Doctoral dissertation

background-color: #CCE4FF
class: hide-slide-number

###Doctoral dissertation
<br>
##Impact of porcine circovirus-associated disease on classical swine fever vaccines and the strategies for application in field farms
<br>
Adviser: Maw-Sheng Chien  
Student: Jing-Yuan Chen<br>  
Graduate Institute of Veterinary Pathobiology,<br> College of Veterinary Medicine,<br>National Chung Hsing University  
2021/5/21 (updated: 2021-05-20)

---

## Outlines

<ul style="font-size: 16pt;">
<li style="color: #2984FF; font-weight: 900;"> Introduction</li>
<ul>
<li> Classical swine fever virus
<li> Porcine circoviruses
</ul><br>
<li style="color: #2984FF; font-weight: 900;">Section I</li>
  <ul>
  <li>The impact of porcine circovirus associated diseases on live attenuated classical swine fever vaccine in field farm applications
  </ul><br>
<li style="color: #2984FF; font-weight: 900;">Section II</li>
  <ul>
  <li>Evaluation of classical swine fever E2 (CSF-E2) subunit vaccine efficacy in the prevention of virus transmission and impact of maternal derived antibody
interference in field farm applications
  </ul><br>
<li style="color: #2984FF; font-weight: 900;">Section III</li>
  <ul>
  <li>Evaluation the induced immune responses and protective efficacy of CSFV-PCV2 bivalent vaccine
  </ul><br>
<li style="color: #2984FF; font-weight: 900;"> Conclusion and discussion</li>
</ul>

???

[xaringanExtra 教學](https://pkg.garrickadenbuie.com/xaringanExtra/#/README?id=xaringanextra)  
日本 2021年的豬瘟疫情 ([Tichigi ken](Tochigi_2021.pdf))

---

class: middle, center ,inverse

# Classical swine fever virus

???
Virus taxonomy

- *Flaviviridae*
- *Pestiviruses*  
  A: BVDV-1  
  B: BVDV-2  
  C: <font style="color: red;">CSFV</font>  
  D: BDV  
  K: <font style="color:#2984FF;">APPV</font>

.footnote[
BVDV: bovine viral diarrhea virus;  
CSFV: classical swine fever virus;  
BDV: border disease virus;  
APPV: atypical porcine pestivirus;  
<font style="font-size: 6pt; text-align: left;">
Reference DOI:  
1: 10.1146/annurev-animal-022513-114209  
2: 10.1099/jgv.0.000873
</font>
]

---

## CSFV

<ul style="font-size: 20pt;">
<li>Flaviviridae</li>
<li>Pestiviruses</li>
<li>1 serotype  
<li style="color: #01B468;">5 genotypes</li>
<ol type="1" style="font-size: 20pt;">
<li style="color: #2984FF; font-weight: 900;"> Ancient, virulent or vaccine strains<br></li>(1.1 ~ 1.7)  
<li style="color: #FF5151; font-weight: 900;"> Predominant epidemic strains<br></li>(2.1 ~ 2.7)  
<li> Taiwan, Japan  
<li> Great Britain/1964  
<li> Korea 1998-1999
</ol>
</ul>

<font style="font-size: 10pt; position: absolute; bottom: 5pt; color: #8e8e8e;">
Reference DOI: 10.1016/j.virusres.2020.198151
</font>

---

## Courses of CSFV infection

<font style="font-size: 10pt; position: absolute; bottom: 5pt; color: #8e8e8e;">
Reference DOI: 10.3390/v9040086
</font>

.panelset[
.panel[.panel-name[Acute infection　　　] 
<img src="Ch1/004AcuteCSF.1.jpg", alt="acuteCSF.1", style="zoom: 45%;">

<div style="width: 380pt; position: absolute; left: 0pt;">
<b style="color: #2984FF;">Incubation</b>: 7-10 days<p>
<div style="position: absolute; top: 0pt; left: 180pt;">
<b style="color: #2984FF;">Atypical</b>: 1~3 weeks
<ul style="font-size: 14pt;">
<li> Hyperpyrexia
<li> Inappetence
<li> Depression
<li> Conjunctivitis
<li> Hunched back
<li> Wasting
<li> Respiratory signs
<li> Gastrointestinal signs
</ul>
</div>
</div>

<div style="position: absolute; top: 0pt; left: 50pt;">
<b style="color: #2984FF;">Typical</b>: 2~4 weeks<p>
<ul style="font-size: 14pt; border: 2px #FF5151 solid; border-radius: 10px;">
<li> Ataxia
<li> Paresis and paralysis
<li> Convulsion
<li> Cyanosis
<li> Petechiae (< 3 cm)
<li> Ecchymoses (> 10 cm)
</ul>
</div>
<div style="position: absolute; top: 0pt; left: 250pt;">
<b style="color: #2984FF;">Secondary infection</b>
<ul style="font-size: 14pt;">
<li> Pneumonia (purulent/fibrinous)
<li> Enteritis 
</ul>
</div>
</div>
]

.panel[.panel-name[Chronic infection　　　]

<div style="width: 600pt; position: absolute; left: 10pt;">
<b style="color: #2984FF;">Incubation</b>: up to 1 month
<div style="position: absolute; top: 0pt; left: 220pt;">
<b style="color: #2984FF;">Atypical signs</b>: 1-3 months<br>
<ul style="font-size: 14pt;">
<li> Remittent fever
<li> Depression
<li> Stunted growth
<li> Wasting
<li> Diffuse dermatitis
<li> Respiratory signs
<li> Gastrointestinal signs
</ul>
</div>

<div style="position: absolute; top: 0pt; left: 150pt;">
<div style="width: 300pt; position: absolute; left: 310pt;">
<b style="color: #2984FF;">Viral shedding</b>: 1-3 months<p>
<b style="color: #2984FF;">Secondary infection</b>:<br>
<ul style="font-size: 14pt;">
<li>Pneumonia (purulent/fibrinous)
<li>Enteritis 
</ul>
</div>
</div>
]

.panel[.panel-name[Persistent infection　　]

<img src="Ch1/007persistent.1.jpg" alt="persistent.1" style="zoom: 45%;">
<b style="color: #FFFFFF; position: absolute; top: 130pt; left: 170pt; border: 2px transparent solid; border-radius: 10px; background-color: #2984FF;">Congenital</b>
<b style="color: #FFFFFF; position: absolute; top: 130pt; left: 495pt; border: 2px transparent solid; border-radius: 10px; background-color:	#01B468;">Postnatal</b>

<ul style="position: absolute; top:140pt; left: 70pt; width: 350pt; font-size: 14pt;">
<li>Transplacental transmission

<li>Viral virulence
<ul>
<li style="color: #8e8e8e;">Virulent/moderate/low</li>
</ul>

<li>Time of infection
<ul>
<li style="color: #8e8e8e;">Early/mid/late gestation
</ul>
<li style="color: #8e8e8e;"><u>Abortion</u>, stillbirth, mummification, malformations, birth of weak, <u>apparently healthy</u>
<li style="color:#FF5151; font-weight: 900;">No antibody response and lifelong viremia
</ul>

<ul style="position: absolute; top: 140pt; left: 410pt; width: 350pt; font-size: 14pt;">
<li>0~3 week-old
<li>Clinically healthy / atypical signs
<li style="color:#FF5151; font-weight: 900;">Immunosuppression</li>
  <ul>
  <li>Innate: IFN-α
    <ul>
    <li> <1weeks <font style="font-weight: 900; font-size: 25pt;">↑</font>, >6 weeks <font style="font-weight: 900; font-size: 25pt;">↓</font>
    </ul>
  <li>Adaptive immune response (absence)
  </ul>
<li style="color: #FF5151; font-weight: 900;">Viral replication and shedding
</ul>
<div style="border: 2px #FF79BC solid; border-radius: 10px; width: 400pt; height: 35pt; position: absolute; top: 330pt; left: 200pt; line-height: 35pt; box-shadow: 3px 3px 12px gray; text-align: center; z-index: 1; background-color: rgba(255,255,255,1);">Viral reservoir and spread virus among populations</div>
]]

</div>

---
class: middle, center, inverse

# Epidemilolgy of CSFV

---

## Epidemiology of CSFV

- 1800s, Midwestern and Southern regions of the United States

- 1860,  US and Europe

- 1960s, Worldwide

<div style="position: absolute; bottom: 80pt; left: 80pt; width: 300pt; font-size: 10pt; border: 1px #8e8e8e solid; border-radius: 10px;">
<b style="color: #adadad; font-size: 12pt;">Gray</b>: no information/ never reported/ disease absent<br>
<b style="color: #FF7575; font-size: 12pt;">Pink</b>: disease present<br>
<b style="color: #8600FF; font-size: 12pt;">Purple</b>: disease present and localized
</div>

.footnote[
<font style="font-size: 10pt; color: #8e8e8e;">
Reference DOI: 10.3390/pathogens9080658
</font>
]

<div style="border: 2px #2984FF solid; border-radius: 10px; position: absolute; top: 145pt; left: 250pt; text-align: center; width: 300pt; box-shadow: 3px 3px 12px gray; background-color: #F0FFF0; height: 20pt; line-height: 20pt;">
Non-vaccination stamping out policy
</div>

---

## Epidemiology in Asia

<div style="position: absolute; bottom: 50pt; left: 40pt; width: 300pt; font-size: 10pt; border: 1px #adadad solid; border-radius: 10px;">
<b style="color: #adadad; font-size: 12pt;">Gray</b>: no information/ never reported/ disease absent<br>
<b style="color: #FF7575; font-size: 12pt;">Pink</b>: disease present<br>
<b style="color: #8600FF; font-size: 12pt;">Purple</b>: disease present and localized
</div>

<div style="position: absolute; top: 100pt; left: 400pt; font-size: 18pt;">
<b>Genotype 1</b>
<ul>
<li>2014~, Jeju island, <font style="color: #01B468;">South Korea</font><br>(LOM-derived  strain)
</ul>
<b>Genotype 2.1d</b>
<ul>
<li>2014~, several provinces, <font style="color:#9F4D95;">China</font><br><font style="color: #8e8e8e;">(Shandong, Jilin, Heilongjiang and Jiangsu)</font>
<li>2016,  Gyeonggi, <font style="color: #01B468;">South Korea</font><br><font style="color: #8e8e8e;">(YC16CS strain, domestic pigs)</font>
<li>2017~2019, <font style="color: #01B468;">South Korea</font><br><font style="color: #8e8e8e;">(CW17WB strain, wild boar)</font>
<li>2018~,  Gifu, <font style="color: #7373b9;">Japan</font>
</ul></div>

<font style="font-size: 10pt; width: 200pt; position: absolute; bottom: 50pt; left: 710pt; color: #8e8e8e;">
Reference DOI:<br>1: 10.3390/pathogens9080658<br>2: 10.3390/pathogens9040244
</font>

<div style="border: 2px #2984FF solid; border-radius: 10px; background-color:#A6FFFF; position: absolute; bottom: 50pt; left: 400pt; width: 300pt; text-align: center; height: 20pt; line-height: 20pt; box-shadow: 3px 3px 12px gray; font-size: 18pt;">Preventative-vaccination</div>

---
class: middle, center, inverse

# CSFV vaccines

---

## CSFV vaccines

<table style="width: 800pt; border-radius: 10px; text-align: left; box-shadow: 3px 3px 12px gray;">
<thead style="font-size: 20pt;">
<tr>
<th style="width:100pt;">Parameters
<th style="width:200pt;">LPC<sup style="font-size:10pt;">1</sup> vaccine
<th style="width:200pt;">CSF-E2 subunit vaccine
</tr>
</thead>
<tbody>

<tr>
<td>Antigen
<td>Infective, replicating live attenuated virus<br><font style="font-size: 12pt; color: gray;">(E<sup>rns</sup>, E1, E2 )</font>
<td>E2 subunit protein
</tr>

<tr>
<td>Immune response
<td>Humoral and cellular mediated immunity
<td>Antibody response
</tr>

<tr>
<td>Cross protection
<td>Yes
<td>Yes <span style="font-size: 12pt; color: gray;">1.1, 2.1, 2.2<br>(Gong et al., 2019, Tran et al., 2020)</span>
</tr>

<tr>
<td>Market share
<td style="font-size: 18pt; color: #FF5151;">95.97%</td>
<td>4.03%
</tr>

</tbody>
<tfoot>
<tr>
<td colspan=3 style="font-size:10pt;">1. Lapinized Philippines Coronel strain<br>2. Differentiate infected pigs from vaccinated animals
</tr>
</tfoot>
</table>

<font style="font-size: 10pt; position: absolute; bottom: 5pt; color: #8e8e8e;">
<ol>
Reference DOI:
<li>10.1016/j.vetmic.2019.108403  
<li>10.7774/cevr.2020.9.1.26
<li><a href="http://beingservice.nvri.gov.tw/module/web/module/Guest/TGuest.aspx">動物用生物藥品檢定綜合服務網</a>
<li><a herf="https://www.naif.org.tw/main.aspx">財團法人中央畜產會</a>
</ol></font>

---

## Factors associated with vaccine efficacy

<div style="width: 350pt; position: absolute; top: 100pt; left: 150pt; z-index: 1;">
<u style="color: #2984FF; font-size: 20pt;">LPC vaccine</u>
<ul>
<li>Viral loads per dose
<li>Master seed strains, manufacture procedures 
<ul>
<li>Batches and manufacturers 
</ul>
<li>Vaccination routes
<li style="color: #FF5151; font-weight: 900; font-size: 18pt;">Maternal derived antibody (MDA)</li>
</ul>
</div>
<img src="Ch1/pigsuck.jpg" alt="pigsuck" style="zoom: 30%; position: absolute; left: 100pt; top: 550pt;">

<div style="width: 400pt; position: absolute; top: 300pt; left: 150pt; z-index: 1;">
<u style="color: #2984FF; font-size: 20pt;">CSF-E2 subunit vaccine</u>
<ul>
<li>Master seed strains
<li style="color: #FF5151; font-weight: 900; font-size: 18pt;">Lack of clinical application information</li>
</ul>
</div>
<img src="Ch1/shot.jpg" alt="shot" style="zoom: 30%; position: absolute; left: 100pt; bottom: 200pt;">

<div style="width: 300pt; position: absolute; top: 100pt; left: 500pt; z-index: 1;">
<u style="color: #2984FF; font-size: 20pt;">Universal factors</u>
<ul>
<li>Stability of vaccine batches
<li>Cold chain of transportation/ storage
<li>Vaccination program
<li style="color: #FF5151; font-weight: 900; font-size: 18pt;">Concurrent infection
</ul></div>

<font style="font-size: 10pt; color: #8e8e8e; position: absolute; bottom: 5pt;">
Data source: <a herf = "https://www.pig333.com/articles/prrsv-interaction-with-other-respiratory-pathogens_7443/">PRRSV: Interaction with other respiratory pathogens</a><br>Images courtesy of Dr. Patrick G. Halbur.
</font>

???

<!--

## Multifactorial disease

<div style="border: 2px #2984FF solid; border-radius: 10px; width: 100pt;height:40pt; text-align: center; position: absolute; bottom: 180pt; left: 440pt;">
Virulence, strain, dose
</div>

<div style="border: 2px #2984FF solid; border-radius: 10px; width: 180pt; text-align: center; position: absolute; bottom: 60pt; left: 20pt;">
Age, breed,<br>genetic background,<br>immunological status
</div>

<div style="border: 2px #2984FF solid; border-radius: 10px; width: 150pt; text-align: center; position: absolute; bottom: 70pt; left: 500pt;">
Hygiene,<br>concurrent infection
</div>
-->

---

## Concurrent infectoin

<font style="font-size: 10pt; color: #8e8e8e; position: absolute; bottom: 5pt;">Reference DOI: 10.1186/s13567-020-00807-8
</font>

---

class: middle, center, inverse

# Porcine circoviruses

---

## Porcine circoviruses

<tbody style="line-height: 15pt;">
<tr>
<td style="text-align: left;">Discovery<br><font style="font-size: 10pt; color: #8e8e8e; line-height: 1pt;">Retrospective</font>
<td>1974<br><font style="font-size: 10pt; color: #8e8e8e; line-height: 1pt;">1974</font>
<td>1997<br><font style="font-size: 10pt; color: #8e8e8e; line-height: 1pt;">1962</font>
<td>2016<br><font style="font-size: 10pt; color: #8e8e8e; line-height: 1pt;">1993</font>
<td>2019<br><font style="font-size: 10pt; color: #8e8e8e; line-height: 1pt;">Not known</font>
</tr>
<!--
<tr>
<td>Circulation of the most recent ancestor
<td>1907-1933
<td>1907-1933
<td>1802-2013
<td>Not known
</tr>

<tr>
<td style="text-align: left;">Earliest detection
<td>1974
<td>1962
<td>1993
<td>Not known
</tr>
-->
<tr>
<td style="text-align: left;">Genome size<br><font style="font-size: 10pt; color: #8e8e8e; line-height: 1pt;">(nt)</font>
<td>1758-1760
<td>1766-1769
<td>1999-2001
<td>1770
</tr>

<tr>
<td style="text-align: left;">Evolution rate<br><font style="font-size: 10pt; color: #8e8e8e; line-height: 1pt;">(substitutions/site/year)</font>
<td>1.15×10<sup style="font-size: 12pt;">-5</sup>
<td style="color: #FF5151; font-size: 18pt;">1.2×10<sup style="font-size: 12pt;">-3</sup>
<td>2.35×10<sup style="font-size: 12pt;">-5</sup>~<br>1.69×10<sup style="font-size: 12pt;">-3</sup>
<td>Not known
</tr>

<tr>
<td style="text-align: left;">Distribution
<td>Global<br><font style="font-size: 10pt; color: #8e8e8e; line-height: 1pt;">(cell culture)</font>
<td style="color: #FF5151; font-size: 18pt;">Global</td>
<td>Global
<td>China
</tr>

<tr>
<td style="text-align: left;">Prevanlence
<td>Low
<td style="color: #FF5151; font-size: 18pt;">High</td>
<td>High
<td>Not known
</tr>

<tr>
<td style="text-align: left;">Genotypes
<td>
<td style="color: #FF5151; font-size: 18pt;">a-e<br><font style="color: #8e8e8e; font-weight: 300">suspected: f-h</td>
<td>
<td>
</tr>

</tbody>
</table>

<font style="font-size: 10pt; position: absolute; bottom: 5pt; color: #8e8e8e;">
Reference DOI: 10.1016/j.virusres.2020.198044
</font>

---

## Prevalence of PCV2 in Taiwan

<font style="font-size: 10pt; position: absolute; bottom: 5pt; color: #8e8e8e;">Reference DOI: 10.1038/s41598-019-47209-1</font>

<div style="border: 2px #2984FF dotted; border-radius: 10px; box-shadow: 3px 3px 12px gray; background-color: #F0FFF0;"><b style="color: #2984FF;">Tsai et al., 2019</b><br>Phylogeographic and genetic characterization of porcine circovirus type 2 in Taiwan from 2001–2017</div>

???
淋巴結在切片下見到壞死的病灶，並且在單核細胞中存在的嗜鹼性包涵體。(A & D)
在肺臟則有見到單核炎症細胞浸潤及間質性肺炎的情形。在免疫組織化學染色下，也可在肺臟及淋巴結看到 PCV2 抗原陽性反應。

---

## Porcine circovirus-associated disease (PCVAD)

<div style="width: 800pt; position: absolute; z-index: 1; line-height: 35pt; top: 90pt;">
<ul>
<li style="font-size: 18pt; color: #2984FF; font-weight: 900;">PCV2-systemic disease (PCV2-SD)</li>
<ul>
<li>Formerly "postweaning multisystemic wasting syndrome" (PMWS)
</ul>
<li style="font-size: 18pt; color: #01B468; font-weight: 900;">PCV2-subclinical infection (PCV2-SI)</li>
<li style="font-size: 18pt;">PCV2-reproductive disease (PCV2-RD)</li>
<li style="font-size: 18pt;">PVC2 dermatitis and nephropathy syndrome (PDNS)
</ul>
</div>

<font style="font-size: 10pt; position: absolute; bottom: 10pt; color: #8e8e8e;">
Reference DOI: 10.1016/j.virusres.2011.10.007<br>
Picture source: <a href="https://www.agromeat.com">Agromeat</a></font>

---

## PCV2-systemic disease

<table style="border-radius: 10px; box-shadow: 3px 3px 12px gray;">
<thead>
<tr>
<th>Clinical signs
<th>Pathological changes
<th>Diagnosis criteria
</tr>
</thead>
<tbody>
<td><img src="Ch2/SD_pig.jpg" alt="sd pig" style="zoom: 40%;">
<td><img src="Ch2/SD_path.jpg" alt="sd path" style="zoom: 150%;">
<td>
<ul style="font-size: 20pt;">
<li>Clinical signs
<li>Moderate-severe lymphoid depletion
<li>Moderate-high among of PCV2 in tissues<br>(>10<sup style="font-size: 12pt;">5-6</sup>)
</ul>
</tbody>
</table>

<font style="font-size: 10pt; position: absolute; bottom: 20pt; color: #8e8e8e;">
Reference DOI:<br>
1: 10.1016/j.virusres.2011.10.007<br>
2: 10.1586/14760584.2015.983084<br>
</font>

<!---

## Prevalencr of PCV2

<font style="font-size: 6pt; position: absolute; bottom: 20pt; left: 50pt;">Reference DOI: 10.1016/j.micpath.2020.104490</font>
-->

---

## PCV2 vaccines

<table style="width: 820pt; position: absolute; left: 40pt; bottom: 80pt; border-radius: 10px; box-shadow: 3px 3px 12px gray;">
<thead style="font-size: 14pt;">
<tr>
<th style="width: 5pt;">Type</th>
<th style="">Antigen type</th>
<th style="width: 300pt;">Vaccines (Vender)</th>
<th style="10pt">Antigen genotypes</th>
</tr>
</thead>
<tbody style="font-size: 14pt;">

<tr>
<td rowspan="4">Whole virus
<td rowspan="2" style="background-color: #CCE5FF;">Inactivated
<td>Circovac® (Ceva)
<td>PCV2a
</tr>

<tr>
<td>Suigen® PCV2 (Virbac)
<td>PCV2d
</tr>

<tr>
<td rowspan="2">Chimeric
<td>Fostera® Gold PCV (Zoetis US, inactivated)
<td>PCV1-2a
</tr>

<tr>
<td>Suvaxyn® Circo (Zoetis EU, inactivated)
<td>PCV1-2a
</tr>

<tr>
<td rowspan="5" style="background-color: #CCE5FF;">Subunit
<td rowspan="4" style="background-color: #CCE5FF; color: #FF5151; font-weight: 900;">Baculovirus-insect cell expression system
<td>Ingelvac CircoFLEX® (Boehringer Ingelheim)
<td>PCV2a
</tr>

<tr>
<td>Procilis® PCV (MSD)
<td>PCV2a
</tr>

<tr>
<td>Circumvent® PCV (Merk)
<td>PCV2a
</tr>

<tr>
<td>CircoGard® (Pharmgate)
<td>PCV2b
</tr>

<tr>
<td>Mycoplasma hyopneumoniae expression vector (Nexhyon)
<td>Mhyosphere® PCV ID (HIPRA)
<td>?
</tr>

</tbody>
</table>

---

## PCV2 vaccination in field farms

<font style="font-size: 10pt; position: absolute; bottom: 20pt; color: #8e8e8e;">
Appendix I. Supplementary data Fig S1.
</font>

<div style="position: absolute; top: 100pt;">
<ul>
<li style="font-size: 18pt;">One dose of PCV2 subunit vaccine at 3–4 weeks old
</ul>
</div>

???

<div style="border: 2px #FF5151 solid; border-radius: 10px; width: 150pt; position: absolute; left: 80pt; bottom: 100pt; text-align: center; animation: fadeInUp; animation-duration: 1s;">Leaky vaccine</div>

---

## Leaky vaccine

<ul style="position: absolute; top: 100pt;">
<li style="color: #2984FF; font-weight: 900; font-size: 18pt;"> Protective efficacy</li>
<ul style="font-size: 16pt;">
  <li> Homo/heterologous genotypes infection
  <li> Against severe clinical syndromes
  <li> Reduce viral replication and viremia level
</ul></ul><br>

<font style="font-size: 10pt; position: absolute; bottom: 20pt; color: #8e8e8e;">
Reference DOI:<br>
1: 10.1016/j.virusres.2011.10.007<br>
2: 10.1586/14760584.2015.983084<br>
</font>

<ul style="position: absolute; top: 200pt;">
<li style="color: #FF5151; font-weight: 900; font-size: 18pt;"> Cannot eliminate virus completely</li>
<ul style="font-size: 16pt;">
<li>Positive selection acting<br>- virus evolution<p>
<li><font style="color: #2984FF;">PCV2a</font> / 
    <font style="color: #01B468;">PCV2b</font> / 
    <font style="color: #FF5151;">PCV2d</font>
</ul>
</ul>

<table style="font-size: 14pt; position: absolute; left: 100pt; bottom: 70pt; width: 400pt; text-align: center; border-radius: 10px; box-shadow: 3px 3px 12px grey;">
<thead style="line-height: 5pt;">
<tr>
<th>Genotypes
<th>Before 2012
<th>2013~2018
</tr>
</thead>

<tr>
<td>Total
<td>22
<td>24
</tr>

</tbody>
</table>

???
- Leaky vaccine
  - Reduce viremia / clinical signs
  - Positive selection acting - virus evolution

---

## PCV2-subclinical infection

<table style="width: 800pt; border-radius: 10px; box-shadow: 3px 3px 12px gray; position: absolute; top: 150pt; left: 50pt;">
<thead>
<tr>
<th style="width: 250pt;">Clinical signs
<th style="width: 250pt;">Pathological changes
<th style="width: 300pt;">Diagnosis criteria
</tr>
</thead>

<tbody style="">
<tr>
<td valign="top">
<ul style="font-size: 18pt;">
<li style="font-weight: 900;">Lack of clinical signs
<li style="color: #8e8e8e;">Decreased ADWG
</ul>
<td valign="top">
<ul style="font-size: 18pt;">
<li style="font-weight: 900;">Lack of pathological changes
<li style="color: #8e8e8e;">Mild lymphoid depletion
</ul>
<td valign="top">
<ul style="font-size: 18pt;">
<li style="color: #FF5151; font-weight: 900;">Very hard for diagnosis</li>
<li>Persistent infection with low viral load (<font style="color: #FF5151;"><10<sup style="font-size: 12pt;">5</sup></font>)
<li>Viral shedding<br>(continuously or intermittently)
</ul>
</tr>
</tbody>
</table>

<font style="font-size: 10pt; position: absolute; bottom: 20pt; color: #8e8e8e;">
Reference DOI:<br>
1: 10.1016/j.virusres.2011.10.007<br>
2: 10.1586/14760584.2015.983084<br>
</font>

---

## Goals of vaccination in field farms

<ul>
<li style="color: #2984FF; font-size: 20pt;">Reduce pathogens propagation or damage</li>
  <ul>
  <li>Against clinical syndrome
  <li>reduce viral replication
  </ul>
  <br>
<li style="color: #2984FF; font-size: 20pt;">Limit pathogen circulation</li>
   <ul>
   <li> Reproductive herd 
   <li> Permanent reservoir and source of reinfection
   </ul>
   <br>
<li style="color: #2984FF; font-size: 20pt;">Head towards <u>eradication</u> at the herd level</li>
</ul>

Leaky vaccine → PCVAD → LPC vaccine → Vaccine efficacy?

</div>

---

## Aim of this study

<ul style="font-size: 16pt;">
<li style="color: #2984FF; font-size: 20pt;"> Evaluate the impact of PCVAD on live attenuated CSF vaccine (Chapter 2)</li>
<ul>
<li>Conventional pigs
<li>Vaccination-challenge model
</ul><br>
<li style="color: #2984FF; font-size: 20pt;"> Interference of MDA and application of CSF vaccines (Chapter 3)</li>
<ul>
<li>Live attenuated CSF vaccine
<li>CSF-E2 subunit vaccine
</ul><br>
<li style="color: #2984FF; font-size: 20pt;"> Efficacy of CSFV-PCV2 bivalent vaccine (Chapter 4)</li>
</ul>

---
name: SecI
class: middle, inverse

## Section I 
<div style="font-size: 20pt; color: #FFFFFF;">
The impact of porcine circovirus-associated disease on live attenuated classical swine fever vaccine in field farm applications</div>

---

## Artificial induced PCVAD in conventional pigs

<div style="border: 2px #8e8e8e dashed; border-radius: 10px; width: 190pt; color: #8e8e8e; text-align: left; font-size: 12pt; position: absolute; top: 10pt; right: 10pt;">
Section I:<br>Impact of PCVAD on LPC vaccine
</div>

<table style="width: 800pt; border-radius: 10px; box-shadow: 3px 3px 12px gray; position: absolute; top: 90pt; left: 60pt;">
<thead style="line-height: 10pt;">
<tr>
<th rowspan="2">Group (n)
<th colspan="2">Weeks of age
</tr>
<tr>
<th>3
<th>7
</tr>
</thead>

<tbody>
<tr>
<td style="text-align: center;">A (4)
<td>PCV2 vaccination
<td rowspan="2" style="color: #FF5151; font-weight: 900; font-size: 20pt;">PCV2 challenge
</tr>
<tr>
<td style="text-align: center;">B (4)
<td>Placebo
</tr>
</tbody>
</table>

---

## Impact of PCVAD on LPC vaccine

<table style="width: 800pt; border-radius: 10px; box-shadow: 3px 3px 12px gray; position: absolute; top:90pt; left: 60pt; font-size: 14pt; line-height: 9pt;">
<thead style="line-height: 5pt;">
<tr>
<th rowspan="2">Group (n)
<th colspan="4">Weeks of age
</tr>
<tr>
<th>3
<th>6
<th>7
<th>9
</tr>
</thead>
<tbody style="line-height: 10pt;">
<tr>
<td style="text-align: center;">C (6)
<td>PCV2 vaccination
<td rowspan="2" style="color: #01B468;">LPC vaccination
<td rowspan="2" style="color: #FF5151; font-weight: 900;">PCV2 challenge
<td rowspan="2" style="color: #01B468;">LPC vaccination
</tr>
<tr>
<td style="text-align: center;">D (4)
<td>Placebo
</tr>
</tbody>
</table>

<div style="position: absolute; top: 160pt; width: 800pt;">
.panelset[
.panel[.panel-name[PCV2 antibody & viremia]
<img src="Ch2/Fig2.2ab.jpg" alt="Fig2.2ab" style="height: 280pt; position: absolute; left: 10pt;">
]
.panel[.panel-name[ADWG & pathological changes]
<img src="Ch2/Fig2.2cd.jpg" alt="Fig2.2cd" style="height: 280pt; position: absolute; left: 50pt;">
]
.panel[.panel-name[CSFV-specific neutralizing antibody]

<div style="position: absolute; left: 500pt; width: 280pt;">
<ul style="font-size: 18pt;">
<font style="color: #2984FF; font-size: 20pt;">Impact of PCVAD</font>
<li>C: 4.17±0.38 log<sub style="font-size: 12pt;">2</sub> <font style="color: #FF5151;">(PCV2-SI)</font>
<li>D: 4.50±1.26 log<sub style="font-size: 12pt;">2</sub> <font style="color: #2984FF;">(PCV2-SD)</font>

</ul>
</div>
]]
</div>

???
<img src="Ch2/Fig2.2.jpg", alt="Fig2.2" style="zoom: 48%; position: absolute; bottom: 5pt; left: 200pt;">

---

## Impact of PCV2-SI in field farms

<table style="width: 800pt; border-radius: 10px; box-shadow: 3px 3px 12px gray; position: absolute; top:90pt; left: 60pt; font-size: 15pt; line-height: 9pt;">
<thead style="line-height: 9pt;">
<tr>
<th rowspan="2">Group (n)
<th colspan="3">Weeks of age
</tr>
<tr>
<th>3
<th>12
<th>15
</tr>
</thead>
<tbody style="line-height: 10pt;">
<tr>
<td style="text-align: center;">E (8)
<td>PCV2 vaccination
<td rowspan="2">LPC vaccination
<td rowspan="2">LPC vaccination
</tr>
<tr>
<td style="text-align: center;">F (12)
<td>Placebo
</tr>
</tbody>
</table>

<div style="position: absolute; top: 165pt; width: 800pt;">
.panelset[
.panel[.panel-name[CSFV-specific neutralizing antibody]
<img src="Ch2/Fig2.3a.jpg" alt="Fig2.3a" style="height: 250pt; width: 450pt; position: absolute; left: 0pt; z-index: 1;">

<div style="position: absolute; left: 460pt; top: 80pt;">
<font style="font-size: 20pt; color: #2984FF;">Conversion of CSFV-specific NA</font>
<ul style="font-size: 16pt;">
<li>Group E: 7.44 ± 0.22 log<sub style="font-size: 12pt;">2</sub>
<li>Group F: 5.08 ± 0.47 log<sub style="font-size: 12pt;">2</sub>
</ul>
</div>
]
.panel[.panel-name[Serum PCV2 viral load]
<img src="Ch2/Fig2.3b.jpg" alt="Fig2.3b" style="height: 250pt; width: 480pt; position: absolute; left: 0pt;">

<div style="position: absolute; left: 450pt; top: 80pt;">
<font style="color: #2984FF; font-size: 20pt;">18 weeks old</font>
<ul style="font-size: 16pt;">
<li>Group E: 0.43 ± 0.28 log<sub style="font-size: 12pt;">10</sub>
<li>Group F: 1.31 ± 0.42 log<sub style="font-size: 12pt;">10</sub>
</ul>
</div>
]
.panel[.panel-name[PCV2-SI and CSFV-specific NA level]
<img src="Ch2/Fig2.3c.1.png" alt="Fig2.3c" style="height: 250pt; width: 450pt; position: absolute; left:0pt;">

<div style="position: absolute; left: 480pt; top: 60pt; font-size: 15pt;">
<b style="color: #2984FF;">Group E</b>
<ul>
<li>PCV2 DNA detected in 2 pigs
<li>All pigs with CSFV NA level > 1:32
</ul>
<b style="color: #2984FF;">Group F</b>
<ul>
<li> 50% pigs were PCV2 DNA positive
  <ul>
  <li>83.33% (5/6) > 10<sup style="font-size: 6pt;">2</sup> copies/μL
  </ul>
<li>Only 2 pigs showed protective CSFV NA level
</ul>
</div>

]]
</div>

---

## Summary I

<div style="position: absolute; top: 100pt; left: 50pt; z-index: 1; font-size: 18pt; width: 800pt; line-height: 25pt;">
<ul>
<li>A vaccination-challenge animal model was conducted to mimic different statuses of PCVAD.</li><br>
<li>The leaky efficacy of PCV2 subunit vaccine was demonstrated with a conventional pig model after PCV2 challenge.</li><br>
<li>The efficacy of live attenuated CSFV vaccine may be <font style="background-color: #4EFEB3;">compromised in pigs with PCVAD</font>.
<ul>
<li>PCV2-SD
<li>PCV2-SI
</ul><br>
<li>Serum PCV2 viral load <font style="background-color: #4EFEB3;">< 10<sup style="font-size: 12pt;">2</sup> copies/μL</font> may not interfer LPC vaccine induced neutralizing antibody.
</ul>
</div>

???
<img src="Ch2/AUC.png" alt="AUC" style="zoom: 15%;">

<font style="font-size: 10pt;color:#8e8e8e;">
Reference DOI: 10.1186/s12889-018-5709-x
</font>

---
name: secII
class: middle, inverse

## Section II 
<div style="font-size: 20pt; color: #FFFFFF;">
Evaluation of classical swine fever E2 (CSF-E2) subunit vaccine efficacy in the prevention of virus transmission and impact of maternal derived antibody interference in field farm applications</div>

---

## Experimental design

---

## Protective efficacy of CSF-E2 subunit vaccine

<div style="width: 800pt; position: absolute; top: 70pt;">
.panelset[
.panel[.panel-name[Schema]
<img src="Ch3/Trial1.1.jpg", alt="Trial1.1", style="zoom: 100%; position: absolute; left: 0pt;">
]
.panel[.panel-name[Gross lesions]
<img src="Ch3/FigS4_atof.jpg", alt="FigS4_atof", style="height: 250pt; position: absolute; left: 150pt;">
]
.panel[.panel-name[Histopathological changes]
<img src="Ch3/FigS4_gtoi.jpg", alt="FigS4_gtoi", style="height: 250pt; position: absolute; left: 150pt;">
]
.panel[.panel-name[Hematology]
<img src="Ch3/Fig3.2cd.jpg", alt="Fig3.2", style="height: 250pt; position: absolute; left: 10pt; z-index: 3;">
]
.panel[.panel-name[CSFV-specific NA and viremia]
<img src="Ch3/Fig3.2ab.jpg", alt="Fig3.2", style="height: 260pt; position: absolute; left: 20pt; z-index: 3;">
]
]
</div>

<table style="width: 500pt; border-radius: 10px; box-shadow: 3px 3px 12px gray; position: absolute; top:400pt; left: 200pt; font-size: 14pt; line-height:9pt;">
<thead style="line-height: 5pt;">
<tr>
<th rowspan="2" style="width: 100pt;">Groups (n)
<th colspan="2">Weeks of age
</tr>

<tr>
<td style="text-align: center;">A (4)
<td>CSF-E2 vaccine
<td style="font-weight: 900; color: #FF5151;">CSFV challenge<sup style="font-size: 10pt;">i</sup>
</tr>

<tr>
<td style="text-align: center;">B (4)
<td>--
<td>Cohabitation at 4 DPC<sup style="font-size: 10pt;">ii</sup>
</tr>

<tr>
<td style="text-align: center;">C (2)
<td>Placebo
<td style="font-weight: 900; color: #FF5151;">CSFV challenge
</tr>

</tbody>
</table>

<font style="font-size: 8pt; position: absolute; bottom: 50pt; z-index:2;">
i: ALD strain, 1 × 10<sup style="font-size: 6pt;">5</sup> TCID<sub>50</sub><br>
ii: Days post challenge
</font>

???

**Pathological changes in group C**

在急性病毒感染的狀態下，可能見到耳翼及四肢末端發紺，
在扁桃腺看到多發的膿瘍，腎臟潮紅及消化道出血，腸繫膜淋巴結腫大。

切片下則可看到，瀰漫性血管內凝血，特別在皮膚、耳翼及腎臟的微血管，扁桃腺則可以看到膿瘍，
脾臟局部性壞死及淋巴結巴球流失及出血。

---

##Vaccination on sows

<div style="position:absolute; top: 70pt; width: 800pt;">
.panelset[
.panel[.panel-name[Schema]
<img src="Ch3/Trial2.1.jpg", alt="Trial2.1", style="height: 250pt; position: absolute;">
]
.panel[.panel-name[Sows vaccination]
<img src="Ch3/Fig3.3ab.jpg", alt="Fig3.3ab" style="height: 250pt; position: absolute; left: 20pt;">
<div style="border: 2px #2984FF solid; border-radius: 10px; font-size: 18pt; box-shadow: 3px 3px 12px grey; position: absolute; top: 50pt; left: 650pt; text-align: left; width: 180pt; height: 80pt; line-height: 20pt;">
<ul>
  <li>D (CSF-E2): 6%
  <li>E (LPC): 42%
</ul>
</div>
]
.panel[.panel-name[Maternal derived antibody in piglets]
<img src="Ch3/Fig3.3c.jpg" alt="Fig3.3c" style="height: 260pt; position: absolute; left: 100pt;">
<div style="position: absolute; left: 480pt; top: 50pt;">
<b style="color: #2984FF;">CSF-E2 subunit vaccine</b>
<ul>
<b style="color: #2984FF;">Sows</b>
<li>Reduced CSFV RNA positive ratio
<li>Consistent antibody level distribution</li><br>
<b style="color: #2984FF;">Offspring</b>
<li>Consistent antibody level distribution
<li>Long-lasting MDA
</ul>
</div>
]]
</div>

<table style="width: 500pt; box-shadow: 3px 3px 12px gray; border-radius: 10px; position: absolute; top:400pt; left: 200pt; font-size: 14pt; line-height: 9pt;">
<thead style="line-height: 5pt;">
<tr>
<th style="width: 100pt; text-align: center;">Groups (n)
<th style="width: 100pt;">Vaccines
<th style="width: 300pt;">Description
</tr>
</thead>

<tbody style="line-height: 10pt;">
<tr>
<td style="text-align: center;">D (25)
<td>CSF-E2
<td>-5 and -3 weeks before parturition
</tr>

<tr>
<td style="text-align: center;">E (35)
<td>LPC
<td>Once before insemination
</tr>

<tbody>
<tr>
<td style="text-align: center;">F (20)
<td>
<td>offspring from group D (5 sows)
</tr>

<tr>
<td style="text-align: center;">G (20)
<td>
<td>offspring from group E (5 sows)
</tr>

</tbody>
</table>

---

## Interference of MDA on CSFV vaccines

<div style="width: 800pt; position: absolute; top: 70pt;">
.panelset[
.panel[.panel-name[Schema]
<img src="Ch3/Trial3.jpg" alt="Trial3" style="height: 220pt; position: absolute; left: 10pt;">
]
.panel[.panel-name[Dynamic of CSFV-specific antibody]
<img src="Ch3/Fig3.4.jpg" alt="Fig3.4" style="width: 450pt; height: 250pt;; position: absolute; left: 10pt;">

<div style="position: absolute; left: 480pt; top: 50pt;">
<b style="color: #2984FF;">At 20 weeks old</b>
<ul>
<li>H (CSF-E2): 72.94 ± 3.56%, <font style="font-weight: 900; color: #1F77B4FF;">CV 11.95%</font><br>(6/6, 100%)<br><br>
<li>I (LPC): 57.21 ± 10.82%, <font style="color: #2CA02CFF; font-weight: 900;">CV 40.31%</font><br>(5/6, 83.33%)<br><br>
<li>J (Control): 14.38 ± 3.89%, <font style="font-weight: 900; color: #FF7F0EFF;">CV 66.29%</font><br>(0/6, 0%)
</ul>
</div>
]]
</div>

<table style="width: 500pt; border-radius: 10px; box-shadow: 3px 3px 12px gray; position: absolute; top: 400pt; left: 200pt; font-size: 14pt; line-height: 9pt">
<thead style="line-height: 5pt;">
<tr>
<th rowspan="2" style="width: 100pt;">Groups (n)
<th colspan="2" style="width: 400pt;">Weeks of age
</tr>
<tr>
<th style="width: 200pt;">3, 6
<th>16<sup style="font-size: 10pt;">*</sup>
</tr>
</thead>

<tr>
<td style="text-align: center;">H (6)
<td>CSF-E2 vaccination
<td rowspan="3" style="font-weight: 900; color: #01B468; font-size: 20pt;">LPC boost</td>
</tr>

<tr>
<td style="text-align: center;">I (6)
<td>LPC vaccination

</tr>

<tr>
<td style="text-align: center;">J (6)
<td>Placebo

</tr>

</tbody>
</table>

---

## Application of CSFV vaccines

<div style="width: 800pt; position: absolute; top: 70pt;">
.panelset[
.panel[.panel-name[Schema]
<img src="Ch3/Trial4.jpg" alt="Trial4" style="zoom: 80%; position: absolute; left: 50pt;">
]
.panel[.panel-name[Dynamic of CSFV-specific antibody]
<img src="Ch3/Fig3.5.jpg" alt="Fig3.5" style="width: 440pt; height: 260pt; position: absolute; left: 20pt;">

<div style="position: absolute; left: 480pt; top: 50pt;">
<b style="color: #2984FF;">At 20 weeks old</b>
<ul>
<li>K (CSF-E2): 85.60 ± 2.43%<br>(10/10, 100%)<br><br>
<li>L (LPC): 72.39 ± 2.77%<br>(10/10, 100%)<br><br>
<li>M (Control): 17.70 ± 2.22%<br>(0/10, 0%)
</ul>
</div>
]]
</div>

<table style="width: 500pt; border-radius: 10px; box-shadow: 3px 3px 12px gray; position: absolute; top: 400pt; left: 200pt; line-height: 9pt; font-size: 14pt;">
<thead style="line-height: 5pt;">
<tr>
<th rowspan="2" style="width: 100pt;">Groups (n)
<th colspan="2">Weeks of age
</tr>

<tbody style="line-height: 10pt;">
<tr>
<td style="text-align: center;">K (10)
<td>CSF-E2 vaccination
<td style="background-color: #adadad;">
</tr>

<tr>
<td style="text-align: center;">L (10)
<td style="background-color: #adadad;">
<td>LPC vaccination
</tr>

<tr>
<td style="text-align: center;">M (10)
<td>Placebo
<td style="background-color: #adadad;">
</tr>
</tbody>
</table>

---

## Summary II

<div style="font-size: 18pt; line-height: 25pt;">
<ul>
<li>The efficacy of CSF-E2 subunit vaccine was demonstrated via a challenge-cohabitation trial.</li><br>
<li>CSF-E2 subunit vaccine immunization <font style="background-color: #4EFBE3">prevents viral transmission among pigs</font> after challenge.</li><br>
<li>CSF-E2 subunit vaccine significantly <font style="background-color: #4EFBE3">reduces CSFV RNA detection</font> via saliva monitoring in sows.</li><br>
<li><font style="background-color: #4EFBE3">No maternally derived antibody interference</font> on early CSF-E2 immunization in piglets was noticed.</li><br>
<li>CSF-E2 subunit vaccine provokes a <font style="background-color: #4EFBE3">long-lasting protective immunity</font> in field farm applications.
</ul>
</div>

???

<div style="width: 400pt;>
<b style="color: #2984FF; font-weight:900;"> Choicing CSFV vaccines</b>
<ul>
<li> Epidemiology of the disease</li><br>
<li> Affected animal population
  <ul>
  <li> Domestic pigs
  <li> Wild boar
  </ul><br>
<li> Production and farming types</li><br>
<li> Economic situation</li>
<font style="font-size: 6pt;">
Reference DOI: 10.1016/j.virusres.2020.198151
</font>
</div>

---
name: secIII
class: middle, inverse

## Section III 
<div style="font-size: 20pt; color: #FFFFFF;">
Evaluation the induced immune responses and protective efficacy of CSFV-PCV2 bivalent vaccine</div>

---

## Clinical courses

<div style="width: 300pt; height: 380pt; position: absolute; left:100pt; top: 100pt;">
<b style="font-weight: 900;">PCV2 infection</b>
<ul>
<li style="color:#2984FF;">Systemic disease</li>
  <ul>
  <li>Wasting, weight loss, paleness of skin, dyspnea, diarrhea,
  </ul><br>
<li style="color:#2984FF;">Subclinical infection</li>
  <ul>
  <li>Predominant
  <li>Lack of clinical signs and barely low virus detection in pigs
  </ul>
</ul>
</div>

<div style="width: 300pt; height: 380pt; position: absolute; left:440pt; top: 100pt;">
<b style="font-weight: 900;">CSFV infection</b>
<ul>
<li style="color: #2984FF;">Chronic infection</li>
  <ul>
  <li>Low virulence strain infection
  <li>Depression, weakness, anorexia, conjunctivitis
  </ul><br>
<li style="color: #2984FF;">Persistent infection</li>
  <ul>
  <li>Congenital/postnatal
  <li>Lack of clinical signs
  </ul>
</ul>
</div>

<div style="border: 2px #2984FF solid; border-radius: 10px; width: 300pt; font-size: 25pt; text-align: center; position: absolute; left: 100pt; top: 350pt; animation: fadeInUp; animation-duration: 1s;">
Leaky vaccine
</div>

<div style="border: 2px #2984FF solid; border-radius: 10px; width: 300pt; font-size: 25pt; text-align: center; position: absolute; left: 480pt; top: 350pt; animation: fadeInUp; animation-duration: 1s;">
Disease/pathogen elimination?
</div>

---

## Bivalent vaccine

---

## Experimental design

---

## Bivalent vaccine induced immune responses

<table style="width: 500pt; border-radius: 10px; box-shadow: 3px 3px 12px gray; position: absolute; top: 85pt; left: 250pt; font-size: 14pt; line-height: 9pt;">
<thead style="line-height: 5pt;">
<tr>
<th style="text-align: center; width: 100pt;">Groups (n)
<th>Vaccines
</tr>
</thead>

<tbody style="line-height: 10pt;">
<tr>
<td style="text-align: center;">A1 (5)
<td>Bivalent vaccine
</tr>

<tr>
<td style="text-align: center;">A2 (5)
<td>PCV2 calpsid subunit vaccine
</tr>

<tr>
<td style="text-align: center;">A3 (5)
<td>CSF-E2 subunit vaccine
</tr>

<tr>
<td style="text-align: center;">A4 (5)
<td>Placebo
</tr>
</tbody>
</table>

<font style="font-size: 10pt; position: absolute; bottom: 5pt; z-index: 1;">*: booster immunization at 2 weeks after prime vaccination</font>

<div style="width: 800pt; position: absolute; top: 175pt;">
.panelset[
.panel[.panel-name[CSFV-specific immune response]

<img src = "Ch4/Fig4.2ac.jpg", alt="Fig4.2ac" style = "height: 220pt; width:600pt;position: absolute; left:-30pt;">
<img src = "Ch4/Fig4.2e.jpg", alt="Fig4.2e" style = "height: 120pt; position: absolute; z-index: 1; top: 100pt; right:-40pt;">
]
.panel[.panel-name[PCV2-specific immune response]

<img src = "Ch4/Fig4.2bd.jpg", alt="Fig4.2bd" style = "height: 220pt; width:600pt;position: absolute; left:-30pt;">
<img src = "Ch4/Fig4.2f.jpg", alt="Fig4.2f" style = "height: 120pt; position: absolute; z-index: 1; top: 100pt; right:-40pt;">
]]
</div>

---

## Bivalent vaccine induced immune response

<table style="width: 600pt; border-radius: 10px; box-shadow: 3px 3px 12px gray; position: absolute; top: 85pt; left: 180pt; font-size: 14pt; line-height: 9pt;">
<thead style="line-height: 5pt;">
<tr>
<th style="text-align: center; with: 150pt;">Groups (n)
<th style="width: 300pt;">Vaccines
<th style="width: 150pt;">Programs
</tr>
</thead>

<tbody style="line-height: 10pt;">
<tr>
<td style="text-align: center;">B1 (5)
<td>PCV2 capsid subnit vaccine
<td rowspan="4">4 and 6 weeks old<p>(2 doses)
</tr>

<tr>
<td style="text-align: center;">B2 (5)
<td>Bivalent vaccine
</tr>

<tr>
<td style="text-align: center;">B3 (5)
<td>CSF-E2 subunit vaccine (W/O/W)<sup style="font-size: 8pt;">i</sup>
</tr>

<tr>
<td style="text-align: center;">B4 (5)
<td>CSF-E2 subunit vaccine (W/O)<sup style="font-size: 8pt;">ii</sup>
</tr>
</tbody>
</table>

<font style="font-size: 8pt; position: absolute; bottom: 20pt;">
Adjuvant: <br>
i: water-in-oil-in-water<br>
ii: water-in-oil
</font>

<div style="width: 800pt; position: absolute; top: 175pt;">
.panelset[
.panel[.panel-name[CSFV-specific immune response]
<img src="Ch4/Fig4.3acd.jpg" alt="Fig4.3acd" style="height: 200pt;">
]
.panel[.panel-name[PCV2-specific immune response]
<img src="Ch4/Fig4.3bef.jpg" alt="Fig4.3bef" style="height: 200pt;">
]]
</div>

---

## Protective efficacy of bivanelt vaccine

<div style="width: 800pt; position: absolute; top:70pt;">
.panelset[
.panel[.panel-name[Schema]
<img src="Ch4/trialC1.jpg" alt="trialC1" style="height: 250pt; position: absolute; left: 80pt;">
]
.panel[.panel-name[PCV2 portion]
<img src="Ch4/Fig4.4eg.jpg" alt="Fig4.4eg" style="height: 250pt; position: absolute; left: 10pt;">
]
.panel[.panel-name[Clinical signs]
<img src="Ch4/Fig4.4abc.jpg" alt="Fig4.4abc" style="height: 220pt;">
]
.panel[.panel-name[CSFV portion]
<img src="Ch4/Fig4.4df.jpg" alt="Fig4.4df" style="height: 250pt; position: absolute; left: 10pt;">
]
.panel[.panel-name[Histopathological changes]
<img src="Ch4/Fig4.4h.jpg" alt="Fig4.4h" style="height: 250pt; position: absolute; left: 30pt;">

]]
</div>

<table style="width: 550pt; border-radius: 10px; box-shadow: 3px 3px 12px gray; position: absolute; top: 400pt; left: 180pt; font-size: 14pt; line-height:9pt;">
<thead style="line-height: 5pt;">
<tr>
<th style="text-align: center; with: 150pt;">Groups (n)
<th style="width: 200pt;">Vaccines
<th style="width: 200pt;">Challenge / cohabitation
</tr>
</thead>

<tbody style="line-height: 10pt">
<tr>
<td style="text-align: center;">C1a (4)
<td>Bivalent vaccine (1 dose)
<td rowspan="3" style="color: #FF5151; font-weight: 900; font-size: 20pt;">CSFV and PCV2</td>
</tr>

<tr>
<td style="text-align: center;">C2a (4)
<td>Bivalent vaccine (2 doses)

</tr>

<tr>
<td style="text-align: center;">C3 (4)
<td>Placebo (2 doses)

</tr>

<tr>
<td style="text-align: center;">C4 (4)
<td>-
<td>Sentinel pigs
</tr>
</tbody>
</table>

???
PCV2的抗體及病毒偵測部份先講，用此確認豬隻為 PCV2-SI 感染的情況

<div style="position: absolute; left: 430pt; top: 50pt;">
<ul>
<li>Antigen-specific immune response</li><br>
<li>No/mild clinical signs and hyperpyrexia</li><br>
<li>Less histopathological changes</li><br>
<li style="color: #FF5151; font-weight: 900;">Preventing viral shedding</li>
</ul>
</div>

---

## Against horizontal transmission

<div style="width: 800pt; position: absolute; top: 70pt;">
.panelset[
.panel[.panel-name[Schema]
<img src="Ch4/trialC2.jpg" alt="trialC2" style="height: 220pt; position: absolute; left: 10pt;">
]
.panel[.panel-name[Clinical signs]
<img src="Ch4/Fig4.5abc.jpg" alt="Fig4.5abc" style="height: 220pt;">
]
.panel[.panel-name[CSFV portion]
<img src="Ch4/Fig4.5df.jpg" alt="Fig4.5df" style="height: 250pt; position: absolute; left: 15pt;">
]
.panel[.panel-name[Histopathological changes]
<img src="Ch4/Fig4.5h.jpg" alt="Fig4.5h" style="height: 250pt; position: absolute; left: 30pt;">
<img src="Ch4/HE2.jpg" alt="HE2" style="zoom: 30%; position: absolute; top: 220pt; right: 80pt; ">

]]
</div>

<table style="width: 550pt; border-radius: 10px; box-shadow: 3px 3px 12px gray; position: absolute; top: 400pt; left: 200pt; font-size: 14pt; line-height: 9pt;">
<thead style="line-height: 5pt;">
<tr>
<th style="text-align: center; width: 100pt;">Groups (n)
<th style="width: 200pt;">Vaccines
<th>Challenge / cohabitation
</tr>
</thead>

<tbody stlye="line-height: 10pt;">
<tr>
<td style="text-align: center;">C1b (4)
<td>Bivalent vaccine (1 dose)
<td rowspan="2" style="font-size: 20pt;">Cohabitation
</tr>

<tr>
<td style="text-align: center;">C2b (4)
<td>Bivalent vaccine (2 doses)
</tr>

<tr>
<td style="text-align: center;">C3 (4)
<td>Placebo (2 doses)
<td style="color: #FF5151; font-weight: 900;">CSFV and PCV2</td>
</tr>
</tbody>
</table>

???
PCV2的抗體及病毒偵測部份帶過就好，由於豬隻一進來就有 PCV2 了
.panel[.panel-name[PCV2 portion]
<img src="Ch4/Fig4.5eg.jpg" alt="Fig4.5eg" style="height: 220pt; position: absolute; left: 80pt;">
]

---

## Vaccination on conventional pigs

<div style="width: 800pt; position: absolute; top:70pt;">
.panelset[
.panel[.panel-name[CSFV-specific antibody]
<img src="Ch4/Fig4.6ab.jpg" alt="Fig4.6ab" style="height: 280pt; position: absolute; left: 0pt;">

]
.panel[.panel-name[PCV2-specific antibody and viral load]
<img src="Ch4/Fig4.6cd.jpg" alt="Fig4.6cd" style="height: 280pt; position: absolute; left: 0pt;">

]
.panel[.panel-name[Peripheral lymph nodes]
<img src="Ch4/Fig4.6e.jpg" alt="Fig4.6e" style="height: 220pt;position: absolute; left: 65pt; z-index: 3;"><img src="Ch4/Fig4.6f.jpg" alt="Fig4.6f" style="height: 220pt; position: absolute; left: 400pt; z-index: 3;">

<table style="width: 600pt; border-radius: 10px; box-shadow: 3px 3px 12px gray; position: absolute; top:420pt; left: 180pt; font-size: 14pt; line-height: 9pt;">
<thead style="line-height: 5pt;">
<tr>
<th style="text-align: center; width: 100pt;">Groups (n)
<th style="width: 300pt;">Vaccines
<th>Challenge
</tr>
</thead>

<tbody style="line-height: 10pt;">
<tr>
<td style="text-align: center;">D1 (5)
<td>Bivalent vaccine (1 dose, 4 week-old)
<td rowspan="2" style="color: #FF5151; font-weight: 900; font-size: 15pt;">PCV2 <font style="color: #8e8e8e;">(at 8 week-old)</font>
</tr>

<tr>
<td style="text-align: center;">D2 (5)
<td>Placebo (1 dose, 4 week-old)
</tr>
</tbody>
</table>

---

## Summary III

<ul style="font-size: 18pt; line-height: 30pt;">
<li>The novel CSFV-PCV2 bivalent vaccine elicits <font style="background-color:#4EFBE3;">antigen-specific immune response</font> on immunized animals.</li><br>

<li>The CSFV-PCV2 bivalent vaccine induces <font style="background-color:#4EFBE3;">complete protective efficacy</font> against CSFV challenge on immunized pigs and prevent horizontal transmission.</li><br>
  
<li>The CSFV-PCV2 bivalent vaccine can induce <font style="background-color:#4EFBE3;">adequate immune response on PCV2-SI pigs</font>, demonstrating its potential for application in field farms.
</ul>
  
---
class: middle, inverse
name: conclusion

# Conclusions

---

## The old viruses

<font style="font-size: 6pt; position: absolute; bottom: 50pt; color: #8e8e8e;">
Reference DOI:<br>1: 10.1016/j.bbrc.2018.01.141<br>2: 10.1016/j.virusres.2020.198151
</font>
<ul style="position: absolute; z-index: 1;">
<li style="font-size: 20pt;"> CSFV and PCV2</li><br>
<li style="font-size: 20pt;"> Vaccination</li>
  <ul>
  <li> Subunit vaccine
  <li> Live attenuated vaccine
  </ul>
</ul>
<img src="Ch5/normalImm.jpg" alt="normalImm" style=" width: 300pt; height: 300pt; position: absolute; top: 100pt; left: 180pt;">

???
在感染後一個月內

<div style="position: absolute; top: 100pt; left: 520pt; font-size: 18pt;">
<ul>
<b style="color: #2984FF; font-weight:900;">CSFV</b>
<li>DIVA
<li>Chronic/persistent infection
</ul>

<ul>
<b style="color: #2984FF; font-weight:900;">PCV2</b>
<li>Decoy epitope of PCV2</ul>

???
在使用活毒減毒疫苗方面，無法經由 DIVA 簡單的區分疫苗毒及野外毒。因此在有慢性或持續性感染的病例存在時，牧場豬群受到病毒感染的風險提升，時間也拖長了！

在 PCV2 部份，已知大部份的豬隻可能都是長期 PCV2-SI 的狀態。

---

## Asymptomatic infection

<font style="font-size: 6pt; position: absolute; bottom: 50pt; color: #8e8e8e;">
Reference DOI:<br>1: 10.1186/s12889-018-5709-x<br>2: 10.1016/j.virusres.2020.198151
</font>

<div style="position: absolute; font-size: 18pt;">
<ul>
<li> Chronic/persistent infection
<li> Subclinical infection
</ul>
</div>
<img src="Ch2/AUC.png" alt="AUC" style="height: 280pt; position: absolute; bottom:50pt; left: 200pt;">

---

## Conclusions

<li> The leaky efficacy of PCV2 subunit vaccine was demonstrated with a vaccination-challenge animal model.

<li> The efficacy of LPC vaccine may be compromised in PCVAD.
  
<li> The CSF-E2 subunit vaccine elicits complete protective efficacy against CSFV challenge and prevent viral transmission.

<li> The CSF-E2 subunit vaccine shows high safety and flexibility to be utilized on all ages of pigs in field farms and elicits high, concentrated and prolonged antibody distribution.
<ul><li> Comprehensive protective efficacy and batch management</ul>

<li> The CSFV-PCV2 bivalent vaccine can elicit antigen-specific immune response on vaccinated animals.

<li> The CSFV-PCV2 bivalent vaccine elicits adequate protective efficacy against challenge of CSFV and PCV2 even on pigs with PCV2-SI.

</ul>

---

## Future works

<div style="border: 2px #2984FF solid; border-radius: 10px; position: absolute; left: 55pt; top: 300pt; width: 200pt;">
<ul>
<li>PRRSV
<li>PCV2
<li>PCV3
<li>PCV4?
<li>Ohter emerging/reemerging
</ul>
</div>

<div style="border: 2px #2984FF solid; border-radius: 10px; position: absolute; right: 35pt; top: 300pt;">
<ul>
<i>
<li>Actionbacillus pleuropneumoniae
<li>Mycoplasma hyopneumoniae
<li>Haemophillus parasuis</i>
<li>Ohter emerging/reemerging
</ul>
</div>

<font style="font-size: 10pt; position: absolute; bottom: 20pt; color: #8e8e8e;">Reference DOI: 10.1186/s13567-020-00807-8
</font>

---

## Acknowledgement

---
class: middle, center

# Thank you!
<img src="Ch5/healthy.jpg" alt="healthy" style="zoom:50%; border: 2px #2984FF solid; border-radius: 40px; box-shadow: 3px 3px 12px grey; position: absolute; bottom: 80pt; left: 650pt;">