我国汉坦病毒基因型和基因亚型的分布研究

为了搞清全国汉坦病毒的基因型和亚型的分布情况,广泛收集了全国各地汉坦病毒毒株、阳性病人血清和阳性鼠肺,并应用RT-PCR的方法,应用汉坦病毒型特异性引物,对这些不同来源的阳性标本中汉坦病毒型特异性M和S片段进行扩增和测序,并与其它已知病毒序列进行比较,以明确其型别和亚型及其在全国的分布情况.结果表明:我国HFRS各疫区仍然为HTNV和SEOV两型病毒,但亚型分布差异较大,其中HTNV可分为9个亚型,SEOV则有4～6个亚型.Q32株的部分M和S片段分属于H9和H2亚型,是一个基因重排病毒,而Nc167株在系统发生上与其它HTNV明显不同,比较核苷酸序列发现,其M片段与其它HTNV的同源性在71.3%～76.7%之间,S片段与其它HTNV的同源性只有52.3%～57.8%,可能是一个新型病毒.     

Dobrava-Belgrade hantavirus from Germany shows receptor usage and innate immunity induction consistent with the pathogenicity of the virus in humans

Background

Dobrava-Belgrade virus (DOBV) is a European hantavirus causing hemorrhagic fever with renal syndrome (HFRS) in humans with fatality rates of up to 12%. DOBV-associated clinical cases typically occur also in the northern part of Germany where the virus is carried by the striped field mouse (Apodemus agrarius). However, the causative agent responsible for human illness has not been previously isolated.

Methodology/Principal Findings

Here we report on characterization of a novel cell culture isolate from Germany obtained from a lung tissue of “spillover” infected yellow necked mouse (A. flavicollis) trapped near the city of Greifswald. Phylogenetic analyses demonstrated close clustering of the new strain, designated Greifswald/Aa (GRW/Aa) with the nucleotide sequence obtained from a northern German HFRS patient. The virus was effectively blocked by specific antibodies directed against β3 integrins and Decay Accelerating Factor (DAF) indicating that the virus uses same receptors as the highly pathogenic Hantaan virus (HTNV). In addition, activation of selected innate immunity markers as interferon β and λ and antiviral protein MxA after viral infection of A549 cells was investigated and showed that the virus modulates the first-line antiviral response in a similar way as HTNV.

Conclusions/Significance

In summary, our study reveals novel data on DOBV receptor usage and innate immunity induction in relationship to virus pathogenicity and underlines the potency of German DOBV strains to act as human pathogen.     

Analysis of immune responses against nucleocapsid protein of the Hantaan virus elicited by virus infection or DNA vaccination

Even though neutralizing antibodies against the Hantaan virus (HTNV) has been proven to be critical against viral infections, the cellular immune responses to HTNV are also assumed to be important for viral clearance. In this report, we have examined the cellular and humoral immune responses against the HTNV nucleocapsid protein (NP) elicited by virus infection or DNA vaccination. To examine the cellular immune response against HTNV NP, we used H-2K(b) restricted T-cell epitopes of NP. The NP-specific CD8(+) T cell response was analyzed using a (51)Cr-release assay, intracellular cytokine staining assay, enzyme-linked immunospot assay and tetramer binding assay in C57BL/6 mice infected with HTNV. Using these methods, we found that HTNV infection elicited a strong NP-specific CD8(+) T cell response at eight days after infection. We also found that several different methods to check the NP-specific CD8(+) T cell response showed a very high correlation among analysis. In the case of DNA vaccination by plasmid encoding nucleocapsid gene, the NP-specific antibody response was elicited 2 approximately 4 weeks after immunization and maximized at 6 approximately 8 weeks. NP-specific CD8(+) T cell response reached its peak 3 weeks after immunization. In a challenge test with the recombinant vaccinia virus expressing NP (rVV-HTNV-N), the rVV-HTNV-N titers in DNA vaccinated mice were decreased about 100-fold compared to the negative control mice.     

Monitoring Neutralization Property Change of Evolving Hantaan and Seoul Viruses with a Novel Pseudovirus-Based Assay

The Hantaan virus(HTNV) and Seoul virus(SEOV) mutants have accumulated over time. It is important to determine whether their neutralizing epitopes have evolved, thereby making the current vaccine powerless. However, it is impossible to determine by using traditional plaque reduction neutralization test(PRNT), because it requires large numbers of live mutant strains. Pseudovirus-based neutralization assays(PBNA) were developed by employing vesicular stomatitis virus(VSV) backbone incorporated with HTNV or SEOV glycoproteins(VSVDG*-HTNVG or VSVDG*-SEOVG). 56 and 51 single amino acid substitutions of glycoprotein(GP) in HTNV and SEOV were selected and introduced into the reference plasmid. Then the mutant pseudoviruses were generated and tested by PBNA. The PBNA results were highly correlated with PRNT ones with R2 being 0.91 for VSVDG*-HTNVG and 0.82 for VSVDG*-SEOVG. 53 HTNV mutant pseudoviruses and 46 SEOV mutants were successfully generated. Importantly, by using PBNA, we found that HTNV or SEOV immunized antisera could neutralize all the corresponding 53 HTNV mutants or the 46 SEOV mutants respectively. The novel PBNA enables us to closely monitor the effectiveness of vaccines against large numbers of evolving HTNV and SEOV. And the current vaccine remains to be effective for the naturally occurring mutants.     

Indirect Immunofluorescence Assay for the Simultaneous Detection of Antibodies against Clinically Important Old and New World Hantaviruses

In order to detect serum antibodies against clinically important Old and New World hantaviruses simultaneously, multiparametric indirect immunofluorescence assays (IFAs) based on biochip mosaics were developed. Each of the mosaic substrates consisted of cells infected with one of the virus types Hantaan (HTNV), Puumala (PUUV), Seoul (SEOV), Saaremaa (SAAV), Dobrava (DOBV), Sin Nombre (SNV) or Andes (ANDV). For assay evaluation, serum IgG and IgM antibodies were analyzed using 184 laboratory-confirmed hantavirus-positive sera collected at six diagnostic centers from patients actively or previously infected with the following hantavirus serotypes: PUUV (Finland, n = 97); SEOV (China, n = 5); DOBV (Romania, n = 7); SNV (Canada, n = 23); ANDV (Argentina and Chile, n = 52). The control panel comprised 89 sera from healthy blood donors. According to the reference tests, all 184 patient samples were seropositive for hantavirus-specific IgG (n = 177; 96%) and/or IgM (n = 131; 72%), while all control samples were tested negative. In the multiparametric IFA applied in this study, 183 (99%) of the patient sera were IgG and 131 (71%) IgM positive (accordance with the reference tests: IgG, 96%; IgM, 93%). Overall IFA sensitivity for combined IgG and IgM analysis amounted to 100% for all serotypes, except for SNV (96%). Of the 89 control sera, 2 (2%) showed IgG reactivity against the HTNV substrate, but not against any other hantavirus. Due to the high cross-reactivity of hantaviral nucleocapsid proteins, endpoint titrations were conducted, allowing serotype determination in >90% of PUUV- and ANDV-infected patients. Thus, multiparametric IFA enables highly sensitive and specific serological diagnosis of hantavirus infections and can be used to differentiate PUUV and ANDV infection from infections with Murinae-borne hantaviruses (e.g. DOBV and SEOV).     

Detection of Hantaan virus RNA from anti‐Hantaan virus IgG seronegative rodents in an area of high endemicity in Republic of Korea

Hantaan virus (HTNV), of the family Bunyaviridae, causes hemorrhagic fever with renal syndrome (HFRS) in humans. Although the majority of epidemiologic studies have found that rodents are seropositive for hantavirus‐specific immunoglobulin, the discovery of hantavirus RNA in seronegative hosts has led to an investigation of the presence of HTNV RNA in rodents captured in HFRS endemic areas. HTNV RNA was detected in seven (3.8%) of 186 anti‐HTNV IgG seronegative rodents in Republic of Korea (ROK) during 2013–2014. RT‐qPCR for HTNV RNA revealed dynamic virus–host interactions of HTNV in areas of high endemicity, providing important insights into the epidemiology of hantaviruses.     

The expression and genetic immunization of chimeric fragment of Hantaan virus M and S segments

Hemorrhagic fever with renal syndrome (HFRS), which is characterized by severe symptoms and high mortality, is caused by hantavirus. There are still no effective prophylactic vaccines directed to HFRS until now. In this research, we fused expressed G2 fragment of M segment and 0.7kb fragment of S segment. We expect it could be a candidate vaccine. Chimeric gene G2S0.7 was first expressed in prokaryotic expression system pGEX-4T. After inducing expressed fusion proteins, GST-G2S0.7 was induced and its molecular weight was about 100kDa. Meanwhile, the fusion protein kept the activity of its parental proteins. Further, BALB/c mice were vaccinated by the chimeric gene. ELISA, cell microculture neutralization test in vitro were used to detect the humoral immune response in immunized BALB/c mice. Lymphocyte proliferation assay was used to detect the cellular immune response. The results showed that the chimeric gene could simultaneously evoke specific antibody against nucleocapsid protein (NP) and glycoprotein (GP). And the immunized mice of every group elicited neutralizing antibodies with different titers. But the titers were low. Lymphocyte proliferation assay results showed that the stimulation indexes of splenocytes of chimeric gene to NP and GP were significantly higher than that of control. It suggested that the chimeric gene of Hantaan virus containing G2 fragment of M segment and 0.7kb fragment of S segment could directly elicit specific anti-Hantaan virus humoral and cellular immune response in BALB/c mice.     

A New Approach to AIDS Research and Prevention: The Use of Gene-Mutated HIV-1/SIV Chimeric Viruses for Anti-HIV-1 Live-Attenuated Vaccines

The lack of a suitable animal model is a major obstacle to developing anti-HIV-1 vaccines. We successfully generated an SIVmac/HIV-1 chimeric virus (SHIV) (designated as NM-3rN) that contains the HIV-1 env gene and is infectious to macaque monkeys. Challenging the vaccinated macaque monkeys with NM-3rN, we developed an evaluation system for anti-HIV-1 Env-targeted vaccines. For the purpose of making the vaccine, a series of gene-mutated SHIVs were constructed. The monkeys vaccinated with these SHIVs had long-term anti-virus immunities without manifesting the disease, and became resistant to a challenge inoculation with NM-3rN. The sera from a monkey showed that, after the vaccination, the neutralizing antibodies not only against the parental HIV-1 but also against an antigenically different HIV-1 were raised. In vivo experiments confirmed that the vaccinated monkeys were protected from the challenge inoculum of an antigenically different SHIV-MN. Vaccination of monkeys with the attenuated SHIVs showed that further gene-deletion of the SHIV resulted in less immunogenicity. Nevertheless, the attenuated SHIVs had a vaccine effect against the challenge inoculation. In addition to specific immunities including neutralizing antibodies and cytotoxic T cells, a more complicated immune mechanism induced by live vaccine appears to play a role in this protection. Our data suggest that the live vaccine can induce strong and wide-range immunity against HIV-1. These SHIVs should contribute to understanding the pathogenicity of AIDS and to the development of future anti-HIV-1 live vaccines for humans.     

Differential antiviral response of endothelial cells after infection with pathogenic and nonpathogenic hantaviruses

Hantaviruses represent important human pathogens and can induce hemorrhagic fever with renal syndrome (HFRS), which is characterized by endothelial dysfunction. Both pathogenic and nonpathogenic hantaviruses replicate without causing any apparent cytopathic effect, suggesting that immunopathological mechanisms play an important role in pathogenesis. We compared the antiviral responses triggered by Hantaan virus (HTNV), a pathogenic hantavirus associated with HFRS, and Tula virus (TULV), a rather nonpathogenic hantavirus, in human umbilical vein endothelial cells (HUVECs). Both HTNV- and TULV-infected cells showed increased levels of molecules involved in antigen presentation. However, TULV-infected HUVECs upregulated HLA class I molecules more rapidly. Interestingly, HTNV clearly induced the production of beta interferon (IFN-beta), whereas expression of this cytokine was barely detectable in the supernatant or in extracts from TULV-infected HUVECs. Nevertheless, the upregulation of HLA class I on both TULV- and HTNV-infected cells could be blocked by neutralizing anti-IFN-beta antibodies. Most strikingly, the antiviral MxA protein, which interferes with hantavirus replication, was already induced 16 h after infection with TULV. In contrast, HTNV-infected HUVECs showed no expression of MxA until 48 h postinfection. In accordance with the kinetics of MxA expression, TULV replicated only inefficiently in HUVECs, whereas HTNV-infected cells produced high titers of virus particles that decreased after 48 h postinfection. Both hantavirus species, however, could replicate equally well in Vero E6 cells, which lack an IFN-induced MxA response. Thus, delayed induction of antiviral MxA in endothelial cells after infection with HTNV could allow viral dissemination and contribute to the pathogenesis leading to HFRS.     

Wild-Type Puumala Hantavirus Infection Induces Cytokines, C-Reactive Protein, Creatinine, and Nitric Oxide in Cynomolgus Macaques

Hantaviruses cause two severe human diseases: hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). Approximately 200,000 cases are reported annually, and there is to date no specific treatment available. A major obstacle in studying the medical aspects of HFRS and HPS has been the lack of an adequate animal model. Here we show that infection of cynomolgus macaques by wild-type Puumala hantavirus resulted in typical signs of HFRS including lethargy, anorexia, proteinuria, and/or hematuria, in addition to cytokine (interleukin 6 [IL-6], IL-10, and tumor necrosis factor alpha), C-reactive protein, creatinine, and nitric oxide responses. Viral RNA was detected in plasma from days 3 to 7 postinoculation until days 24 to 28 postinoculation, infectious virus was recovered, and the virus-specific immune responses (immunoglobulin M [IgM], IgG, and neutralizing antibodies) mimicked those seen in humans. The results indicated that the monkey model will provide a valuable tool for studies of pathogenesis, candidate vaccines, and antivirals for hantavirus disease.     

Evaluation of envelope vaccines derived from the South African subtype C human immunodeficiency virus type 1 TV1 strain

Human immunodeficiency virus type 1 (HIV-1) subtype C infections are on the rise in Sub-Saharan Africa and Asia. Therefore, there is a need to develop an HIV vaccine capable of eliciting broadly reactive immune responses against members of this subtype. We show here that modified HIV envelope (env) DNA vaccines derived from the South African subtype C TV1 strain are able to prime for humoral responses in rabbits and rhesus macaques. Priming rabbits with DNA plasmids encoding V2-deleted TV1 gp140 (gp140TV1DeltaV2), followed by boosting with oligomeric protein (o-gp140TV1DeltaV2) in MF59 adjuvant, elicited higher titers of env-binding and autologous neutralizing antibodies than priming with DNA vaccines encoding the full-length TV1 env (gp160) or the intact TV1 gp140. Immunization with V2-deleted subtype B SF162 env and V2-deleted TV1 env together using a multivalent vaccine approach induced high titers of oligomeric env-binding antibodies and autologous neutralizing antibodies against both the subtypes B and C vaccine strains, HIV-1 SF162 and TV1, respectively. Low-level neutralizing activity against the heterologous South African subtype C TV2 strain, as well as a small subset of viruses in a panel of 13 heterologous primary isolates, was observed in some rabbits immunized with the V2-deleted vaccines. Immunization of rhesus macaques with the V2-deleted TV1 DNA prime/protein boost also elicited high titers of env-binding antibodies and moderate titers of autologous TV1 neutralizing antibodies. The pilot-scale production of the various TV1 DNA vaccine constructs and env proteins described here should provide an initial platform upon which to improve the immunogenicity of these subtype C HIV envelope vaccines.     

Active and passive vaccination against hantavirus pulmonary syndrome with Andes virus M genome segment-based DNA vaccine

Hantavirus pulmonary syndrome (HPS) is a rapidly progressing human disease with one of the highest case fatality rates (30 to 50%) of any acute viral disease known. There are no vaccines, effective antiviral drugs, or immunologics to prevent or treat HPS. In an attempt to develop HPS medical countermeasures, we constructed an expression plasmid, pWRG/AND-M, that contains the full-length M genome segment of Andes virus (ANDV), a South American hantavirus. Transfection experiments in cell culture indicated that both the G1 and G2 glycoproteins are expressed from pWRG/AND-M. Rhesus macaques vaccinated by gene gun with pWRG/AND-M developed remarkably high levels of neutralizing antibodies that not only neutralized ANDV but also cross-neutralized other HPS-associated hantaviruses, including Sin Nombre virus. To determine if the antibodies elicited in the monkeys could confer protection, we performed a series of passive-transfer experiments using a recently described lethal HPS animal model (i.e., adult Syrian hamsters develop HPS and die within 10 to 15 days after challenge with ANDV). When injected into hamsters 1 day before challenge, sera from the vaccinated monkeys either provided sterile protection or delayed the onset of HPS and death. When injected on day 4 or 5 after challenge, the monkey sera protected 100% of the hamsters from lethal disease. These data provide a proof of concept for a gene-based HPS vaccine and also demonstrate the potential value of a postexposure immunoprophylactic to treat individuals after exposure, or potential exposure, to these highly lethal hantaviruses.     

Evaluation of the protective immunogenicity of the N, P, M, NV and G proteins of infectious hematopoietic necrosis virus in rainbow trout oncorhynchus mykiss using DNA vaccines.

The protective immunogenicity of the nucleoprotein (N), phosphoprotein (P), matrix protein (M), non-virion protein (NV) and glycoprotein (G) of the rhabdovirus infectious hematopoietic necrosis virus (IHNV) was assessed in rainbow trout using DNA vaccine technology. DNA vaccines were produced by amplifying and cloning the viral genes in the plasmid pCDNA 3.1. The protective immunity elicited by each vaccine was evaluated through survival of immunized fry after challenge with live virus. Neutralizing antibody titers were also determined in vaccinated rainbow trout Oncorhynchus mykiss fry (mean weight 2 g) and 150 g sockeye salmon Oncorhynchus nerka. The serum from the 150 g fish was also used in passive immunization studies with naive fry. Our results showed that neither the internal structural proteins (N, P and M) nor the NV protein of IHNV induced protective immunity in fry or neutralizing antibodies in fry and 150 g fish when expressed by a DNA vaccine construct. The G protein, however, did confer significant protection in fry up to 80 d post-immunization and induced protective neutralizing antibodies. We are currently investigating the role of different arms of the fish immune system that contribute to the high level of protection against IHNV seen in vaccinated fish.     

Towards a safe, effective vaccine for Rift Valley fever virus

Rift Valley fever virus (RVFV) is an important animal and human threat and leads to longstanding morbidity and mortality in susceptible hosts. Since no therapies currently exist to treat Rift Valley fever, it remains a public and animal health priority to develop safe, effective RVFV vaccines (whether for animals, humans, or both) that provide long-term protective immunity. In the evaluated article, Bhardwaj and colleagues describe the creation and testing of two successful vaccine strategies against RVFV, a DNA plasmid vaccine expressing Gn coupled to C3d, and an alpha-virus replicon vaccine expressing Gn protein. Both vaccines elicited strong neutralizing antibody responses, prevented morbidity and mortality in RVFV-challenged mice, and enabled protection of naive mice via passive antibody transfer from vaccinated mice. Both DNA and replicon RVFV vaccines have previously been shown to protect against RVFV challenge, but these results allow for direct comparison of the two methods and evaluation of a combined prime-boost method. The results also highlight the specific humoral and cell-mediated immune responses to vaccination.     

Immune serum produced by DNA vaccination protects hamsters against lethal respiratory challenge with Andes virus

Hantavirus pulmonary syndrome (HPS) is a highly pathogenic disease (40% case fatality rate) carried by rodents chronically infected with certain viruses within the genus Hantavirus of the family Bunyaviridae. The primary mode of transmission to humans is thought to be inhalation of excreta from infected rodents; however, ingestion of contaminated material and rodent bites are also possible modes of transmission. Person-to-person transmission of HPS caused by one species of hantavirus, Andes virus (ANDV), has been reported. Previously, we reported that ANDV injected intramuscularly causes a disease in Syrian hamsters that closely resembles HPS in humans. Here we tested whether ANDV was lethal in hamsters when it was administered by routes that more accurately model the most common routes of human infection, i.e., the subcutaneous, intranasal, and intragastric routes. We discovered that ANDV was lethal by all three routes. Remarkably, even at very low doses, ANDV was highly pathogenic when it was introduced by the mucosal routes (50% lethal dose [LD₅₀], ~100 PFU). We performed passive transfer experiments to test the capacity of neutralizing antibodies to protect against lethal intranasal challenge. The neutralizing antibodies used in these experiments were produced in rabbits vaccinated by electroporation with a previously described ANDV M gene-based DNA vaccine, pWRG/AND-M. Hamsters that were administered immune serum on days −1 and +5 relative to challenge were protected against intranasal challenge (21 LD₅₀). These findings demonstrate the utility of using the ANDV hamster model to study transmission across mucosal barriers and provide evidence that neutralizing antibodies produced by DNA vaccine technology can be used to protect against challenge by the respiratory route.     

Efficacy of DNA and fowlpox virus priming/boosting vaccines for simian/human immunodeficiency virus

Further advances are required in understanding protection from AIDS by T-cell immunity. We analyzed a set of multigenic simian/human immunodeficiency virus (SHIV) DNA and fowlpox virus priming and boosting vaccines for immunogenicity and protective efficacy in outbred pigtail macaques. The number of vaccinations required, the effect of DNA vaccination alone, and the effect of cytokine (gamma interferon) coexpression by the fowlpox virus boost was also studied. A coordinated induction of high levels of broadly reactive CD4 and CD8 T-cell immune responses was induced by sequential DNA and fowlpox virus vaccination. The immunogenicity of regimens utilizing fowlpox virus coexpressing gamma interferon, a single DNA priming vaccination, or DNA vaccines alone was inferior. Significant control of a virulent SHIV challenge was observed despite a loss of SHIV-specific proliferating T cells. The outcome of challenge with virulent SHIV(mn229) correlated with vaccine immunogenicity except that DNA vaccination alone primed for protection almost as effectively as the DNA/fowlpox virus regimen despite negligible immunogenicity by standard assays. These studies suggest that priming of immunity with DNA and fowlpox virus vaccines could delay AIDS in humans.     

Dengue-1 envelope protein domain III along with PELC and CpG oligodeoxynucleotides synergistically enhances immune responses

The major weaknesses of subunit vaccines are their low immunogenicity and poor efficacy. Adjuvants can help to overcome some of these inherent defects with subunit vaccines. Here, we evaluated the efficacy of the newly developed water-in-oil-in-water multiphase emulsion system, termed PELC, in potentiating the protective capacity of dengue-1 envelope protein domain III. Unlike aluminum phosphate, dengue-1 envelope protein domain III formulated with PELC plus CpG oligodeoxynucleotides induced neutralizing antibodies against dengue-1 virus and increased the splenocyte secretion of IFN-γ after in vitro re-stimulation. The induced antibodies contained both the IgG1 and IgG2a subclasses. A rapid anamnestic neutralizing antibody response against a live dengue virus challenge was elicited at week 26 after the first immunization. These results demonstrate that PELC plus CpG oligodeoxynucleotides broaden the dengue-1 envelope protein domain III-specific immune responses. PELC plus CpG oligodeoxynucleotides is a promising adjuvant for recombinant protein based vaccination against dengue virus.     

DNA vaccination of mice with a plasmid encoding Puumala hantavirus nucleocapsid protein mimics the B-cell response induced by virus infection

Inoculation of naked DNA has been applied for the development of prophylactic and therapeutic vaccines against different viral infections. To study the humoral immune response induced by DNA vaccination we cloned the entire nucleocapsid protein-encoding sequence of the Puumala hantavirus strain Vranica/H?lln?s into the CMV promoter-driven expression unit of the plasmid pcDNA3, generating pcDNA3-VR1. A single dose injection of 50 microg of plasmid DNA into each M. tibialis anterior of BALB/c mice induced a high-titered antibody response against the nucleocapsid protein as documented 6 and 11 weeks after immunisation. PEPSCAN analysis of a serum pool of the pcDNA3-VR1-vaccinated animals revealed antibodies reacting with epitopes covering the whole nucleocapsid protein. The epitope-specificity of the immune response induced by DNA vaccination seems to reflect the antibody response in experimentally virus-infected bank voles (the natural host of the Puumala virus) and humans. The data suggest that DNA vaccination could be used for the identification of highly immunogenic epitopes in viral proteins.     

Specificity of DNA vaccines against the U and M genogroups of infectious hematopoietic necrosis virus (IHNV) in rainbow trout (Oncorhynchus mykiss)

Infectious hematopoietic necrosis virus (IHNV) is a fish rhabdovirus that causes significant mortality in salmonid species. In North America IHNV has three major genogroups designated U, M, and L. Host-specificity of the M and U genogroups of IHNV has been established both in the field and in experimental challenges, with M isolates being more prevalent and more virulent in rainbow trout (Oncorhynchus mykiss), and U isolates being more prevalent and highly virulent in sockeye salmon (Oncorhynchus nerka). In this study, efficacy of DNA vaccines containing either M (pM) or U (pU) virus glycoprotein genes was investigated during intra- and cross-genogroup challenges in rainbow trout. In virus challenges at 7 days post-vaccination (early antiviral response), both pM and pU were highly protective against either M or U IHNV. In challenges at 28 days post-vaccination (specific antiviral response), both pM and pU were protective against M IHNV but the homologous pM vaccine was significantly more protective than pU in one of two experiments. At this stage both pM and pU induced comparably high protection against U IHNV challenge. Correlates of protection were also investigated by assessing the expression of the interferon-stimulated gene Mx-1 and the production of neutralizing antibodies (NAbs) following pM or pU DNA vaccination. Mx-1 gene expression, measured at 4 and 7 days post-vaccination as an indicator of the host innate immune response, was found to be significantly higher after pM than pU vaccination in some cases. Neutralizing antibody was produced in response to the two vaccines, but antibody titers did not show consistent correlation with protection. The results show that the rainbow trout innate and adaptive immune responses have some ability to distinguish between the U and M genogroup IHNV, but overall the pM and pU vaccines were protective against both homologous and cross-genogroup challenges.     

逆转录-聚合酶链反应结合限制性片段长度多态性分析对我国肾综合征出血热病毒分型的研究(英文)

建立准确、快速、灵敏的汉坦病毒基因分型方法,可弥补传统血清学方法的不足,对防治该病毒所致的肾综合征出血热(HFRS)具有重要意义。本试验采用逆转录－聚合酶反应(RT-PCR)和限制性片段长度多态性(RFLP)和限制性片段长度多态性(RFLP)方法,对流行于我国的两型汉坦病毒代表株??汉滩型(HTNV)76－118和汉城型(SEOV)R22株进行基因分析。根据病毒DNA序列的电脑软件分析,不同HFRSV囊膜糖蛋白编码基因M节段1199~1497间核苷酸序列上RsaI、TaqI和HindIII的酶切位点存在差异(Fig.1), 可用于进行限制性内切酶基因多态性分析,以确定HFRV的型别。首先,以一对引物扩增该片段(Fig.2)。然后,分别用这三种内切酶(Fig.3,4,5)进行酶切分型。共分析了从我国不同地区,不同宿主分离的毒株18株,及国际标准毒株2株,酶切图谱显示,这些毒株可以被分为三组(Table.1)：9株可定为HTNV型,8株可定为SEOV型,3株无法确定其型别(X型)。该法分型结果与血清学经典的空斑减数中和试验分型结果基本一致,说明该酶切分型方法具有一定的可行性。