Multiplex Reverse Transcription-PCR for Simultaneous Surveillance of Influenza A and B Viruses.

Influenza A and B viruses are the causative agents of annual influenza epidemics that can be severe, and influenza A viruses intermittently cause pandemics. Sequence information from influenza virus genomes is instrumental in determining mechanisms underpinning antigenic evolution and antiviral resistance. However, due to sequence diversity and the dynamics of influenza virus evolution, rapid and high-throughput sequencing of influenza viruses remains a challenge. We developed a single-reaction influenza A/B virus (FluA/B) multiplex reverse transcription-PCR (RT-PCR) method that amplifies the most critical genomic segments (hemagglutinin [HA], neuraminidase [NA], and matrix [M]) of seasonal influenza A and B viruses for next-generation sequencing, regardless of viral type, subtype, or lineage. Herein, we demonstrate that the strategy is highly sensitive and robust. The strategy was validated on thousands of seasonal influenza A and B virus-positive specimens using multiple next-generation sequencing platforms.

Application of a targeted-enrichment methodology for full-genome sequencing of Dengue 1-4, Chikungunya and Zika viruses directly from patient samples.

The frequency of epidemics caused by Dengue viruses 1-4, Zika virus and Chikungunya viruses have been on an upward trend in recent years driven primarily by uncontrolled urbanization, mobility of human populations and geographical spread of their shared vectors, Aedes aegypti and Aedes albopictus. Infections by these viruses present with similar clinical manifestations making them challenging to diagnose; this is especially difficult in regions of the world hyperendemic for these viruses. In this study, we present a targeted-enrichment methodology to simultaneously sequence the complete viral genomes for each of these viruses directly from clinical samples. Additionally, we have also developed a customized computational tool (BaitMaker) to design these enrichment baits. This methodology is robust in its ability to capture diverse sequences and is amenable to large-scale epidemiological studies. We have applied this methodology to two large cohorts: a febrile study based in Colombo, Sri Lanka taken during the 2009-2015 dengue epidemic (n = 170) and another taken during the 2016 outbreak of Zika virus in Singapore (n = 162). Results from these studies indicate that we were able to cover an average of 97.04% ± 0.67% of the full viral genome from samples in these cohorts. We also show detection of one DENV3/ZIKV co-infected patient where we recovered full genomes for both viruses.

Dissecting Japan's Dengue Outbreak in 2014.

Despite Japan's temperate climate, a dengue outbreak occurred in Tokyo for the first time in over 70 years in 2014. We dissected this dengue outbreak based on phylogenetic analysis, travel interconnectivity, and environmental drivers for dengue epidemics. Comparing the available dengue virus 1 (DENV1) E gene sequence from this outbreak with 3,282 unique DENV1 sequences in National Center for Biotechnology Information suggested that the DENV might have been imported from China, Indonesia, Singapore, or Vietnam. With travelers arriving into Japan, Guangzhou (China) may have been the source of DENV introduction, given that Guangzhou also reported a large-scale dengue outbreak in 2014. Coinciding with the 2014 outbreak, Tokyo's climate conditions permitted the amplification of Aedes vectors and the annual peak of vectorial capacity. Given suitable vectors and climate conditions in addition to increasing interconnectivity with endemic areas of Asia, Tokyo's 2014 outbreak did not come as a surprise and may foretell more to come.

Molecular determinants of plaque size as an indicator of dengue virus attenuation.

The development of live viral vaccines relies on empirically derived phenotypic criteria, especially small plaque sizes, to indicate attenuation. However, while some candidate vaccines successfully translated into licensed applications, others have failed safety trials, placing vaccine development on a hit-or-miss trajectory. We examined the determinants of small plaque phenotype in two dengue virus (DENV) vaccine candidates, DENV-3 PGMK30FRhL3, which produced acute febrile illness in vaccine recipients, and DENV-2 PDK53, which has a good clinical safety profile. The reasons behind the failure of PGMK30FRhL3 during phase 1 clinical trial, despite meeting the empirically derived criteria of attenuation, have never been systematically investigated. Using in vitro, in vivo and functional genomics approaches, we examined infections by the vaccine and wild-type DENVs, in order to ascertain the different determinants of plaque size. We show that PGMK30FRhL3 produces small plaques on BHK-21 cells due to its slow in vitro growth rate. In contrast, PDK53 replicates rapidly, but is unable to evade antiviral responses that constrain its spread hence also giving rise to small plaques. Therefore, at least two different molecular mechanisms govern the plaque phenotype; determining which mechanism operates to constrain plaque size may be more informative on the safety of live-attenuated vaccines.

Analysis of Dengue Virus Genetic Diversity during Human and Mosquito Infection Reveals Genetic Constraints.

Dengue viruses (DENV) cause debilitating and potentially life-threatening acute disease throughout the tropical world. While drug development efforts are underway, there are concerns that resistant strains will emerge rapidly. Indeed, antiviral drugs that target even conserved regions in other RNA viruses lose efficacy over time as the virus mutates. Here, we sought to determine if there are regions in the DENV genome that are not only evolutionarily conserved but genetically constrained in their ability to mutate and could hence serve as better antiviral targets. High-throughput sequencing of DENV-1 genome directly from twelve, paired dengue patients' sera and then passaging these sera into the two primary mosquito vectors showed consistent and distinct sequence changes during infection. In particular, two residues in the NS5 protein coding sequence appear to be specifically acquired during infection in Ae. aegypti but not Ae. albopictus. Importantly, we identified a region within the NS3 protein coding sequence that is refractory to mutation during human and mosquito infection. Collectively, these findings provide fresh insights into antiviral targets and could serve as an approach to defining evolutionarily constrained regions for therapeutic targeting in other RNA viruses.