Posts tagged influenza

Scientist Spotlight: Brett Pickett, Ph.D.

The son of a dentist, Brett Pickett grew up in Salt Lake City, Utah focused initially on a career in the family business (his siblings are hygienists and an oral surgeon). Brett believed from an early age that he would follow in his father’s footsteps. He enrolled in Brigham Young University committed to dental school. It was not until Brett’s zoology major was canceled that he became a student of microbiology, where he began researching antibiotic resistance genes in gut microbiota. Dental school was out. Brett received his B.S. in microbiology and continued his studies at the University of Alabama at Birmingham (UAB).

Brett Pickett, Ph.D.

While working in UAB’s bacteria pathogenesis labs, Brett’s path would take another detour as he encountered West Nile, Hepatitis C, and Dengue viruses in his work. He also began to cultivate an interest in computers, technology, and statistics as it related to biological data. These experiences have led to him to his current field of research: viral bioinformatics.

In 2010, Brett moved to the University of Texas Southwestern Medical Center at Dallas to begin his postdoctoral research with Dr. Richard Scheuermann (presently the Director of JCVI La Jolla). Working with Richard, Brett began to shift his focus on how a virus behaves to examining how the human host is responding to being infected. While at UT Southwestern, Brett worked with Richard and his team to identify and develop new statistical, analysis, and visualization tools for the National Institutes of Health (NIH)-funded Viral Pathogen Resource Bioinformatics Database (ViPR). In 2012, Brett moved his family to La Jolla to be a part of JCVI’s informatics team. During this time, his work focused on enhancing the Virus Pathogen Resource and Influenza Research Database bioinformatics resource centers.

Brett stepped away from JCVI for a brief period to work at Thomson Reuters. There he analyzed “-omics” data with pathway analysis and network-building tools, together with drugs and protein target information to better understand viral infection, differences between pathogenic and commensal bacteria, oncology, and other therapeutic areas. This experience allowed him to gain a better understanding of human genetics, disease profiling, and biomarker identification before returning to research at JCVI in 2016.

At JCVI, Brett continues to work on cutting-edge science. He appreciates “the access to collaborators to solve big problems,” and Brett’s efforts are addressing the world’s biggest health challenges. He recently received funding from the US Agency for International Development (USAID) to develop a method for differentiating antibodies against Zika and other closely-related viruses in human patients.

Brett lives in San Diego with his wife and five children. When he is not in the lab, Brett enjoys golf, waterskiing, playing the piano, and visiting the beach with his family. His children, ranging in ages from 1-11, want to be scientists or doctors when they grow up. While there may be no dentists in this generation either, it is clear Brett’s children will have inspirational and accomplished footsteps in which to follow.

Study Signals Bat Flu Unlikely to Jump to Humans

Bats species harbor a large number of viruses that cause human disease.  So, when the first influenza sequences from Guatemalan little yellow-shouldered bats were uncovered in 2009, the question arose of whether bat influenza viruses pose a threat to human health.  A collaborative project between JCVI and Kansas State University was recently published in PLoS Pathogens to address this question.

H1N1 influenza virus particles

Image Credit: National Institute of Allergy and Infectious Diseases (NIAID)

The approach employed cutting-edge synthetic biology approaches and demonstrated that, while the sequences of the bat influenza virus of the subtype H17N10 are viable, they are unable to infect human cells. Additional experiments clearly indicated that these bat virus sequences are not able to reassort with other influenza A and B viruses known to infect humans. Therefore, the potential for a pandemic bat influenza entering the human population is extremely unlikely.

David Wentworth, the former Director of Viral Programs at JCVI, was the lead investigator for this study.  Additional authors from JCVI include Tim Stockwell, Wei Wang, Xudong Lin, Bin Zhou (now at NYU), and Reed Shabman.

For additional information see the press release.

JCVI Viral Finishing Pipeline: a Winning Combination of Advanced Sequencing Technologies, Software Development and Automated Data Processing

JCVI viral projects are supported by the NIAID Genomic Sequencing Center for Infectious Disease (GSCID). The viral sequencing and finishing pipeline at JCVI combines next generation sequencing technologies with automated data processing. This allowed us to complete over 1,800 viral genomes in the last 12 months, and almost 8,800 genomes since 2005.

Viral Projects at JCVI

JIRA Viral Sample Tracking Workflow

Our NextGen pipeline, which utilizes SISPA-generated libraries with Roche/454 and Illumina sequencing, enables us to complete a wide variety of viral genomes including challenging samples. Automated assembly pipeline employs CLCbio command-line tools and JCVI cas2consed, a cas to ace assembly format conversion tool. Our complimentary Sanger pipeline software is currently being integrated with the NextGen pipeline. This will improve our data processing and will allow us to use validation software (autoTasker) more efficiently.

Assembly of Repetitive Viral Genomes

Genome Organization of Varicella-Zoster

Assembly of Novel Viral Genomes

CLC Assembly Viewer Representation

Promoter of Bat Genome

Promoter of Bat Genome

During the past year we have found that novel viruses, repetitive genomes, and mixed infection samples could not be easily integrated with our high-throughput assembly pipeline. We have developed an assembly and finishing process that utilizes components of the high-throughput pipeline and combines them with manual reference selection and editing. Using this approach we completed novel adenovirus genomes and mixed-infection avian influenza genomes, and improved assemblies of previously unknown arbovirus genomes. We are currently working on optimizing and automating this new pipeline.

Assembly of Mixed Viral Genomes

Consed Representation of Mixed Viral Sample

Consed Representation of Mixed Viral Sample

Repetitive genomes have long been known to present great challenges during assembly and finishing. We are presenting a new approach to assembly and finishing of repetitive varicella genome that is based on separating it into overlapping PCR amplicons followed by merging sequenced amplicons during assembly.

To streamline our viral pipelines, we have fully integrated them with JCVI’s LIMS and JIRA Workflow Management to create a semi-automated tracking interface that follows the progress of viral samples from acquisition through to NCBI submission. This allows us to process a large volume of samples with limited manual interaction and, at the same time, gives us flexibility to work on challenging and novel genomes.

Acknowledgements

The JCVI Viral Genomics Group is supported by federal funds from the National Institute of Allergy and Infectious Disease, the National Institutes of Health, and the Department of Health and Human Services under contracts no. HHSN272200900007C.

Bat coronavirus project is collaboration with Kathryn Holmes and Sam Dominguez, University of Colorado Medical Center.

The authors would like to thank members of the Viral Genomics and Informatics group at JCVI.

References

Viral genome sequencing by random priming methods. Djikeng A, Halpin R, Kuzmickas R, Depasse J, Feldblyum J, Sengamalay N, Afonso C, Zhang X, Anderson NG, Ghedin E, Spiro DJ. BMC Genomics. 2008 Jan 7;9:5A virus discovery method incorporating DNase treatment and its application to the identification of two bovine parvovirus species.  Allander T, Emerson SU, Engle RE, Purcell RH, Bukh J.

Note

This post is based on a poster by Nadia Fedorova, Danny Katzel, Tim Stockwell, Peter Edworthy, Rebecca Halpin, and David E. Wentworth.

Scientist Spotlight: Meet David Wentworth

During the height of the H1N1 Flu pandemic, David Wentworth was running a microbial genetics laboratory at the Wadsworth Center, New York State Department of Health (NYSDOH) where he was instrumental in developing a method to amplify influenza genomes regardless of strain using “universal primers” or short strands of DNA that recognize conserved segments across the genomes of many different flu strains. This amplification process was developed to generate recombinant influenza A viruses (the most common flu type affecting humans and animals) that could be used for the production of new vaccines. From a clinical swab it took his team 9-12 days to develop vaccine seed stocks. It was this work that first brought Dave to JCVI’s attention.

Several years ago Dave began collaborations with JCVI scientists to sequence human and avian influenza viruses. The collaborations intensified two years ago when all pandemic flu samples (or suspected flu samples) were first sent to Dave’s lab so the virus could be amplified in sufficient quantities for sequencing using his new amplification pipeline. The amplification took only a day and then isolated, non-infectious, DNA was sent to JCVI for sequencing. JCVI was the natural choice for this work since we are host to the government-funded “Influenza Genome Sequencing Project,” with the goal of sequencing large numbers of viral genomes to help scientists worldwide to understand how flu viruses evolve and cause disease. JCVI researchers then deposited influenza sequences into GenBank within two days of receiving DNA from Dave’s lab, enabling researchers worldwide to track what strains are circulating and how they are evolving. JCVI has sequenced over 75% of the influenza genomes in GenBank, the NIH public repository for sharing genetic sequencing data.

Influenza Genome Amplification Directly From Clinical Specimens

Influenza Genome Amplification Directly From Clinical Specimens (Zhou, B., M. E. Donnelly, D. T. Scholes, K. St.George, M. Hatta, Y. Kawaoka, and D. E. Wentworth. 2009. J.Virol. 83:10309-10313.).

Dave was soon invited for a talk at JCVI. “The opportunities at JCVI were to help build the [viral genomics] program. And already good, quality people are here studying viruses with a focus on viral evolution and sequencing analysis,” Dave remarked. “Being part of generating that information, I think makes you have a better feel for the biology.” The capabilities for viral sequencing combined with IFX strengths and the interest in viral evolution at JCVI was a draw for Dave and he soon joined the team. Moreover, there are opportunities at JCVI to work with collaborators who send specimens from various regions of the world for sequencing so that we can “more deeply understand the mutations that contribute to virulence,” he said. He is particularly interested in antigenic drift (how viruses escape immunity) that contributes to the “annual influenza escape,” which is critical in developing vaccine strains.

New Live Attenuated Vaccine Approaches

New Live Attenuated Vaccine Approaches. Figure shows influenza RNA polymerase activity (GFP) at various temperatures. Mutations engineered into the genome (PB1-Mut3, PB2-Mut4) synergize and inhibit replication at higher temperatures of the lung (37 C) or fever (39 C).

The need for new and improved methods to develop vaccines, coupled with the advances in synthetic genomics developed at JCVI led to the formation last year by JCVI and the company Synthetic Genomics Inc. of a new company, Synthetic Genomics Vaccines Inc. (SGVI). JCVI scientists, through SGVI, are working on a three-year collaboration agreement with Novartis to apply synthetic genomics tools and technologies to accelerate the production of the influenza seed strains required for vaccine manufacturing. The agreement, supported by an award from the U.S. Biomedical Advanced Research and Development Authority (BARDA), could ultimately lead to a more timely and effective response to seasonal and pandemic influenza outbreaks. The idea is to create viruses de novo or synthesize genes critical for its antigenicity and put these in normal vaccine strains for production. The goal of the work at SGVI is to synthesize a virus in one week, or rather a seed stock, which still needs to be amplified in big fermenters. New seed stocks take 3-4 weeks to produce which is currently a rate liming step.

You don’t hear too many people singing its praises and saying “I love the flu!” as Dave has remarked, but put in context, his enthusiasm for his work shines through best when talking about his love of teaching. He gets excited teaching young scientists about virology, especially helping them to understand the important areas to study, and where the research will lead to solve a major problem. “The rewarding part of being a mentor is to see all of the people who have found their niche – it might not be bench research but they are still carrying knowledge with them.”

David Wentworth DEW checking a hive in the late Spring.

David Wentworth DEW checking a hive in the late Spring.

Aside from spending time with his family, Dave enjoys a hobby started by his dad – to cultivate honey bees. A community gardens group at a middle school in Albany, NY was looking for bees to pollinate their plants. Dave spearheaded the effort and used it as a learning tool for kids, who helped feed honey to caterpillars and moths. He also used to give lectures on bee cultivation and has taught college courses in animal science. Dave’s enthusiasm for science among his students and peers could be considered infectious, just like the subject of his research!