**1. Introduction**

The study and monitoring of wildlife diseases are key points for establishing conservational policies for wild fauna.

Wildlife are often in double risk from disease due to the high number of infectious diseases of wildlife origin that affects humans and (or) domestic animals and also the opposite, humans and domestic animals' diseases that could affect the wildlife. Likewise, due to habitat changes, the introduction of pathogens from domestic animals as well as other actions with long-term adverse effects for the conservation of species needs to have supervising.

As in other diseases, some wildlife animal species could be at risk for transmissible spongiform encephalopathies (TSEs), acting as a potential prion reservoir, threatening the livestock and public health.

TSEs are fatal neurodegenerative diseases characterized by the accumulation of an abnormal isoform, partially resistant to the enzymatic digestion, of the cellular prion protein (PrPc ), usually designated by PrPsc or prion. Due to its conformational arrangement, it is very resistant to common inactivation procedures used on conventional infectious agents. As PrPc is host-encoded by the *PRNP* gene, polymorphisms in this gene can act upon the susceptibility or the resistance to TSEs.

The most common and well-known diseases of this group are scrapie in small ruminants, bovine spongiform encephalopathy (BSE), Creutzfeldt-Jakob disease (CJD) in humans, and chronic wasting disease (CWD) in cervids.

Scrapie and CWD are recognized as natural transmitted TSEs, so wildlife can be naturally affected by these two TSEs.

Since scrapie was identified in mouflon sheep (*Ovis musimon*) [1], wild species of sheep and goats, like Iberian wild goat (*Capra pyrenaica*), and Pyrenean chamois (*Rupicapra P. pyrenaica*) can also be infected by scrapie as well as by BSE. Furthermore, according to some studies, European wild ruminants have a *PRNP* genetic background that is compatible with TSE susceptibility [2, 3].

Albeit limited, some countries, namely, Estonia, Finland, Spain, and Cyprus, reported negative results on samples tested for TSE in other wild animal species mink (*Mustela lutreola*), fox (genus *Vulpes*), raccoon dog (*Nyctereutes procyonoides*), bison (*Bison bison*), and Cyprus mouflon (*Ovis gmelini ophion*) [4].

Considering that CWD is a TSE affecting several cervid species, a very contagious disease with an efficient horizontal transmission, appearing to be enzootic and to be expanding both geographically and in prevalence [5], all the sections in this chapter are focused in order to better characterize the epidemiology, transmission, pathogenesis, diagnosis, genetics, and control of this disease.

## **2. Molecular basis of transmissible spongiform encephalopathies (TSEs)**

#### **2.1 Biology of the etiologic agent**

Initially, TSEs were thought to be caused by "slow viruses" (reviewed in [6]). However, as the agent causing scrapie was not deactivated by both chemical and physical procedures, which modify or destroy nucleic acids, it was suggested that this infectious agent was not harboring nucleic acids. Thus, in 1967 Griffith proposed a model in which the scrapie agent could be a protein, but it was Prusiner in 1982, after confirming that procedures used to modify or destroy proteins deactivated the scrapie agent, who published that the etiologic agents of TSEs were proteinaceous infectious particles, called prions [6, 7].

According to this protein-only hypothesis, TSEs are caused by the conversion of the physiological cellular prion protein (PrPc ) into a pathogenic misfolded isoform (designated PrPsc) that is able to propagate by recruiting and transforming more PrPc , by an increase in β-sheet structure and a propensity to aggregate into oligomers (reviewed [6, 8]). Moreover, this conformational change confers to PrPsc a greater insolubility in nonionic detergents, high resistance to heat and chemical sterilization, and partial resistance to protease digestion—the truncated

**105**

*TSE Monitoring in Wildlife Epidemiology, Transmission, Diagnosis, Genetics and Control*

in this chapter) and is normally present in the cell surface in different organs and tissues of mammals and other vertebrates but with high expression levels in the central and peripheral nervous systems. It is a glycosylphosphatidylinositol (GPI)-anchored glycoprotein of 33–35 kDa with a C-terminal globular domain, an N-terminal flexible tail, a single disulfide bond, and an α-helix content. The N-terminal tail includes two charged clusters, the octarepeat region and a hydrophobic domain. At the C-terminus, in the globular domain upstream of the

sialylated GPI anchor, there are two N-glycosylation sites [11]. Thus, PrPc

Regarding the physiological function of the PrPc

putative sites of glycosylation, and three glycoforms of PrP can be described: di-, mono-, and non-glycosilated PrP. The relative proportions of these glycoforms and the size of the unglycosylated PrPsc fragment are dependent on the strain of prion

nevertheless, there are several proposed roles; ones are supported by compatible results of different experiments (neuronal excitability; glutamate receptor function, neurite outgrowth; neuroprotection; copper, zinc, iron, and lactate metabolism; and peripheral myelin maintenance), while others are yielding inconsistent results (synaptic transmission and plasticity, memory formation, stabilization of sleep and circadian rhythm, calcium homeostasis, and toxicity elicited by oligo-

the molecular mechanisms behind prion strains are still not known, despite all the

secondary, tertiary, and quaternary structures, to adopt the PrPsc conformation,

comprehend this molecular process, it is essential to know the architecture of PrPsc. As extensively reviewed by Requena and Wille [9, 10], distinct molecular models have been proposed for PrPsc (27–30 kDa): (1) a four-stranded β-sheet plus two C-terminal α-helices [13, 14]; (2) an antiparallel, intertwined β-helix structure [15]; (3) a parallel β-helix [16]; (4) a fold modeled on the human TATA-box-binding protein containing a five-stranded β-sheet, with the C-terminal α-helices [17]; (5) a "spiral model" with three α-helices of the original structure and four β-strands [18]; (6) a unit consisting of a left-handed four-rung parallel β-helical fold for the N-terminal part and α-helical state for the C-terminal portion, assembled as a trimer [19]; (7) a PrP primary structure based onto the β-helical template and the C-terminal α-helices [20]; (8) a β-helical architecture with a threefold domain in the trimeric unit and α-helical structure for the C-terminal portion [21]; (9) a two-rung β-helix with the C-terminal α-helices [22]; (10) a parallel in-register intermolecular β-sheet (PIRIBS) architecture [23, 24]; and (11) a parallel β-helix extended to the

Nevertheless, none of these models have explained all the experimental results. Recently, methodologies like cryo-electron microscopy and X-ray fiber diffraction have pointed out a four-rung β-solenoid as the basic structural element structure of PrPsc [26], though the available structural information is still limited to determine

To explain strain diversity of prions, the prion-only hypothesis considers that, in the absence of a nucleic acid, the variety of PrPsc conformers and its mixture

PrP 27–30 kDa (reviewed in [8–10]). Until now, this latter feature has been used for diagnostic purposes, being PrPsc a diagnostic marker for these diseases (see Section

is host-encoded by the *PRNP* gene (see Section 2.3. Prion protein gene,

has two

, it has not been clarified yet;

, with the same primary but different

, followed by refolding. To thoroughly

is well studied and identified, the structure of PrPsc,

converts into PrPsc in a posttranslational process, and

*DOI: http://dx.doi.org/10.5772/intechopen.85797*

6 Diagnosis of CWD, in this chapter).

PrPc

(reviewed in [12]).

meric species) [11].

While the structure of PrPc

the mechanism by which PrPc

experimental attempts [8–10].

PrPsc, as a physical template, compels PrPc

probably on a complete unfolding of PrPc

C-terminal portion of the PrP molecule [25].

the PrPsc architecture in atomic details (reviewed in [10]).

#### *TSE Monitoring in Wildlife Epidemiology, Transmission, Diagnosis, Genetics and Control DOI: http://dx.doi.org/10.5772/intechopen.85797*

PrP 27–30 kDa (reviewed in [8–10]). Until now, this latter feature has been used for diagnostic purposes, being PrPsc a diagnostic marker for these diseases (see Section 6 Diagnosis of CWD, in this chapter).

PrPc is host-encoded by the *PRNP* gene (see Section 2.3. Prion protein gene, in this chapter) and is normally present in the cell surface in different organs and tissues of mammals and other vertebrates but with high expression levels in the central and peripheral nervous systems. It is a glycosylphosphatidylinositol (GPI)-anchored glycoprotein of 33–35 kDa with a C-terminal globular domain, an N-terminal flexible tail, a single disulfide bond, and an α-helix content. The N-terminal tail includes two charged clusters, the octarepeat region and a hydrophobic domain. At the C-terminus, in the globular domain upstream of the sialylated GPI anchor, there are two N-glycosylation sites [11]. Thus, PrPc has two putative sites of glycosylation, and three glycoforms of PrP can be described: di-, mono-, and non-glycosilated PrP. The relative proportions of these glycoforms and the size of the unglycosylated PrPsc fragment are dependent on the strain of prion (reviewed in [12]).

Regarding the physiological function of the PrPc , it has not been clarified yet; nevertheless, there are several proposed roles; ones are supported by compatible results of different experiments (neuronal excitability; glutamate receptor function, neurite outgrowth; neuroprotection; copper, zinc, iron, and lactate metabolism; and peripheral myelin maintenance), while others are yielding inconsistent results (synaptic transmission and plasticity, memory formation, stabilization of sleep and circadian rhythm, calcium homeostasis, and toxicity elicited by oligomeric species) [11].

While the structure of PrPc is well studied and identified, the structure of PrPsc, the mechanism by which PrPc converts into PrPsc in a posttranslational process, and the molecular mechanisms behind prion strains are still not known, despite all the experimental attempts [8–10].

PrPsc, as a physical template, compels PrPc , with the same primary but different secondary, tertiary, and quaternary structures, to adopt the PrPsc conformation, probably on a complete unfolding of PrPc , followed by refolding. To thoroughly comprehend this molecular process, it is essential to know the architecture of PrPsc. As extensively reviewed by Requena and Wille [9, 10], distinct molecular models have been proposed for PrPsc (27–30 kDa): (1) a four-stranded β-sheet plus two C-terminal α-helices [13, 14]; (2) an antiparallel, intertwined β-helix structure [15]; (3) a parallel β-helix [16]; (4) a fold modeled on the human TATA-box-binding protein containing a five-stranded β-sheet, with the C-terminal α-helices [17]; (5) a "spiral model" with three α-helices of the original structure and four β-strands [18]; (6) a unit consisting of a left-handed four-rung parallel β-helical fold for the N-terminal part and α-helical state for the C-terminal portion, assembled as a trimer [19]; (7) a PrP primary structure based onto the β-helical template and the C-terminal α-helices [20]; (8) a β-helical architecture with a threefold domain in the trimeric unit and α-helical structure for the C-terminal portion [21]; (9) a two-rung β-helix with the C-terminal α-helices [22]; (10) a parallel in-register intermolecular β-sheet (PIRIBS) architecture [23, 24]; and (11) a parallel β-helix extended to the C-terminal portion of the PrP molecule [25].

Nevertheless, none of these models have explained all the experimental results. Recently, methodologies like cryo-electron microscopy and X-ray fiber diffraction have pointed out a four-rung β-solenoid as the basic structural element structure of PrPsc [26], though the available structural information is still limited to determine the PrPsc architecture in atomic details (reviewed in [10]).

To explain strain diversity of prions, the prion-only hypothesis considers that, in the absence of a nucleic acid, the variety of PrPsc conformers and its mixture

*Wildlife Population Monitoring*

prion protein (PrPc

of species needs to have supervising.

threatening the livestock and public health.

conventional infectious agents. As PrPc

naturally affected by these two TSEs.

**2.1 Biology of the etiologic agent**

also the opposite, humans and domestic animals' diseases that could affect the wildlife. Likewise, due to habitat changes, the introduction of pathogens from domestic animals as well as other actions with long-term adverse effects for the conservation

As in other diseases, some wildlife animal species could be at risk for transmissible spongiform encephalopathies (TSEs), acting as a potential prion reservoir,

TSEs are fatal neurodegenerative diseases characterized by the accumulation of an abnormal isoform, partially resistant to the enzymatic digestion, of the cellular

tional arrangement, it is very resistant to common inactivation procedures used on

Scrapie and CWD are recognized as natural transmitted TSEs, so wildlife can be

Since scrapie was identified in mouflon sheep (*Ovis musimon*) [1], wild species of sheep and goats, like Iberian wild goat (*Capra pyrenaica*), and Pyrenean chamois (*Rupicapra P. pyrenaica*) can also be infected by scrapie as well as by BSE. Furthermore, according to some studies, European wild ruminants have a *PRNP* genetic background that is compatible with TSE susceptibility [2, 3].

Albeit limited, some countries, namely, Estonia, Finland, Spain, and Cyprus, reported negative results on samples tested for TSE in other wild animal species mink (*Mustela lutreola*), fox (genus *Vulpes*), raccoon dog (*Nyctereutes procyonoides*),

Considering that CWD is a TSE affecting several cervid species, a very contagious disease with an efficient horizontal transmission, appearing to be enzootic and to be expanding both geographically and in prevalence [5], all the sections in this chapter are focused in order to better characterize the epidemiology, transmis-

**2. Molecular basis of transmissible spongiform encephalopathies (TSEs)**

Initially, TSEs were thought to be caused by "slow viruses" (reviewed in [6]). However, as the agent causing scrapie was not deactivated by both chemical and physical procedures, which modify or destroy nucleic acids, it was suggested that this infectious agent was not harboring nucleic acids. Thus, in 1967 Griffith proposed a model in which the scrapie agent could be a protein, but it was Prusiner in 1982, after confirming that procedures used to modify or destroy proteins deactivated the scrapie agent, who published that the etiologic agents of TSEs were

According to this protein-only hypothesis, TSEs are caused by the conversion

, by an increase in β-sheet structure and a propensity to aggregate

isoform (designated PrPsc) that is able to propagate by recruiting and transform-

into oligomers (reviewed [6, 8]). Moreover, this conformational change confers to PrPsc a greater insolubility in nonionic detergents, high resistance to heat and chemical sterilization, and partial resistance to protease digestion—the truncated

) into a pathogenic misfolded

morphisms in this gene can act upon the susceptibility or the resistance to TSEs. The most common and well-known diseases of this group are scrapie in small ruminants, bovine spongiform encephalopathy (BSE), Creutzfeldt-Jakob disease

(CJD) in humans, and chronic wasting disease (CWD) in cervids.

bison (*Bison bison*), and Cyprus mouflon (*Ovis gmelini ophion*) [4].

sion, pathogenesis, diagnosis, genetics, and control of this disease.

proteinaceous infectious particles, called prions [6, 7].

of the physiological cellular prion protein (PrPc

), usually designated by PrPsc or prion. Due to its conforma-

is host-encoded by the *PRNP* gene, poly-

**104**

ing more PrPc

may result in different prion strains. Each prion strain presents a specific disease phenotype (incubation times, clinical signs, histopathological lesions, PrPsc deposition patterns in the brain, and PrPsc biochemical characteristics) which is exactly preserved upon serial passage within the same host genotype. Nevertheless, the molecular mechanisms by which a range of PrPsc conformers would be produced and selected have not been yet established (reviewed in [8]).

In relation to chronic wasting disease (CWD) prion strains, two prevalent CWD prion strains (CWD1 and CWD2) were identified in North America based on transmission in transgenic cervid (TgCerPrP) mice of several CWD isolates from different cervid species and geographic areas. CWD1 and CWD2 showed distinct incubation time, clinical signs, and neuropathologic profile but with indistinguishable electrophoretic migration patterns of di-, mono-, and non-glycosyl forms of PrPsc [27], reviewed in [28]. These bioassay results showed that elk may be infected with either CWD1 or CWD2 strains, while in deer CWD1/CWD2 strain mixture can be present ([27], reviewed in [28]) (**Figure 1**).

Regarding the CWD prion strain(s) responsible for the outbreaks of the disease in new geographic areas—South Korea, Norway, and Finland—the available data is still limited. The strain characterization of the Korean CWD cases in elk in 2001 and 2004 suggested a single strain responsible for the outbreaks imported from Canada [29], without identifying if it was CWD1, CWD2, or both. The biochemical analysis and immunohistochemistry (IHC) distribution of PrPsc from Norway reindeer revealed a pattern indistinguishable from North America isolates [30]. Remarkably, in CWD-affected moose in Norway, a different phenotype was observed in both PrPsc distribution and biochemical features, suggesting a presence of a different type of CWD prion strain in moose from this country (designated Nor-16CWD) [31] (**Figure 1**).

Novel cervid prion strains have been experimentally generated by adaptation of prions from other species, for instance, scrapie (SSBP1, an American classical scrapie isolate) [28] and BSE [32], demonstrating that cervid species can also be susceptible to other prions. Moreover, CWD prions easily adapt to new species (see Section 4), including sheep, cattle, and squirrel monkeys. Thus, there is a putative risk of development of novel CWD-related prion disease in livestock by grazing in CWD-contaminated pasture (**Figure 1**). Lastly, the ability of the CWD prions to cross the human species barrier has to be further evaluated, but amino acid residues (residues 165–175) in the β2–α2 loop sequence of human PrPc can constitute a species barrier to its conversion by CWD prions [33], reviewed in [28].

#### **2.2 Prions and the deviations in the central dogma of molecular biology**

According to the central dogma of molecular biology, first published in 1958 and revisited later [34], heritable information is stored in DNA, expressed as RNA, and translated into protein. Nevertheless, this paradigm has been updated by many aspects of the regulation of gene expression, namely, by the identification and characterization of alternative splicing, alternative promoters, alternative polyadenylation events, and the increasing number of noncoding RNAs (ncRNAs) with critical importance in the regulation of messenger RNA (mRNA) [35] and the discovery of "prions": prion proteins can adopt multiple conformations, at least one of which has the capacity to self-template [36, 37] (**Figure 2**).

#### **2.3 Prion protein gene (PRNP)**

The astounding improvement in genetic tools and bioinformatic programs/ algorithms and the incredible amount of data deposited freely in the main scientific

**107**

**Figure 1.**

*and img.linkfrog.com).*

*TSE Monitoring in Wildlife Epidemiology, Transmission, Diagnosis, Genetics and Control*

databases allow us to use comparative genomics in an effective manner. **Figure 3** contains the representation of the *PRNP* gene in *Homo sapiens*, used as a reference and establishing a comparison with other animal species according to the Ensembl database. This gene is constituted by two exons, although a single exon is responsible for the open reading frame (ORF) and the remaining sequence contains untranslated and regulatory regions. Some animal species have the same distribution, while others contain a single exon (e.g., *Canis lupus familiaris*, *Felis catus*, and *Ovis aries*), three exons (*Cervus elaphus* and *Odocoileus virginianus* from *Cervidae* family), or four exons (like *Bos taurus),* according to Ensembl database. Nevertheless, a high level of conservation at the coding sequence and correspond-

*Summary of CWD prions and transmission. Like North America-CWD-isolates with PrPres biochemical and PrPres distribution similar to that described in North America CWD cases. natural transmission; putative transmission; experimental transmission; potential spread of CWD prions or decrease of PrPres environmental reservoir at the carcass site due to scavenging process, (silhouettes and pictures from freepik.com* 

The fundamental event in the pathogenesis of TSE is not a primary structure

same amino acid sequence, but differences in the secondary structure originate the tertiary and quaternary structures that dictate the PrPsc with new physicochemical properties, namely, insolubility in nondenaturing detergents and partial resistance to proteolysis. It is therefore important to identify the specific codons and amino

Rongyan and collaborators [39] compared the *PRNP* gene sequences among 83 species and reinforced a remarkable degree of conservation among the mammalian sequences. In order to confirm this statement, a DNA, RNA, and protein comparison of *PRNP* among humans, bovine, ovine, caprine, and deer was performed. According to this comparison, human *PRNP* is less similar compared to the others, which is understandable since they are phylogenetically more distant species. However, the protein comparison showed a high similarity between all these species

In order to add supplementary information especially regarding wild species, the PrP protein sequences from 13 different species were compared and are presented in

) into

and PrPsc share the

modification but the conversion of the normal cellular prion protein (PrPc

ing protein sequence is maintained (as confirmed in **Figure 4**).

acids with relevant importance in the protein structure dynamics.

the misfolded pathogenic isoform (PrPsc) [38]. In fact, PrPc

(above 90%), indicating a high conservation of PrP.

*DOI: http://dx.doi.org/10.5772/intechopen.85797*

*TSE Monitoring in Wildlife Epidemiology, Transmission, Diagnosis, Genetics and Control DOI: http://dx.doi.org/10.5772/intechopen.85797*

#### **Figure 1.**

*Wildlife Population Monitoring*

[31] (**Figure 1**).

may result in different prion strains. Each prion strain presents a specific disease phenotype (incubation times, clinical signs, histopathological lesions, PrPsc deposition patterns in the brain, and PrPsc biochemical characteristics) which is exactly preserved upon serial passage within the same host genotype. Nevertheless, the molecular mechanisms by which a range of PrPsc conformers would be produced

In relation to chronic wasting disease (CWD) prion strains, two prevalent CWD

Regarding the CWD prion strain(s) responsible for the outbreaks of the disease in new geographic areas—South Korea, Norway, and Finland—the available data is still limited. The strain characterization of the Korean CWD cases in elk in 2001 and 2004 suggested a single strain responsible for the outbreaks imported from Canada [29], without identifying if it was CWD1, CWD2, or both. The biochemical analysis and immunohistochemistry (IHC) distribution of PrPsc from Norway reindeer revealed a pattern indistinguishable from North America isolates [30]. Remarkably, in CWD-affected moose in Norway, a different phenotype was observed in both PrPsc distribution and biochemical features, suggesting a presence of a different type of CWD prion strain in moose from this country (designated Nor-16CWD)

Novel cervid prion strains have been experimentally generated by adaptation of prions from other species, for instance, scrapie (SSBP1, an American classical scrapie isolate) [28] and BSE [32], demonstrating that cervid species can also be susceptible to other prions. Moreover, CWD prions easily adapt to new species (see Section 4), including sheep, cattle, and squirrel monkeys. Thus, there is a putative risk of development of novel CWD-related prion disease in livestock by grazing in CWD-contaminated pasture (**Figure 1**). Lastly, the ability of the CWD prions to cross the human species barrier has to be further evaluated, but amino acid residues

can constitute a spe-

(residues 165–175) in the β2–α2 loop sequence of human PrPc

the capacity to self-template [36, 37] (**Figure 2**).

**2.3 Prion protein gene (PRNP)**

cies barrier to its conversion by CWD prions [33], reviewed in [28].

**2.2 Prions and the deviations in the central dogma of molecular biology**

According to the central dogma of molecular biology, first published in 1958 and revisited later [34], heritable information is stored in DNA, expressed as RNA, and translated into protein. Nevertheless, this paradigm has been updated by many aspects of the regulation of gene expression, namely, by the identification and characterization of alternative splicing, alternative promoters, alternative polyadenylation events, and the increasing number of noncoding RNAs (ncRNAs) with critical importance in the regulation of messenger RNA (mRNA) [35] and the discovery of "prions": prion proteins can adopt multiple conformations, at least one of which has

The astounding improvement in genetic tools and bioinformatic programs/ algorithms and the incredible amount of data deposited freely in the main scientific

prion strains (CWD1 and CWD2) were identified in North America based on transmission in transgenic cervid (TgCerPrP) mice of several CWD isolates from different cervid species and geographic areas. CWD1 and CWD2 showed distinct incubation time, clinical signs, and neuropathologic profile but with indistinguishable electrophoretic migration patterns of di-, mono-, and non-glycosyl forms of PrPsc [27], reviewed in [28]. These bioassay results showed that elk may be infected with either CWD1 or CWD2 strains, while in deer CWD1/CWD2 strain mixture

and selected have not been yet established (reviewed in [8]).

can be present ([27], reviewed in [28]) (**Figure 1**).

**106**

*Summary of CWD prions and transmission. Like North America-CWD-isolates with PrPres biochemical and PrPres distribution similar to that described in North America CWD cases. natural transmission; putative transmission; experimental transmission; potential spread of CWD prions or decrease of PrPres environmental reservoir at the carcass site due to scavenging process, (silhouettes and pictures from freepik.com and img.linkfrog.com).*

databases allow us to use comparative genomics in an effective manner. **Figure 3** contains the representation of the *PRNP* gene in *Homo sapiens*, used as a reference and establishing a comparison with other animal species according to the Ensembl database. This gene is constituted by two exons, although a single exon is responsible for the open reading frame (ORF) and the remaining sequence contains untranslated and regulatory regions. Some animal species have the same distribution, while others contain a single exon (e.g., *Canis lupus familiaris*, *Felis catus*, and *Ovis aries*), three exons (*Cervus elaphus* and *Odocoileus virginianus* from *Cervidae* family), or four exons (like *Bos taurus),* according to Ensembl database. Nevertheless, a high level of conservation at the coding sequence and corresponding protein sequence is maintained (as confirmed in **Figure 4**).

The fundamental event in the pathogenesis of TSE is not a primary structure modification but the conversion of the normal cellular prion protein (PrPc ) into the misfolded pathogenic isoform (PrPsc) [38]. In fact, PrPc and PrPsc share the same amino acid sequence, but differences in the secondary structure originate the tertiary and quaternary structures that dictate the PrPsc with new physicochemical properties, namely, insolubility in nondenaturing detergents and partial resistance to proteolysis. It is therefore important to identify the specific codons and amino acids with relevant importance in the protein structure dynamics.

Rongyan and collaborators [39] compared the *PRNP* gene sequences among 83 species and reinforced a remarkable degree of conservation among the mammalian sequences. In order to confirm this statement, a DNA, RNA, and protein comparison of *PRNP* among humans, bovine, ovine, caprine, and deer was performed. According to this comparison, human *PRNP* is less similar compared to the others, which is understandable since they are phylogenetically more distant species. However, the protein comparison showed a high similarity between all these species (above 90%), indicating a high conservation of PrP.

In order to add supplementary information especially regarding wild species, the PrP protein sequences from 13 different species were compared and are presented in **Figure 4**. The high level of conservation suggests the preservation of some important functional characteristics of PrP through evolution.

According to NCBI-SNP database (accessed in December 2018), 3683 variations in the human *PRNP* gene were presented. Some of these variations are located in the coding sequence and originate modifications in the protein. Once PrP is highly conserved, the variations already described in humans can be used to predict variations in other species. In order to simplify this process, all the missense and nonsense mutations associated with prion diseases described in humans are presented in **Figure 2**.

Variations in PrP sequences exist between species and also between individuals of the same species. It was already demonstrated that this fact can influence the susceptibility to prion infection and ultimately to disease. Chronic wasting disease appear to have a higher potential of transmissibility than other forms of prion disease, and it has been confirmed that some genetic variations are associated with lower rates of infection and slower progression of clinical manifestations [40]. A total of 17 polymorphic sites have been reported in the PrP in *Cervidae* species.

Historically, O'Rourke and collaborators [41] were the first authors that reported that *PRNP* gene from *Cervus elaphus* was polymorphic at codon 132 encoding methionine (M) or leucine (L). This codon is equivalent to codon 129 in

#### **Figure 2.**

*Updated vision of the classical central dogma of the molecular biology. A DNA sequence can originate multiple RNAs (by using different promoters, by alternative polyadenylation and alternative splicing events). Some of these RNAs can be degraded by nonsense mediated decay (NMD), normally if they contain a premature termination codon (PTC). Other RNAs are not translated but still have a possible regulatory function (noncoding RNAs, ncRNA) and supplementary RNAs are translated originating a different protein, with similar or unrelated function comparing to the canonical protein. The prion postulation assumes that an abnormal protein conformation (PrPsc) propagates itself via an autocatalytic mode by recruiting the normal cellular isoform (PrPc) as a substrate and acting as the disease transmissible agent. This misfolding can be reversible and prion proteins have the ability to interact with nucleic acids (DNA and RNA) and other polyanions (as lipids).*

**109**

**Figure 3.**

*to create this scheme.*

*TSE Monitoring in Wildlife Epidemiology, Transmission, Diagnosis, Genetics and Control*

*Organization of the Homo sapiens PRNP gene. From the two exons, only exon 2 is codifying for the PrP protein (black square). The coding sequence is presented below with the top lines showing the nucleotide sequence and the bottom lines showing the amino acid sequence. Missense and nonsense mutations in PrP associated with human prion diseases are marked in red. Information retrieved from Ensembl and NCBI databases were used* 

the human protein encoding either M or valine (V) [42]. According to Collinge and colleagues [43] and Palmer and collaborators [44], this variation has an important impact on human prion disease presentation. In the same direction, regarding CWD, O'Rourke and collaborators [45, 46] indicated that the L132 allele protected against this disease in *Cervidae*. Although all *PRNP* genotypes can be affected with CWD, there are some polymorphisms that appear to result in longer incubation periods in some species. The polymorphisms Q95H and G96S are related to the reduction of the risk of infection [47]. S96S or G96S and G95H

*DOI: http://dx.doi.org/10.5772/intechopen.85797*

*TSE Monitoring in Wildlife Epidemiology, Transmission, Diagnosis, Genetics and Control DOI: http://dx.doi.org/10.5772/intechopen.85797*

#### **Figure 3.**

*Wildlife Population Monitoring*

**Figure 2**.

*Cervidae* species.

**Figure 4**. The high level of conservation suggests the preservation of some impor-

According to NCBI-SNP database (accessed in December 2018), 3683 variations in the human *PRNP* gene were presented. Some of these variations are located in the coding sequence and originate modifications in the protein. Once PrP is highly conserved, the variations already described in humans can be used to predict variations in other species. In order to simplify this process, all the missense and nonsense mutations associated with prion diseases described in humans are presented in

Variations in PrP sequences exist between species and also between individuals of the same species. It was already demonstrated that this fact can influence the susceptibility to prion infection and ultimately to disease. Chronic wasting disease appear to have a higher potential of transmissibility than other forms of prion disease, and it has been confirmed that some genetic variations are associated with lower rates of infection and slower progression of clinical manifestations [40]. A total of 17 polymorphic sites have been reported in the PrP in

Historically, O'Rourke and collaborators [41] were the first authors that reported that *PRNP* gene from *Cervus elaphus* was polymorphic at codon 132 encoding methionine (M) or leucine (L). This codon is equivalent to codon 129 in

*Updated vision of the classical central dogma of the molecular biology. A DNA sequence can originate multiple RNAs (by using different promoters, by alternative polyadenylation and alternative splicing events). Some of these RNAs can be degraded by nonsense mediated decay (NMD), normally if they contain a premature termination codon (PTC). Other RNAs are not translated but still have a possible regulatory function* 

*(noncoding RNAs, ncRNA) and supplementary RNAs are translated originating a different protein, with similar or unrelated function comparing to the canonical protein. The prion postulation assumes that an abnormal protein conformation (PrPsc) propagates itself via an autocatalytic mode by recruiting the normal cellular isoform (PrPc) as a substrate and acting as the disease transmissible agent. This misfolding can be reversible and prion proteins have the ability to interact with nucleic acids (DNA and RNA) and other polyanions (as lipids).*

tant functional characteristics of PrP through evolution.

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**Figure 2.**

*Organization of the Homo sapiens PRNP gene. From the two exons, only exon 2 is codifying for the PrP protein (black square). The coding sequence is presented below with the top lines showing the nucleotide sequence and the bottom lines showing the amino acid sequence. Missense and nonsense mutations in PrP associated with human prion diseases are marked in red. Information retrieved from Ensembl and NCBI databases were used to create this scheme.*

the human protein encoding either M or valine (V) [42]. According to Collinge and colleagues [43] and Palmer and collaborators [44], this variation has an important impact on human prion disease presentation. In the same direction, regarding CWD, O'Rourke and collaborators [45, 46] indicated that the L132 allele protected against this disease in *Cervidae*. Although all *PRNP* genotypes can be affected with CWD, there are some polymorphisms that appear to result in longer incubation periods in some species. The polymorphisms Q95H and G96S are related to the reduction of the risk of infection [47]. S96S or G96S and G95H

#### **Figure 4.**

*Alignment of PrP protein sequences among 13 different species. T-coffee was the multiple alignment tool used from EMBL-EBI (https://www.ebi.ac.uk/Tools/msa/tcoffee/) and Genedoc version 2.7.000 was the multiple sequence alignment editor. The accession numbers of each species considering a short name (by order of presentation in the alignment) are: Hs\_ENSP00000368752.4; Bt\_ENSBTAP00000043233.2; Oa\_ENSOARP00000004991.1; Ch\_ NP\_001301176; Rr\_UniProt\_spQ5XVM4; Cc\_AY639096; Ce\_UniProt\_ spP67987; Cn\_UniProt\_trQ6DN38; Ov\_UniProt\_trQ7JIQ1; Oh\_UniProt\_trQ6VS46; Aa\_UniProt\_trQ693S2; Rtg\_UniProt\_trQ3Y673; Dd\_UniProt\_trQ7YSF3. Besides human (the reference), the four following species belong to Bovidae family and the last eight species belong to Cervidae family (highlighted with a rectangle frame). The arrows locate the seventeen polymorphic amino acids described in Cervidae.*

seem to produce a reduced susceptibility, with longer survivor period [48], being underrepresented in CWD-affected populations [45]. Regarding S225F polymorphism there is a differential susceptibility to experimental oral exposure and incubation periods [45, 49].

**Table 1** presents the information concerning the amino acidic variations reported until 2018, with the references and functional implications in the susceptibility to CWD.
