Given the profound impact of Zika infection on the neurodevelopment in newborns, a model of the fetal brain to support in situ studies of infection would be ideal. For ethical reasons, however, living human brain tissue is very difficult to come by, thus requiring the development of alternative methods for studying human neurons. In 2012, Sir John B. Gurdon and Shinya Yamanaka were awarded the Nobel prize in physiology or medicine for their ground-breaking discovery that mature cells can be reprogrammed to become pluripotent. This has led to a plethora of methods being developed to create more physiologically relevant cellular models for drug discovery research. New protocols to derive neuronal cells (cortical neurons, motor neurons and glial cells, like astrocytes) and iPSC-derived brain organoids may result in the generation of relevant cellular models to elucidate underlying mechanisms associated to fetal infection and neurological complications (Lanko et al, 2017; Quian et al, 2017), especially given the intrinsic immaturity related to cells/tissues derived from iPSCs (Suzuki et al, 2016, McNutt, et al, 2015). These approaches can offer relevant insights on the development of the microcephaly condition, but likely multiple models will be needed to coalesce a clear picture of defective neurodevelopment. The Lanko protocol is limited to single populations and to intercellular molecular analysis but amenable to high-throughput screening; while the Quian model can more accurately recapitulate the fetal brain architecture and composition, with the caveat that the methods for derivation can be more complex and much slower to perform (Quian et al, 2017).
and the method of the year goes to?
In 2013 Nature Methods' choice for Method of the Year was single-cell sequencing, as they saw its ability to transform many areas of biology and medicine. Single-cell sequencing involves isolating a single cell and examining its nucleic acid information using next-generation sequencing technologies, to fully characterize cells and provide powerful insights into cellular differences. The field of single-cell genomics is advancing rapidly, yet researchers face multiple challenges, including reproducibility, sensitivity, scalability and cost, particularly when large numbers of cells are analyzed. In more recent years, automation and miniaturization of RNA-Seq protocols has been shown to help to address these limitations. This year, a group of researchers from Stanford University employed single cell RNA-seq with the highly sensitive and accurate Smart-seq2 protocol to examine developmental heterogeneity of microglia and brain myeloid cells (Barres et al, 2018). This study showed better insights into the functions which are carried out by subsets of microglia during different stages of development and adulthood or within specific brain regions. They found that the majority of adult microglia with homeostatic signatures are remarkably similar in transcriptomes, regardless of brain region. By contrast, postnatal microglia represent a more heterogeneous population.
However, conventional approaches ignore heterogeneity in virus abundance across cells, resulting in the lack of a thorough understanding of the host response to the viral infection. Another group of researchers from the Chan Zuckerberg Biohub examined a new approach to probe the host transcriptome together with intracellular viral RNA at the single cell level, viscRNA-Seq (virus-inclusive single cell RNA-Seq). They applied viscRNA-Seq to monitor dengue and Zika virus infection in cultured cells and discovered extreme heterogeneity in virus abundance. Using this variation to identify host factors that showed complex dynamics and a high degree of specificity for either virus, they discovered novel proviral and antiviral factors. viscRNA-Seq is a powerful approach to assess the genome-wide virus-host dynamics at single cell level (Quake et al, 2018).
Employing automation and miniaturization of scRNA-Seq protocols, including viscRNA-Seq, has significantly decreased both the cost and labor required for single-cell transcriptome studies, making analysis of hundreds to thousands of single cells feasible.
I trust the irony is still not lost on you that both Quake and Barres are using their TTP Labtech mosquito liquid handling instruments to investigate zika virus infections!
All of these methods have helped to bring us closer to identifying potential therapeutic interventions for Zika virus infection and prevention, but we’re not there yet. Stay tuned as we continue to explore this research area and find out if there are other notable contributions being made on the journey to Zika eradication.
Did you miss the previous blogs to this journey - catch up below
>> part 1: Once bitten – twice shy! A look at the mosquito-borne flavivirus Zika
>> part 2: The Zika journey continues – have you been bitten by the mosquito?