
2018 ◽  
Vol 6 (3) ◽  
pp. 46 ◽  
Author(s):  
Rory de Vries ◽  
Sander Herfst ◽  
Mathilde Richard
Keyword(s):  
Avian Influenza ◽  
Influenza A Virus ◽  
Influenza A ◽  
Vaccine Development ◽  
Influenza Pandemic ◽  
Broad Host Range ◽  
Influenza A Viruses ◽  
Vaccination Strategies ◽  
Pandemic Virus ◽  
Wide Range

Influenza A viruses can infect a wide range of hosts, creating opportunities for zoonotic transmission, i.e., transmission from animals to humans, and placing the human population at constant risk of potential pandemics. In the last hundred years, four influenza A virus pandemics have had a devastating effect, especially the 1918 influenza pandemic that took the lives of at least 40 million people. There is a constant risk that currently circulating avian influenza A viruses (e.g., H5N1, H7N9) will cause a new pandemic. Vaccines are the cornerstone in preparing for and combating potential pandemics. Despite exceptional advances in the design and development of (pre-)pandemic vaccines, there are still serious challenges to overcome, mainly caused by intrinsic characteristics of influenza A viruses: Rapid evolution and a broad host range combined with maintenance in animal reservoirs, making it near impossible to predict the nature and source of the next pandemic virus. Here, recent advances in the development of vaccination strategies to prepare against a pandemic virus coming from the avian reservoir will be discussed. Furthermore, remaining challenges will be addressed, setting the agenda for future research in the development of new vaccination strategies against potentially pandemic influenza A viruses.

scholarly journals Method comparison of targeted influenza A virus typing and whole-genome sequencing from respiratory specimens of companion animals

2020 ◽  
pp. 104063872093387
Author(s):  
Patrick K. Mitchell ◽  
Brittany D. Cronk ◽  
Ian E. H. Voorhees ◽  
Derek Rothenheber ◽  
Renee R. Anderson ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Influenza A ◽  
Sequence Data ◽  
Companion Animals ◽  
The United States ◽  
Quality Data ◽  
Whole Genome ◽  
Influenza A Viruses ◽  
Respiratory Specimens

Epidemics of H3N8 and H3N2 influenza A viruses (IAVs) in dogs, along with recognition of spillover infections from IAV strains typically found in humans or other animals, have emphasized the importance of efficient laboratory testing. Given the lack of active IAV surveillance or immunization requirements for dogs, cats, or horses imported into the United States, serotype prediction and whole-genome sequencing of positive specimens detected at veterinary diagnostic laboratories are also needed. The conserved sequences at the ends of the viral genome segments facilitate universal amplification of all segments of viral genomes directly from respiratory specimens. Although several methods for genomic analysis have been reported, no optimization focusing on companion animal strains has been described, to our knowledge. We compared 2 sets of published universal amplification primers using 26 IAV-positive specimens from dogs, horses, and a cat. Libraries prepared from the resulting amplicons were sequenced using Illumina chemistry, and reference-based assemblies were generated from the data produced by both methods. Although both methods produced high-quality data, coverage profiles and base calling differed between the 2 methods. The sequence data were also used to identify the subtype of the IAV strains sequenced and then compared to standard PCR assays for neuraminidase types N2 and N8.

scholarly journals Matriptase, HAT, and TMPRSS2 Activate the Hemagglutinin of H9N2 Influenza A Viruses

2012 ◽  
Vol 87 (3) ◽  
pp. 1811-1820 ◽  
Author(s):  
Joanna Baron ◽  
Carolin Tarnow ◽  
Deborah Mayoli-Nüssle ◽  
Eva Schilling ◽  
Daniela Meyer ◽  
...  
Keyword(s):  
Cleavage Site ◽  
Influenza A ◽  
Surface Glycoprotein ◽  
Virus Infectivity ◽  
Kidney Cells ◽  
Cleavage Sites ◽  
Influenza A Viruses ◽  
Proteolytic Activation ◽  
Wide Range ◽  
Canine Kidney

ABSTRACTInfluenza A viruses of the subtype H9N2 circulate worldwide and have become highly prevalent in poultry in many countries. Moreover, they are occasionally transmitted to humans, raising concern about their pandemic potential. Influenza virus infectivity requires cleavage of the surface glycoprotein hemagglutinin (HA) at a distinct cleavage site by host cell proteases. H9N2 viruses vary remarkably in the amino acid sequence at the cleavage site, and many isolates from Asia and the Middle East possess the multibasic motifs R-S-S-R and R-S-R-R, but are not activated by furin. Here, we investigated proteolytic activation of the early H9N2 isolate A/turkey/Wisconsin/1/66 (H9-Wisc) and two recent Asian isolates, A/quail/Shantou/782/00 (H9-782) and A/quail/Shantou/2061/00 (H9-2061), containing mono-, di-, and tribasic HA cleavage sites, respectively. All H9N2 isolates were activated by human proteases TMPRSS2 (transmembrane protease, serine S1 member 2) and HAT (human airway trypsin-like protease). Interestingly, H9-782 and H9-2061 were also activated by matriptase, a protease widely expressed in most epithelia with high expression levels in the kidney. Nephrotropism of H9N2 viruses has been observed in chickens, and here we found that H9-782 and H9-2061 were proteolytically activated in canine kidney (MDCK-II) and chicken embryo kidney (CEK) cells, whereas H9-Wisc was not. Virus activation was inhibited by peptide-mimetic inhibitors of matriptase, strongly suggesting that matriptase is responsible for HA cleavage in these kidney cells. Our data demonstrate that H9N2 viruses with R-S-S-R or R-S-R-R cleavage sites are activated by matriptase in addition to HAT and TMPRSS2 and, therefore, can be activated in a wide range of tissues what may affect virus spread, tissue tropism and pathogenicity.

scholarly journals Aptamer Profiling of A549 Cells Infected with Low-Pathogenicity and High-Pathogenicity Influenza Viruses

Viruses ◽  
2019 ◽  
Vol 11 (11) ◽  
pp. 1028 ◽  
Author(s):  
Kevin M. Coombs ◽  
Philippe F. Simon ◽  
Nigel J. McLeish ◽  
Ali Zahedi-Amiri ◽  
Darwyn Kobasa
Keyword(s):  
Influenza A ◽  
Human Lung ◽  
A549 Cells ◽  
Influenza Viruses ◽  
Post Translational Modification ◽  
Emerging Pathogens ◽  
Influenza A Viruses ◽  
Human Lung Cells ◽  
Wide Range ◽  
High Pathogenicity

Influenza A viruses (IAVs) are important animal and human emerging and re-emerging pathogens that are responsible for yearly seasonal epidemics and sporadic pandemics. IAVs cause a wide range of clinical illnesses, from relatively mild infections by seasonal strains, to acute respiratory distress during infections with highly pathogenic avian IAVs (HPAI). For this study, we infected A549 human lung cells with lab prototype A/PR/8/34 (H1N1) (PR8), a seasonal H1N1 (RV733), the 2009 pandemic H1N1 (pdm09), or with two avian strains, an H5N1 HPAI strain or an H7N9 strain that has low pathogenicity in birds but high pathogenicity in humans. We used a newly-developed aptamer-based multiplexed technique (SOMAscan®) to examine >1300 human lung cell proteins affected by the different IAV strains, and identified more than 500 significantly dysregulated cellular proteins. Our analyses indicated that the avian strains induced more profound changes in the A549 global proteome compared to all tested low-pathogenicity H1N1 strains. The PR8 strain induced a general activation, primarily by upregulating many immune molecules, the seasonal RV733 and pdm09 strains had minimal effect upon assayed molecules, and the avian strains induced significant downregulation, primarily in antimicrobial response, cardiovascular and post-translational modification systems.

scholarly journals Single-Reaction Genomic Amplification Accelerates Sequencing and Vaccine Production for Classical and Swine Origin Human Influenza A Viruses

2009 ◽  
Vol 83 (19) ◽  
pp. 10309-10313 ◽  
Author(s):  
Bin Zhou ◽  
Matthew E. Donnelly ◽  
Derek T. Scholes ◽  
Kirsten St. George ◽  
Masato Hatta ◽  
...  
Keyword(s):  
Influenza A ◽  
Reverse Genetics ◽  
High Throughput Sequencing ◽  
Genomic Analysis ◽  
H3n2 Virus ◽  
Influenza A Viruses ◽  
Genomic Amplification ◽  
Reverse Transcription Pcr ◽  
Virus Subtype ◽  
Single Reaction

ABSTRACT Pandemic influenza A viruses that emerge from animal reservoirs are inevitable. Therefore, rapid genomic analysis and creation of vaccines are vital. We developed a multisegment reverse transcription-PCR (M-RTPCR) approach that simultaneously amplifies eight genomic RNA segments, irrespective of virus subtype. M-RTPCR amplicons can be used for high-throughput sequencing and/or cloned into modified reverse-genetics plasmids via regions of sequence identity. We used these procedures to rescue a contemporary H3N2 virus and a swine origin H1N1 virus directly from human swab specimens. Together, M-RTPCR and the modified reverse-genetics plasmids that we designed streamline the creation of vaccine seed stocks (9 to 12 days).

scholarly journals Influenza A virus avian-derived PB1 gene has evolved to match its codon usage to interferon-altered human tRNA pools

2017 ◽  
Author(s):  
Bartram L Smith ◽  
Guifang Chen ◽  
Claus O Wilke ◽  
Robert M Krug
Keyword(s):  
Codon Usage ◽  
Influenza A Virus ◽  
Influenza A ◽  
High Throughput Sequencing ◽  
Regulation Of Gene Expression ◽  
Human Cells ◽  
Influenza A Viruses ◽  
Synonymous Mutations ◽  
Avian Virus

Influenza A viruses cause an annual highly contagious respiratory disease in humans, and are responsible for periodic human pandemics that have high mortality rates. Pandemic influenza A viruses can result from reassortment of one or more gene segments between a human and an avian virus. These avian virus gene segments need to adapt to humans post introduction. The role of synonymous mutations in this adaptation is not known. Here we focus on the human adaptation of the synonymous codons of the avian virus PB1 gene of the 1968 H3N2 pandemic virus. We generate recombinant H3N2 viruses differing only in codon usage of PB1 mRNA, and demonstrate that the codon usage of recent virus isolates enhances replication in interferon-treated human cells rather than in untreated cells, thereby partially alleviating the interferon-induced antiviral state. High-throughput sequencing of tRNA pools explains this virus phenotype: the levels of some tRNAs differ between interferon-treated and untreated human cells; and the codon usage of H3N2 PB1 mRNA has been evolving over time to match the tRNA pools in interferon-treated human cells. Consequently, our results identify a previously unknown mechanism by which influenza A virus counteracts the host interferon-induced antiviral response and highlight the important role of tRNA pools in the regulation of gene expression.

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