A Porcine Model of Zika Virus Infection to Profile the In Utero Interferon Alpha Response.

Pigs are highly relevant to model human in utero Zika virus (ZIKV) infection because both species have similar physiology, genetics, immunity, fetal brain development, and postnatal brain growth. The virus causes persistent in utero infection and replicates in the fetal brain, fetal membranes, and placenta. Subclinical persistent in utero infection in mid-gestation also increases interferon alpha (IFN-α) levels in fetal blood plasma and amniotic fluid. Moreover, we demonstrated altered IFN-α responses in porcine offspring affected with subclinical in utero ZIKV infection. Elevated levels of in utero type I interferons were suggested to play a role in fetal pathology. Thus, the porcine model may provide an understanding of ZIKV-induced immunopathology in fetuses and sequelae in offspring, which is important for the development of targeted interventions. Here, we describe surgery, ultrasound-guided in utero injection, postoperative monitoring, sampling, and cytokine testing protocols.

S/MAR Element Facilitates Episomal Long-Term Persistence of Adeno-Associated Virus Vector Genomes in Proliferating Cells.

Adeno-associated virus (AAV) vectors are one of the most frequently applied gene transfer systems in research and human clinical trials. Since AAV vectors do not possess an integrase activity, application is restricted to terminally differentiated tissues if transgene expression is required long term. To overcome this limitation and to generate AAV vectors that persist episomally in dividing cells, AAV vector genomes were equipped with a scaffold/matrix attachment region (S/MAR). After a mild antibiotic selection, cells transduced with AAV-S/MAR established colonies that maintained long-term transgene expression (>50 population doublings) from replicating AAV vector episomes in the absence of further selection. Unexpectedly, with a lesser but still significant efficiency, the control vector (AAV-ΔS/MAR), a standard single-stranded AAV vector, also established stable transgene-expressing colonies, most of which were maintained as replicating episomes rather than integrated vector genomes. Thus, based on the result in HeLa cells, it is concluded that AAV vector genomes per se possess the ability to establish episomal maintenance in proliferating cells, a feature that can be enhanced by incorporation of a foreign genomic element such as an S/MAR element.