Sequence variability of Borna disease virus open reading frame II found in human peripheral blood mononuclear cells.

A cDNA fragment of the Borna disease virus (BDV) open reading frame II (ORF-II), which encodes a 24-kDa phosphoprotein (p24 [P protein]), was amplified from total RNA of peripheral blood mononuclear cells (PBMC) from three psychiatric inpatients. The amplified cDNA fragments were cloned, sequenced, and analyzed. A total of 15 clones, 5 from each patient, were studied. Intrapatient divergencies of the BDV ORF-II nucleotide sequence were 4.2 to 7.3%, 4.8 to 7.3%, and 2.8 to 7.1% for the three patients, leading to differences of 7.7 to 14.5%, 10.3 to 17.1%, and 6.0 to 16.2%, respectively, in the deduced amino acid sequence for BDV p24. Interpatient divergencies among the 15 clones were 5.9 to 12.7% at the nucleotide level and 12.8 to 28.2% at the amino acid level. Thus, in p24, BDV in human PBMC of the patients undergoes mutation at high rates in vivo. Additionally, we found that the nucleotide sequence of the 15 human BDV ORF-II cDNA clones differed from those of the horse strains V and He/80-1 by 4.2 to 9.3%. However, comparison of the consensus amino acid sequence deduced from the 15 human clones with those of the horse strains revealed no human-specific amino acid residue, suggesting that the BDV infecting humans may be related to that infecting horses.     

我国新分离森林脑炎病毒E基因特征分析

为了解分离自黑龙江省大兴安岭林区全沟硬蜱中的DXAL-5、12、13、16、18,21共6株森林脑炎(TBE)病毒E蛋白基因特征并确定病毒基因型,应用RT-PCR技术对6株病毒E蛋白基因进行体外扩增、克隆、测序.结果发现,6株病毒E蛋白基因的核苷酸序列长均为1 488 bp,推导的氨基酸序列长均为496 aa.与TBE参考毒株E蛋白基因进行比较,这6株病毒与远东亚型同源性最高,其次是西伯利亚亚型,与欧洲亚型同源性最差;在决定亚型特征的氨基酸位点多数属于TBE病毒远东亚型.E蛋白基因推导的氨基酸种系发生树分析表明,6株病毒均在远东亚型分枝内.因此就E蛋白基因而言,DXAL-5、12、13、16、18、21株均属于TBE病毒的远东亚型.新分离毒株与Senzhang株同源性较高,种系发生关系也比较接近,推测疫苗株对新分离毒株仍具有很好的保护作用.但是在E蛋白的A、B和C抗原决定区内,6株病毒均有不同程度的氨基酸改变,这些突变有可能影响E蛋白的功能.     

Molecular Epidemiology of Japanese Encephalitis Virus in Okinawa

In order to elucidate the molecular characteristics of Japanese encephalitis (JE) virus in Okinawa, 23 strains of JE virus isolated in a 25-year span were sequenced for the 240 nucleotides of the C-preM junction region and 111 nucleotides of the E gene region and compared with those of reference strains isolated in mainland Japan. The results of phylogenic analysis showed that although all the Okinawan isolates showed more than 96% homology in the nucleotide sequence in each region, they were chronologically divided into two groups: the old group (nine strains) and a new group (14 strains). On the other hand, in a comparison with reference strains in mainland Japan, the Okinawan isolates showed more than 94% nucleotide sequence homology in both regions, indicating that the Okinawan strains belong to the same genotype as that of JE strains in mainland Japan. The nucleotide homology of the old group was relatively higher than that of the new group. Among the 14 strains in the new group, 13 strains were isolated from mosquitoes collected from a pig farm from 1986 through 1992. These strains showed higher nucleotide divergence than the old group strains, isolated from mosquitoes and swine sera collected at several sites, in both regions. A nucleotide substitution at the position 1920 in the E gene was identified in three isolates. This substitution generated an asparagine-proline-threonine sequence capable of serving as an attachment site of carbohydrate.     

Borna Disease Virus-Induced Neurological Disorder in Mice: Infection of Neonates Results in Immunopathology

Borna disease virus (BDV) is a neurotropic nonsegmented negative-stranded RNA virus that persistently infects warm-blooded animals. In horses and other natural animal hosts, infections with BDV cause meningoencephalitis and behavioral disturbances. Experimental infection of adult mice takes a nonsymptomatic course, an observation previously believed to indicate that this animal species is not suitable for pathogenesis studies. We now demonstrate that BDV frequently induces severe neurological disease in infected newborn mice. Signs of neurological disease were first observed 4 to 6 weeks after intracerebral infection. They included a characteristic nonphysiological position of the hind limbs at an early stage of the disease and paraparesis at a later stage. Histological examination revealed large numbers of perivascular and meningeal inflammatory cells in brains of diseased mice and, unexpectedly, no increase in immunoreactivity to glial fibrillar acidic protein. The incidence and severity of BDV-induced disease varied dramatically among mouse strains. While only 13% of the infected C57BL/6 mice showed disease symptoms, which were mostly transient, more than 80% of the infected MRL mice developed severe neurological disorder. In spite of these differences in susceptibility to disease, BDV replicated to comparable levels in the brains of mice of the various strains used. Intracerebral infections of newborn β2-microglobulin-deficient C57BL/6 and MRL mice, which both lack CD8⁺ T cells, did not result in meningoencephalitis or neurological disease, indicating that the BDV-induced neurological disorder in mice is a cytotoxic T-cell-mediated immunopathological process. With this new animal model it should now be possible to characterize the disease-inducing immune response to BDV in more detail.     

浙江省麻疹病毒的基因特征分析

目的分析浙江省流行的麻疹病毒(MV)的基因特征,为更好地防控麻疹提供科学参考。方法从GenBank检索并下载浙江省所有麻疹病毒株、中国麻疹疫苗株和WHO推荐参考株的血凝素蛋白(H)和核蛋白(N)的基因组序列,利用MEGA 6.0软件进行比对分析,构建种系进化树,确定浙江省麻疹病毒流行的基因型别,并进行同源性和基因变异分析。结果浙江省麻疹病毒以H1基因型为主(27株),其他基因型为辅(2株B3型)。H1a亚型(21/27)占绝对优势,其次为H1b亚型(6/27)。浙江省所有毒株间H蛋白的氨基酸同源性为96.9%~100.0%,与疫苗株Shanghai-191和Changchun-47的同源性均为95.0%~96.0%。浙江省所有毒株间N蛋白羧基末端的氨基酸同源性为82.7%~100.0%,与疫苗株Shanghai-191的同源性为85.8%~89.5%,与疫苗株Changchun-47的同源性为87.3%~91.0%。结论麻疹病毒H1基因型为浙江省麻疹流行的优势株,与现行参考疫苗株(A基因型)差异较大,因此针对麻疹病毒H1基因型疫苗的研制是今后浙江省麻疹病毒防控的关键。     

Antigenic and genetic characterization of a novel hemagglutinin subtype of influenza A viruses from gulls.

Influenza A virus isolates from ring-billed, Franklin, blackback, and herring gulls in the United States possess a hemagglutinin (HA) distinct from the 12 reference HA subtypes. Serological assays (hemagglutination inhibition and double-immunodiffusion) with specific antisera to reference strains and to a representative gull isolate showed that the HA of the gull virus was not antigenically related to that of any known subtype. The gull virus did not replicate in ducks or chickens but did replicate in ferrets. Comparison of the nucleotide sequences (and deduced amino acid sequences) of the 3' 20% of the HA genes of these viruses indicates that the gull viruses represent a genetically distinct group. We propose that this HA, which has been detected only in gull isolates thus far, be called the H13 subtype.     

Molecular epidemiology of 58 new African human T-cell leukemia virus type 1 (HTLV-1) strains: identification of a new and distinct HTLV-1 molecular subtype in Central Africa and in Pygmies.

To gain new insights on the origin, evolution, and modes of dissemination of human T-cell leukemia virus type I (HTLV-1), we performed a molecular analysis of 58 new African HTLV-1 strains (18 from West Africa, 36 from Central Africa, and 4 from South Africa) originating from 13 countries. Of particular interest were eight strains from Pygmies of remote areas of Cameroon and the Central African Republic (CAR), considered to be the oldest inhabitants of these regions. Eight long-term activated T-cell lines producing HTLV-1 gag and env antigens were established from peripheral blood mononuclear cell cultures of HTLV-1 seropositive individuals, including three from Pygmies. A fragment of the env gene encompassing most of the gp21 transmembrane region was sequenced for the 58 new strains, while the complete long terminal repeat (LTR) region was sequenced for 9 strains, including 4 from Pygmies. Comparative sequence analyses and phylogenetic studies performed on both the env and LTR regions by the neighbor-joining and DNA parsimony methods demonstrated that all 22 strains from West and South Africa belong to the widespread cosmopolitan subtype (also called HTLV-1 subtype A). Within or alongside the previously described Zairian cluster (HTLV-1 subtype B), we discovered a number of new HTLV-1 variants forming different subgroups corresponding mainly to the geographical origins of the infected persons, Cameroon, Gabon, and Zaire. Six of the eight Pygmy strains clustered together within this Central African subtype, suggesting a common origin. Furthermore, three new strains (two originating from Pygmies from Cameroon and the CAR, respectively, and one from a Gabonese individual) were particularly divergent and formed a distinct new phylogenetic cluster, characterized by specific mutations and occupying in most analyses a unique phylogenetic position between the large Central African genotype (HTLV-1 subtype B) and the Melanesian subtype (HTLV-1 subtype C). We have tentatively named this new HTLV-1 genotype HTLV-1 subtype D. While the HTLV-1 subtype D strains were not closely related to any known African strain of simian T-cell leukemia virus type 1 (STLV-1), other Pygmy strains and some of the new Cameroonian and Gabonese HTLV-1 strains were very similar (>98% nucleotide identity) to chimpanzee STLV-1 strains, reinforcing the hypothesis of interspecies transmission between humans and monkeys in Central Africa.     

Enhanced neurovirulence of borna disease virus variants associated with nucleotide changes in the glycoprotein and L polymerase genes

Borna disease virus (BDV) infection produces a variety of clinical diseases, from behavioral illnesses to classical fatal encephalitis (i.e., Borna disease [BD]). Since the genomes of most BDV isolates differ by less than 5%, host factors are believed responsible for much of the reported variability in disease expression. The contribution of BDV genomic differences to variation in BD expression is largely unexplored. Here we compared the clinical outcomes of rats infected with one of two related BDV variants, CRP3 or CRNP5. Compared to rats inoculated with CRP3, adult and newborn Lewis rats inoculated with CRNP5 had more severe and rapidly fatal neurological disease, with increased damage to the hippocampal pyramidal neurons and rapid infection of brain stem neurons. To identify possible virus-specific contributions to the observed variability in disease outcome, the genomes of CRP3 and CRNP5 were sequenced. Compared to CRP3, there were four nucleotide changes in the CRNP5 variant, two each in the G protein and in the L polymerase, resulting in four amino acid changes. These results suggest that small numbers of genomic differences between BDV variants in the G protein and/or L polymerase can contribute to the variability in BD outcomes.     

Pathogenesis of Borna Disease Virus: Granulocyte Fractions of Psychiatric Patients Harbor Infectious Virus in the Absence of Antiviral Antibodies

Borna disease virus (BDV) causes acute and persistent infections in various vertebrates. During recent years, BDV-specific serum antibodies, BDV antigen, and BDV-specific nucleic acid were found in humans suffering from psychiatric disorders. Furthermore, viral antigen was detected in human autopsy brain tissue by immunohistochemical staining. Whether BDV infection can be associated with psychiatric disorders is still a matter of debate; no direct evidence has ever been presented. In the present study we report on (i) the detection of BDV-specific nucleic acid in human granulocyte cell fraction from three different psychiatric patients and (ii) the isolation of infectious BDV from these cells obtained from a patient with multiple psychiatric disorders. In leukocyte preparations other than granulocytes, either no BDV RNA was detected or positive PCR results were obtained only if there was at least 20% contamination with granulocytes. Parts of the antigenome of the isolated virus were sequenced, demonstrating the close relationship to the prototype BDV strains (He/80 and strain V) as well as to other human virus sequences. Our data provide strong evidence that cells in the granulocyte fraction represent the major if not the sole cell type harboring BDV-specific nucleic acid in human blood and contain infectious virus. In contrast to most other reports of putative human isolates, where sequences are virtually identical to those of the established laboratory strains, this isolate shows divergence in the region previously defined as variable in BDV from naturally infected animals.     

Detection of a newly described pestivirus of Pyrenean chamois (Rupicapra pyrenaica pyrenaica) in France

A pestivirus was detected and characterized in chamois (Rupicapra pyrenaica pyrenaica) originating from the French part of the Pyrenees. Phylogenetic analysis of the pestivirus was done on the basis of a fragment from the 5' noncoding region including 22 published nucleotide sequences of different pestivirus strains. Our strain was grouped within the clade of border disease viruses (BDV). However, it had an intermediate position between clade BDV and classical swine fever viruses representing a basal position to BDV strains of domestic sheep. Our strain was grouped as a sister unit to a novel pestivirus (Chamois-1) recently described from chamois in Spain. Therefore, we postulate that this virus occurs in the entire population of Pyrenean chamois. On the basis of the phylogenetic grouping of this isolate, a postulated cross-species transmission of pestivirus from domestic sheep to chamois via shared pastures seems to be unlikely.     

Borna disease virus multiplication in mouse organotypic slice cultures is site-specifically inhibited by gamma interferon but not by interleukin-12

Borna disease virus (BDV) induces a nonpurulent CD4- and CD8-T-cell-dependent meningoencephalitis in susceptible animals. Upon intracerebral infection, BDV replicates in the mouse central nervous system (CNS), but only a few mouse strains develop neurological disorder. The antiviral T cells appear to suppress BDV replication by a noncytolytic mechanism. Since BDV does not replicate in standard mouse cell cultures, the putative role of gamma interferon (IFN-gamma) in virus control could not be tested experimentally. Here, we report that mouse organotypic slice cultures can be used to elucidate the complex interactions of BDV, the CNS, and the immune system. We show that BDV replicated in various cell types of mouse cerebellar slice cultures in vitro. In infected slice cultures, a moderate upregulation of the chemokine genes CCL5 and CXCL10 was observed, while expression of various neural genes as well as other chemokine and cytokine genes was not altered. IFN-gamma inhibited the multiplication of BDV in cerebellar and hippocampal slice cultures in a dose-dependent manner. However, while complete suppression of BDV was observed in cerebellar slice cultures, inhibition was incomplete in hippocampal slice cultures. Kinetic studies indicated that IFN-gamma protects noninfected cells from infection rather than clearing the virus from infected cells. These results demonstrate that BDV can replicate in cultured neural cells of the mouse if organ integrity is well preserved. They further show that IFN-gamma is a powerful inhibitor of BDV in the absence of blood-borne leukocytes in mouse cerebellar slice cultures.     

Adaptation of Borna disease virus to new host species attributed to altered regulation of viral polymerase activity

Borna disease virus (BDV) can persistently infect the central nervous system of a broad range of mammalian species. Mice are resistant to infections with primary BDV isolates, but certain laboratory strains can be adapted to replicate in mice. We determined the molecular basis of adaptation by studying mutations acquired by a cDNA-derived BDV strain during one brain passage in rats and three passages in mice. The adapted virus propagated efficiently in mouse brains and induced neurological disease. Its genome contained seven point mutations, three of which caused amino acid changes in the L polymerase (L1116R and N1398D) and in the polymerase cofactor P (R66K). Recombinant BDV carrying these mutations either alone or in combination all showed enhanced multiplication speed in Vero cells, indicating improved intrinsic viral polymerase activity rather than adaptation to a mouse-specific factor. Mutations R66K and L1116R, but not N1398D, conferred replication competence of recombinant BDV in mice if introduced individually. Virus propagation in mouse brains was substantially enhanced if both L mutations were present simultaneously, but infection remained mostly nonsymptomatic. Only if all three amino acid substitutions were combined did BDV replicate vigorously and induce early disease in mice. Interestingly, the virulence-enhancing effect of the R66K mutation in P could be attributed to reduced negative regulation of polymerase activity by the viral X protein. Our data demonstrate that BDV replication competence in mice is mediated by the polymerase complex rather than the viral envelope and suggest that altered regulation of viral gene expression can favor adaptation to new host species.     

Borna disease virus accelerates inflammation and disease associated with transgenic expression of interleukin-12 in the central nervous system

Targeted expression of biologically active interleukin-12 (IL-12) in astrocytes of the central nervous system (CNS) results in spontaneous neuroimmunological disease of aged mice. Borna disease virus (BDV) can readily multiply in the mouse CNS but does not trigger disease in most strains. Here we show that a large percentage of IL-12 transgenic mice developed severe ataxia within 5 to 10 weeks after infection with BDV. By contrast, no disease developed in mock-infected IL-12 transgenic and wild-type mice until 4 months of age. Neurological symptoms were rare in infected wild-type animals, and if they occurred, these were milder and appeared later. Histological analyses showed that the cerebellum of infected IL-12 transgenic mice, which is the brain region with strongest transgene expression, contained large numbers of CD4(+) and CD8(+) T cells as well as lower numbers of B cells, whereas other parts of the CNS showed only mild infiltration by lymphocytes. The cerebellum of diseased mice further showed severe astrogliosis, calcifications and signs of neurodegeneration. BDV antigen and nucleic acids were present in lower amounts in the inflamed cerebellum of infected transgenic mice than in the noninflamed cerebellum of infected wild-type littermates, suggesting that IL-12 or IL-12-induced cytokines exhibited antiviral activity. We propose that BDV infection accelerates the frequency by which immune cells such as lymphocytes and NK cells enter the CNS and then respond to IL-12 present in the local milieu causing disease. Our results illustrate that infection of the CNS with a virus that is benign in certain hosts can be harmful in such normally disease-resistant hosts if the tissue is unfavorably preconditioned by proinflammatory cytokines.     

Antigenic presentation of heterologous epitopes engineered into the outer surface-exposed helix 4 loop region of human papillomavirus L1 capsomeres

The molecular characterization of isolates of citrus tristeza virus (CTV) from eight locations in Mexico was undertaken by analyzing five regions located at the opposite ends of the virus genome. Two regions have been previously used to study CTV variability (coat protein and p23), while the other three correspond to other genomic segments (p349-B, p349-C and p13). Our comparative nucleotide analyses included CTV sequences from different geographical origins already deposited in the GenBank databases. The largest nucleotide differences were located in two fragments located at the 5' end of the genome (p349-B and p349-C). Phylogenetic analyses on those five regions showed that the degree of nucleotide divergence among strains tended to correlate with their pathogenicity. Two main groups were defined: mild, with almost no noticeable effects on the indicator plants and severe, with drastic symptoms. Mild isolates clustered together in every analyzed ORF sharing a genetic distance below 0.022, in contrast with the severe isolates, which showed a more disperse distribution and a genetic distance of 0.276. Analyses of the p349-B and p349-C regions evidenced two lineages within the severe group: severe common subgroup (most of severe isolates) and severe divergent subgroup (T36-like isolates). This study represents the first attempt to analyze the genetic variability of CTV in Mexico by constructing phylogenetic trees based on new genomic regions that use group-specific nucleotide and amino acid sequences. These results may be useful to implement specific assays for strain discrimination. Moreover, it would be an excellent reference for the CTV situation in México to face the recent arrival of brown citrus aphid.     

Mechanism of the antiviral action of 1-beta-D-arabinofuranosylcytosine on Borna disease virus

Borna disease virus (BDV) is a nonsegmented, negative-stranded RNA virus that causes neurological diseases in a variety of warm-blooded animal species. Recently, we showed that the nucleoside analog 1-beta-D-arabinofuranosylcytosine (Ara-C) was a potent inhibitor of BDV. This finding was surprising for an RNA virus, since Ara-C is a DNA polymerase inhibitor. Thus, we sought to better define the mechanism of action of Ara-C on BDV. Here, we show that (i) this effect is specific for an arabinoside ring carrying a cytosine base, (ii) it requires phosphorylation of the nucleotide, and (iii) it can be reversed by an excess of cytidine. Using the recently described minigenome assay for BDV, we provide evidence suggesting that Ara-C may act as a competitive inhibitor of the BDV replication complex.     

Computational reconstruction of Bole1a, a representative synthetic hepatitis C virus subtype 1a genome

Hepatitis C virus (HCV) research is hampered by the use of arbitrary representative isolates in cell culture and immunology. The most replicative isolate in vitro is a subtype 2a virus (JFH-1); however, genotype 1 is more prevalent worldwide and represents about 70% of infections in the United States, and genotypes differ from one another by 31% to 33% at the nucleotide level. For phylogenetic and immunologic analyses, viruses H77 and HCV-1 (both subtype 1a) are commonly used based on their historic importance. In an effort to rationally design a representative subtype 1a virus (Bole1a), we used Bayesian phylogenetics, ancestral sequence reconstruction, and covariance analysis on a curated set of 390 full-length human HCV 1a sequences from GenBank. By design, Bole1a contains variations present in widely circulating strains and matches more epitope-sized peptides in a full-genome comparison to subtype 1a isolates than any other sequence studied. Parallel analyses confirm that selected epitopes from the Bole1a genome were able to elicit a robust T cell response. In a proof of concept for infectivity, the envelope genes (E1 and E2) of Bole1a were expressed in an HIV pseudoparticle system containing HCV envelope genes and HIV nonenvelope genes with luciferase expression. The resulting Bole1a pseudoparticle robustly infected Hep3B cells. In this study, we demonstrate that a rationally designed, fully synthetic HCV genome contains representative epitopes and envelope genes that assemble properly and mediate entry into target cells.     

肾综合征出血热病毒Gou3株M片段的分子特性及序列的比较分析

为测定我国肾综合征出血热病毒Ｇｏｕ３株的Ｍ片段全基因序列,了解该株分子基础,并分析其瑟其他病毒株的差异,将提取的感染了病毒的Ｖｅｒｏ－Ｅ６细胞总ＲＮＡ进行逆转录ＰＣＲ扩增,产物直接或纯化后克隆Ｔ载体并测定序列,结果测得Ｇｏｕ３株Ｍ片段的全基因序列共３６５１个核苷 ,最大读码框架从４７到３４４８,共编码１１３４个氨基酸。     

两种博尔纳病病毒株持续感染PC-12细胞模型的建立

目的构建两种博尔纳病病毒株持续感染的PC-12细胞模型,为研究博尔纳病病毒感染致病机制及比较两种病毒株致病特点的差别提供工具。方法将BDV Strain V株和Hu株分别感染PC-12细胞系,并进行传代培养,最后通过Real time FQ RT-PCR、Western blot、间接免疫荧光方法进行病毒核酸和蛋白的检测。结果在培养传代6代后,两种病毒株感染的PC-12细胞均可检测到BDV核酸及蛋白。结论 BDV Strain V株和Hu株均可在PC-12细胞中复制和表达,两种博尔纳病病毒株持续感染PC-12细胞模型构建成功。