利用细菌人工染色体技术构建整合F基因的重组MDV疫苗株*

目的:预防马立克氏病病毒(MDV)和新城疫病毒(NDV)混合感染鸡引起的疾病,构建表达NDV F蛋白的MDV疫苗株CVI988 BAC重组载体,并包装成重组病毒,为疫苗免疫提供更多的重组疫苗选择。方法:首先利用PCR扩增带有卡那霉素(Kanamycin,Kana)抗性基因片段的F基因,采用同源重组的方法将其整合到CVI988 BAC上,进一步诱导I-SceI表达敲除Kana基因而获得重组质粒CVI988 BAC-F。通过磷酸钙法转染鸡胚成纤维细胞获得重组病毒。结果:Western blot和间接免疫荧光实验证实重组病毒能够表达F蛋白。病毒生长曲线和蚀斑大小测定结果表明,F基因的插入不影响病毒的体外增殖。结论:利用BAC技术成功构建了整合F基因的重组MDV病毒CVI988 BAC-F,为MDV重组疫苗研发,防控NDV与MDV共感染奠定了基础。     

表达禽流感病毒M2基因的重组马立克氏病病毒的构建

将禽流感病毒M2基因克隆于真核表达质粒pIRES-EGFP中,使其位于pCMV启动子的调控下,并与绿色荧光蛋白基因（EGFP）串联后,将上述串联基因插入到含MDV CVI988的非必需区US基因的重组质粒pUS2中,构建带标记的重组质粒,然后将此重组质粒转染感染了MDV CVI988的鸡胚成纤维细胞,利用同源重组的方法,筛选了表达禽流感病毒M2基因的重组病毒MDV1。经PCR、Dot-blotting,Western-blotting等实验的结果表明,禽流感病毒M2基因的确插入到MDV1（CVI988）基因组中并获得表达。重组MDV1免疫1日龄SPF鸡21天后,用ELISA可检测到M2蛋白的特异性抗体。接种了重组病毒rMDV的鸡体内针对H9N2疫苗血凝素的抗体滴度(p<0.05)明显提高,以禽流感病毒AIV A/Chicken/Guangdong/00（H9N2）攻毒后进行病毒重分离试验的结果发现,重组病毒能有效地降低病毒的排出量(p<0.01),说明该重组病毒可以用于防制禽流感的免疫。     

表达强毒pp38决定簇的马立克病病毒疫苗毒CVI988点突变株的构建和特性

用鸡马立克病病毒（ＭＤＶ）强毒ＧＡ株的３８ｋＤ磷蛋白（ｐｐ３８）基因克隆ＤＮＡ转染Ｉ型弱毒疫苗ＣＡＩ９８８／Ｒｉｓｐｅｎｓ株ＭＤＶ感染的鸡胚成纤维细胞,再用能识别Ｉ型强毒ｐｐ３８的单克隆抗体Ｈ１９做免疫荧光试验,筛选到能在ｐｐ３８基因上表达强毒株特异性抗原决定簇的定向点突变弱毒株ＣＶＩ／ｒｐｐ３８。用＾３５Ｓ－蛋氨酸标记的细胞裂解物做免疫沉淀反应表明,单抗Ｈ１９不能识别天然ＣＶＩ９８８株ＭＤＶ中的     

火鸡疱疹病毒细菌人工染色体的构建

火鸡疱疹病毒(HVT)为一种-疱疹病毒,因其与马立克氏病病毒(MDV)抗原相关性而被广泛用作预防马立克氏病(MD)的活疫苗.[目的]本研究的目的是构建HVT全基因组感染性细菌人工染色体(BAC).[方法]利用Eco-gpt(黄嘌呤鸟嘌呤磷酸核糖转移酶)基因和BAC载体pBeloBAC11的基本功能序列,构建重组病毒转移载体Pgab-gpt-BAC11.通过将Pgab-gpt-BAC11与HVT感染细胞总DNA共转染原代鸡胚成纤维细胞(CEF),待出现病毒噬斑后,利用霉酚酸(MPA)阻断核酸代谢途径,经过筛选获得纯化的重组病毒purified-Rhvt.提取purified-Rhvt感染细胞总DNA电转化大肠杆菌DH10B感受态细胞,在氯霉素抗性平板上筛选阳性克隆,并用酶切和PCR方法对其进行鉴定.随机选取BAC克隆提取BAC DNA转染次代CEF,完成HVT重组病毒的拯救.[结果]经过6轮筛选后获得纯化的重组病毒,并筛选到25个BAC分子克隆化病毒.其中BAC6、BAC8和BAC10再次启动病毒感染,产生与野生型HVT感染CEF相似的病毒噬斑形态,说明已经获得拯救出的HVT重组病毒.[结论]本研究构建了HVT全基因组感染性细菌人工染色体,建立了HVT反向遗传操作技术平台.     

Development of a new hydrogen peroxide–based vaccine platform

Safe and effective vaccines are crucial for maintaining public health and reducing the global burden of infectious disease. Here we introduce a new vaccine platform that uses hydrogen peroxide (H(2)O(2)) to inactivate viruses for vaccine production. H(2)O(2) rapidly inactivates both RNA and DNA viruses with minimal damage to antigenic structure or immunogenicity and is a highly effective method when compared with conventional vaccine inactivation approaches such as formaldehyde or β-propiolactone. Mice immunized with H(2)O(2)-inactivated lymphocytic choriomeningitis virus (LCMV) generated cytolytic, multifunctional virus-specific CD8(+) T cells that conferred protection against chronic LCMV infection. Likewise, mice vaccinated with H(2)O(2)-inactivated vaccinia virus or H(2)O(2)-inactivated West Nile virus showed high virus-specific neutralizing antibody titers and were fully protected against lethal challenge. Together, these studies demonstrate that H(2)O(2)-based vaccines are highly immunogenic, provide protection against a range of viral pathogens in mice and represent a promising new approach to future vaccine development.     

一株具有急性致瘤特性的马立克氏病病毒的分离与鉴定

应用鸭胚成纤维细胞(DEF)从免疫过CVI988/Rispens疫苗株患马立克氏病(MD)的三黄鸡中分离到一株马立克氏病病毒(MDV)(命名为YL040920株)。从该分离株蚀斑克隆获得的9个克隆在蚀斑形成时间及其形态大小上均无明显差别,表明它较为单一;应用聚合酶链式反应(PCR)技术扩增并测定了毒株的致瘤相关基因meq的核苷酸序列,并与其他MDV-1参考毒株的序列进行比较分析,发现其序列具有MDV-1强毒株的特征;用基于抗MDV-1 MEQ蛋白的单克隆抗体3G12E6的免疫荧光试验(FA)对毒株的DEF培养物进行检测,发现有特异性的荧光定位于细胞核内;应用毒株感染霞烟鸡,最早在接种后(PI)21d即可诱发明显的内脏器官,在各器官肿瘤中以心脏、肝脏和皮肤的肿瘤发生率最高;用禽肿瘤病三重PCR鉴别诊断技术对毒株的DEF培养物以及感染鸡的内脏器官组织进行检测,均能扩增到MDV-1强毒株的特异性带,而网状内皮增生症病毒(REV)和禽白血病病毒(ALV)的检测则均为阴性。研究的结果表明,分离株YL040920为单一的MDV-1强毒株,无REV、ALV以及疫苗株CVI988/Rispens的混杂,并具有以心脏、肝脏和皮肤肿瘤为主的急性致瘤特性。     

Extended Preclinical Safety,Efficacy and Stability Testing of a Live-attenuated Chikungunya Vaccine Candidate

We recently described a new, live-attenuated vaccine candidate for chikungunya (CHIK) fever, CHIKV/IRES. This vaccine was shown to be well attenuated, immunogenic and efficacious in protecting against CHIK virus (CHIKV) challenge of mice and nonhuman primates. To further evaluate its preclinical safety, we compared CHIKV/IRES distribution and viral loads in interferon-α/β receptor-incompetent A129 mice to another CHIK vaccine candidate, 181/clone25, which proved highly immunogenic but mildly reactive in human Phase I/II clinical trials. Compared to wild-type CHIK virus, (wt-CHIKV), both vaccines generated lower viral loads in a wide variety of tissues and organs, including the brain and leg muscle, but CHIKV/IRES exhibited marked restrictions in dissemination and viral loads compared to 181/clone25, and was never found outside the blood, spleen and muscle. Unlike wt-CHIKV, which caused disrupted splenic architecture and hepatic lesions, histopathological lesions were not observed in animals infected with either vaccine strain. To examine the stability of attenuation, both vaccines were passaged 5 times intracranially in infant A129 mice, then assessed for changes in virulence by comparing parental and passaged viruses for footpad swelling, weight stability and survival after subcutaneous infection. Whereas strain 181/clone25 p5 underwent a significant increase in virulence as measured by weight loss (from <10% to >30%) and mortality (from 0 to 100%), CHIKV/IRES underwent no detectible change in any measure of virulence (no significant weight loss and no mortality). These data indicate greater nonclinical safety of the CHIKV/IRES vaccine candidate compared to 181/clone25, further supporting its eligibility for human testing.     

An overlapping bacterial artificial chromosome system that generates vectorless progeny for channel catfish herpesvirus

Herpesviruses are important pathogens of humans and other animals. Herpesvirus infectious clones that can reconstitute phenotypically wild-type (wt) virus are extremely valuable tools for elucidating the roles of specific genes in virus pathophysiology as well as for making vaccines. Ictalurid herpesvirus 1 (channel catfish herpesvirus [CCV]) is economically very important and is the best characterized of the herpesviruses that occur primarily in bony fish and amphibians. Here, we describe the cloning of the hitherto recalcitrant CCV genome as three overlapping subgenomic bacterial artificial chromosomes (BACs). These clones allowed us to regenerate vectorless wt CCVs with a phenotype that is indistinguishable from that of the wt CCV from which the BACs were derived. To test the recombinogenic systems, we next used the overlapping BACs to construct a full-length CCV BAC by replacing the CCV ORF5 with the BAC cassette and cotransfecting CCO cells. The viral progeny that we used to transform Escherichia coli and the resulting BAC had only one of the 18-kb terminal repeated regions. Both systems suggest that one of the terminal repeat regions is lost during the replicative stage of the CCV life cycle. We also demonstrated the feasibility of introducing a targeted mutation into the CCV BAC infectious clone by constructing a CCV ORF12 deletion mutant and showed that ORF12 encodes a nonessential protein for virus replication. This is the first report of the generation of an infectious BAC clone of a member of the fish and amphibian herpesviruses and its use to generate recombinants.     

Cloning of the koi herpesvirus genome as an infectious bacterial artificial chromosome demonstrates that disruption of the thymidine kinase locus induces partial attenuation in Cyprinus carpio koi

Koi herpesvirus (KHV) is the causative agent of a lethal disease in koi and common carp. In the present study, we describe the cloning of the KHV genome as a stable and infectious bacterial artificial chromosome (BAC) clone that can be used to produce KHV recombinant strains. This goal was achieved by the insertion of a loxP-flanked BAC cassette into the thymidine kinase (TK) locus. This insertion led to a BAC plasmid that was stably maintained in bacteria and was able to regenerate virions when permissive cells were transfected with the plasmid. Reconstituted virions free of the BAC cassette but carrying a disrupted TK locus (the FL BAC-excised strain) were produced by the transfection of Cre recombinase-expressing cells with the BAC. Similarly, virions with a wild-type revertant TK sequence (the FL BAC revertant strain) were produced by the cotransfection of cells with the BAC and a DNA fragment encoding the wild-type TK sequence. Reconstituted recombinant viruses were compared to the wild-type parental virus in vitro and in vivo. The FL BAC revertant strain and the FL BAC-excised strain replicated comparably to the parental FL strain. The FL BAC revertant strain induced KHV infection in koi carp that was indistinguishable from that induced by the parental strain, while the FL BAC-excised strain exhibited a partially attenuated phenotype. Finally, the usefulness of the KHV BAC for recombination studies was demonstrated by the production of an ORF16-deleted strain by using prokaryotic recombination technology. The availability of the KHV BAC is an important advance that will allow the study of viral genes involved in KHV pathogenesis, as well as the production of attenuated recombinant candidate vaccines.     

Comparative efficacy of subtype AE simian-human immunodeficiency virus priming and boosting vaccines in pigtail macaques

Vaccination against AIDS is hampered by great diversity between human immunodeficiency virus (HIV) strains. Heterologous B-subtype-based simian-human immunodeficiency virus (SHIV) DNA prime and poxvirus boost vaccine regimens can induce partial, T-cell-mediated, protective immunity in macaques. We analyzed a set of DNA, recombinant fowlpox viruses (FPV), and vaccinia viruses (VV) expressing subtype AE HIV type 1 (HIV-1) Tat, Rev, and Env proteins and SIV Gag/Pol in 30 pigtail macaques. SIV Gag-specific CD4 and CD8 T-cell responses were induced by sequential DNA/FPV vaccination, although lower FPV doses, VV/FPV vaccination, and DNA vaccines alone were not as consistently immunogenic. The SHIV AE DNA prime, FPV boost regimens were significantly less immunogenic than comparable B-subtype SHIV vaccination. Peak viral load was modestly (0.4 log10 copies/ml) lower among the AE subtype SHIV-immunized animals compared to controls following the virulent B subtype SHIV challenge. Protection from persistent high levels of viremia and CD4 T-cell depletion was less in AE subtype compared to B subtype SHIV-vaccinated macaques. Gag was highly immunodominant over the other AE subtype SHIV vaccine proteins after vaccination, and this immunodominance was exacerbated after challenge. Interestingly, the lower level of priming of immune responses did not blunt postchallenge Gag-specific recall responses, despite more modest protection. These studies suggest priming of T-cell immunity to prevent AIDS in humans is possible, but differences in the immunogenicity of various subtype vaccines and broad cross-subtype protection are substantial hurdles.     

Newcastle Disease Virus Fusion Protein Is the Major Contributor to Protective Immunity of Genotype-Matched Vaccine

Virulent strains of Newcastle disease virus (NDV) can cause devastating disease in chickens worldwide. Although the current vaccines are substantially effective, they do not completely prevent infection, virus shedding and disease. To produce genotype-matched vaccines, a full-genome reverse genetics system has been used to generate a recombinant virus in which the F protein cleavage site has been changed to that of avirulent vaccine virus. In the other strategy, the vaccines have been generated by replacing the F and HN genes of a commercial vaccine strain with those from a genotype-matched virus. However, the protective efficacy of a chimeric virus vaccine has not been directly compared with that of a full-genome virus vaccine developed by reverse genetics. Therefore, in this study, we evaluated the protective efficacy of genotype VII matched chimeric vaccines by generating three recombinant viruses based on avirulent LaSota (genotype II) strain in which the open reading frames (ORFs) encoding the F and HN proteins were replaced, individually or together, with those of the circulating and highly virulent Indonesian NDV strain Ban/010. The cleavage site of the Ban/010 F protein was mutated to the avirulent motif found in strain LaSota. In vitro growth characteristics and a pathogenicity test indicated that all three chimeric viruses retained the highly attenuated phenotype of the parental viruses. Immunization of chickens with chimeric and full-length genome VII vaccines followed by challenge with virulent Ban/010 or Texas GB (genotype II) virus demonstrated protection against clinical disease and death. However, only those chickens immunized with chimeric rLaSota expressing the F or F plus HN proteins of the Indonesian strain were efficiently protected against shedding of Ban/010 virus. Our findings showed that genotype-matched vaccines can provide protection to chickens by efficiently preventing spread of virus, primarily due to the F protein.     

表达IBDV VP2融合蛋白的重组MDV的构建及其免疫特性

将增强型绿色荧光蛋白基因(eGFP)与鸡传染性法氏囊病病毒(IBDV)的VP2基因融合,插入马立克氏病毒(MDV)CVI988/Rispens的非必需区US10片段中,成功构建表达VP2融合蛋白的MDVCVI988转移载体pUC18-US10-VP2。将转移载体质粒与CVI988/Rispens疫苗毒共转染鸡胚成纤维细胞(CEF),筛选获得表达VP2融合蛋白的重组MDV(rMDV)。聚合酶链式反应(PCR)和间接免疫荧光实验(IFA)证明,rMDV传至第31代仍能稳定表达VP2融合蛋白。用rMDV免疫SPF鸡,进行IBDV攻毒保护试验,1日龄SPF鸡分别用1000PFU、2000PFU、5000PFU的rMDV进行免疫,33日龄用100LD50的IBDVJS超强毒进行攻毒,鸡的免疫保护率分别为50%、60%、80%。值得注意的是,5000PFU的rMDV一次免疫1日龄SPF鸡,其法氏囊组织病理损伤等级与IBD中等毒力活疫苗常规二次免疫相当(2·0/1·5),其保护效果无显著差异(p>0·05),而与非重组病毒免疫组相比较,保护效果差异显著(P<0·01),这表明构建的表达IBDVVP2融合蛋白的rMDV可以有效地为SPF鸡提供免疫保护作用。     

Diva vaccines that reduce virus transmission.

This brief review deals with the effect of diva (Differentiating Infected from VAccinated individuals) vaccines (also termed marker vaccines) on transmission of herpesviruses and pestiviruses in swine and cattle. Pseudorabies and bovine herpesvirus 1 diva vaccines have been demonstrated to reduce transmission of wild-type virus in populations of pigs and cattle in the laboratory as well as in the field. A subunit diva vaccine based on the immunodominant E2 protein of classical swine fever virus that is expressed in the baculovirus system may reduce transmission of wild-type virus among pigs and also transmission from mother to foetuses. A similar diva vaccine against bovine virus diarrhoea infections protected sheep against transplacental transmission of antigenically homologous wild-type virus. Diva vaccines along with their companion diagnostic tests can play a role in control of infections, ultimately leading to eradication of viruses.     

Live bivalent vaccine for parainfluenza and influenza virus infections

Influenza and human parainfluenza virus infections are of both medical and economical importance. Currently, inactivated vaccines provide suboptimal protection against influenza, and vaccines for human parainfluenza virus infection are not available, underscoring the need for new vaccines against these respiratory diseases. Furthermore, to reduce the burden of vaccination, the development of multivalent vaccines is highly desirable. Thus, to devise a single vaccine that would elicit immune responses against both influenza and parainfluenza viruses, we used reverse genetics to generate an influenza A virus that possesses the coding region for the hemagglutinin/neuraminidase ectodomain of parainfluenza virus instead of the influenza virus neuraminidase. The recombinant virus grew efficiently in eggs but was attenuated in mice. When intranasally immunized with the recombinant vaccine, all mice developed antibodies against both influenza and parainfluenza viruses and survived an otherwise lethal challenge with either of these viruses. This live bivalent vaccine has obvious advantages over combination vaccines, and its method of generation could, in principle, be applied in the development of a "cocktail" vaccine with efficacy against several different infectious diseases.     

Construction of Recombinant HVT Expressing PmpD,and Immunological Evaluation against Chlamydia psittaci and Marek’s Disease Virus

Chlamydia psittaci (C. psittaci) is an obligate intracellular zoonotic pathogen that can be transmitted to humans from birds. No efficacious commercial vaccine is available for clearing chlamydial infection due to lack of potential vaccine candidates and effective delivery vehicles. Herpesvirus of turkeys (HVT) is an efficacious commercially available vaccine against Marek’s Disease virus (MDV). In this study, a recombinant HVT-delivered vaccine against C. psittaci and Marek’s disease was developed and examined. The 5''-terminus of pmpD gene (pmpD-N) encoding the N-terminal fragment of polymorphic membrane protein D of C. psittaci was inserted into a nonessential region of HVT genome using reverse genetics based on an infectious bacterial artificial chromosome (BAC) clone of HVT. The recombinant virus (rHVT-pmpD-N) was recovered from primary chicken embryo fibroblast (CEF) cells by transfection of modified HVT BAC DNA containing the pmpD-N gene. The rHVT-pmpD-N construct was confirmed to express PmpD-N by immunoblot and immunofluorescence. The rHVT-pmpD-N was stable during 20 passages in vitro. The growth kinetics of rHVT-pmpD-N was comparable to that of parental HVT in vitro and in vivo. One-day-old SPF chickens inoculated subcutaneously with rHVT-pmpD-N displayed increased PmpD-specific antibody levels and a vigorous PmpD-specific lymphocyte proliferation response using HVT vector or CEF cells as control. Furthermore, the percentage of CD4+ cells was significantly elevated in rHVT-pmpD-N-immunized birds as compared to the parental HVT. All chickens vaccinated with rHVT-pmpD-N or parental HVT were protected completely against challenge with a very virulent strain of Marek’s Disease virus (MDV) RB-1B. Post challenge with C. psittaci CB7 strain, a significant decrease in respiratory distress, lesions and Chlamydia load was found in the rHVT-pmpD-N-vaccinated group compared to the parental HVT. In conclusion, our study suggests that the rHVT-pmpD-N live vaccine may be viable as a candidate dual vaccine that provides protection against both very virulent MDV and C. psittaci.     

Broadening the Heterologous Cross-Neutralizing Antibody Inducing Ability of Porcine Reproductive and Respiratory Syndrome Virus by Breeding the GP4 or M genes

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most economically important swine pathogens, which causes reproductive failure in sows and respiratory disease in piglets. A major hurdle to control PRRSV is the ineffectiveness of the current vaccines to confer protection against heterologous strains. Since both GP4 and M genes of PRRSV induce neutralizing antibodies, in this study we molecularly bred PRRSV through DNA shuffling of the GP4 and M genes, separately, from six genetically different strains of PRRSV in an attempt to identify chimeras with improved heterologous cross-neutralizing capability. The shuffled GP4 and M genes libraries were each cloned into the backbone of PRRSV strain VR2385 infectious clone pIR-VR2385-CA. Three GP4-shuffled chimeras and five M-shuffled chimeras, each representing sequences from all six parental strains, were selected and further characterized in vitro and in pigs. These eight chimeric viruses showed similar levels of replication with their backbone strain VR2385 both in vitro and in vivo, indicating that the DNA shuffling of GP4 and M genes did not significantly impair the replication ability of these chimeras. Cross-neutralization test revealed that the GP4-shuffled chimera GP4TS14 induced significantly higher cross-neutralizing antibodies against heterologous strains FL-12 and NADC20, and similarly that the M-shuffled chimera MTS57 also induced significantly higher levels of cross-neutralizing antibodies against heterologous strains MN184B and NADC20, when compared with their backbone parental strain VR2385 in infected pigs. The results suggest that DNA shuffling of the GP4 or M genes from different parental viruses can broaden the cross-neutralizing antibody-inducing ability of the chimeric viruses against heterologous PRRSV strains. The study has important implications for future development of a broadly protective vaccine against PRRSV.     

Protection Induced in Broiler Chickens following Drinking-Water Delivery of Live Infectious Laryngotracheitis Vaccines against Subsequent Challenge with Recombinant Field Virus

Infectious laryngotracheitis virus (ILTV) causes acute upper respiratory tract disease in chickens. Attenuated live ILTV vaccines are often used to help control disease, but these vaccines have well documented limitations, including retention of residual virulence, incomplete protection, transmission of vaccine virus to unvaccinated birds and reversion to high levels of virulence following bird-to-bird passage. Recently, two novel ILTV field strains (class 8 and 9 ILTV viruses) emerged in Australia due to natural recombination between two genotypically distinct commercial ILTV vaccines. These recombinant field strains became dominant field strains in important poultry producing areas. In Victoria, Australia, the recombinant class 9 virus largely displaced the previously predominant class 2 ILTV strain. The ability of ILTV vaccines to protect against challenge with the novel class 9 ILTV strain has not been studied. Here, the protection induced by direct (drinking-water) and indirect (contact) exposure to four different ILTV vaccines against challenge with class 9 ILTV in commercial broilers was studied. The vaccines significantly reduced, but did not prevent, challenge virus replication in vaccinated chickens. Only one vaccine significantly reduced the severity of tracheal pathology after direct drinking-water vaccination. The results indicate that the current vaccines can be used to help control class 9 ILTV, but also indicate that these vaccines have limitations that should be considered when designing and implementing disease control programs.     

用电穿孔方法研究 H5N1 禽流感病毒DNA 疫苗对动物的免疫保护作用

为了研究 H5N1 DNA 疫苗对小鼠和鸡的保护效率,用 H5N1 禽流感病毒 HA DNA 疫苗免疫 BALB/c 小鼠和 SPF 鸡 . 小鼠和鸡分别经电穿孔和肌肉注射免疫两次,间隔为 3 周 . 二次免疫后,用致死量的同源病毒进行攻毒实验 . 空白对照组在攻毒后全部死亡,而经电穿孔免疫的小鼠和鸡均获得了完全的保护,并能有效地抑制病毒在小鼠肺脏和鸡泄殖腔的繁殖 . 同时,电穿孔免疫的小鼠和鸡均产生了高水平的特异性抗体 . 经电穿孔免疫的小鼠攻毒后 CTL 反应明显加强 . 这些结果表明, HA DNA 疫苗能有效地保护小鼠和鸡对禽流感病毒的感染,同时也表明电穿孔免疫是 DNA 疫苗免疫的有效途径之一 .