Recombinant infectious bronchitis coronavirus Beaudette with the spike protein gene of the pathogenic M41 strain remains attenuated but induces protective immunity

We have replaced the ectodomain of the spike (S) protein of the Beaudette strain (Beau-R; apathogenic for Gallus domesticus chickens) of avian infectious bronchitis coronavirus (IBV) with that from the pathogenic M41 strain to produce recombinant IBV BeauR-M41(S). We have previously shown that this changed the tropism of the virus in vitro (R. Casais, B. Dove, D. Cavanagh, and P. Britton, J. Virol. 77:9084-9089, 2003). Herein we have assessed the pathogenicity and immunogenicity of BeauR-M41(S). There were no consistent differences in pathogenicity between the recombinant BeauR-M41(S) and its apathogenic parent Beau-R (based on snicking, nasal discharge, wheezing, watery eyes, rales, and ciliostasis in trachea), and both replicated poorly in trachea and nose compared to M41; the S protein from the pathogenic M41 had not altered the apathogenic nature of Beau-R. Both Beau-R and BeauR-M41(S) induced protection against challenge with M41 as assessed by absence of recovery of challenge virus and nasal exudate. With regard to snicking and ciliostasis, BeauR-M41(S) induced greater protection (seven out of nine chicks [77%]; assessed by ciliostasis) than Beau-R (one out of nine; 11%) but less than M41 (100%). The greater protection induced by BeauR-M41(S) against M41 may be related to the ectodomain of the spike protein of Beau-R differing from that of M41 by 4.1%; a small number of epitopes on the S protein may play a disproportionate role in the induction of immunity. The results are promising for the prospects of S-gene exchange for IBV vaccine development.     

Recombinant avian infectious bronchitis virus expressing a heterologous spike gene demonstrates that the spike protein is a determinant of cell tropism

A recombinant infectious bronchitis virus (IBV), BeauR-M41(S), was generated using our reverse genetics system (R. Casais, V. Thiel, S. G. Siddell, D. Cavanagh, and P. Britton, J. Virol. 75:12359-12369, 2001), in which the ectodomain region of the spike gene from IBV M41-CK replaced the corresponding region of the IBV Beaudette genome. BeauR-M41(S) acquired the same cell tropism phenotype as IBV M41-CK in four different cell types, demonstrating that the IBV spike glycoprotein is a determinant of cell tropism.     

The Replicase Gene of Avian Coronavirus Infectious Bronchitis Virus Is a Determinant of Pathogenicity

We have previously demonstrated that the replacement of the S gene from an avirulent strain (Beaudette) of infectious bronchitis virus (IBV) with an S gene from a virulent strain (M41) resulted in a recombinant virus (BeauR-M41(S)) with the in vitro cell tropism of the virulent virus but that was still avirulent. In order to investigate whether any of the other structural or accessory genes played a role in pathogenicity we have now replaced these from the Beaudette strain with those from M41. The recombinant IBV was in effect a chimaeric virus with the replicase gene derived from Beaudette and the rest of the genome from M41. This demonstrated that it is possible to exchange a large region of the IBV genome, approximately 8.4 kb, using our transient dominant selection method. Recovery of a viable recombinant IBV also demonstrated that it is possible to interchange a complete replicase gene as we had in effect replaced the M41 replicase gene with the Beaudette derived gene. Analysis of the chimaeric virus showed that it was avirulent indicating that none of the structural or accessory genes derived from a virulent isolate of IBV were able to restore virulence and that therefore, the loss of virulence associated with the Beaudette strain resides in the replicase gene.     

Development and characterization of a recombinant infectious bronchitis virus expressing the ectodomain region of S1 gene of H120 strain

Infectious bronchitis (IB), caused by infectious bronchitis virus (IBV), is a highly contagious chicken disease, and can lead to serious economic losses in poultry enterprises. The continual introduction of new IBV serotypes requires alternative strategies for the production of timely and safe vaccines against the emergence of variants. Modification of the IBV genome using reverse genetics is one way to generate recombinant IBVs as the candidates of new IBV vaccines. In this study, the recombinant IBV is developed by replacing the ectodomain region of the S1 gene of the IBV Beaudette strain with the corresponding fragment from H120 strain, designated as rBeau-H120(S1e). In Vero cells, the virus proliferates as its parental virus and can cause syncytium formation. The peak titer would reach 10^5.9 50 % (median) tissue culture infective dose/mL at 24 h post-infection. After inoculation of chickens with the recombinant virus, it demonstrated that rBeau-H120(S1e) remained nonpathogenic and was restricted in its replication in vivo. Protection studies showed that vaccination with rBeau-H120 (S1e) at 7-day after hatch provided 80 % rate of immune protection against challenge with 10³ 50 % embryos infection dose of the virulent IBV M41 strain. These results indicate that rBeau-H120 (S1e) has the potential to be an alternative vaccine against IBV based on excellent propagation property and immunogenicity. This finding might help in providing further information that replacement of the ectodomain fragment of the IBV Beaudette S1 gene with that from a present field strain is promising for IBV vaccine development.     

鸡传染性支气管炎病毒纤突蛋白的目的基因点突变对其免疫原性的影响

反转录聚合酶链反应扩增鸡传染性支气管炎病毒中国流行株的主要免疫原纤突蛋白Ｓ１基因,将其插入载体ｐＵＣ１８的ＢａｍＨⅠ／ＨｉｎｄⅢ位点,在大肠杆菌中实现目的的基因的分子克隆。经克隆化Ｓ１基因的限制性酶切片多段多态性分析和Ｓｏｕｔｈｅｒｎ杂交之后,双脱氧链终止法测定其５‘端高变区核苷酸序列,并以此与ＧｅｎｅＢａｎｋ中的参考毒株Ｍａｓｓａｃｈｕｓｓｅｔｔｓ４１相应序列作比较,分析其同源性。     

Binding of avian coronavirus spike proteins to host factors reflects virus tropism and pathogenicity

The binding of viruses to host cells is the first step in determining tropism and pathogenicity. While avian infectious bronchitis coronavirus (IBV) infection and avian influenza A virus (IAV) infection both depend on α2,3-linked sialic acids, the host tropism of IBV is restricted compared to that of IAV. Here we investigated whether the interaction between the viral attachment proteins and the host could explain these differences by using recombinant spike domains (S1) of IBV strains with different pathogenicities, as well as the hemagglutinin (HA) protein of IAV H5N1. Protein histochemistry showed that S1 of IBV strain M41 and HA of IAV subtype H5N1 displayed sialic acid-dependent binding to chicken respiratory tract tissue. However, while HA bound with high avidity to a broad range of α2,3-linked sialylated glycans, M41 S1 recognized only one particular α2,3-linked disialoside in a glycan array. When comparing the binding of recombinant IBV S1 proteins derived from IBV strains with known differences in tissue tropism and pathogenicity, we observed that while M41 S1 displayed binding to cilia and goblet cells of the chicken respiratory tract, S1 derived from the vaccine strain H120 or the nonvirulent Beaudette strain had reduced or no binding to chicken tissues, respectively, in agreement with the reduced abilities of these viruses to replicate in vivo. While the S1 protein derived from the nephropathogenic IBV strain B1648 also hardly displayed binding to respiratory tract cells, distinct binding to kidney cells was observed, but only after the removal of sialic acid from S1. In conclusion, our data demonstrate that the attachment patterns of the IBV S proteins correlate with the tropisms and pathogenicities of the corresponding viruses.     

Reactivation of human immunodeficiency virus type 2 in macaques after simian immunodeficiency virus SIVmac superinfection.

By superinfection of human immunodeficiency virus type 2 (HIV-2) strain HIV-2ben-infected macaques with simian immunodeficiency virus (SIV) strain SIVmac, we investigated the mutual influences of an apathogenic and a pathogenic virus in vivo. Four rhesus and two cynomolgus monkeys were infected with HIV-2ben in 1988 and 1989, respectively. Virus could be reisolated from five of six animals 6 weeks after infection. The monkeys remained healthy over the next 2 to 3 years. PCR for viral RNA became negative, and virus could no longer be reisolated by coculture. All six macaques were superinfected with the pathogenic SIVmac251/32H. Subsequently, five monkeys became persistently viremic, while one animal was protected against the SIVmac infection. In the peripheral blood mononuclear cells and cocultures of the five viremic animals, DNA from both HIV-2 and SIVmac was present. The plasma contained RNA from both viruses. Thus, superinfection with SIVmac activated HIV-2. A proliferative T-cell response against both HIV-2 and SIVmac was measured in all animals after superinfection. Such a response was regularly seen after infection with the apathogenic HIV-2 but never when the pathogenic SIVmac alone was administered. While naive control monkeys inoculated with SIVmac251/32H regularly develop AIDS-like symptoms soon after infection and have to be killed, none of the preinfected animals has developed AIDS-like symptoms, but two of six animals developed tumors. After the SIVmac challenge, however, apoptotic lymphocytes were detected in the peripheral blood mononuclear cells of all animals. Thus, the presence of an apathogenic viral variant seems to retard the disease occurring after infection with a pathogenic virus rather than to confirm total protection. This partial protection appears to depend on a specific proliferative T-cell response early after infection.     

肾型IBVS1基因重组BCG菌株的构建及其免疫原性研究

S1基因是传染性支气管炎病毒(IBV)的主要保护性抗原基因,应用RT-PCR技术特异地扩增嗜肾型传染性支气管炎病毒X株的S1基因,经序列分析证实为S1基因,将其连接到含有人结核分枝杆菌启动子hsp70基因和堪萨斯分枝杆菌α信号肽基因的表达载体pRR3上,从而构建了大肠杆菌-分枝杆菌穿梭表达质粒pR-α-S1,再电转化至BCG中,形成重组菌株rBCG-S1。热激后S1蛋白在耻垢分枝杆菌M.smegmatismc2155中高效表达,并且通过ELISA和Western blot检测能够被抗IBVS1蛋白的单克隆抗体识别。将106CFU的重组菌株rBCG-S1皮下注射免疫6周龄的SPF鸡,动物保护试验结果表明重组菌株rBCG-S1对鸡具有一定的免疫保护效果,能够保护SPF鸡抵抗IBVX毒株强毒攻击。血凝抑制试验表明血凝抑制抗体滴度能够显著增加。构建的重组菌株为今后开发新型鸡传染性支气管炎基因工程弱毒疫苗奠定了基础。     

一株鸭源传染性支气管炎病毒的分离鉴定及结构蛋白基因和血清型分析

【目的】对从广西某鸭场发生呼吸道感染的11天龄樱桃谷肉鸭分离到的病毒株进行鉴定,并探索此鸭源病毒分离株的遗传变异情况。【方法】通过血凝试验、鸡胚接种实验、3?端非编码区(3'UTR)基因扩增与序列测定对分离株进行鉴定,并对该分离株的结构基因S1、E、M和N分别进行序列测定以及相似性、系统进化树分析和血清型鉴定。【结果】血凝试验为阴性,接种鸡胚盲传5代后出现侏儒胚,3?UTR基因测序结果表明为传染性支气管炎病毒(IBV)序列。该分离株S蛋白的裂解位点为RRSRR,S1、E、M和N基因与IBV毒株H120、4/91、LTD3核苷酸相似性分别为:78.6%–99.7%、85.4%–100.0%、91.6%–93.2%、86.7%–91.7%。除N基因存在点突变外,S1、E和M基因均存在氨基酸的突变、插入和(或)缺失。系统进化树分析显示,其S1基因属于4/91型,E、M和N基因均为LDT3型。血清型分析表明,该分离株的血清型不同于疫苗株H120和4/91。【结论】此鸭源病毒分离株为IBV,且该分离株的基因型与血清型均发生了变异。本研究结果暗示禽类传染性支气管炎的防控面临着更严峻的挑战。     

Comparative Efficacy of Hemagglutinin,Nucleoprotein, and Matrix 2 Protein Gene-Based Vaccination against H5N1 Influenza in Mouse and Ferret

Efforts to develop a broadly protective vaccine against the highly pathogenic avian influenza A (HPAI) H5N1 virus have focused on highly conserved influenza gene products. The viral nucleoprotein (NP) and ion channel matrix protein (M2) are highly conserved among different strains and various influenza A subtypes. Here, we investigate the relative efficacy of NP and M2 compared to HA in protecting against HPAI H5N1 virus. In mice, previous studies have shown that vaccination with NP and M2 in recombinant DNA and/or adenovirus vectors or with adjuvants confers protection against lethal challenge in the absence of HA. However, we find that the protective efficacy of NP and M2 diminishes as the virulence and dose of the challenge virus are increased. To explore this question in a model relevant to human disease, ferrets were immunized with DNA/rAd5 vaccines encoding NP, M2, HA, NP+M2 or HA+NP+M2. Only HA or HA+NP+M2 vaccination conferred protection against a stringent virus challenge. Therefore, while gene-based vaccination with NP and M2 may provide moderate levels of protection against low challenge doses, it is insufficient to confer protective immunity against high challenge doses of H5N1 in ferrets. These immunogens may require combinatorial vaccination with HA, which confers protection even against very high doses of lethal viral challenge.     

Contributions of the viral genetic background and a single amino acid substitution in an immunodominant CD8+ T-cell epitope to murine coronavirus neurovirulence

The immunodominant CD8+ T-cell epitope of a highly neurovirulent strain of mouse hepatitis virus (MHV), JHM, is thought to be essential for protection against virus persistence within the central nervous system. To test whether abrogation of this H-2Db-restricted epitope, located within the spike glycoprotein at residues S510 to 518 (S510), resulted in delayed virus clearance and/or virus persistence we selected isogenic recombinants which express either the wild-type JHM spike protein (RJHM) or spike containing the N514S mutation (RJHM(N514S)), which abrogates the response to S510. In contrast to observations in suckling mice in which viruses encoding inactivating mutations within the S510 epitope (epitope escape mutants) were associated with persistent virus and increased neurovirulence (Pewe et al., J Virol. 72:5912-5918, 1998), RJHM(N514S) was not more virulent than the parental, RJHM, in 4-week-old C57BL/6 (H-2b) mice after intracranial injection. Recombinant viruses expressing the JHM spike, wild type or encoding the N514S substitution, were also selected in which background genes were derived from the neuroattenuated A59 strain of MHV. Whereas recombinants expressing the wild-type JHM spike (SJHM/RA59) were highly neurovirulent, A59 recombinants containing the N514S mutation (SJHM(N514S)/RA59) were attenuated, replicated less efficiently, and exhibited reduced virus spread in the brain at 5 days postinfection (peak of infectious virus titers in the central nervous system) compared to parental virus encoding wild-type spike. Virulence assays in BALB/c mice (H-2d), which do not recognize the S510 epitope, revealed that attenuation of the epitope escape mutants was not due to the loss of a pathogenic immune response directed against the S510 epitope. Thus, an intact immunodominant S510 epitope is not essential for virus clearance from the CNS, the S510 inactivating mutation results in decreased virulence in weanling mice but not in suckling mice, suggesting that specific host conditions are required for epitope escape mutants to display increased virulence, and the N514S mutation causes increased attenuation in the context of A59 background genes, demonstrating that genes other than that for the spike are also important in determining neurovirulence.     

Assembly and immunogenicity of baculovirus-derived infectious bronchitis virus–like particles carrying membrane,envelope and the recombinant spike proteins

Objective

To assemble infectious bronchitis virus (IBV)-like particles bearing the recombinant spike protein and investigate the humoral immune responses in chickens.

Results

IBV virus-like particles (VLPs) were generated through the co-infection with three recombinant baculoviruses separately encoding M, E or the recombinant S genes. The recombinant S protein was sufficiently flexible to retain the ability to self-assemble into VLPs. The size and morphology of the VLPs were similar to authentic IBV particles. In addition, the immunogenicity of IBV VLPs had been investigated. The results demonstrated that the efficiency of the newly generated VLPs was comparable to that of the inactivated M41 viruses in eliciting IBV-specific antibodies and neutralizing antibodies in chickens via subcutaneous inoculation.

Conclusions

This work provides basic information for the mechanism of IBV VLP formation and develops a platform for further designing IBV VLP-based vaccines against IBV or other viruses.

     

传染性支气管炎病毒江苏分离株核蛋白基因序列测定与同源性分析

将本室鸡传染性支气管炎病毒（IBV）江苏省地方分离肾型毒株JS／95／03接种鸡胚,分离、纯化病毒,提取单股RNA做为反转录－聚合酶链反应（RT－PCR）的扩增模板。用Genbank公开序列多重比较后设计一对引物,使用单管RT－PCR方法,物异扩增IBV核蛋白（N）基因5'端854bp的片段,扩增产物纯化后测,序列分析表明,IBV N基因也存在较大变异,此毒株与呼吸型疫苗株M41序列同源性最高。     

传染性支气管炎病毒江苏分离株 核蛋白基因序列测定与同源性分析

将本室鸡传染性支气管炎病毒（IBV）江苏省地方分离肾型毒株JS/95/03接种鸡胚,分离、纯化病毒,提取单股RNA做为反转录-聚合酶链反应（RT-PCR）的扩增模板。用Genbank公开序列多重比较后设计一对引物,使用单管RT-PCR方法,特异扩增IBV核蛋白（N）基因5'端854 bp的片段。扩增产物纯化后测序,序列分析表明,IBVN基因也存在较大变异,此毒株与呼吸型疫苗株M41序列同源性最高。     

Induction of heterosubtypic immunity to influenza virus by intranasal immunization

Recovery from live influenza virus infection is known to induce heterosubtypic immunity. In contrast, immunity induced by inactivated vaccines is predominantly subtype specific. In this study, we investigated the heterosubtypic protective immunity induced by inactivated influenza virus. Intranasal immunization of mice with inactivated influenza virus A/PR8 (H1N1) provided complete protection against the homologous virus and a drift virus within the same subtype, A/WSN (H1N1), but not against the heterosubtypic virus A/Philippines (H3N2). However, coadministration of inactivated virus with cholera toxin as an adjuvant conferred complete heterosubtypic protection, without observed illness, even under conditions of CD4⁺ or CD8⁺ T-cell depletion. Analysis of immune correlates prior to challenge and postchallenge indicated that humoral immune responses with cross-neutralizing activity in lungs and in sera play a major role in conferring protective immunity against heterosubtypic challenge. This study has significant implications for developing broadly cross-reactive vaccines against newly emerging pathogenic influenza viruses.     

Characterization of the 2012 Highly Pathogenic Avian Influenza H7N3 Virus Isolated from Poultry in an Outbreak in Mexico: Pathobiology and Vaccine Protection

In June of 2012, an H7N3 highly pathogenic avian influenza (HPAI) virus was identified as the cause of a severe disease outbreak in commercial laying chicken farms in Mexico. The purpose of this study was to characterize the Mexican 2012 H7N3 HPAI virus (A/chicken/Jalisco/CPA1/2012) and determine the protection against the virus conferred by different H7 inactivated vaccines in chickens. Both adult and young chickens intranasally inoculated with the virus became infected and died at between 2 and 4 days postinoculation (p.i.). High virus titers and viral replication in many tissues were demonstrated at 2 days p.i. in infected birds. The virus from Jalisco, Mexico, had high sequence similarity of greater than 97% to the sequences of wild bird viruses from North America in all eight gene segments. The hemagglutinin gene of the virus contained a 24-nucleotide insert at the hemagglutinin cleavage site which had 100% sequence identity to chicken 28S rRNA, suggesting that the insert was the result of nonhomologous recombination with the host genome. For vaccine protection studies, both U.S. H7 low-pathogenic avian influenza (LPAI) viruses and a 2006 Mexican H7 LPAI virus were tested as antigens in experimental oil emulsion vaccines and injected into chickens 3 weeks prior to challenge. All H7 vaccines tested provided ≥90% protection against clinical disease after challenge and decreased the number of birds shedding virus and the titers of virus shed. This study demonstrates the pathological consequences of the infection of chickens with the 2012 Mexican lineage H7N3 HPAI virus and provides support for effective programs of vaccination against this virus in poultry.     

Vaccination of rhesus macaques with recombinant Mycobacterium bovis bacillus Calmette-Guérin Env V3 elicits neutralizing antibody-mediated protection against simian-human immunodeficiency virus with a homologous but not a heterologous V3 motif

Although the correlates of vaccine-induced protection against human immunodeficiency virus type 1 (HIV-1) are not fully known, it is presumed that neutralizing antibodies (NAb) play a role in controlling virus infection. In this study, we examined immune responses elicited in rhesus macaques following vaccination with recombinant Mycobacterium bovis bacillus Calmette-Guérin expressing an HIV-1 Env V3 antigen (rBCG Env V3). We also determined the effect of vaccination on protection against challenge with either a simian-human immunodeficiency virus (SHIV-MN) or a highly pathogenic SHIV strain (SHIV-89.6PD). Immunization with rBCG Env V3 elicited significant levels of NAb for the 24 weeks tested that were predominantly HIV-1 type specific. Sera from the immunized macaques neutralized primary HIV-1 isolates in vitro, including HIV-1BZ167/X4, HIV-1SF2/X4, HIV-1CI2/X4, and, to a lesser extent, HIV-1MNp/X4, all of which contain a V3 sequence homologous to that of rBCG Env V3. In contrast, neutralization was not observed against HIV-1SF33/X4, which has a heterologous V3 sequence, nor was it found against primary HIV-1 R5 isolates from either clade A or B. Furthermore, the viral load in the vaccinated macaques was significantly reduced following low-dose challenge with SHIV-MN, and early plasma viremia was markedly decreased after high-dose SHIV-MN challenge. In contrast, replication of pathogenic SHIV-89.6PD was not affected by vaccination in any of the macaques. Thus, we have shown that immunization with an rBCG Env V3 vaccine elicits a strong, type-specific V3 NAb response in rhesus macaques. While this response was not sufficient to provide protection against a pathogenic SHIV challenge, it was able to significantly reduce the viral load in macaques following challenge with a nonpathogenic SHIV. These observations suggest that rBCG vectors have the potential to deliver an appropriate virus immunogen for desirable immune elicitations.     

Protection against High-Dose Highly Pathogenic Mucosal SIV Challenge at Very Low Serum Neutralizing Titers of the Antibody-Like Molecule CD4-IgG2

Passive transfer studies using monoclonal or polyclonal antibodies in the macaque model have been valuable for determining conditions for antibody protection against immunodeficiency virus challenge. Most studies have employed hybrid simian/human immunodeficiency virus (SHIV) challenge in conjunction with neutralizing human monoclonal antibodies. Passive protection against SIV, particularly the pathogenic prototype virus SIVmac239, has been little studied because of the paucity of neutralizing antibodies to this virus. Here, we show that the antibody-like molecule CD4-IgG2 potently neutralizes SIVmac239 in vitro. When administered by an osmotic pump to maintain concentrations given the short half-life of CD4-IgG2 in macaques, the molecule provided sterilizing immunity/protection against high-dose mucosal viral challenge to a high proportion of animals (5/7 at a 200 mg dose CD4-IgG2 and 3/6 at a 20 mg dose) at serum concentrations below 1.5 μg/ml. The neutralizing titers of such sera were predicted to be very low and indeed sera at a 1∶4 dilution produced no neutralization in a pseudovirus assay. Macaque anti-human CD4 titers did develop weakly at later time points in some animals but were not associated with the level of protection against viral challenge. The results show that, although SIVmac239 is considered a highly pathogenic virus for which vaccine-induced T cell responses in particular have provided limited benefit against high dose challenge, the antibody-like CD4-IgG2 molecule at surprisingly low serum concentration affords sterilizing immunity/protection to a majority of animals.     

Nucleocapsid or spike protein-specific CD4+ T lymphocytes protect against coronavirus-induced encephalomyelitis in the absence of CD8+ T cells.

To investigate the antiviral CD4+ T cell response in coronavirus MHV-JHM-induced encephalomyelitis, spleen and thymic lymphocytes from diseased rats were stimulated in culture with virus Ag, expanded and tested for their specificity to viral proteins and nucleocapsid (N) and spike (S) proteins that had been expressed in bacteria. A strong T cell response specific for N was measurable during acute disease, whereas S-specific T cells were only detectable in rats with a later onset of disease. CD4+ T cell lines with specificity for virus and either N or S protein were established and their influence on the course of a mouse hepatitis virus-JHM infection was investigated. All lines were of the CD4+ phenotype. Both N and S protein-specific CD4+ T cells conferred protection to infected Lewis rats and reduced the amount of infectious virus in the central nervous system. After transfer of CD4+ T cells and challenge with virus, an increase in the antiviral IgM response occurred, but neutralizing antibodies were not detectable during the period of virus clearance. Previous CD8+ cell depletion did not abrogate protection mediated by CD4+ T cell line transfer.