Abstract
Pseudorabies virus (PRV) initially replicates in the porcine upper respiratory tract. It easily invades the mucosae and submucosae for subsequent spread throughout the body via blood vessels and nervous system. In this context, PRV developed ingenious processes to overcome different barriers such as epithelial cells and the basement membrane. Another important but often overlooked barrier is the substantial mucus layer which coats the mucosae. However, little is known about how PRV particles interact with porcine respiratory mucus. We therefore measured the barrier properties of porcine tracheal respiratory mucus, and investigated the mobility of nanoparticles including PRV in this mucus. We developed an in vitro model utilizing single particle tracking microscopy. Firstly, the mucus pore size was evaluated with polyethylene glycol coupled (PEGylated) nanoparticles and atomic force microscope. Secondly, the mobility of PRV in porcine tracheal respiratory mucus was examined and compared with that of negative, positive and PEGylated nanoparticles. The pore size of porcine tracheal respiratory mucus ranged from 80 to 1500 nm, with an average diameter of 455±240 nm. PRV (zeta potential: −31.8±1.5 mV) experienced a severe obstruction in porcine tracheal respiratory mucus, diffusing 59-fold more slowly than in water. Similarly, the highly negatively (−49.8±0.6 mV) and positively (36.7±1.1 mV) charged nanoparticles were significantly trapped. In contrast, the nearly neutral, hydrophilic PEGylated nanoparticles (−9.6±0.8 mV) diffused rapidly, with the majority of particles moving 50-fold faster than PRV. The mobility of the particles measured was found to be related but not correlated to their surface charge. Furthermore, PEGylated PRV (-13.8±0.9 mV) was observed to diffuse 13-fold faster than native PRV. These findings clearly show that the mobility of PRV was significantly hindered in porcine tracheal respiratory mucus, and that the obstruction of PRV was due to complex mucoadhesive interactions including charge interactions rather than size exclusion.
Highlights
Herpesviruses are double-stranded DNA viruses with the potential to cause severe diseases in different species
Atomic force microscopy (AFM) images were obtained in ambient conditions in air using a multimode scanning probe microscope (Digital Instruments, Santa Barbara, CA, USA) equipped with a Nanoscope IIIa controller. 5 mm65 mm scans were recorded in tapping mode using a silicon cantilever (OTESPA, Veeco, CA, USA)
Diameter 124.660.5 232.662.4 538.067.5 unravel the invasion of Pseudorabies Virus (PRV) through the mucus layer, we have set up an in vitro model of porcine tracheal respiratory mucus and analyzed the mobility of PRV through it
Summary
Herpesviruses are double-stranded DNA viruses with the potential to cause severe diseases in different species. Many members of the subfamily Alphaherpesvirinae initially replicate in the epithelial cells of the respiratory and/or genital mucosae [1,2,3]. After local replication in the epithelial cells, these viruses gain access to the connective tissue and find their way to blood vessels and nerve endings for spread throughout the host [4,5,6,7]. Different important barriers of the host try to hamper PRV invasion. It has been shown that in order to invade, different alphaherpesviruses including PRV use proper processes to pass an important barrier underneath the epithelium, the basement membrane [2,8,9]. Prior to epithelial cell entry, a mucus layer that coats the epithelium and serves as the first line of defense, has to be overcome by the virus [10]
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