Induction of protective immunity in swine by recombinant bamboo mosaic virus expressing foot-and-mouth disease virus epitopes

<p>Abstract</p> <p>Background</p> <p>Plant viruses can be employed as versatile vectors for the production of vaccines by expressing immunogenic epitopes on the surface of chimeric viral particles. Although several viruses, including tobacco mosaic virus, potato virus X and cowpea mosaic virus, have been developed as vectors, we aimed to develop a new viral vaccine delivery system, a bamboo mosaic virus (BaMV), that would carry larger transgene loads, and generate better immunity in the target animals with fewer adverse environmental effects.</p> <p>Methods</p> <p>We engineered the BaMV as a vaccine vector expressing the antigenic epitope(s) of the capsid protein VP1 of foot-and-mouth disease virus (FMDV). The recombinant BaMV plasmid (pBVP1) was constructed by replacing DNA encoding the 35 N-terminal amino acid residues of the BaMV coat protein with that encoding 37 amino acid residues (T¹²⁸-N¹⁶⁴) of FMDV VP1.</p> <p>Results</p> <p>The pBVP1 was able to infect host plants and to generate a chimeric virion BVP1 expressing VP1 epitopes in its coat protein. Inoculation of swine with BVP1 virions resulted in the production of anti-FMDV neutralizing antibodies. Real-time PCR analysis of peripheral blood mononuclear cells from the BVP1-immunized swine revealed that they produced VP1-specific IFN-γ. Furthermore, all BVP1-immunized swine were protected against FMDV challenge.</p> <p>Conclusion</p> <p>Chimeric BaMV virions that express partial sequence of FMDV VP1 can effectively induce not only humoral and cell-mediated immune responses but also full protection against FMDV in target animals. This BaMV-based vector technology may be applied to other vaccines that require correct expression of antigens on chimeric viral particles.</p

Development and evaluation of a loop-mediated isothermal amplification method for rapid detection and differentiation of two genotypes of porcine circovirus type 2

BackgroundPorcine circovirus type 2 (PCV2) is one of the major swine viral diseases and has caused significant economic loss to pig producers. PCV2 has been divided into two major genotypes: PCV2a, PCV2b. A loop-mediated isothermal amplification (LAMP) method was developed for the detection and differentiation of PCV2a and PCV2b in clinical samples.MethodsLAMP-specific primer sets were designed based on six PCV2a and six PCV2b reference isolates. To determine the analytical specificity of LAMP, DNA samples extracted from 36 porcine virus isolates were tested by LAMP, including eight PCV2a, 11 PCV2b, four PCV type 1, two porcine parvovirus, three pseudorabies virus, and eight porcine reproductive and respiratory virus. To evaluate the analytical sensitivity of the assay, 10-fold serial dilutions of PCV2a and PCV2b recombinant plasmids were performed to prepare the dilutions at concentration from 106 to 1 copy(ies)/μL, and each dilution was tested by both LAMP and nested polymerase chain reaction (nested PCR). A total of 168 clinical samples were analyzed by both LAMP and nested PCR, and the relative sensitivity and specificity of LAMP compared to nested PCR were calculated.ResultsUsing different primer sets of LAMP, LAMP could be completed within 50 minutes. This method was found to be highly analytically specific for PCV2a and PCV2b; only the target gene was detected without cross-reaction. The analytical sensitivity of LAMP for PCV2a and PCV2b were 10 copies/μL, demonstrating analytical sensitivity comparable to that obtained using nested PCR. In addition, the sensitivity and specificity of LAMP relative to those of nested PCR were 97.7% and 100.0%, respectively. The percentage of observed agreement was 98.2%, and the κ statistic was 0.949.ConclusionLAMP is a rapid, specific, and sensitive diagnostic method for the detection and differentiation of PCV2a and PCV2b in clinical samples

Differentiation of Foot-and-Mouth Disease-Infected pigs from Vaccinated Pigs Using Antibody-Detecting Sandwich ELISA

The presence of serum antibodies for nonstructural proteins of the foot-and-mouth disease virus (FMDV) can differentiate FMDV-infected animals from vaccinated animals. In this study, a sandwich ELISA was developed for rapid detection of the foot-and-mouth disease (FMD) antibodies; it was based on an Escherichia coli-expressed, highly conserved region of the 3ABC nonstructural protein of the FMDV O/TW/99 strain and a monoclonal antibody derived from the expressed protein. The diagnostic sensitivity of the assay was 98.4%, and the diagnostic specificity was 100% for naïve and vaccinated pigs; the detection ability of the assay was comparable those of the PrioCHECK and UBI kits. There was 97.5, 93.4 and 66.6% agreement between the results obtained from our ELISA and those obtained from the PrioCHECK, UBI and CHEKIT kits, respectively. The kappa statistics were 0.95, 0.87 and 0.37, respectively. Moreover, antibodies for nonstructural proteins of the serotypes A, C, Asia 1, SAT 1, SAT 2 and SAT 3 were also detected in bovine sera. Furthermore, the absence of cross-reactions generated by different antibody titers against the swine vesicular disease virus and vesicular stomatitis virus (VSV) was also highlighted in this assay's specificit

Detection of porcine circovirus type 1 in commercial porcine vaccines by loop-mediated isothermal amplification

A loop-mediated isothermal amplification (LAMP) method with a real-time monitoring system was developed for the detection of porcine circovirus type 1 (PCV1) in commercial swine vaccines. This method was highly specific for PCV1. No cross-reaction to porcine circovirus type 2, porcine parvovirus, pseudorabies virus, classical swine fever virus, and porcine reproductive and respiratory syndrome virus was observed. The analytical sensitivity of the LAMP for PCV1 DNA was 10 copies/μl in the case of positive recombinant plasmid comparable to that obtained from the nested polymerase chain reaction (nested PCR). Furthermore, 25 commercial swine vaccines were tested by both the LAMP and the nested PCR, and three of them were tested positive for PCV1 DNA. These results indicate that PCV1 DNA can be real-time detected by the LAMP; the method was highly specific, sensitive, and rapid for the detection of PCV1 DNA, particularly in commercial swine vaccines

Analysis of the Amino Acid Sequence of Fo Cleavage Site at

新城雞病在本省曾有三次大爆發，本試驗是針對其分離株 16 株及疫 苗株 5 株，加上 Sato 強毒株與沙烏地阿拉伯的分離株 3 株，共 25 株 病毒，進行反轉錄-聚合增幅融合蛋白前軀物 F0 蛋白切割位的核酸片 段，並將產物進行直接定序，再把所得之核酸序列，以電腦 GCG 軟體進 行序列比對，並以 PAUP 軟體得 parsimony 方法，進行系統演化樹的建 立，結果發現，這 25 株新城雞病病毒的核酸序列大致相同，但在演化樹 的分析上，則大略可分成臺灣分離株 3 群、沙烏地阿拉伯分離株、疫苗 毒株及 Sato 株各 1 群等 6 大群，其中有各 1 株的臺灣分離株病毒， 自成 1 群，其間差異懸殊，並與其他的臺灣分離株相去甚遠，這可能是 RNA 病毒變異所造成；另一方面，將此等核酸序列，以電腦 GCG 軟體的 轉譯程式，轉換成氨基酸序列後發現，除了 1 株臺灣分離株病毒的 F0 蛋白切割位上，不具有二對鹼性氨基酸外，其餘各株則都含有二對鹼性氨 基酸，此外，所有臺灣分離株的第 117 個氨基酸均為苯氨基丙酸，所以 都是屬於強毒株；而疫苗毒株的 F0 蛋白切割位上，則都具有單一的鹼性 氨基酸，且其第 117 個氨基酸是為白氨酸，所以是屬於弱毒株，至於沙 烏地阿拉伯分離株與 Sato 株，則均顯現強毒株型的氨基酸序列。There were three major outbreaks of Newcastle disease in Taiwan. Sixteen Taiwanese isolates from these outbreaks, together with 5 vaccine strains, 3 Saudi Arabian isolates and the Sato strain, were subject to RT-PCR (reverse transcription- polymerase chain reaction) amplification and direct DNA sequencing. The sequences obtained were then compiled by GCG software and analyzed by PAUP (phylogenetic analysis using parsimony) to construct the phylogenetic tree of these viruses. The tree was found to contain six main groups. The first three groups are composed of Taiwanese isolates in three different groups exhibited extensive sequence variations, which might be due to the accumulation of mutations during the replication of RNA viruses. Moreover, amino-acid sequences predicted from the nucleotide sequences by the GCG software revealed that all Taiwanese isolates, except one, have two pairs of basic amino acids in their F0 cleavage site. In addition, the residue in the position 117 of F0 protein fromall Taiwanese isolates was found to be phenylalanine, indicating that all these isolates are virulent viruses. For the vaccine strains analyzed, the residue in the position 117 was leucine, the overall sequences revealed that they are avirulent strains. Finally, three Saudi Arabian isolates, and the Sato strain all showed typical amino- acid sequences of virulent viruses

Control technology and molecular epidemiology of foot-and-mouth disease in Taiwan

口蹄疫是偶蹄類動物的一種高度傳染性疾病，其致病原是屬於具有單股RNA的小核醣核酸病毒科(Picornaviridae)口瘡病毒屬(Aphthovirus)中之口蹄疫病毒(FMDV)。該病毒具有七種不同血清型，分別為O、A、C、南非一型(SAT 1)、南非二型(SAT 2)、南非三型(SAT 3)及亞洲一型(Asia 1)，各血清型病毒之間並無交叉保護作用。 歷史記載臺灣曾發生過二次口蹄疫疫情，第一次是發生在1913年到1916年之間，而第二次則在1924年到1929年之間，該二次疫情都是以撲殺策略得到疫情的控制與撲滅。直到1997年3月，台灣再度發生口蹄疫疫情，而終止其將近68年口蹄疫非疫區的狀態。引起這次疫情的口蹄疫病毒是O血清型親豬源病毒株，只會在豬隻引起典型的水疱病變，此病毒株稱為O/TW/97 病毒或是中國株(cathay strain)病毒。接著在1999年於金門島的中國黃牛被檢出另一株口蹄疫病毒，是O血清型泛亞洲型病毒株，此株病毒在田間並不會引起中國黃牛的水疱病變，此病毒株稱為O/TW/99 病毒，在1999年12月至2000年2月間，也造成臺灣乳牛及羊隻的散發性口蹄疫疫情。而O/TW/97 及 O/TW/99 口蹄疫病毒株最主要的不同點就是其感染的宿主範圍不同，其原因是O/TW/97 比 O/TW/99 口蹄疫病毒在3A基因處少了30個鹼基對。 因為口蹄疫與豬瘟是臺灣最重要的二種豬隻病毒性疾病，而這二種疾病都是以全面性的疫苗注射作為控制的手段，為顧及操作之便捷性及減少免疫注射造成之緊迫，我們進行豬隻同時免疫口蹄疫及豬瘟二種疫苗的試驗。這個試驗的結果顯示，豬隻同時免疫口蹄疫及豬瘟二種疫苗，對其抗體生成之影響並不顯著；此外，所有免疫豬瘟疫苗的豬隻，在以豬瘟強毒攻擊後，於臨床上均為正常。因此，豬隻是可以同時免疫口蹄疫及豬瘟疫苗，做法上，可以同時將該二種疫苗於豬隻背頸部不同位置分別注射或是混合後於同一位置注射，此一試驗結果應用在口蹄疫及豬瘟疫苗的免疫，有助於人力的精簡。 此外，雖然臺灣自1997年至2007年施行全面性的口蹄疫疫苗免疫注射，在1998年至2009年間仍發生零星的口蹄疫疫情。由於口蹄疫病毒具有高度的抗原變異性，尤其在有疫苗抗原存在的壓力下，口蹄疫病毒族群更能加速演化，並獲得適應性。為了解親豬源口蹄疫病毒在歷經長達10年使用疫苗免疫的區域中所衍生的變化，我們分析從1998年到2009年間在臺灣分離到的口蹄疫病毒之遺傳與抗原特性。在遺傳特性上，由口蹄疫病毒分離株的VP1基因核酸序列分析結果顯示，該等口蹄疫分離株病毒均衍生自1997年的口蹄疫病毒，但是2009年的分離株病毒已發生多處變異；另一方面，就抗原特性而言，以血清中和試驗也證實2009年分離株病毒在抗原性上有顯著的改變。 由於臺灣地區2009年所分離的口蹄疫病毒在抗原性上已有顯著的改變，而且其核酸變異性已達10%。為了評估在臺灣地區已使用超過10年的口蹄疫疫苗株（O/TW-205/98）對於2009年新分離株病毒的保護效力，我們乃進行市售口蹄疫(O/TW-205/98)不活化疫苗的效力試驗。經以市售口蹄疫不活化疫苗免疫10頭8週齡無口蹄疫抗體之第二代SPF小豬，再於4週後以2009年口蹄疫臺灣分離株（O/TW-258/2009）病毒進行攻毒試驗。結果顯示，免疫組豬隻在攻毒後，除了均未出現臨床症狀，該等豬隻檢體也未檢出病毒或呈現血清口蹄疫非結構性蛋白抗體陽轉現象。因此，口蹄疫(O/TW-205/98)不活化疫苗對2009年新分離株病毒仍具有保護作用，所以台灣地區2009年之口蹄疫疫情，並非導因於疫苗失效，現行疫苗應可持續使用。但是從另一觀點而言，口蹄疫之防治不能僅靠疫苗，適當的口蹄疫病毒抗體及抗原監測計畫，配合現場獸醫師之訓練與對於口蹄疫疫情之即時回報，都是控制與清除口蹄疫之必要措施。Foot-and-mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals. It is caused by FMD virus (FMDV), which belongs to the genus Aphthovirus, family Picornaviridae with a single-stranded RNA. FMDV has seven distinct serotypes, namely O, A, C, SAT 1, SAT 2, SAT 3, and Asia 1. Each of which does not provide immunity against another. There were two records of FMD epidemic in Taiwan in the past. The first record of the FMD epidemic was in 1913, which lasted until 1916. The second one was in 1924, which continued to 1929. Both epidemics were controlled and eradicated by a stamping out policy. Unfortunately, the situation, 68 years free from FMD, ended in March 1997. The FMDV isolated in this outbreak was a serotype-O virus that caused typical vesicular lesions only in swine but not in bovine. This FMDV strain was named as the O/TW/97 strain or a cathay strain. In 1999, a Pan Asia topotype serotype O FMDV was detected in the Chinese yellow cattle in Kinmen Island and the virus did not develop vesicular lesions under field conditions. The virus was isolated and named O/TW/99 FMDV. The virus caused sporadic outbreaks in dairy cattle and goats from late December 1999 to February 2000. The major difference between O/TW/97 and O/TW/99 FMDV strains is their respective ranges of hosts, owing to a 30-base pairs nucleotide deletion within the 3A region of the O/TW/97 FMDV strain. Since FMD and Classical Swine Fever (CSF) are the two most important viral diseases of swine in Taiwan. Both of them have been controlled by mandatory vaccination. For the convenience of practice and reduction of the stress of vaccination concerns, we conduct a study to evaluate the efficacy of simultaneous vaccination of piglets against FMD and CSF. In this study, we found that antibody responses to FMD and CSF were not significantly influenced by the simultaneous vaccination of the two vaccines. Furthermore, piglets vaccinated with CSF, either alone or together with FMD, remained clinically normal after the challenge, demonstrating that piglets can be simultaneously vaccinated with FMD and CSF vaccines, either by separate inoculations or by a single inoculation of mixed vaccines. This finding is useful for establishing a labor-saving program for vaccination against FMD and CSF. A blanket vaccination against FMDV was conducted in Taiwan between 1997 and 2007 but sporadic outbreaks of FMD occurred between 1998 and 2009. Since FMDV contains a high antigenic variability, especially under a vaccine pressure, the virus has a great potential to evolve and adapt. To understand how the changes of the porcinophilic FMDV has evolved in an area in which long-term mandatory vaccination was practiced. We analyzed the genetic and antigenic characterics of FMD viruses isolated in Taiwan between 1998 and 2009. Sequence analyses of the VP1 genes showed that the viruses isolated in Taiwan in 1998-2009 were derived from the viruses isolated in Taiwan in 1997. However, substantial mutations were found in the viruses isolated in 2009. On the other hand, serum neutralization tests confirmed that viruses isolated in 2009 had a significant change in the antigenicity. Since the FMDV isolated in Taiwan in 2009 has about 10% nucleotide differences in VP1 from that of the FMDV isolated during 1997-1998 and has a significant change in the antigenicity, it is necessary to evaluate the protective efficacy of currently used vaccine (O/TW-205/98). To assess the current vaccine, which is used over 10 years in Taiwan, against challenge infection with O/TW-258/2009, ten 8-week-old SPF pigs were vaccinated with the vaccine prepared from O/TW-205/98 and then challenged with O/TW-258/2009 at four weeks post vaccination. The results showed that all the vaccinated pigs remained healthy after the challenge. No FMD virus could be detected in the vaccinated pigs by RT-PCR after challenge and no seroconversion to non-structural protein (NSP) antibody positive was observed in the vaccinated pigs. Demonstrating that the currently used FMD vaccine (O/TW-205/98) confers protection of pigs against challenge infection with O/TW-258/2009. Therefore, the FMD outbreak in Taiwan in 2009 was not attributed to the loss of efficacy of the vaccine. The vaccine made of O/TW-205/98 FMDV still can be used for the prevention and control of FMD. However, to vaccinate pigs against FMDV is not the only way for the prevention and control of FMD. In conclusion, the essential measures to the control and eradication of FMD include conducting a proper surveillance program for the detection both of anti-FMDV antibodies and FMDV in the field, working in cooperation with a well-trained clinical veterinarian and promulgating the epidemic of FMD immediately.Table of Contents 摘要 i Abstract iv Table of Contents vii List of Tables ix List of Figures x List of Publications xi CHAPTER I: INTRODUCTION 1 1. FOOT-AND-MOUTH DISEASE VIRUS 2 2. FMD IN TAIWAN 4 3. PREVENTION AND CONTROL OF FMD 8 REFERENCES 11 CHAPTER II: EFFICACY OF SIMULTANEOUS VACCINATION OF PIGLETS AGAINST FOOT-AND-MOUTH DISEASE AND CLASSICAL SWINE FEVER 17 ABSTRACT 18 INTRODUCTION 19 MATERIALS AND METHODS 21 RESULTS 24 DISCUSSION 27 REFERENCES 33 ACKNOWLEDGEMENTS 35 CHAPTER III: GENETIC AND ANTIGENEIC CHARACTERIZATION OF FOOT-AND-MOUTH DISEASE VIRUSES ISOLATED IN TAIWAN BETWEEN 1998 AND 2009 36 ABSTRACT 37 INTRODUCTION 38 MATERIALS AND METHODS 40 RESULTS 44 DISCUSSION 48 REFERENCES 57 CHAPTER IV: PROTECTIVE EFFICACY OF FOOT-AND-MOUTHDISEASE VACCINE (O/TW-205/98) AGAINST CHALLENGE WITH THE VIRUS ISOLATED IN TAIWAN IN 2009 61 ABSTRACT 62 INTRODUCTION 63 MATERIALS AND METHODS 66 RESULT 70 DISCUSSION 72 REFERENCES 81 CHAPTER V: SUMMARY OF DISCUSSION 8

Desriping Hyperion Imagery Using Spline Interpolation

Abstract: This paper presents an alternative approach for removing striping patterns of Hyperion hyperspectral imagery. In this study, a cubic spline-based curve-fitting algorithm was used to estimate gray values of striping pixels in an image. The idea is to treat each line of a Hyperion image as a piece-wise spline curve in each spectral band. After identifying pixels affected by the striping noises, the noise-free pixels are collected and used as samples to construct a cubic Hermite spline curve that passes all of the control points (samples). The original gray values of striping covered pixels can then be approximated from the spline curve. Applying this procedure to an image line by line or column by column, a striping-free image can be reconstructed accordingly

Tone production and perception and intelligibility of produced speech in Mandarin-speaking cochlear implanted children

<p><i>Objective</i>: This study explored tone production, tone perception and intelligibility of produced speech in Mandarin-speaking prelingually deaf children with at least 5 years of cochlear implant (CI) experience. Another focus was on the predictive value of tone perception and tone production as they relate to speech intelligibility. <i>Design</i>: Cross-sectional research. <i>Study sample</i>: Thirty-three prelingually deafened children aged over eight years with over five years of experience with CI underwent tests for tone perception, tone production, and the Speech Intelligibility Rating (SIR). A Pearson correlation and a stepwise regression analysis were used to estimate the correlations among tone perception, tone production, and SIR scores. <i>Results</i>: The mean scores for tone perception, tone production, and SIR were 76.88%, 90.08%, and 4.08, respectively. Moderately positive Pearson correlations were found between tone perception and production, tone production and SIR, and tone perception and SIR (<i>p</i> < 0.01, <i>p</i> < 0.01 and <i>p</i> < 0.01, respectively). In the stepwise regression analysis, tone production, as the major predictor, accounted for 29% of the variations in the SIR (<i>p</i> < 0.01). <i>Conclusions</i>: Mandarin-speaking cochlear-implanted children with sufficient duration of CI use produce intelligent speech. Speech intelligibility can be predicted by tone production performance.</p