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1.
Figure 1

Figure 1. MV F expression in infected neurons. From: Neurokinin-1 enables measles virus trans-synaptic spread in neurons.

48 hpi with MV-Ed (MOI=3), primary CD46+ neurons and Vero cells were fixed for immunostaining or solubilized in protein sample buffer as described. The percent of infected cells was determined by immunochemistry, after which the protein lysate volume equivalent to 105 infected cells was then subjected to Western blot analysis using an anti-F antibody. Size standards are indicated at right. Shown is one representative experiment of three.

Nina Makhortova, et al. Virology. ;362(1):235-244.
2.
Figure 2

Figure 2. Fusion inhibitory peptide prevents MV spread in primary neurons. From: Neurokinin-1 enables measles virus trans-synaptic spread in neurons.

Vero cells and CD46+ neurons were plated onto coverslips as described, and infected with MV-Ed, MOI=0.1. Six hpi, FIP was added to achieve a final concentration of 200 μM, and slips were fixed and immunostained 3 days thereafter. (A) and (B): Vero cells; (C) and (D): neurons. (A) and (C): DMSO added; (B) and (D): FIP added. Original magnification = 400X. Shown are fields from one representative experiment of three at this virus and dose concentration. No differences were noted using MOIs ranging from 0.1-3.

Nina Makhortova, et al. Virology. ;362(1):235-244.
3.
Figure 5

Figure 5. Infection in neurokinin-1 knockout neonatal mice. From: Neurokinin-1 enables measles virus trans-synaptic spread in neurons.

(A) Survival curves following MV challenge in CD46+/NK-1+ neonates (black triangles, n=11); CD46-/NK-1+ neonates (white circles, n=20); and CD46+/NK-1 KO neonates (black circles, n=12). Shown is one representative experiment of three. NSE-CD46+ neonates were chosen because these mice are permissive for MV infection and develop CNS disease within 1 week post-challenge (Lawrence et al., 1999). (B) RT-PCR analysis for MV-nucleoprotein of total brain RNA, harvested from neonatal CD46+/NK-1+ and CD46+/NK-1 KO mice at 6 dpi with MV-Ed, as described in Materials and Methods.

Nina Makhortova, et al. Virology. ;362(1):235-244.
4.
Figure 3

Figure 3. FIP prevents de novo infection in neurons. From: Neurokinin-1 enables measles virus trans-synaptic spread in neurons.

Vero cells (A-G) and primary CD46+ neurons (H-N) were plated on coverslips as described. FIP (200 μM) (D-G; K-N), or an equivalent volume of DMSO (A-C; H-J) were added and, after a 1 hr incubation, cells were infected with MV-Ed, MOI=3. Coverslips were fixed and immunostained 1-3 dpi. For the washout experiments (G and N), at 3 dpi the cells were washed and replaced with fresh media without FIP. Samples were then collected 48 hours post-FIP removal, equivalent to 5 dpi. Representative fields from one of four independent experiments are shown. Original magnification = 100X.

Nina Makhortova, et al. Virology. ;362(1):235-244.
5.

Figure 4. Effect of neuropeptide transmitters on MV spread. From: Neurokinin-1 enables measles virus trans-synaptic spread in neurons.

(A) Amino acid sequences of neuropeptides used. Gray boxes indicate active sites of the neurotachykinins and homology with FIP. (B) Inhibitory effect of each neuropeptide on MV spread. Neurons were infected with MV-Ed, and neuropeptides were added to a final concentration of 200 μM; for Substance P, three doses were tested: 200, 100 and 50 μM. Samples were collected 3 dpi and counted; values reflect the percent infection as compared with the DMSO control. The horizontal line indicates the initial level of infection after ∼1 round of replication (24 hpi). Because the peptides were added after infection, this represents the maximal achievable inhibitory effect. Shown is one representative experiment of five.

Nina Makhortova, et al. Virology. ;362(1):235-244.
6.

Figure 6. Weight loss, morbidity and MV infection levels in untreated and aprepitant-treated NSE-CD46+/RAG-2 KO mice. From: Neurokinin-1 enables measles virus trans-synaptic spread in neurons.

(A) Mice were infected as described and administered aprepitant orally one day prior to infection (double dose), one day after infection, and every other day thereafter. Mice were monitored daily, and scored from 0-4, based on the defined criteria (see Materials and Methods). Average values (obtained between days 6 and 10) are shown. Mice were also weighed on the day before infection to establish individual baselines, and every other day thereafter. Maximal weight changes are shown as percent weight loss or gain. (B) Mice were infected as described, and collected between 10-12 days post-infection. Shown are immunohistochemical results from untreated (a-c) and aprepitant-treated (d-i) mice. (d-f): sick animals at collection; (g-i): healthy animals at collection. Original magnification=20X.

Nina Makhortova, et al. Virology. ;362(1):235-244.

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