Characterization of Porcine Enterocytes and Their Susceptibility to Rotavirus

Characterization of porcine enterocytes and their susceptibility to rotavirus

Word count: 8979

Yana Rommens Student number: 01710629

Supervisor: Prof. Dr. Hans Nauwynck Supervisor: Dr. Sebastiaan Theuns Supervisor: PhD candidate: Nick Vereecke

A dissertation submitted to Ghent University in partial fulfilment of the requirements for the degree of Master of Veterinary Medicine

Academic year: 2019 - 2020

Ghent University, its employees and/or students, give no warranty that the information provided in this thesis is accurate or exhaustive, nor that the content of this thesis will not constitute or result in any infringement of third-party rights. Ghent University, its employees and/or students do not accept any liability or responsibility for any use which may be made of the content or information given in the thesis, nor for any reliance which may be placed on any advice or information provided in this thesis.

Preamble Part of this thesis was supposed to be based on research under the supervision of the Department of Virology. In April 2020, three weeks of experiments were planned to try to shed light on the complicated pathways and morphology of enterocytes in the small intestines of neonatal and older piglets. Finding differences in morphology, expression of receptors and several other factors could be a useful in finding exactly what makes young animals more susceptible to rotavirus.

Due to the Covid-19 pandemic all research projects were cancelled and this thesis is now a literature study. As not much is known about the exact reasons why young animals are more sensitive to rotavirus, the second part is much shorter than intended as a result of the absence of data from my own research project.

Preface I would like to thank Prof. Dr. Hans Nauwynck for giving me the chance to get to know the lab of virology in the first semester. I would also like to express my thanks to Dr. Sebastiaan Theuns and PhD- candidate Nick Vereecke for their guidance and explanation of the different techniques used in the lab and allowing me to learn them. Their never seizing enthusiasm for their work motivated me and I learned so much. I would also like to thank them for finding the time to correct my thesis and helping me along with their suggestions. In addition to this, I am also grateful for the help of technical assistent Marthe Pauwels who found the time to help me with the different techniques.

Finally, I would also like to thank my friends for always offering support. Not only in these last months, but throughout the whole process.

Table of contents

1. Summary ...... 6 1.1 English summary ...... 6 1.2 Dutch summary ...... 6

2. Introduction ...... 7

3. Rotavirus ...... 8 3.1 Structure of the virus ...... 8 3.1.1 Genome ...... 8 3.1.2 Viral particle ...... 8 3.2 Classification...... 9 3.3 Epidemiology ...... 10 3.3.1 Zoonotic potential ...... 11 3.4 Replication cycle ...... 11 3.5 Pathogenesis ...... 13 3.6 Pathology and clinical signs...... 13 3.6.1 Macroscopic lesions ...... 13 3.6.2 Histology ...... 14 3.7 Immunology ...... 14 3.7.1 Innate immune response ...... 14 3.7.2 Acquired immune response ...... 15 3.7.3 Passive immune response ...... 15 3.8 Prevention ...... 15 3.8.1 Hygiene measures ...... 16 3.9 Vaccination ...... 16

4. Characteristics of the small intestine ...... 18 4.1 General anatomy of the digestive tract ...... 18 4.2 Structure of the small intestine ...... 18 4.2.1 Cells of the epithelium ...... 18 4.2.2 Cell-cell adhesions ...... 19 4.2.3 Cell-matrix adhesions ...... 20 4.3 Changes in small intestine due to aging ...... 21 4.3.1 Postpartum ...... 21 4.3.2 Weaning ...... 21 4.4 Age-dependent susceptibility...... 22 4.4.1 Influence of TLR3 ...... 22 4.4.2 Inflammasome ...... 22 4.4.2 Maturing of gastro-intestinal tract and cells ...... 23

5. Discussion ...... 24

6. References ...... 25

1. Summary 1.1 English summary One of the most important problems in piglets is diarrhea. In the gestation unit and shortly after weaning piglets are utmost susceptible to it. Diarrhea can have multiple causes such as viruses, bacteria and parasites. Porcine rotaviruses (RVs) are often a cause of acute viral gastro-enteritis (Theuns, 2015). According to a study conducted by Debouck and Pensaert (1983) the virus is present in almost every farm and will infect almost every pig in the course of their life.

The target cells for rotaviruses are the enterocytes of the small intestines. The epithelium of the small intestine consists of different cell types, each with their own function. In addition to this, cell-cell adhesions form a barrier against pathogens including rotaviruses since receptors are found on the basolateral sides of enterocytes and with intact cell-cell adhesions these receptors are less easy to reach. Other factors such as Toll-like receptor 3 seem to play an important role insusceptibility of enterocytes to rotavirus. The intestines are a complex and intriguing part of the gastro-intestinal system and further extensive research is necessary to discover and comprehend all possible receptors, pathways and other factors and mechanisms. Understanding all this could lead to a better healthcare and therefore better production results in intensive farming.

1.2 Dutch summary Een van de belangrijkste problemen bij biggen is diarree. In de kraamstal en na het spenen zijn biggen er zeer gevoelig aan. Diarree kan veroorzaakt worden door verschillende pathogenen, zoals virussen, bacteriën en parasieten. Porciene rotavirussen zijn vaak de oorzaak van acute gastro-intestinale enteritis (Theuns, 2015). Volgens een studie van Debouck en Pensaert (1983) is het virus aanwezig op bijna elk bedrijf en zullen zo goed als alle dieren geïnfecteerd worden.

De doelwitcellen van het rotavirus zijn de enterocyten van de dunne darm. Het epitheel van de dunne darm is opgebouwd uit verschillende soorten cellen, elk met hun eigen functie. Verder vormen cel-cel adhesies een belangrijke barrière tegen pathogenen en dus ook rotavirus, waarvan de receptor zich op het baso-laterale gedeelte van de cel bevindt. Met intacte cel-cel adhesies zijn deze receptoren dus moeilijker te bereiken. Andere factoren, zoals onder andere Toll-like receptor 3, blijken een belangrijke rol te spelen in de gevoeligheid van enterocyten voor rotavirus. De dunne darm is een complex en intrigerend deel van het gastro-intestinale stelsel en verder uitgebreid onderzoek is nodig om alle receptoren, pathways en andere mechanismen te ontdekken en begrijpen. Het volledig in kaart brengen van al deze factoren leidt tot het verbeteren van de gezondheidszorg en welzijn van dieren en dus ook tot het verbeteren van de productieresultaten in de industriële veehouderij.

2. Introduction Pig farming in Belgium has gone through a major change in the last century. Since the 1950’s, our country is completely self-sustaining and has become one of the leading countries in export of pork products worldwide. Intensive pig farming takes up the largest part of the agricultural sector in Belgium1. Small influences can have considerable impact on profitability in this sector and financial losses are often just around the corner. Therefore maintaining an optimal health status on pig farms is one of the components fundamental to keeping profitability and animal welfare at the highest level (Honeyman, 1996). One of the most important problems in piglets is diarrhea. In the gestation unit and post weaning piglets are utmost susceptible to it. Diarrhoea can have multiple causes such as bacteria, parasites and viruses. Porcine rotaviruses (RVs) are often a cause of acute viral gastro-enteritis (Theuns, 2015). According to a study conducted by Debouck and Pensaert (1983) the virus is present in almost every farm and will infect almost every pig in the course of their life. In piglets, rotavirus A, B and C are associated with diarrhea (Chepngeno et al., 2019). As to this date, the prevention of rotavirus is difficult due to the fact that knowledge gaps exists in multiple areas necessary to fully understand the pathways that RVs use to infect their hosts. Further insights in immunity, entry factors, distribution among populations, improved cell culture methods to gain relevant research results and multiple other factors are imperative in creating effective vaccines. In this thesis the basics of RVs are explained, as well as a brief summary situating the current knowledge of the characteristics of enterocytes and the changes they undergo in their maturation. Causing them to be more resilient to RV infection and clinical presentation of the disease.

1 Brochure varkensloket, aandoeningen bij varkens, afdeling duurzame landbouwontwikkeling (https://www.varkensloket.be/Portals/63/Documents/aandoeningen_bij_varkens.pdf). Last seen on 1/4/2020

3. Rotavirus 3.1 Structure of the virus 3.1.1 Genome Rotavirus is a non-enveloped double-stranded RNA virus and belongs to the family of Reoviridae. The genome consists of 11 gene segments which encodes for 6 structural (VP1 – VP4, VP6 and VP7) and 6 non-structural proteins (NSP1 – NSP6). Each gene segment encodes for one protein except for the 11th segment, which codes for both NSP5 and NSP6 (Tuanthap et al., 2019). The non-structural proteins are responsible for both the replication of the genome, virion assembly, pathogenesis and for antagonizing the innate immune responses of the host (Crawford et al., 2017).

3.1.2 Viral particle Rotavirus derives its name from the wheel-like virion observed by electron microscopy (Vlasova et al, 2017). A concentric triple-layered icosahedral protein capsid encases the gene segments. The core layer of the virus consists of a shell, formed by VP2. The gene segments along with VP1 and VP3 are situated within the core layer. The inner layer consists of VP6 proteins and the outer layer is formed by VP7 and VP4. A schematic representation of the rotavirus particle is given in Figure 1.

Fig. 1 Build-up of the Rotavirus particle A) shows the general structure of a rotavirus particle and places of the corresponding viral proteins (VPs). VP2 forming the inner shell and VP1 interacting with it from the inside. VP3 is not represented in the model. The middle layer of VP6 is visible with around it the VP7 outer shell with protruding VP4 spikes. B) shows a detailed conformation of a VP4 spike with the VP8* domain attached on top of the VP5* domain. They are connected with VP6 of the inner layer through a pore in the VP7. Trp indicates the position of the proteolysis by trypsin (Rodríguez et al., 2019).

Core layer Inside this VP2 shell, each gene segment is associated with one VP1 and a VP3 protein. VP1 is the RNA- dependent RNA polymerase, an essential enzyme for replication of the positive RNA-strands of the virus (Vidal et al., 2018). This VP1 protein is associated with a capping enzyme (VP3). VP3 will form a cap at the 5’ end of transcribed positive stranded rotavirus RNA. Interactions between VP2 and VP1 are necessary for the integrity of the inner layer surface and are also important for the activation of RNA-dependent RNA polymerase (Rodríguez et al., 2019). Together, the gene segments, VP1/VP3 complex and VP2 are known as the single layered particle (SLP). Together with NSP1, VP3 portrays a role in evading the innate immune system and will be discussed later on (Ogden et al., 2014; Rodríguez et al., 2019).

Inner layer The VP6 proteins are situated in the inner layer and are the largest antigenic mass within the virion (Bishop, 1996). They interact with the core and the outer layer. This part of the viral structure is

responsible for translation and can activate infection when ssRNA is released in the cytoplasm of the host cell. Together with the SLP and the VP6, a double layered particle (DLP) is formed.

Outer layer VP7 and VP4 in the outer layer are responsible for adhesion to and entry in the host cell. VP4-spikes protrude from the outer VP7 shell layer. VP4 is responsible for adhesion and entry in the host cell but before this is possible and the viral particle can become infectious, conformational changes are necessary. These changes are caused by proteolysis under the influence of trypsin that is released from the pancreas into the gastro-intestinal tract of animals. The cleavage of VP4 results in two domains VP5* and VP8*. These domains remain associated by hydrophobic interactions. The VP8* domain sits on top of the VP5* domain and they are connected with VP6 of the inner layer through a pore in the VP7 (Theuns, 2015). The VP8* is a globular domain and plays a role in the attachment to carbohydrates on the the viral receptor. This domain varies between strains causing some genotypes to be more susceptible depending on the affinity of the interaction between the different carbohydrates (Liu et al., 2017; Pang et al., 2018; Sun et al., 2018).

3.2 Classification It is necessary to define organisms in order to study them in an organized manner. Each new entity is given a name and classified according to their characteristics. This process is called taxonomy and the basics where established in 1735 by Carolinus Linnaeus with his Systema Naturae. For viruses, a committee was founded in 1966 to universalize the classification of viruses and naming of virus taxa. This International Committee on Taxonomy of Viruses (ICTV) is responsible for developing guidelines in naming taxa, approving proposed taxonomy and names. In light of this, rotavirus taxonomy is as follows. Rotavirus is part of the subfamily of Sedoreovirinae which is in its turn part of the Reoviridae family Lefkowitz et al., 2017). This tree of viral families is further elaborated in Figure 2.

Fig 2. Taxonomy of Rotaviruses Taxonomy starts its hierarchy from broad to narrow, so the genus Rotavirus belongs to the realm of Riboviria, kingdom of Orthornavirae, phylum of Duplomaviricota, class of Resentoviricetes, order of Reovirales, family of Reoviridae, subfamily of Sedoreovirinae. Officially 9 species of Rotavirus are acknowledged (Lefkowitz et al., 2017).

Based on VP6 protein relationships, rotavirus is classified into at least 9 official species A-J, without an E species. Group A, B and C are the major causes of clinical cases of diarrhea in pigs (Vlasova et al., 2017). Within each rotavirus species, VP7 and VP4 induce neutralizing antibodies and are used to type Rotaviruses within their groups. This is called serologic typing. VP4 determines the P serotype and VP 7 the G serotype (e.g. G2P4) However, due to the high diversity of strains, serotyping is not used in practice. It is replaced by genotyping VP7 en VP4. (Todd et al., 2010). VP7 is a glycoprotein and the different genotypes are referred to as ‘G’ followed by a specific number. VP4 is a protease-sensitive protein and the different genotypes are represented by ‘P’ also followed by a specific number in brackets. Together these form the base of the dual-genotyping system. As an example, G1P[8] is a correct way to address the VP4/VP7 genotype constellation of a major human rotavirus strain. A lot of different genotype have been described for both humans and animals. So far 15 G genotypes en 27 P genotypes have been detected. Because of the high genetic diversity the dual-typing system was extended to a full-genome classification system. This is widely used for rotavirus A strains, but knowledge on non-RVA strains is more limited. In this system nucleotide percent identity cut-off values are established for all gene segments. In this system, each of the viral genes VP7-VP4-VP6-VP1-VP2- VP3-NSP1-NSP2-NSP3-NSP4-NSP5 are represented by Gx-P[x]-Ix-Rx-Cx-Mx-Ax-Nx-Tx-Ex-Hx genotypes (Matthijnssens et al., 2008). Complete genome analysis of RVA strains was performed at the Laboratory of Virology, Faculty of Veterinary Medicine, UGhent. This analysis showed high variety in VP4 and VP7, but a stable composition of the remaining gene segments. This could indicate that the stable part plays an important role in the viral replication of RVs (Theuns et al., 2015). A Rotavirus Classification Working Group (RCWG) was formed to maintain proper classification of different strains of RVA (Vlasova et al., 2017).

For RVC, a comparable system with classification based on nucleotide sequences was established (Chepngeno et al., 2019). Ten G genotypes, seven P genotypes and seven I genotypes are thus far discovered for RVC. Porcine RVC mostly belong to G1, G3 and G5 – G10. Due to the difficulty of adapting RVB and RVC strains to cell culture and the limited and variable shedding in feces, serological characterization is not possible. This problem is bypassed with next generation sequencing (NGS). Full genomes can be analyzed using this technique. Since only two RVC genomes are fully analyzed, further information through NGS might lead to interesting insights in epidemiology and infectivity of RVC (Theuns et al., 2016). Results thus far suggest that porcine RVB genotypes may be host species- and region-specific (Vlasova et al., 2017).

3.3 Epidemiology Infections with rotavirus A (RVA) are present worldwide, but are not always associated with diarrhea. Worldwide prevalence rates of RVA in pigs range from 3.3% - 67.3%, but is strongly influenced by the diagnostics that are being used. Season does not influence the prevalence and certain genotypes appear to re-emerge from time to time. On farm-level the prevalence of RVA ranges from 61% - 74%. In Belgium G2 – G5, G9 and G11 and P[6], P[7], P[13], P[23], and P[27] genotypes were isolated from pigs with and without symptoms. The most common genotype combination worldwide is G5P[7]. Rotavirus C (RVC) is also present worldwide and associations with diarrhea are observed. It appears to be a common pathogen in conditions with poor hygiene and is spread by infected animals via the feces. Chepngeno and colleagues found that RVC is more frequently found in suckling piglets. And although both non-symptomatic piglets and piglets with diarrhea can shed viral particles in the feces, diarrheic piglets shed a significantly higher amount. Continuous screening is necessary to obtain an accurate view of the prevalence (Chepngeno et al., 2019). For RVB, there is little data concerning prevalence,

pathogenic potential and genetic characteristics but RVB is often found in older pigs (Chepngeno et al., 2019; Vlasova et al., 2017).

3.3.1 Zoonotic potential Pigs are reservoirs for zoonotic RVs. It is likely that emerging human RV are created by gene re- assortment by porcine species. When two different RV strains infect the same cell, a new genetic variant may be formed with a combination of gene segments from both strains. This process is called antigenic/genetic shift. In addition to this, interspecies transmission is also sporadically observed. Most human RVA strains do not need sialic acid for cellular attachment but use histo-blood group antigens (HBGA). Similar polymorphic HBGA are found in pigs (e.g. A and H antigens). These H antigens are recognized by P[6] of RVA strains and may be the reason why these strains are often transmitted between humans and pigs (Vlasova et al., 2017). These interspecies transmission are more frequently observed in developing countries as people there live in closer proximity with animals (Vlasova et al., 2017). Since genetic expression of HBGAs can differ among populations, it might also explain why some regions appear to be more susceptible to developing new strains (Crawford et al., 2017). Most interspecies transmissions are dead end, they will therefore not spread from human to human all though it is possible that some new strains will have this aptitude (Theuns, 2015). In contrast to the many reports of RVA strains having zoonotic potential, there is no evidence that RVC strains present the same zoonotic characteristics (Vlasova et al., 2017).

3.4 Replication cycle In order for the virus to multiply, it needs to infect a host cell. Several steps are required for rotaviral particle to enter a cell and replicate itself. Figure 3 shows a schematic overview of the different steps necessary for the virus to multiply.

Fig 3. Replication cycle of rotavirus. The figure shows the attachment to the viral receptor followed by receptor mediated endocytosis, release of the double layered particle and dsRNA transcription. Translation of mRNA to create viral proteins and replication of the viral genome. In viroplasms a new double-layered particle is formed. These double-layered particle are internalized by NSP4 that is present as a receptor in the endoplasmatic reticulum (ER). A bilipid envelope surrounds the double-

layered particle after passing through the ER. Incorporation of VP4 and VP7 replace the bilipid layer. Subsequently, two different mechanisms for the release of viral particles has been observed. Depending on the cell culture used viral particles are either release by cell lysis or by budding. This is another example that demonstrates that pathways might possibly be different in porcine enterocytes. (Desselberg, 2014).

Entrapment of rotavirus in the mucus layer Once the VP4 spike on the outer shell of the virus is proteolyzed by trypsin, conformational changes divide it into two domains, VP5* and VP8*. One of these domains, VP8*, is used to attach itself to glycans present in the intestinal mucus. This first attachment prevents the elimination of viral particles by gastro-intestinal movement (Rodríguez, 2017).

Attachment to viral receptor Initial attachment to the host cell is established though a connection of VP8* and glycans. Depending on the RVA strain, specific molecules are used for attachment. Some rotavirus strains use sialic acids (SA) of the host cell to form a bond with VP8*. These SA are monosaccharides and are found on the terminal branches of N-glycans, O-glycans and gangliosides. However, some strains appear to be independent of these terminal SA for their attachment. In this case, it is suggested that the VP8* domain binds with internal sialic residues present on gangliosides (Rodríguez, 2017).

Receptor-mediated endocytosis Internalization of the viral particle is accomplished by endocytosis. Depending on the viral strain, endocytosis is a clathrin-mediated depending on cholesterol and dynamin, while others are clathrin- and caveolin independent, but still use cholesterol and dynamin. Most research on endocytosis was performed in kidney epithelial cell line MA104 cells derived from an African Green Monkey. Consequently, it is uncertain that the same pathways take place in porcine small intestinal enterocytes (Rodríguez, 2017).

Release of double-layered particle Once the particle is located in the endosome, low Ca2+ -concentration dissolves the VP7 and VP4 layer. A double-layered particle remains and double-layered particles are released in the cytoplasm (Ludert et al., 1987).

RNA transcription As a result of the disengagement of the VP7 layer, VP6 and VP2 encounter some conformational changes causing type 1 channels to widen which makes the core of the particle accessible for nucleotides and ions. The dsRNA segments can now begin transcription to mRNA. An enzymatic transcription complex of VP1, the RNA-dependent RNA polymerase and VP3 produces a ssRNA transcript from each of the eleven gene segments (Jenni et al., 2019).

Translation This mRNA is then used as a template to produce viral proteins. At the 3’ end, NSP3 recognizes a translation enhancer and binds to host cell initiating factors such as eIF4G and eIF4E. The interaction of NSP3 and eIF4E downregulates host cell protein translation allowing the viral protein syntheses to take over from normal cell activities (Desselberger, 2014).

Viroplasm assembly NSP2 and NSP 5 activate the formation of cytoplasmatic inclusion bodies (viroplasms). NSP2 promotes the depolymerization of microtubuli to recruit the tubulin in the viroplasm structure and will form complexes with VP1 and VP2 (Criglar et al., 2014; Martin et al, 2010).

Incorporation of transcriptionally active double-layered particle in viroplasm Due to interactions between different VP2 proteins in the viroplasm, single layered particles are formed. The inner capsid layer is formed when the VP2 layer is enclosed by VP6 resulting in a double- layered particle.

Budding through endoplasmatic reticulum and release of viral particles These double-layered particle are internalized by NSP4 that is present as a receptor in the endoplasmatic reticulum (ER). A bilipid envelope surrounds the double-layered particle after passing through the ER. Incorporation of VP4 and VP7 replace the bilipid layer. Subsequently, two different mechanisms for the release of viral particles has been observed. Depending on the cell culture used viral particles are either release by cell lysis or by budding. This is another example that demonstrates that pathways might possibly be different in porcine enterocytes (Taylor et al., 1996).

3.5 Pathogenesis The virus is transmitted through fecal-oral route. When viral particles are ingested, the RV replication occurs inside the enterocytes and entero-endocrine cells of the small intestine (Lundgren and Swenson, 2001). Replication of the virus takes place in the apical cells on the intestinal vili. As a result of the destruction of the absorptive enterocytes, malabsorption followed by osmotic diarrhea is seen in infected animals. The concentration of intracellular calcium increases, which will disrupt the cytoskeleton and tight junctions, and increase permeability of the enterocytes. In addition to this rotavirus produces an enterotoxin (NSP4) that will further disrupt the electrolyte imbalance and enhances secretory diarrhea due to Ca2+ efflux from the endoplasmatic reticulum through a phospholipase C-dependent mechanism (Vlasova et al., 2017). The increased levels of Ca2+ in the cytoplasm activate Ca-dependent chloride channels which leads to an increased secretion of chloride in the intestinal lumen. This creates an osmotic gradient and water is attracted to the lumen. Hence, creating a secretory diarrhea. This secretory diarrhea is further complicated by the activation of the enteric nervous system. 5-hydroxytriptamine (serotonin) is released from entero-endocrine cells due to the cytoplasmatic increased Ca2+ concentration. Elevated serotonin concentrations activate the enteric nerves that influence the motility of the small intestine. This rise in concentration stimulates motility. In addition to this, 5-HT also stimulates vagal nerves associated with nausea and vomiting. Another factor that is influenced by rotavirus is the gastric emptying, which is delayed by RV infection. The precise mechanisms that are responsible for this delay are not yet uncovered but suggestions have been made that it is altered by either increased secretion of gastric hormones or activation of gastric neuronal pathways. A combination of both is possible as well (Crawford et al., 2017).

3.6 Pathology and clinical signs 3.6.1 Macroscopic lesions Lesions from RVA, RVB and RVC are similar, therefore macroscopic examination cannot be used to determine rotavirus species. The intestines are dilated and filled with watery, yellow/grey feces. The intestinal wall is thinner than in non-infected animals and the stomach can be dilated (Diseases of swine, 2019).

3.6.2 Histology Jejunal and ileal epithelial cells swell during the early stages of the infection and this will become more pronounced as the infection progresses. Short after infection, the epithelial cells of the jejunum and ileum are shed into the lumen. As a result the villi become shorter and thinner, the crypts are elongated and cellular hyperplasia is visible. In addition to this, the villi are covered by abnormal cuboidal cells. Clinical signs can be observed before histological chances take place, which indicates that clinical presentation cannot only be explained by histological changes (Diseases of Swine, 2019). Even though disease can be subclinical, it is important to keep in mind that production losses are still present. Other factors such as diet, coinfection and vitamin A deficiencies have an influence on the severity and duration of the diarrhea associated with RV. Especially vitamin A deficiency seems to have an important influence. Vitamin A is involved in the synthesis of the secretory component (SC), which is found on the cell membrane of enterocytes and facilitates transportation of IgA to the intestinal lumen. Deficiencies can lead to a failed production of the SC and therefore less antibodies will reach the lumen (Ahmed et al., 1991). So deficiencies lead to more severe diarrhea, higher viral titers in feces and more intestinal damage. It can also alter B cell immune responses after vaccination which can lead to less immunologic protection and lower immunoglobulin levels (Diseases of swine, 2019).

3.7 Immunology 3.7.1 Innate immune response Pattern recognition receptors (PRRs) are the first to notice infections such as rotavirus. These receptors are present on enterocytes and cells of the immune system. Examples of PRRs are ATP-dependent RNA helicase DDX58, also known as RIG-I, which recognizes 5’ triphosphate uncapped RNA and interferon- induced helicase C domain-containing protein 1 (IFIH1 or MDA5) which recognizes dsRNA. Both can induce a type I and type III interferon responses. Other PRRs are Toll-like receptors (TLRs). Where myeloid differentiation primary response protein MYD88, an adaptor protein, that presents a role in TLR signaling. The precise pathway is unknown (Crawford et al., 2017). An increased expression of Toll- like receptor 3 generate an improved protection as well (Theuns, 2015). TLR 3 and retinoic acid- inducible gene-like receptors (RLRs) such as Rig-I and Mda5 present in the cytosol will recognize dsRNA molecules. For its pathway, TLR3 uses the TIR-domain which contains the adaptor inducing interferon- β (Trif) adaptor molecule. Both of the TLR and RLR pathway activate NF-κB, a MAP-kinase, and IRF3 which will lead to the induction of proinflammatory and antiviral reponse genes.In addition to this it is important that INF-α production in the body is balanced as an imbalance could lead to more severe clinical symptoms. NSP1 will serve as a suppressor of type 1 INF responses which will cause an abolishment of the host’s innate immune response (Vlasova et al., 2017). In a study of Pitt and colleagues (2017) it was found that TLR3, which is MYD88 independent, is expressed at higher levels in older mice and might be part of the explanation for the reason why adult animals are less susceptible to RV infection. An antiviral (IFN-α) and pro-inflammatory cytokines (such as IL-2) are produced by plasmacytoid dendritic cells. This will cause an inhibition of the replication of the virus and stimulate other immune cells such as natural killer cells. These produce granzymes, perforines and TNF-α which can lyse infected cells. Plasmacytoic dendritic cells and natural killer cells play an important role in reducing infection and severity of clinical disease.

3.7.2 Acquired immune response Acquired immunity has a humeral and a cellular component. Both prevent and react to infections in their own specific way. As the humeral immune response is important to construct neutralizing antibodies created by B-cells. Viral antigens are presented to T helper cells (Th cells) and this will activate them. The Th cells secrete IFN-γ and will activate B cells (Vlasova et al., 2017). B cell lymphocytes in the small intestine are mainly located in the lamina propria and the Peyer’s patches. The B cells start producing IgA antibodies and differentiate into plasma cells. These plasma cells produce massive amounts of IgA which will thereafter be transported to the intestinal lumen. This takes place through internalization of IgAs and transcytosis. When IgA is passing through, a secretory piece is added. This is necessary to stabilize the antibody and protect it against the hostile environment in the gastro-intestinal lumen. VP7 and VP4 are the main proteins that induce neutralizing antibodies, but antibodies against VP6 and VP2 are also a part of the immune response (Theuns, 2015).

3.7.3 Passive immune response Piglets are born without having received antibodies in utero. This means that after birth both IgG and IgA need to be transferred to the piglets though the colostrum of the sow. In the piglets IgA will provide a local protection and will keep receiving these antibodies throughout the lactation period. These IgA antibodies are present in the milk of the sow post infection. The infection will trigger the activation of the acquired immune response and B cells are activated in the lymphoid tissue of the small intestine. Part of these activated B cells migrate to the mammary gland where they become plasma cells. The gut-mammary-sIgA axis is necessary to provide the milk of the sow with antibodies. The secretory piece of the IgA (sIgA ) is added when the antibodies are transported into the milk and are necessary to protect them from the harsh environmental conditions of the gastro-intestinal tract. At weaning the lactogenic immunity stops abruptly and combined with other factors such as stress the animals will become sensitive to infection again. They no longer receive IgA antibodies through the sow’s milk and their own acquired immune system is not yet able to effectively fight of infections. This causes an immunity gap which will leave weaned piglet vulnerable to infection. Whilst RVA is more prevalent in older suckling and weaned piglets, RVC is more common in suckling piglets. This could suggest that maternal lactogenic immunity is insufficient. It is necessary to develop an antibody detection test (e.g. antibody detection Enzyme-Linked Immuno Sorbent Assay (ELISA)) to evaluate the influence of maternal immunity on the course of the disease. In a study of Chepngeno and colleagues (2019), it was demonstrated that gilts had significantly more diarrheic litters, higher mortality rates following infection and lower concentration of IgG and IgA in colostrum than multiparous sows. Effectivity of protection against RVA through colostrum is linked with antibody titers since litters with higher IgA and IgG titers in the serum of sows proved to be healthier. For RVC this correlation was not found, suggesting that systemic antibodies do not contribute to the protection of piglets. In the same study, multiparous sow also had higher titers of IgG and IgA in the milk suggesting that transfer of antibodies in the milk and the transfer of plasma cells from the gut to the mammary gland might be influenced by parity as well.

3.8 Prevention The transmission of RV can occur through direct contact or via the environment since the virus is very resistant (Lundgren and Svensson, 2001). Apart from hygiene measures, there are not many available products to prevent infection. Only a few vaccines are available in Belgium but practical experience leads to the conclusion that these are not always as effective as advertised. Another method to prevent infection is the use of spray dried plasma. This contains immunoglobulin, growth factors, biological

active peptides and other factors that protect the intestines. A passive immunity is simulated with this technique and infected animals will not show symptoms but can still excrete virus in the feces (Corl et al., 2007).

3.8.1 Hygiene measures Maintaining proper hygiene is one of the most important strategies for preventing disease in general. Since the use of antibiotics in treatment is reduced to prevent resistance, it is of the utmost priority to keep infection pressure in the environment as low as possible. Since low infection pressure will lead to less disease or diseases remaining subclinical. Good hygiene not only helps reduce infection with RV, but other pathogens will be eliminated as well. As most Belgian farms work with an all-in-all-out system, it is advised to clean at the end of each production round. Each compartment has to be cleaned and disinfected. Organic materials such as feces should first be removed since pathogen can survive for extended periods of time in these organic materials and the material itself will also reduce the efficacy of most disinfectants. After the removal of organic matter, cleaning with detergent can start. It is recommended to let the detergent soak in order to eliminate more pathogens. After this soaking period, the stables have to be cleaned with water (high pressure). The next step in the process is to disinfect. Finally, the surfaces should be allowed to dry, as this is highly effective as even more pathogens will not survive the draught. However, in the field the cleaning and disinfection protocol is often neglected. Since rotaviruses are considerably resistant and stable in the environment, specific methods should be used to properly inactivate the virus particle. According to a study of Estes and colleagues (1997), the virus is resistant to both chloroform and ether. It can be deactivated by the use of products with acidic or alkalic pH, by 5M EDTA and 5M EGTA. Infectivity decreases 99% by heating up to 50°C for 30 min.

In order to further decease the heating time to 15min it is possible to add 2M MgCl2/CaCl2/NaCl. MgSO4 has a stabilizing effect on RV infectivity. Further, sodium chloride, iodophors, and quaternary ammonium disinfectants are not suited for RV inactivation. While inactivation of the virus is obtained in two hours by exposure to10% formal (Theuns, 2015). Chandler-Bostock and Mellitus (2015) tested the efficacy of several disinfectants. A general conclusion of this study was that the presence of organic matter significantly reduced the efficacy of disinfecting. So the importance of removing organic matter and cleaning with detergent prior to using disinfectants is not to be underestimated. As possible products for disinfection Bi-OO-cyst was found to be most effective along with Virkon S and Vanadox.

3.9 Vaccination Increasing the lactogenic immunity can be obtained by vaccinating the sow, while vaccines that boost active immunity are administered to the young pigs. Availability is Belgium is quite limited. Only one vaccine is registered (FIXR ROTA COLI) by Kernfarm. This vaccine contains an inactivated RVA and induces neutralizing antibodies. It is unknown which strain is present in the vaccine. The vaccine also contains E. Coli with different virulence factors. The vaccine is administered intramuscular and 2 injections of 2 cc are needed if the sows did not yet receive a primo-vaccination. The doses are given with a 2-4 week interval and the last one a least 2 weeks before farrowing. When the sows are revaccinated only one dose is needed 2-4 weeks before farrowing2. Since it is an inactivated vaccine and administered intramuscular, IgA production will not be stimulated. This is seen in the field when efficacy of the vaccine is not always as advertised. Before this vaccine was registered, veterinarians in the field used bovine rotavirus vaccines such as Rotavec Corona, but in Belgian legislation this is now no longer allowed since the registration of the species specific porcine vaccine. Different commercial

2 Vetcompendium, https://www.vetcompendium.be/nl/node/5508, last seen on 20/5/2020

vaccines are worldwide available, but are not registered in the EU. Some attenuated vaccines might even contribute to the development of new strains by gene re-assortment of vaccine strains and wild strains which can spread in the population (Vlasova et al., 2017). Monitoring of genotypes is not only useful for creating a general image of circulating strains, but will in addition shed a light on whether vaccines have an influence on RV evolution (Crawford et al., 2017).

4. Characteristics of the small intestine 4.1 General anatomy of the digestive tract The digestive tract consists of different sections, each with their own morphology and functions. The oral cavity is where it all starts and the following components are pharynx, esophagus, stomach, duodenum, jejunum, ileum, caecum, colon, rectum and anus. Together the duodenum, jejunum and ileum make up the small intestine. Since RV infections take place in the small intestine, these will be discussed in more detail further on. Both gallbladder and the exocrine part of the pancreas have ducts through which their exceptions can reach the duodenum (Helander and Fändriks, 2014).

4.2 Structure of the small intestine The lumen of the small intestine consists of specialized epithelium and is in direct contact with the environment. This indicates that, apart from its primary function such as absorption of nutrients, it is also a barrier that protects the organism against intestinal pathogens. This barrier is formed by special structure that ensures cell-cell adhesion. The epithelial cells are structured on finger-like protrusions which are called villi. These villi amplify the surface through which absorption can take place. Towards the serosal side, the villi form crypts. The epithelium consists of five different cell types: absorptive enterocytes, hormone-secreting endocrine cells, goblet cells, Paneth cells and M-cells (Junqueira et al., 2007).

Fig 4. Electon microscope image of the vili of the small intestine A) shows an image of vili in the duodenum. B) shows an image of vili in the jejunum (Helander and Fändriks, 2014).

4.2.1 Cells of the epithelium Enterocytes Within the crypts of Lieberkühn, stem cells differentiate into mature enterocytes and move alongside the villi to the top. Morphologically, these cells are cylindrical with a brush border on top. The brush border forms the microvilli which will increase the absorption surface even more. The enterocytes at the top of the villi are shed through apoptosis and as a result the complete epithelium is renewed every 4-5 days (Hall et al.,1994).

Entero-endocrine cells Different types of entero-endocrine cells are found throughout the whole length of the digestive tract and have different morphology and functions depending on their location. They are characterized by granules present at the basal side of the cell (Grube and Forssmann, 1979).

Goblet cells Goblet cells are found scattered along the villi and crypts. They produce mucus which will protect the enterocytes against damaging or corrosive agents and will ease the passage of content through the digestive tract. Goblet cells have a lifespan of approximately 3 days (Knoop and Newberry, 2018).

Paneth cells These cells are present in the bottom of the crypts and secrete specific proteins which influence microbial flora in the gut. Paneth cells have a longer turn-over of at least 3 weeks. Both Paneth cells and goblet cells are important as a first line of defense against enteric pathogens. The mucus and antimicrobial proteins they produce acts as a physical and biochemical barrier (Cui, 2019). These cells also use the MY88D pathway for activation of TLRs.

M-cells Microfold cells are part of the intestinal immune system. The cells are responsible for the phagocytosis and transcytosis of antigens. Underneath the M-cell, under the basolateral membrane, lymphocytes and phagocytes are located. Transfer of antibodies to these immune cells is an important step in initiating mucosal immune responses (Mabbott et al., 2013)

4.2.2 Cell-cell adhesions To protect the organism from pathogens and toxins, cells use complex protein networks that allow the absorption nutrients, water but keep damaging components in the gut lumen. 4 types of adhesions have so far been discovered in the intestines: desmosomes, tight junctions, adherence junctions and gap junctions, see Figure 5.

Fig. 5 Main cell-cell adhesions. From top to bottom: adherence junctions, that keep cells tightly together. Desmosomes, for the mechanical strength of the epithelium. Tight junctions, that serve as selective permeability junctions. Gap junctions, for intercellular communication (Wei and Huang, 2013).

Desmosomes Desmosomes are responsible for the mechanical strength of the epithelium. They consist of desmoglein 2 and desmocollin 2 which are proteins with an extracellular and intracellular domain. The extracellular domains establish contact with neighboring cells through a Ca2+ -dependent mechanism. The intracellular domain is connected with desmoplakin through plakoglobin and plakofilin, which in turn connects with the cells cytokeratine (Cernatano and Cirillo, 2017).

Tight junctions These intercellular connections are situated on the apical side of the cell and are formed by several proteins originating from different families of transmembrane proteins. The most important ones are claudin, occludin and junction adhesion molecule proteins (JAM proteins). Both claudin and occludin have extracellular loops which interact with loops from other adjoining cells. In contrast, JAM proteins have only one extracellular immunoglobin-like domain. Other tight junctions are formed by cytoplasmatic proteins. These zona occludens proteins are situated under the membrane and form connections with other tight junction proteins via specialized domains. In addition to this, the proteins are also responsible for connecting the transmembrane tight junctions to components of the cytoskeleton of the enterocytes (Zihni et al.,2016 ). These tight junctions serve as selective permeability barriers (Wei and Huang, 2013).

Adherence junctions Another group of proteins, E-cadherines, also contributes to forming an intestinal barrier. The extracellular domain of these transmembrane proteins changes conformation when exposed to Ca2+. This change in conformation allows the protein to form a tight connection with E-cadherines from neighboring cells. The intracellular domain associates with actine present in the cytoskeleton (Theuns, 2010).

Gap junctions Inter-cellular communication of enterocytes is possible because of small hemichannels that connect the cytoplasm from adjoining cells. Through these channels, small signaling molecules, such as ATP and cAMP, can be exchanged. This exchange is known as gap intercellular communication The gap juctions also play an important role in the homeostasis of enterocytes, formation of oral tolerance and innate immune response. The transmembrane proteins that forms these hemichannels are connexins. When epithelial damage occurs, important molecules for enterocyte migration are exchanged though the tunnels (Maes et al., 2015).

4.2.3 Cell-matrix adhesions The extracellular matrix has an important function as a microenvironment that supports epithelial cells in differentiation, migration and proliferation. The basal membrane is situated under the epithelium. Integrins attach the enterocytes to the membrane and serve as receptors that interact with extracellular molecules to mediate these different actions. Integrin α3β1, for example, binds laminin- 5 and is found at the base of epithelial cells. Laminin-5 is a component of the basal membrane (Lussier et al., 2000).

4.3 Changes in small intestine due to aging 4.3.1 Postpartum Since piglets are born without an active adaptive immune system, the first 24-48 hours are of the utmost importance to make sure piglets are provided with passive immunity. Macromolecules, such as immunoglobulins are present in the colostrum and can be absorbed via pinocytosis. After this process, the gut mucosal immune system starts do develop through contact with new antigens. A complex process is used to identify harmful and tolerate non-harmful antigens. Right after birth, the gut starts to change and develop. The maturation is initiated by the increased oxygen, presence of nutrients and hormones such as cortisol and epidermal growth factor. Along with the development of the intestine, the microbiological flora progresses to colonize the lumen. Fetal enterocytes are gradually replaced over the course of 3-4 week by adult enterocytes that have no endocytosis capacity. Not only do the enterocytes lose some activities, certain expressions of enzymes that are situated on the brush border change. An example of an enzyme that is highly active in the gut of newborn piglets is lactose, and its activity will decease when the piglet is weaned. Furthermore, the gastro-intestinal tissues have great need of essential amino acids for their growth and function. Most of the amino acids such as lysine and glutamine are not recruited from the nutrients in the intestinal lumen, but are acquired from the systemic arterial circulation (Diseases of swine, 2019).

4.3.2 Weaning In nature piglets are weaned gradually and the process takes about 15-20 weeks. The contact with the sow steadily decreases and at the same time explorative behavior develops and as a result the intake of solid feed is stimulated. In modern day farm practices, weaning is an utmost stressful period for the piglet and puts an immense pressure on the gastro-intestinal tract since changes occur very abrupt. In intensive livestock farming piglet are weaned at the age of 3-4 week3. The development to a fully functioning intestinal immune system takes up to 9 weeks and weaning before the process in completed will delay the development. This renders them more susceptible to infections. Furthermore, negative effect such as low and variable feed intake, growth stasis and compromised integrity of the small intestinal epithelium are associated with weaning at this age. The sudden change from the sow’s milk to the diet used in the nurturing unit causes villi to shorten and crypts become deeper. The morphological changes will also have an influence on the microvilli which will become shorter as well. Therefor it is important that piglets have the possibility to explore and consume feed in the gestation unit before they are weaned. In addition to this, it is advised to use feeds that are high in milk products, highly digestible and have low levels of anti-nutrient factors. This to maximize feed intake as soon as possible after weaning to prevent growth retardation and diarrhea. Stress in pigs is also responsible for a lower pH in the intestines. As a result, intestinal permeability increases which makes it easier for pathogens or toxins to get into the systemic circulation (Diseases of Swine, 2019). Another factor that increases sensibility to RV infections is the disturbance of the cell-cell adhesions. These adhesions as mentioned before, play an important role in forming a barrier against enteric pathogens. An in vitro study on a primary enterocyte culture system that disrupted these adhesions using EGTA, showed that infection of enterocytes increased as well as the ability of the virus to bind to these enterocytes. This can be explained by the location of a rotavirus receptor on the basolateral side of the cells, which becomes more accessible when cell-cell adhesions are no longer functioning optimally (Cui, 2019).

3 Maes, D., Syllabus ‘bedrijfsbegeleiding varken’, 2019-2020

4.4 Age-dependent susceptibility Rotavirus infections cause more problems in young animals. One of the hypothesis for this occurrence is that the innate immune responses develop further after birth. When rotavirus is ingested, several structural components are recognized by receptors on present on cells of the small intestine. These receptors are a key ingredient that forms one of the basic components to initiate immunity.

4.4.1 Influence of TLR3 TLR 3 and retinoic acid-inducible gene-like receptors (RLRs) such as Rig-I and Mda5 present in the cytosol will recognize dsRNA molecules. For its pathway, TLR3 uses the TIR-domain which contains the adaptor inducing interferon-β (Trif) adaptor molecule. Both of the TLR and RLR pathway activate NF- κB, a MAP-kinase, and IRF3 which will lead to the induction of proinflammatory and antiviral reponse genes. In a study of Pott and colleagues (2012), an oral mouse infection model was used to determine expression of receptors in different age groups. They found a significant difference of quantity of expression of TRL3. In young mice, TLR3 expression is found to be fundamentally lower and as the animals age, the expression multiplies. This correlated with a decreased susceptibility to infection, less viral shedding and a smaller extent of histological damage was observed. Another pathway dependent on the TLR3/Trif mechanism is the expression of the chemokine Rants. This chemokine recruits immune effector cells to the infection site. In addition to this, TLR3 seams to play an important role to activate the immune system when tissue damage occurs. Disruption of tissue due to viral toxins or when virus-infected cells are detected. Until now, the specific location of TLR3 is still unknown due to the fact that antibodies against TLR3 are not yet available. Another factor that is important for correct development of the enterocytes after birth is B-lymphocyte induced maturation protein 1 (Blimp 1). This factor is important to adapt the neonatal enterocytes to suckling conditions. As expression of Blimp 1 decreases, expression of TLR3 increases. Indicating an connection in the regulation of both (Pott et al., 2012).

4.4.2 Inflammasome Another component of the immune system is the inflammasome. This is a complex of cytosolic proteins that helps defend the host against pathogens and regulates inflammatory diseases by activating the pyroptotic cascade. The inflammasome sensors interact with or respond to pattern recognition receptors (PAMPs) and damage recognition receptors. The role of the inflammasome is not yet entirely know, but studies suggest it plays a major role in the activation of the innate immune response. Slowly, parts of these inflammasome pathways and components are uncovered. In a recent study of … a new cytosolic sensor for rotavirus was found. This NLRP9b is part of the NLR (NOD-like receptor)-family. In mice dsRNA is recognized by DHX9 which subsequently binds with NLRP9b and activates the inflammasome in intestinal cells. This activation stimulates the production of pro-inflammatory cytokines such as IL-1β and IL-18. Since rotaviruses have adapted remarkably well to their host, the virus uses a number of techniques to evade the innate immune system. This trickery of the innate immune system might also be the reason younger animals are more sensitive to clinical infection since their acquired immune system is not yet fully functional to back up the innate immune response. For example, NSP1 interferes with the interferon cascade. It prevents the activation of several factors necessary for the production of interferon (INF) and several cytokines. Another viral protein, VP3, encaps the viral mRNA which makes it difficult for RLRs to recognize it as viral RNA and thus efficiently evading a part of the innate immunity. In addition to the capping of the viral RNA, VP3 also antagonizes the interferon pathway by dividing 2′,5′-linked oligoadenylates (2-5A). This is a signaling molecule in the dsRNA-responsive

OAS/RNase L pathway (Morelli et al., 2015). This pathway is involved in an antiviral response stimulated by the production of interferon and leads to the suppression of viral replication by cleaving the viral RNA (Banerjee et al., 2019)

4.4.2 Maturation of gastro-intestinal tract and cells In another study performed in mice, Wolf et al. (1981) found that administering cortisone before weaning increased the speed of maturation of enterocytes and thus making them less susceptible to the virus. Even though this maturation is only partial, it appears to be sufficient to decrease sensitivity to infection. This effect can be explained by the decrease in transportation of macromolecules which are absorbed through pinocytosis. At the time of weaning, this process stops in mice and administering cortisone speeds up the process. Furthermore around the time of weaning, chymotrypsin and trypsin activity increases significantly. As RV VP4 needs trypsin to undergo a conformational change and become infective. This too might explained increased sensibility to the virus at this age. However, this study was performed in mice and rats so relevance to porcine biological changes might need to be researched further (Wolf et al., 1981). Although many factors that influence age-dependent susceptibility to rotavirus may still be unknown, a study of Gelberg (1992) found that piglets raised in an environment free of rotavirus do not develop age dependent resistance until 12 weeks of age indicating that resistance to clinical infection is caused by other factors than age-related changes in enterocytes. This in contrast to studies performed in mice.

As is often observed, older pig infected with rotavirus do not show the same severe symptoms as younger piglets. One of the possible factors that might protect the pig from clinical disease is the faster turn-over of enterocytes. In older animals turn-over of these cells take about 2-3 days, and slower in neonatal animals. This causes damaged cells to be replaced rather quickly. In addition to this, the water absorptive capacity of the colon is more developed in older animals. This is an added factor that reduces diarrhea since additional fluids secreted by the small intestine under the influence of rotavirus infection are resorbed in the colon. Therefore the component of secretary diarrhea is disabled (Gelberg, 1992). Even though rotavirus needs trypsin in order to become infective and this trypsin is not present in neonatal animals and colostrum contains anti trypsin factors, other mechanisms are used by the virus to infect enterocytes at this stage in life. It is suggested by Gelberg (1992) that infection occurs independent of specific viral receptor and is linked to the transport of macromolecules in the first days after birth. Since in the field almost all animals are infected with rotavirus clinically or subclinically, after recurrent infection antibodies develop a higher affinity for the virus neutralizing it more efficiently. In addition to this, higher titers of sIgA might also be available and be present in the lumen of the small intestine which will also contribute to a better protection in older animals (Gelberg,1992).

5. Discussion Almost every article written about rotavirus starts by stating that the virus is one of the most important causes for acute gastro-enteritis in young animals and children. In the pig farming industry healthy animals form the basis of a profitable enterprise and absence of disease is one of the pillars in animal welfare. Therefore, prevention of Rotavirus infections is one of the steps necessary to optimize farming.

A good knowledge of prevalence and disease associated with each rotavirus strain and genotype, as well as understanding the pathways and mechanisms the virus uses to infect host cells and evade the immune system, would help us to create better vaccines and therapeutics. In order to be successful, extensive further research is necessary in every area.

Due to the genetic diversity, and the emerging of new strains, epidemiology becomes dynamic and very complex. Since the virus has a zoonotic potential, keeping track of the genetics of the strains and genotypes using molecular diagnostics and the phenotypical characteristics is crucial. Further research is also imperative when it comes to the entry and internalization of the virus in the host cell. Considering that most research in this area is performed on cell cultures such as MA 104 cells, biological relevance and accuracy of pathways involved might be questioned. To determine the correct mechanisms involved, research on more representative cells will give new and better insights. In addition to this, the exact changes involved in intestinal maturation and in parallel with this, the mechanism for an improved protection against rotavirus infections, should be elucidated. In this context, more extensive research is needed. Better insights in the highly complex gastro-intestinal system would not only result in a better prevention of rotavirus-associated alimentary problems, but would also contribute to a more in-depth understanding of other pathogens. As for the factors that determine susceptibility to rotavirus, some studies have already been performed in rodents. Extrapolation of these results to pigs should be applied with caution and biological relevance should be tested.

Overall, more knowledge should be obtained in every aspect of the viral cycle and mechanisms involved in susceptibility. This will contribute to the development of better vaccines, therapeutics and preventive measures available in the ongoing fight with a pathogen responsible for considerable losses in pig farming worldwide.

6. References Ahmed, F., Jones, D., Jackson, A., 1991. Effect of vitamin A deficiency on the immune respone to epizoontic diarrhoea of infant mice (EDIM) rotavirus infection in mice. British Journal of Nutrition, 65(3), 474-485.

Banerjee, S., Gusho, E., Gaughan, C., Dong, B., Gu, X., Holvey-Bates, E., Talukdar M., Li Y., Weiss S. R., Sicheri F. et al., 2019. OAS-RNase L innate immune pathway mediates the cytotoxicity of a DNA- demethylating drug. Proceedings of the National Academy of Sciences, 116(11), 5071-5076.

Bishop, R.F., 1996. Natural history of human rotavirus infection. Archives of Virology 12, 119-128.

Carvalho, M.F., Gill, D., 2019. Rotavirus vaccine efficacy: current status and areas for improvement. Human Vaccines & Immunotherapeutics, 15(6), 1237-1250.

Celentano, A., Cirillo, N., 2017. Desmosomes in disease: a guide for clinicians. Oral Diseases, 23(2), 157–167.

Corl, B. A., Harrell, R. J., Moon, H. K., Phillips, O., Weaver, E. M., Campbell, J. M., Odle, J., 2007. Effect of animal plasma proteins on intestinal damage and recovery of neonatal pigs infected with rotavirus. The Journal of Nutritional Biochemistry, 18(12), 778-784.

Crawford, S. E., Ramani, S., Tate, J. E., Parashar, U. D., Svensson, L., Hagbom, M., Franco, M. A., Greenberg, H. B., O'Ryan, M., Kang, G., Desselberger, U. et al., 2017. Rotavirus infection. Nature Reviews. Disease primers, 3, 17083.

Criglar, J. M., Hu, L., Crawford, S. E., Hyser, J. M., Broughman, J. R., Prasad, B. V., Estes, M. K., 2014. A novel form of rotavirus NSP2 and phosphorylation-dependent NSP2-NSP5 interactions are associated with viroplasm assembly. Journal of Virology, 88(2), 786-798

Cui, T., 2019. A co-culture system of primary porcine enterocytes/myofibroblasts: investigation the replication characteristics of porcine enteric viruses in their target cell. PhD, PhD thesis in Veterinary Medicine, Faculty of Veterinary Medicine, UGhent, Belgium.

Cui, T., Theuns, S., Desmarets, L.M.B., Xie, J., De Gryse G.M.A., Yang, B., Van den Broeck, W., Nauwynck, H.J., 2018. Establishment of porcine enterocyte/myofibroblast co-cultures for the growth of porcine rota- and coronaviruses. Scientific Reports 8(1), 1-17

Desselberger, U., 2014. Rotaviruses. Virus Research, 190, 75-96.

Estes, M. K., Graham, D. Y., Smith, E. M., Gerba, C. P., 1979. Rotavirus stability and inactivation. Journal of General Virology, 43(2), 403-409.

Grube, D., Forssmann, W. G., 1979. Morphology and function of the entero-endocrine cells. Hormone and Metabolic Research 11(11), 589–606.

Hall, P. A., Coates, P. J., Ansari, B., Hopwood, D., 1994. Regulation of cell number in the mammalian gastrointestinal tract: the importance of apoptosis. Journal of Cell Science, 107(12), 3569-3577.

Helander, H.F., Fändriks, L., 2014. Surface area of the digestive tract - revisited. Scandinavian Journal of Gastroenterology, 49(6), 681–689.

Honeyman, M. S., 1996. Sustainability issues of U.S. swine production. Journal of Animal Science, 74(6), 1410–1417

Jenni, S., Salgado, E. N., Herrmann, T., Li, Z., Grant, T., Grigorieff, N., Trapani, S., Estrozi, L. F., Harrison, S. C., 2019. In situ Structure of Rotavirus VP1 RNA-Dependent RNA Polymerase. Journal of Molecular Biology, 431(17), 3124–3138.

Junqueira, L.C., Carneiro, J., 2010. Het spijsverteringskanaal, In: Functionele Histologie, 11th Edn. Elsevier Gezondheidszorg, Amsterdam, Nederland, pp. 417-428

Knoop, K. A., Newberry, R. D., 2018. Goblet cells: multifaceted players in immunity at mucosal surfaces. Mucosal Immunology, 11(6), 1551–1557.

Liu, Y., Xu, S., Woodruff, A.L., Xia, M., Tan, M., Kennedy, M.A., Jiang, X., 2017. Structural basis of glycan specificity of P[19] VP8*: Implications for rotavirus zoonosis and evolution. PLoS Pathogens, 13(11), e1006707

Ludert, J.E., Michelangeli, F., Gil, F., Liprandi, F., Esparza, J., 1987. Penetration and uncoating of rotaviruses in cultured cells. Intervirology, 27(2), 95-101.

Lussier, C., Basora, N., Bouatrouss, Y., Beaulieu, J.F. 2000. Integrins as mediators of epithelial cell- matrix interactions in the human small intestinal mucosa. Microscopy Research and Technique, 51(2), 169–178.

Mabbott, N.A., Donaldson, D.S., Ohno, H., Williams, I.R., Mahajan, A., 2013. Microfold (M) cells: important immunosurveillance posts in the intestinal epithelium. Mucosal Immunology, 6(4), 666– 677.

Maes, M., Cogliati, B., Crespo Yanguas, S., Willebrords, J., Vinken, M., 2015. Roles of connexins and pannexins in digestive homeostasis. Cellular and Molecular Life Sciences, 72(15), 2809–2821.

Martin, D., Duarte, M., Lepault, J., and Poncet, D., 2010. Sequestration of free tubulin molecules by the viral protein NSP2 induces microtubule depolymerization during rotavirus infection. Journal of Virology, 84(5), 2522-2532.

Matthijnssens, J., Ciarlet, M., Heiman, E., Arijs, I., Delbeke, T., McDonald, S.M., Palumbo A.E., Iturriza- Gómara M., Maes P., Patton J.T. et al., 2008. Full genome-based classification of rotaviruses reveals a common origin between human Wa-Like and porcine rotavirus strains and human DS-1-like and bovine rotavirus strains. Journal of Virology, 82(7), 3204-3219.

Morelli, M., Ogden, K.M., Patton, J.T., 2015. Silencing the alarms: innate immune antagonism by rotavirus NSP1 and VP3. Virology, 479, p. 75-84

Ogden, K.M., Snyder, M.J., Dennis, A.F., Patton, J.T., 2014. Predicted structure and domain organization of rotavirus capping enzyme and innate immune antagonist VP3. Journal of Virology, 88(16), 9072- 9085.

Pang, L.L., Wang, M.X., Sun, X.M., Yuan, Y., Qing, Y., Xin, Y., Zhang, J.Y., Li, D.D., Duan, Z. J., 2018. Glycan binding patterns of human rotavirus P[10] VP8* protein. Virology Journal, 15(1), 161

Pott, J., Stockinger, S., Torow, N., Smoczek, A., Lindner, C., McInerney, G., Bäckhed, F., Baumann, U., Pabst, O., Bleich, A. et al., 2012. Age-dependent TLR3 expression of the intestinal epithelium contributes to rotavirus susceptibility. PLoS pathogens, 8(5), e1002670

Rodríguez J.M., Luque D., 2019 Structural insights into Rotavirus entry, Physical Virology, 45-68

Sun, X., Li, D., Qi, J., Chai, W., Wang, L., Wang, L., Peng, R., Wang, H., Zhang, Q., Pang, L. et al., 2018. Glycan Binding Specificity and Mechanism of Human and Porcine P[6]/P[19] Rotavirus VP8*s. Journal of Virology, 92(14), e00538-18.

Taylor, J.A., O'Brien, J.A., Yeager, M., 1996. The cytoplasmic tail of NSP4, the endoplasmic reticulum‐ localized non‐structural glycoprotein of rotavirus, contains distinct virus binding and coiled coil domains. The EMBO Journal, 15(17), 4469-4476.

Theuns, S., 2015. Porcine rotavirus infection in Belgian piglets and assessment of their relationship with human rotaviruses. PhD, PhD thesis in Veterinary Medicine, Faculty of Veterinary Medicine, UGhent, Belgium.

Theuns, S., 2010. Porcine intestinale mucosacellen voor de studie van het rotavirus. Master thesis Master of Veterinary Medicine, Faculty of Veterinary Medicine, UGhent, Belgium.

Theuns, S., Conceição-Neto, N., Zeller, M., Heylen, E., Roukaerts, I.D., Desmarets, L.M., Van Ranst M., Nauwynck H.J., Matthijnssens, J., 2016. Characterization of a genetically heterogeneous porcine rotavirus C, and other viruses present in the fecal virome of a non-diarrheic Belgian piglet. Infection, Genetics and Evolution, 43, 135-145.

Theuns, S., Heylen, E., Zeller, M., Roukaerts, I. D., Desmarets, L.M., Van Ranst, M., Nauwynck H., Matthijnssens, J., 2015. Complete genome characterization of recent and ancient Belgian pig group A rotaviruses and assessment of their evolutionary relationship with human rotaviruses. Journal of Virology, 89(2), 1043-1057.

Todd, S., Page, N.A., Steele, A.D., Peenze, I., and Cunliffe, N.A., 2010. Rotavirus strain types circulating in Africa: review of studies published during 1997–2006. Journal of Infectious Diseases, 202(Supplement_1), S34-S42.

Tuanthap, S., Vongpunsawad, S., Luengyosluechakul, S., Sakkaew, P., Theamboonlers, A., Amonsin, A., Poovorawan, Y. 2019. Genome constellations of 24 porcine rotavirus group A strains circulating on commercial Thai swine farms between 2011 and 2016. PloS One, 14(1).

Vidal, A., Clilverd, H., Cortey, M., Martín-Valls, G.E., Franzo, G., Darwich, L. and Mateu, E., 2018. Full- genome characterization by deep sequencing of rotavirus a isolates from outbreaks of neonatal diarrhoea in pigs in Spain. Veterinary Microbiology, 227, 12-19.

Vlasova A.N., Amimo J.O., Saif L.J., 2017. Porcine rotaviruses: epidemiology, immune responses and control strategies. Viruses, 9(3), 48.

Wei, Q., Huang, H., 2013. Insights into the role of cell–cell junctions in physiology and disease. In: International Review of Cell and Molecular Biology, first Edn. Academic Press, Oxford, UK, Vol. 306, pp. 187-221.

Wolf, J.L., Cukor, G., Blacklow, N.R., Dambrauskas, R., Trier, J.S., 1981. Susceptibility of mice to rotavirus infection: effects of age and administration of corticosteroids. Infection and Immunity, 33(2), 565-574.

Wolf, M., Vo P.T., Greenberg H.B., 2011. Rhesus rotavirus entry into a polarized epithelium is endocytosis dependent and involves sequential VP4 conformational changes. Journal of Virology, 85(6), 2492-503.

Zihni, C., Mills, C., Matter, K., and Balda, M.S., 2016. Tight junctions: from simple barriers to multifunctional molecular gates. Nature Reviews. Molecular Cell Biology, 17(9), 564–580.

Zimmerman, J.J., Karriker, L.A., Ramirez, A., Schwartz, K.J., Stevenson, G.W., Zhang, J., 2019. Digestive system. In: Diseases of Swine, 11th Edn. Wiley-Blackwell, Hoboken, NJ, USA, pp. 234-242

Zimmerman, J.J., Karriker, L.A., Ramirez, A., Schwartz, K.J., Stevenson, G.W., Zhang, J., 2019. Reoviruses. In: Diseases of Swine, 11th Edn. Wiley-Blackwell, Hoboken, NJ, USA, pp. 715-727