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1.
重组高繁殖力疫苗株H5N2(H5/PR8)病毒的制备和鉴定   总被引：1，自引：1，他引：1  
作为禽流感疫苗株,不但要求其具有良好的免疫原性,而且要求在生产中具有良好的生长特性,现用的A/Turkey/England/N28/73(H5N2)疫苗株,血凝价28,生长滴度较差.本研究利用自然重组法,预制备一株高繁殖力的预备疫苗株,使它的表面基因血凝素(HA)和神经氨酸酶(NA)来源于A/Turkey/England/N28/73(H5N2)毒株,并使其保持原有的良好的免疫原性,内部基因来源于流感病毒PR8,A/Puerto Rico/8/34(H1N1)毒株,并使其重组病毒赋有了流感病毒PR8高繁殖力的特性.实验结果表明,已获得一株高繁殖力的重组流感病毒H5/PR8,血凝价达到211,生长繁殖特性明显增强.抗原性分析显示,H5/PR8重组病毒与亲本毒株H5N2抗原性无明显差异;致病性分析发现,H5/PR8重组病毒的致病能力较亲本毒株H5N2有明显下降.这为疫苗株的改良奠定了坚实的基础,对禽流感灭活疫苗的生产具有重大意义.  相似文献   

2.
Novel reassortant H3N2 swine influenza viruses (SwIV) with the matrix gene from the 2009 H1N1 pandemic virus have been isolated in many countries as well as during outbreaks in multiple states in the United States, indicating that H3N2 SwIV might be a potential threat to public health. Since southern China is the world''s largest producer of pigs, efficient vaccines should be developed to prevent pigs from acquiring H3N2 subtype SwIV infections, and thus limit the possibility of SwIV infection at agricultural fairs. In this study, a high-growth reassortant virus (GD/PR8) was generated by plasmid-based reverse genetics and tested as a candidate inactivated vaccine. The protective efficacy of this vaccine was evaluated in mice by challenging them with another H3N2 SwIV isolate [A/Swine/Heilongjiang/1/05 (H3N2) (HLJ/05)]. Prime and booster inoculation with GD/PR8 vaccine yielded high-titer serum hemagglutination inhibiting antibodies and IgG antibodies. Complete protection of mice against H3N2 SwIV was observed, with significantly reduced lung lesion and viral loads in vaccine-inoculated mice relative to mock-vaccinated controls. These results suggest that the GD/PR8 vaccine may serve as a promising candidate for rapid intervention of H3N2 SwIV outbreaks in China.  相似文献   

3.
In April 2009 a new influenza A/H1N1 strain, currently named "pandemic (H1N1) influenza 2009" (H1N1v), started the first official pandemic in humans since 1968. Several incursions of this virus in pig herds have also been reported from all over the world. Vaccination of pigs may be an option to reduce exposure of human contacts with infected pigs, thereby preventing cross-species transfer, but also to protect pigs themselves, should this virus cause damage in the pig population. Three swine influenza vaccines, two of them commercially available and one experimental, were therefore tested and compared for their efficacy against an H1N1v challenge. One of the commercial vaccines is based on an American classical H1N1 influenza strain, the other is based on a European avian H1N1 influenza strain. The experimental vaccine is based on reassortant virus NYMC X179A (containing the hemagglutinin (HA) and neuraminidase (NA) genes of A/California/7/2009 (H1N1v) and the internal genes of A/Puerto Rico/8/34 (H1N1)). Excretion of infectious virus was reduced by 0.5-3 log(10) by the commercial vaccines, depending on vaccine and sample type. Both vaccines were able to reduce virus replication especially in the lower respiratory tract, with less pathological lesions in vaccinated and subsequently challenged pigs than in unvaccinated controls. In pigs vaccinated with the experimental vaccine, excretion levels of infectious virus in nasal and oropharyngeal swabs, were at or below 1 log(10)TCID(50) per swab and lasted for only 1 or 2 days. An inactivated vaccine containing the HA and NA of an H1N1v is able to protect pigs from an infection with H1N1v, whereas swine influenza vaccines that are currently available are of limited efficaciousness. Whether vaccination of pigs against H1N1v will become opportune remains to be seen and will depend on future evolution of this strain in the pig population. Close monitoring of the pig population, focussing on presence and evolution of influenza strains on a cross-border level would therefore be advisable.  相似文献   

4.
The prevalence and continuous evolution of H9N2 avian influenza viruses in poultry have necessitated the use of vaccines in veterinary medicine. Because of the inadequate growth properties of some strains, additional steps are needed for producing vaccine seed virus. In this study, we generated three H9N2/PR8 reassortant viruses using a total cDNA plasmid-transfection system, as an alternative strategy for developing an avian influenza vaccine for animals. We investigated the vaccine potency of the reassortant viruses compared with the existing vaccine strain which was adapted by the 20th serial passages in embryonated eggs with A/Ck/Kor/01310/01 (H9N2). The H9N2/PR8 reassortant viruses, containing the internal genes of the high-yielding PR8 strain and the surface gene of the A/Ck/Kor/01310/01 strain, could be propagated in eggs to the same extent as existing vaccine strain without additional processing. Similar to vaccine strain, the H9N2/PR8 reassortant viruses induced hemagglutination-inhibiting antibodies in chickens and prevented virus shedding and replication in multiple organs in response to homologous infection. However, due to the continuing evolution and increasing biologic diversity of H9N2 influenza in Korea, the vaccine provided only partial protection against currently isolates. Taken together, our results suggest that the H9N2/PR8 reassortant virus can be used as a seed virus for avian influenza vaccines in poultry farm. Considering the constant genetic changes in H9 strains isolated in Korea, this reverse genetic system may offer a prompt and simple way to change the vaccine seed virus and mitigate the impact of unexpected influenza outbreaks.  相似文献   

5.
Swine influenza monitoring programs have been in place in Italy since the 1990 s and from 2009 testing for the pandemic H1N1/2009 virus (H1N1pdm) was also performed on all the swine samples positive for type A influenza. This paper reports the isolation and genomic characterization of a novel H1N2 swine influenza reassortant strain from pigs in Italy that was derived from the H1N1pdm virus. In May 2010, mild respiratory symptoms were observed in around 10% of the pigs raised on a fattening farm in Italy. Lung homogenate taken from one pig showing respiratory distress was tested for influenza type A and H1N1pdm by two real time RT-PCR assays. Virus isolation was achieved by inoculation of lung homogenate into specific pathogen free chicken embryonated eggs (SPF CEE) and applied onto Caco-2 cells and then the complete genome sequencing and phylogenetic analysis was performed from the CEE isolate. The lung homogenate proved to be positive for both influenza type A (gene M) and H1N1pdm real time RT-PCRs. Virus isolation (A/Sw/It/116114/2010) was obtained from both SPF CEE and Caco-2 cells. Phylogenetic analysis showed that all of the genes of A/Sw/It/116114/2010, with the exception of neuraminidase (NA), belonged to the H1N1pdm cluster. The NA was closely related to two H1N2 double reassortant swine influenza viruses (SIVs), previously isolated in Sweden and Italy. NA sequences for these three strains were clustering with H3N2 SIVs. The emergence of a novel reassortant H1N2 strain derived from H1N1pdm in swine in Italy raises further concerns about whether these viruses will become established in pigs. The new reassortant not only represents a pandemic (zoonotic) threat but also has unknown livestock implications for the European swine industry.  相似文献   

6.
The recent pandemic caused by human influenza virus A(H1N1) 2009 contains ancestral gene segments from North American and Eurasian swine lineages as well as from avian and human influenza lineages. The emergence of this A(H1N1) 2009 poses a potential global threat for human health and the fact that it can infect other species, like pigs, favours a possible encounter with other influenza viruses circulating in swine herds. In Europe, H1N1, H1N2 and H3N2 subtypes of swine influenza virus currently have a high prevalence in commercial farms. To better assess the risk posed by the A(H1N1) 2009 in the actual situation of swine farms, we sought to analyze whether a previous infection with a circulating European avian-like swine A/Swine/Spain/53207/2004 (H1N1) influenza virus (hereafter referred to as SwH1N1) generated or not cross-protective immunity against a subsequent infection with the new human pandemic A/Catalonia/63/2009 (H1N1) influenza virus (hereafter referred to as pH1N1) 21 days apart. Pigs infected only with pH1N1 had mild to moderate pathological findings, consisting on broncho-interstitial pneumonia. However, pigs inoculated with SwH1N1 virus and subsequently infected with pH1N1 had very mild lung lesions, apparently attributed to the remaining lesions caused by SwH1N1 infection. These later pigs also exhibited boosted levels of specific antibodies. Finally, animals firstly infected with SwH1N1 virus and latter infected with pH1N1 exhibited undetectable viral RNA load in nasal swabs and lungs after challenge with pH1N1, indicating a cross-protective effect between both strains.  相似文献   

7.
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8.
Influenza is a viral disease that affects human and several animal species. In Brazil, H1N1, H3N2 and 2009 pandemic H1N1 A(H1N1)pdm09 influenza A viruses (IAV) circulate in domestic swine herds. Wild boars are also susceptible to IAV infection but in Brazil until this moment there are no reports of IAV infection in wild boars or in captive wild boars populations. Herein the occurrence of IAV in captive wild boars with the presence of lung consolidation lesions during slaughter was investigated. Lung samples were screened by RT-PCR for IAV detection. IAV positive samples were further analyzed by quantitative real-time PCR (qRRT-PCR), virus isolation, genomic sequencing, histopathology and immunohistochemistry (IHC). Eleven out of 60 lungs (18.3%) were positive for IAV by RT-PCR and seven out of the eleven were also positive for A(H1N1)pdm09 by qRRT-PCR. Chronic diffuse bronchopneumonia was observed in all samples and IHC analysis was negative for influenza A antigen. Full genes segments of H1N2 IAV were sequenced using Illumina's genome analyzer platform (MiSeq). The genomic analysis revealed that the HA and NA genes clustered with IAVs of the human lineage and the six internal genes were derived from the H1N1pdm09 IAV. This is the first report of a reassortant human-like H1N2 influenza virus infection in captive wild boars in Brazil and indicates the need to monitor IAV evolution in Suidae populations.  相似文献   

9.
为分离流感病毒,本研究将疑似甲型流感患者的咽喉拭子接种SPF鸡胚尿囊腔,经检测收获的鸡胚尿囊液具有血凝活性.采用流感病毒通用引物进行PCR扩增出8个基因节段,经测序与GenBank中登录的序列比对后确定该病毒株为2009年爆发的甲型H1N1流感病毒,并命名为A/Harbin/LM/2009 (H1N1),简称LM株.由于LM分离株在鸡胚中增殖能力比较弱,为提高其在鸡胚中的增殖能力,我们利用反向遗传技术将LM株的HA和NA基因与高增殖特性的人流感病毒细胞高产株PR8的另外6个节段进行“6+2”重配,获得拯救病毒,但该重配病毒血凝价并未显著提高.为进一步鉴定影响病毒增殖能力的基因,本研究将LM株所有节段以“7+1”的方式逐一与PR8的7个片段搭配构成完整的流感病毒基因组,分别得到8个重配病毒.结果显示分离株的PB1、PA、HA基因显著降低了重配病毒在鸡胚细胞中的增殖能力.  相似文献   

10.
In 2009, a novel swine-origin H1N1 influenza A virus (S-OIV), antigenically and genetically divergent from seasonal H1N1, caused a flu pandemic in humans. Development of an effective vaccine to limit transmission of S-OIV in animal reservoir hosts and from reservoir hosts to humans and animals is necessary. In the present study, we constructed and evaluated a vectored vaccine expressing the H1 hemagglutinin of a recent S-OIV isolate using equine herpesvirus 1 (EHV-1) as the delivery vehicle. Expression of the recombinant protein was demonstrated by immunofluorescence and western blotting and the in vitro growth properties of the modified live vector were found to be comparable to those of the parental virus. The EHV-1-H1 vaccine induced an influenza virus-specific antibody response when inoculated into mice by both the intranasal and subcutaneous routes. Upon challenge infection, protection of vaccinated mice could be demonstrated by reduction of clinical signs and faster virus clearance. Our study shows that an EHV-1-based influenza H1N1 vaccine may be a promising alternative for protection against S-OIV infection.  相似文献   

11.
To investigate whether the 2009 pandemic H1N1 influenza A virus was still being transmitted in swine, a total of 1029 nasal swab samples from healthy swine were collected from January to May 2010 in Jiangsu province of China. Eight H1N1 influenza viruses were isolated and identified, and their full length genomes were sequenced. We found that all eight of the H1N1 viruses shared higher than 98.0% sequence identity with the 2009 pandemic virus A/Jiangsu/1/2009 (JS1). In addition, some of these viruses had D225G (3/8) mutations in the receptor binding sites of the hemagglutinin (HA) protein, indicating enhancement of their binding affinity to the sialic α2, 3Gal receptor. In conclusion, the 2009 pandemic H1N1 influenza A virus has retro-infected swine from humans in mainland China, and significant viral evolution is still ongoing in this species.  相似文献   

12.
Beginning in April 2009, a novel H1N1 influenza virus caused acute respiratory disease in humans, first in Mexico and then around the world. The resulting pandemic influenza A H1N1 2009 (pH1N1) virus was isolated in swine in Canada in June 2009 and later in breeder turkeys in Chile, Canada, and the United States. The pH1N1 virus consists of gene segments of avian, human, and swine influenza origin and has the potential for infection in poultry following exposure to infected humans or swine. We examined the clinical events following the initial outbreak of pH1N1 in turkeys and determined the relatedness of the hemagglutinin (HA) gene segments from the pH1N1 to two H1N1 avian influenza (AI) isolates used in commercial turkey inactivated vaccines. Overall, infection of turkey breeder hens with pH1N1 resulted in -50% reduction of egg production over 3-4 weeks. Genetic analysis indicated one H1N1 AI vaccine isolate (Alturkey/North Carolina/17026/1988) contained approximately 92% nucleotide sequence similarity to the pH1N1 virus (A/Mexico/4109/2009); whereas, a more recent AI vaccine isolate (A/ swine/North Carolina/00573/2005) contained 75.9% similarity. Comparison of amino acids found at antigenic sites of the HA protein indicated conserved epitopes at the Sa site; however, major differences were found at the Ca2 site between pH1N1 and A/ turkey/North Carolina/127026/1988. Hemagglutinin-inhibition (HI) tests were conducted with sera produced in vaccinated turkeys in North Carolina to determine if protection would be conferred using U.S. AI vaccine isolates. HI results indicate positive reactivity (HI titer > or = 5 log2) against the vaccine viruses over the course of study. However, limited cross-reactivity to the 2009 pH1N1 virus was observed, with positive titers in a limited number of birds (6 out of 20) beginning only after a third vaccination. Taken together, these results demonstrate that turkeys treated with these vaccines would likely not be protected against pH1N1 and current vaccines used in breeder turkeys in the United States against circulating H1N1 viruses should be updated to ensure adequate protection against field exposure.  相似文献   

13.
WHO declared pandemic of A/H1N1influenza in 2009 following global spread of the newly emerged strain of the virus from swine. Presently there is a dearth of data on the ecology of pandemic influenza H1N1 required for planning of intervention measures in sub Saharan Africa. Herein we report isolation of 2009 pandemic influenza A/H1N1 in an intensive mega piggery farms operation in South West Nigeria.  相似文献   

14.
The efficacy of a commercial swine influenza vaccine based on A/New Jersey/8/76 (H1N1) and A/Port Chalmers/1/73 (H3N2) strains was tested against challenge with an H1N2 swine influenza virus. Influenza virus-seronegative pigs were vaccinated twice with the vaccine when they were four and eight weeks old, or with the same vaccine supplemented with an H1N2 component. Control pigs were left unvaccinated. Three weeks after the second vaccination, all the pigs were challenged intratracheally with the swine influenza strain Sw/Gent/7625/99 (H1N2). The commercial vaccine induced cross-reactive antibodies to H1N2, as detected by the virus neutralisation (VN) assay, but VN antibody titres were 18 times lower than in the pigs vaccinated with the H1N2-supplemented vaccine. The challenge produced severe respiratory signs in nine of 10 unvaccinated control pigs, which developed high H1N2 virus titres in the lungs 24 and 72 hours after the challenge. Vaccination with the commercial vaccine resulted in milder respiratory signs, but H1N2 virus replication was not prevented. Mean virus titres in the pigs vaccinated with the commercial vaccine were 1-5 log10 lower than in the controls at 24 hours but no different at 72 hours. In contrast, the H1N2-supplemented vaccine prevented respiratory disease in most pigs. There was a 4-5 log10 reduction in the mean virus titre at 24 hours in the pigs vaccinated with this vaccine, and no detectable virus replication at 72 hours. These data indicate that the commercial swine influenza vaccine did not confer adequate protection against the H1N2 subtype.  相似文献   

15.
Yang P  Tang C  Luo D  Zhan Z  Xing L  Duan Y  Jia W  Peng D  Liu X  Wang X 《Veterinary microbiology》2010,146(1-2):17-23
The avian H5N1 influenza virus has the potential to cause a new pandemic. The increasing number of recent outbreaks of highly pathogenic avian influenza H5N1 in birds and humans emphasizes the urgent need to develop a potent H5N1 vaccine. Here, we studied the immunogenicity and protective effect of a vaccine prepared from H5N1 inactivated whole virus. This vaccine was intranasally co-administered in mice with phosphate buffered saline, recombinant cholera toxin B subunit (rCTB), cholera toxin (CT), rCTB containing a trace amount of holotoxin (rCTB/CT), polyinosinic:polycytidylic acid double-stranded RNA (polyI:C), or MF59 as an adjuvant. Intranasal administration of H5N1 inactivated whole virus vaccine with rCTB, CT, rCTB/CT, polyI:C, and MF59 elicited an immunological response with both secretory IgA (sIgA) in nasal, lung, and vaginal lavage, and IgG antibody in serum, showing protective immunity against lethal H5N1 infection. Cross-clade protection was also observed in animals immunized with a vaccine derived from Anhui/01/2005(H5N1) with rCTB, CT, rCTB/CT, polyI:C, or MF59 as adjuvants that were subsequently challenged with the A/OT/SZ/097/03 influenza strain.  相似文献   

16.
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17.
为了解猪流感病毒(SIV)的变异情况,我们2009年11月从河北某养殖场采集呈流感症状的猪鼻拭子40份,接种10日龄SPF鸡胚,分离到一株猪流感病毒,通过RT-PCR和血凝抑制试验鉴定为H1N1亚型,命名为A/swine/Hebei/15/2009(H1N1),其全基因序列测定及同源性分析发现,8个基因片段均与2000年左右H1N1人流感病毒有较高的同源性。系统遗传演化显示,该病毒分离株是由2000年人源H1N1流感病毒A/Dunedin/2/2000(H1N1)进化而来。抗原性分析显示该株与甲型H1N1流感病毒和经典H1N1病毒株抗原性差异较大。对小鼠致病性试验表明该病毒株可以直接感染小鼠并导致小鼠轻微临床症状和组织病理学变化,但不致死小鼠,表现为低致病性。  相似文献   

18.
Inactivated influenza virus vaccine prepared from a non-pathogenic influenza virus strain A/duck/Hokkaido/Vac-1/2004 (H5N1) from the virus library conferred protective immunity to chickens against the challenge of antigenically drifted highly pathogenic avian influenza virus (HPAIV), A/whooper swan/Hokkaido/1/2008 (H5N1). The efficacy of the vaccine was comparable to that prepared from genetically modified HPAIV strain deltaRRRRK rg-A/ whooper swan/Mongolia/3/2005 (H5N1), which is more antigenically related to the challenge virus strain, in chickens.  相似文献   

19.
Infection of pigs with influenza A H1N1 2009 virus (A(H1N1)pdm09) was first detected in England in November 2009 following global spread of the virus in the human population. This paper describes clinical and epidemiological findings in the first English pig farms in which A(H1N1)pdm09 influenza virus was detected. These farms showed differences in disease presentation, spread and duration of infection. The factors likely to influence these features are described and relate to whether pigs were housed or outdoors, the age of the pigs, inter-current disease and the management system of the unit. Infection could be mild or clinically inapparent in breeding pigs with more typical respiratory disease being identified later in their progeny. Mortality was low where disease was uncomplicated by environmental stresses or concurrent infections. Where deaths occurred in pigs infected with A(H1N1)pdm09 influenza, they were mainly due to other infections, including streptococcal disease due to Streptococcus suis infection. This paper demonstrates the ease with which A(H1N1)pdm09 virus was transmitted horizontally and maintained in a pig population.  相似文献   

20.
In this study, two highly pathogenic avian influenza (HPAI) H5N8 viruses were isolated from chicken and geese in 2018 and 2019 (Chicken/ME-2018 and Geese/Egypt/MG4/2019). The hemagglutinin and neuraminidase gene analyses revealed their close relatedness to the clade-2.3.4.4b H5N8 viruses isolated from Egypt and Eurasian countries. A monovalent inactivated oil-emulsion vaccine containing a reassortant virus with HA gene of the Chicken/ME-2018/H5N8 strain and a bivalent vaccine containing same reassortant virus plus a previously generated reassortant H5N1 strain (CK/Eg/RG-173CAL/17). The safety of both vaccines was evaluated in specific-pathogen-free (SPF) chickens. To evaluate the efficacy of the prepared vaccines, 2-week-old SPF chickens were vaccinated with 0.5 mL of a vaccine formula containing 108/EID50 /dose from each strain via the subcutaneous route. Vaccinated birds were challenged with either wild-type HPAI-H5N8 or H5N1 viruses separately at 3 weeks post-vaccine. Results revealed that both vaccines induced protective hemagglutination-inhibiting (HI) antibody titers as early as 2 weeks PV (≥5.0 log2). Vaccinated birds were protected clinically against both subtypes (100 % protection). HPAI-H5N1 virus shedding was significantly reduced in birds that were vaccinated with the bivalent vaccine; meanwhile, HPAI-H5N8 virus shedding was completely neutralized in both tracheal and cloacal swabs after 3 days post-infection in birds that had been vaccinated with either vaccine. In conclusion, the developed bivalent vaccine proved to be efficient in protecting chickens clinically and reduced virus shedding via the respiratory and digestive tracts. The applicability of the multivalent avian influenza vaccines further supported their value to facilitate vaccination programs in endemic countries.  相似文献   

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