I completed my PhD on October 2016 at the University of Warwick under the supervision of Professor David J. Evans. I recently joined Dr. Christopher Brook lab as a postdoc at the University of Illinois at Urbana-Champaign (UIUC). My research aims to understand the biological machinery that governs viral evolution. During my PhD I utilized Next Generation Sequencing (NGS) to study recombination - a crucial evolutionary mechanism - in poliovirus. My work with Dr. Brooke focuses on studying the Defective Interfering (DI) particles in Influenza Virus. Understanding how viruses evolve will help us to predict their behaviors and consequently develop 'smart' and more efficient vaccines.
My goal is to succeed in my studies at the UIUC and build a more comprehensive understanding of how viruses evolve in particular, and how evolution works in general.
My name is Fadi AlNaji (image top right). I passed my PhD examination (viva voce) in 20th of July 2016. I did my PhD under the supervision of Professor David Evans (image bottom right). I joined the Evans lab in July 2012 to study genetic recombination between two different serotypes of poliovirus; poliovirus type 1 (PV1) and poliovirus type 3 (PV3). Recombination is an evolutionary mechanism by which viruses co-infecting a cell can generate novel hybrid genomes. These may exhibit changes in tropism or pathogenesis. I studied an extensive panel of recombinant viruses using next generation sequencing (NGS), a high-thoughput technology that allowed me to both quantify and qualify recombination junctions in a mixed virus population.
Recombination contributes to the emergence of modified viruses associated with disease outbreaks. For example in the live-attenuated vaccine, recombination between the PV serotypes - that compose the vaccine - has been documented. Moreover, recombination between live attenuated vaccine and co-circulating viruses can produce virulent hybrid virus. Therefore, understanding recombination and how to control it would help to develop safer vaccines composed of non-recombinogenic and genetically stable strains.
Viruses with RNA genomes constitute the most common types of emerging pathogens amongst humans. Of these, viruses with positive-sense, single-stranded genomes have large population sizes and small genomes and, related to this, error-prone RNA-dependent RNA polymerases (RdRp). During replication, the mutations caused by the RdRp generate diversity, an evolutionary mechanism which enables the viruses to survive under changing environmental pressures. More extensive genetic variation is achieved through recombination between co-infecting viruses.
Studies of the viable recombination molecules from cells co-infected with different serotypes of polioviruses are confounded by the relatively low frequency of the recombinants in comparison to the high levels of parental viruses produced. To overcome this, and other limitations, a system called CRE-REP was developed in Evans lab that enables the recovery of recombinants alone from dually transfected cells. This approach was developed on the assumption that recombination occurs by a by a ‘copy choice’ mechanism, which involves polymerase template switching during negative-strand synthesis.
The CRE-REP system demonstrated that recombination is a biphasic process in which initial recombinants containing limited genomic duplications undergo a secondary process termed ‘resolution’ in which the additional sequences are lost with the consequent increase in viral fitness. Recombinant genomes with additional inserted sequences were termed ‘imprecise’ recombinants, with fully resolved recombinants being termed ‘precise’. Despite the Despite the fact that only imprecise recombinants with insertions were detectable in the CRE-REP assay, recombinants could also theoretically contain deletions (figure 1).
Since the CRE-REP assay involved harvesting virus progeny 24-48 hours post infection and isolating by limit dilution in HeLa cells, it was hypothesized that isolating recombinants at an even earlier stage would decrease the selection and allow detection of a wider range of recombinants. Therefore, in my project, I developed a viability-independent assay involving PCR and NGS sequencing to amplify intertypic recombinants between wild-type poliovirus type 1 and type 3 in the absence of any selection for specific markers (unmodified viruses). For the sake of comparison the targeted region in my project was the same that was targeted by the CRE-REP assay.
The resulting viruses were sequenced, analysed and the recombination junctions were displayed using Parallel Coordinates - a visualisation tool that was developed for this purpose. An example can be seen in figure 2. The lines within the map show the connection between the location from where the RdRp had disassociated from the donor and re-associated to the acceptor. The lines are horizontal (slope = 0) if the recombinants did not contain any insertion or deletion.
Figure 2 Parallel Coordinates map of recombinants amplified from the P2 region of poliovirus.
The detected junction locations were visualized using the Parallel Coordinates tool, which was developed for this project. The PV1 genome (the donor strand) is in white on the left side. The recipient genome is on the right side. The genomes are represented by cartoon drawings referring to the regions of interest (within the P2 region) with the coding regions indicated. The lines between the genomes represent recombinants and connect between the junction locations in both genomes. The red lines denote out-of-frame recombinants, and blue lines indicate in-frame recombinants. The slope of the line reflects the size of inserted or deleted fragments, while the direction of the line refers to the type of recombinants (horizontal line = precise, negative-slope line = deletion, positive-slope line = insertion).
During this project, several hundred recombinants were isolated, sequenced and analysed. As a consequence, several high-quality maps were generated to show the locations and features of all types of recombination (precise, imprecise-deletion and imprecise-insertion). This enabled the subsequent analysis of the recombination junction for RNA sequences or structures that might influence the process. These studies have provided important insights into the recombination process. They have additionally developed methods that will be useful for the analysis of resolution, the second stage of this important fundamental mechanism of virus evolution.