Differential Cellular Tropism of Lentivirus and Adeno-Associated Virus in the Brain of Cynomolgus Monkey

Abstract

Many researchers are using viruses to deliver genes of interest into the brains of laboratory animals. However, certain target brain cells are not easily infected by viruses. Moreover, the differential tropism of different viruses in monkey brain is not well established. We investigated the cellular tropism of lentivirus and adeno-associated virus (AAV) toward neuron and glia in the brain of cynomolgus monkeys (Macaca fascularis). Lentivirus and AAV were injected into putamen of the monkey brain. One month after injection, monkeys were sacrificed, and then the presence of viral infection by expression of reporter fluorescence proteins was examined. Tissues were sectioned and stained with NeuN and GFAP antibodies for identifying neuronal cells or astrocytes, respectively, and viral reporter GFP-expressing cells were counted. We found that while lentivirus infected mostly astrocytes, AAV infected neurons at a higher rate than astrocytes. Moreover, astrocytes showed reactiveness when cells were infected by virus, likely due to virus-mediated neuroinflammation. The Sholl analysis was done to compare the hypertrophy of infected and uninfected astrocytes by virus. The lentivirus infected astrocytes showed negligible hypertrophy whereas AAV infected astrocytes showed significant changes in morphology, compared to uninfected astrocytes. In the brain of cynomolgus monkey, lentivirus shows tropism for astrocytes over neurons without much reactivity in astrocytes, whereas AAV shows tropism for neurons over glial cells with a significant reactivity in astrocytes. We conclude that AAV is best-suited for gene delivery to neurons, whereas lentivirus is the best choice for gene delivery to astrocytes in the brain of cynomolgus monkeys.

Keywords: AAV; astrocyte; lentivirus; monkey; neuron; virus tropism.

Fig. 1. Timeline and schematic diagram of virus injection in cynomolgus monkey. (a) Protocol for stereotaxic surgery and post-mortem experiments. After post-fixation and incubation in sucrose, immunohistochemistry (IHC) was performed. (b) Cynomolgus monkey. Adeno-associated virus (AAV) and lentivirus were infected in putamen of cynomolgus monkeys. AAV preferred to infect neuron (NeuN+), lentivirus preferred to infect astrocytes (GFAP+).

Fig. 2. Infection by lentivirus. (a) Immunostaining of NeuN (red in horizontal line), GFAP (red in vertical line). Lenti-GFP was injected into the putamen region. The fluorescence is detected by confocal microscope imaging. Scale bar, 25 µm. (b) High magnification images of GFP expressing cell. GFP and GFAP are well colocalized. Scale bar, 5 µm. (c) Cell counting analysis for GFP expressing cells of colocalization with NeuN or GFAP. Approximately 60% of GFP expressing cells are colocalized with GFAP. Analysis is performed with ImageJ (n=99: total number of cells).

Fig. 3. Infection by AAV. (a) Immunostaining of NeuN (red in horizontal line), GFAP (red in vertical line). AAV-GFP was injected into the putamen region. The fluorescence is detected by confocal microscope imaging. Scale bar, 25 µm. (b) High magnification images of GFP expressing cell. GFP and NeuN are well colocalized in neuronal cell body. Scale bar, 5 µm. (c) Cell counting analysis for GFP expressing cells of colocalization with NeuN or GFAP. Approximately 80% of GFP expressing cells are colocalized with NeuN. Analysis is performed with ImageJ (n=206: total number of cells).

Fig. 4. Reactiveness of astrocytes induced by virus infection. (a) Immunohistochemistry and sholl analysis images of cell infected by lenti-GFP. (b) Immunohistochemistry and sholl analysis images of cell infected by AAV-GFP. (c) Sum of intersections of uninfected cells and infected cells within region injected lenti-GFP. Analysis was performed by ImageJ. The numbers in each bar are the total number of analyzed cells in each group. Student's t-test (^*p>o.o5). (d) Sum of intersections of uninfected cells and infected cells within region injected AAV-GFP. By ImageJ. Student's t-test (^*p<o.o5, n=total number of cells in each group). (e) Ramification index of uninfected cells and infected cells within region injected lenti-GFP. By ImageJ. Student's t-test (^*p>o.o5, n=total number of cells). (f) Ramification index of uninfected cells and infected cells within region injected AAV-GFP. By ImageJ. Student's t-test (^*p<o.o5, n=total number of cells).