@phdthesis{01JBV096FRRR07Q56A5QHWB76Q,
  abstract     = {{Severe viral gastroenteritis in newborn piglets (less than 3 days old) poses a significant challenge for the pig industry, with mortality rates reaching up to 100% in some cases. However, these rates drop dramatically around the peri-weaning age (3-4 weeks after birth). The primary culprits are porcine enteric coronaviruses, such as transmissible gastroenteritis virus (TGEV), and porcine rotaviruses. The reasons behind this age-dependent decrease in susceptibility are not well understood. Therefore, this doctoral thesis was aimed to investigate the underlying causes of this phenomenon.
Chapter 1 discussed the current knowledge and gaps in this area. Chapter 2 outlined the aims of the doctoral research.
First part of experiments (Chapter 3) focused on intestines in terms of changes occurring in the intestinal morphology, number of mucus-producing cells and the expression of coronavirus receptors APN, DPP4, ACE2, and TMPRSS2 in pigs with aging. Villus height and crypt depth increased with age from 3 days to 3 months in duodenum and ileum but not in mid-jejunum, where the villus height decreased from 580µm at 3 days to 430µm at 3 months. Enterocyte length-to-width ratio increased from 3 days to 3 months in all intestinal regions. The number of mucus-producing cells increased with age in the intestinal villi and crypts. The Brunner’s glands of the duodenum contained the highest concentration of mucus-producing cells. The expression of APN was highest in the small intestinal villi at all ages. DPP4 expression slightly decreased over time in jejunum and ileum; it was highest in the ileal villi of 3-day-old piglets (70.2% of cells). ACE2 and TMPRSS2 positive cells increased with age in jejunal and ileal crypts and were particularly dominant in the ileal crypts (>45% of cells). The expression pattern of the selected coronavirus receptors was very different and not correlated with the age-dependent susceptibility to viral infections. However, the number of mucus-producing cells increased over time and may play an essential role in protecting enteric mucosa against intestinal viruses.
Thus, the second part of experiments (Chapter 4) focused on the intestinal mucus. The age-dependent blocking activity of porcine ex-vivo intestinal mucus against TGEV was observed. Single Particle Tracking (SPT) revealed that TGEV had significantly higher logarithmic diffusion coefficient values in 3-day mucus compared to 3-week mucus. TGEV and charged and uncharged control nanoparticles diffused freely in 3-day mucus while hindered by 3-week mucus in the diffusion model; TGEV mimicked the diffusion behavior of negatively charged carboxylated particles. On ST cells, the virus inoculum made by incubating TGEV with mucus before inoculation of ST cells, the average number of TGEV-infected ST cells per five fields was significantly reduced with 3-week mucus (30.9±11.9) compared with 3-day mucus inoculum (84.6±16.4). These results show that 3-week mucus has a significant TGEV-blocking activity compared to 3-day mucus in free diffusion and infection of the underlying susceptible cells.
This was further explored by examining the age-related physiochemical changes in porcine intestinal mucus and their implications for enteric viral infections. The 3-week mucus exhibited less shear thinning and higher viscosity values from 0.1 to 100 shear rate per second as compared to the 3-day mucus. The mean pore diameter for 3-day mucus (234.56±129.5nm) was higher than that of 3-week mucus (152.60±94.4nm). Furthermore, more than 80% of the pores in 3-day mucus were larger than the average diameter of TGEV/PRCV particles as compared to only 50% in 3-week mucus. Label-free proteomic analysis showed an increased expression of mucin 13, known for negatively regulating the tight junctions in intestinal epithelium, in 3-day-old pigs. In 3-week-old pigs, a higher expression of mucin 2, a type of secreted mucin which is known for inhibiting coronavirus infection, was observed. Aminopeptidase N (APN), the main receptor for TGEV was also upregulated in 3-day mucus. Taken together, it was concluded that the physiochemical differences between the intestinal mucus of 3-day-old and 3-week-old pigs show key changes that could explain the decrease in viral susceptibility of older pigs.
Lastly, Chapter 5 discussed the thesis findings, limitations, and future research pathways.}},
  author       = {{Saleem, Waqar}},
  keywords     = {{TGEV,Mucus,Intestine,Coronavirus infection,Gastroenteritis,Age-dependent infection,Infection block}},
  language     = {{eng}},
  pages        = {{VI, 178}},
  publisher    = {{Ghent University. Faculty of Veterinary Medicine}},
  school       = {{Ghent University}},
  title        = {{Mucus matters : unraveling its role in age-dependent susceptibility to enteric viral infections}},
  year         = {{2024}},
}