Determination of the coding capacity of the M genome segment of nephropathia epidemica virus strain H?lln?s B1 by molecular cloning and nucleotide sequence analysis

The M genome RNA segment of nephropathia epidemica virus (NEV) strain H?lln?s B1 was characterized by molecular cloning and DNA nucleotide sequencing of the corresponding cDNA clones. The size of the M RNA segment is 3682 nucleotides. The 3' and 5' terminal sequences are complementary for 21 bases and their predicted secondary structure is very stable. The viral complementary messenger RNA possesses a single long open reading frame with a coding capacity of 1148 amino acids (polypeptide of 126 kDa). A comparison of the NEV M segment to that of Hantaan virus strain 76-118 reveals 61% sequence homology at the nucleotide level and 53% at the deduced amino acid level. Four out of five potential asparagine-linked glycosylation sites of the encoded glycoproteins have been conserved between NEV and Hantaan M. The isoelectric points (IEP) are nearly identical. Furthermore it was found that 90% of all cysteine residues have been conserved. Putative NEV G1 and G2 are preceded by a short hydrophobic sequence as shown for G1 and G2 of Hantaan virus. Hydrophilicity profiles of the two segments are of striking similarity. These data indicate that NEV- and Hantaan virus M-encoded polypeptides seem to be very similar in structure and function despite the relatively low amino acid sequence homology.     

Coding properties of the S and the M genome segments of Sapporo rat virus: comparison to other causative agents of hemorrhagic fever with renal syndrome

Three serologically distinct groups of hantaviruses have been associated with severe, moderate, and mild forms of hemorrhagic fever with renal syndrome (HFRS). To gain a better understanding of the genetic variation among these viruses, we cloned and sequenced the M and the S genome segments of Sapporo rat virus, an etiologic agent of moderate HFRS, and compared the predicted gene products to those of Hantaan virus, and the H?lln?s strain of Puumala virus, which are etiologic agents of severe and mild HFRS, respectively. The SR-11 S segment consisted of 1769 nucleotides and had an open reading frame (ORF) in the virus-complementary sense RNA with a coding capacity of 429 amino acids. Deduced amino acids from the SR-11 S segment ORF displayed 83% homology with those of Hantaan nucleocapsid (N) protein. Comparison of the S segment ORFs of all three viruses revealed 58% homology. No evidence for additional nonstructural protein(s) encoded by the SR-11 S segment was obtained. The SR-11 M segment consisted of 3651 nucleotides and had an ORF in the virus-complementary sense RNA with a coding capacity of 1134 amino acids. Amino acid sequences predicted from the SR-11 M segment ORF were 75% homologous with those encoding Hantaan G1 and G2 envelope glycoproteins. Comparison of the deduced amino acid sequences of the M segment ORFs of SR-11, Hantaan, and H?lln?s viruses revealed a 43% homology for amino acids constituting the G1 proteins and a 55% homology for amino acids constituting the G2 proteins of the three viruses. The envelope proteins of SR-11 virus were localized within the M segment ORF by amino-terminal sequence analysis of purified G1 and G2. G1 initiated at amino acid 17 and G2 at amino acid 647 within the ORF. Five potential asparagine-linked glycosylation sites were identified in the SR-11 G1 coding sequences, four of which were conserved between Hantaan and SR-11 viruses and three of which were conserved among all three viruses. One potential glycosylation site was identified in the SR-11 G2 coding sequences and was conserved among Hantaan, SR-11 and H?lln?s viruses. Cysteine residues were highly conserved within the M segment ORFs of all three viruses, suggesting a similar structure and function of the G1 and G2 proteins.     

汉滩病毒囊膜糖蛋白G2与核蛋白氨基端在昆虫细胞中的融合表达

目的：在Bac-to-Bac杆状病毒表达系统中，融合表达汉滩病毒的囊膜糖蛋白G2与核蛋白氨基端。方法：构建含有汉滩病毒G2S0．7嵌合基因的表达载体pFBDHTa—G2S0．7，并转化DH10Bac感受态菌。利用其含有的细菌Tn7转座系统，将嵌台基因重组至杆状病毒穿梭质粒hacmid中，并筛选含有G2S0．7嵌合基因的重组杆状病毒，在昆虫细胞中表达该融合蛋白。对表达产物用间接免疫荧光、ELISA和免疫印迹进行检测。结果：构建了的含G2S0．7嵌合基因的重组杆状病毒，感染昆虫细胞后，可表达相应大小的融合蛋白。该蛋白可被抗汉滩病毒核蛋白及糖蛋白G2的特异性单克隆抗体(mAb)所识别。结论：利用杆状病毒表达系统，成功地表达同时具有核蛋白及糖蛋白G2生物学活性的融合蛋白G2S0．7，为进一步研究其免疫学特性奠定了基础。     

汉滩病毒M片段噬菌体表位文库的构建及筛选

目的 通过构建汉滩病毒M片段的噬菌体展示文库，筛选汉滩病毒包膜蛋白的抗原表位。方法 DNaseⅠ随机消化的M片段与载体pComb3连接，转化宿主XL-1Blue，在辅助噬菌体VCSM13存在的条件下，获得M片段的随机噬菌体文库.利用纯化的恢复期病人血清对M片段噬菌体文库进行3轮淘洗，通过ELISA、序列分析对所获得的克隆进行鉴定。并对其中2个阳性克隆的目的片段进行了原核表达和免疫原性分析。结果 筛选获得的5个阳性噬菌体克隆均位于G1蛋白编码区，原核表达的2个阳性：充隆片段蛋白免疫兔，免疫兔血清能与病毒抗原发生特异反应。结论 本技术路线可用于汉滩病毒包膜蛋白抗原表位的研究，为汉滩病毒诊断试剂的研制、亚单位疫苗的设计提供了数据。     

The Hantaan virus glycoprotein precursor is cleaved at the conserved pentapeptide WAASA.

The medium segment of the tripartite negative-stranded RNA genome of hantaviruses encodes for the predicted glycoprotein precursor GPC. We have demonstrated here the expression of the glycoprotein precursor of Hantaan virus following transfection of mammalian cells. The cleavage of the precursor into the glycoproteins G1 and G2 followed the rules for signal peptides and seemed to occur directly at the pentapeptide motif "WAASA." Our data indicate that the signal peptidase complex is responsible for the proteolytic processing of the precursor GPC of Hantaan virus. The comparison of this region of the glycoprotein precursor, including the absolutely conserved WAASA motif, suggests a similar cleavage event for all hantavirus glycoproteins.     

Antibody-dependent enhancement of hantavirus infection in macrophage cell lines

Summary Antibody-dependent enhancement (ADE) of hantavirus infections (strains Hantaan 76–118 and SR-11) was studied using macrophage-like cell lines (J774.1, P388D1, and U937). Significantly higher virus titers (1,000 to 4,000 FFU/ml) were obtained by pretreatment of the virus with immune serum as compared to normal serum (<20 FFU/ml). Monoclonal antibodies (MAbs) to strain Hantaan 76–118 were employed to determine the antigenic determinants responsible for the ADE activity. ADE of the infection occurred with MAbs to both G1 and G2 envelope glycoproteins, but not with MAbs to nucleocapsid protein. Antigenic determinants related to haemagglutination or virus neutralization were found to cause ADE of the infection.     

汉滩病毒M、S基因部分片段嵌合基因真核载体的构建与基因免疫

目的 在本室前期工作的基础上,进一步进行汉滩病毒M基因G2片段与S基因0．7Kb片段嵌合基因基因免疫的研究。方法 构建汉滩病毒76－118株M基因G2片段与S基因5‘端700bp片段的嵌合基因真核表达载体pcDNA3.1-G2S0.7。用该质粒免疫Balb/c小鼠,并用ELISA法及淋巴细胞增殖实验,检测基因免疫后的免疫应答效果。结果 限制性内切酶鉴定结果表明真核表达载体的构建正确。用pcDNA3.1-G2S0.7直接免疫小鼠。可诱导产生抗汉滩病毒核蛋白（NP）及糖蛋白（GP）特异性的抗体,抗体效价分别为1：200及1：80。淋巴细胞增殖实验表明,嵌合基因免疫小鼠脾细胞对NP及GP的增殖指数,均明显高于对照组。结论汉滩病毒M基因G2片段及S基因0．7kb片段的嵌合基因,既可刺激机体产生特异性的抗汉滩病毒体液免疫应答,也可刺激机体产生特异性的细胞免疫应答,本研究为进一步进行汉滩病毒基因疫苗的研究奠定了实验基础。     

Immunoblot analysis of the serological response in Hantavirus infections

Sera from patients with nephropathia epidemica (NE) or Korean hemorrhagic fever (KHF) were tested for specific antibody response to antigens of H?lln?s virus and Hantaan virus strain 76-118. A Vero E6 derived cell line persistently infected with H?lln?s virus strain B1, and Vero E6 cells freshly infected with Hantaan virus type strain 76-118 were used as antigens in the immunofluorescence assay (IFA) and the immunoblot. Blots were prepared from whole cell lysates. The convalescent-phase sera of NE patients tested in this study regularly revealed a marked reaction with a 52 kilodalton (Kd) protein of H?lln?s virus and a 50 Kd protein of Hantaan virus. A convalescent serum from a patient with Korean hemorrhagic fever and a rat antiserum against Hantaan virus could recognize the 50 Kd band of Hantaan virus but showed no apparent reactivity with the 52 Kd component of H?lln?s virus in the standard dilutions. Some sera could additionally identify minor bands in the 55 Kd and/or 67 Kd region of the blots. A one-way cross reactivity between Hantaan and H?lln?s viruses was also evident from the results of the immunofluorescence assays in that NE convalescent sera reacted with both viruses, whereas KHF convalescent or anti-Hantaan sera gave strongly positive results with Hantaan virus but only faint reaction with H?lln?s virus.     

Hantaan virus M RNA: coding strategy, nucleotide sequence, and gene order

The M genome segment of Hantaan virus was molecularly cloned and the nucleotide sequence of cDNA was determined. The virion RNA is 3616 bases long with 3'- and 5'-terminal nucleotide sequences complementary for 18 bases. A single long open reading frame in the viral complementary-sense RNA had the potential to encode 1135 amino acids or a polypeptide of 126,000 Da. Amino-terminal sequences of isolated G1 and G2 envelope glycoproteins were determined, revealing a gene order with respect to message sense RNA of 5'-G1-G2-3'. Mature G1 begins 18 amino acids beyond the first AUG of the open reading frame, preceded by a short, hydrophobic leader sequence. G2 begins at the 649th amino acid of the open reading frame and also follows a hydrophobic sequence. Carboxy termini of G1 and G2 were localized and gene order was verified by immune precipitation of Hantaan proteins with antisera to synthetic peptides generated by using amino acid sequences derived from the cDNA sequence. The antipeptide sera were also reactive by immunoblotting with SDS-denatured G1 and G2. Molecular weights of 64,000 and 53,700 were calculated for the G1 and G2 glycoproteins, respectively, from their predicted amino acid sequences. Five potential asparagine-linked glycosylation sites were contained within the G1 amino acid sequence and two within the G2 sequence. These data are consistent with our previous estimates of the molecular weights and extent of glycosylation of the Hantaan envelope glycoproteins.     

汉坦病毒A9株M基因片段全长cDNA克隆及其在痘苗病毒中的瞬时表达

采取反转录一聚合酶链反应（ＰＣＲ），分３个片段扩增Ｉ型汉坦病毒（野鼠型）中国毒株Ａ９株Ｍ基因片段，分别克隆入ＰＧＥＭ－Ｔ载体中。选择３个正向插入的ＴＡ９ＩＢ、ＴＡｇＣＤ、ＴＡｇＥＡ克隆，选用ＣｌａＩ、ＥｃｏＲＶ进行酶切、连接，获得了１个在ＰＧＥＭ－Ｔ载体中插入３．６ｋｂ的Ａｇ株Ｍ基因片段ｃＤＮＡ克隆，酶切图谱分析证实播入片段正确。将Ａ９Ｍ片段在痘苗病毒／Ｔ７噬菌体ＲＮＡ聚合酶瞬时（Ｔｒａｎｓｉｅｎｔ）表达系统中进行表达，用抗汉坦病毒糖蛋白单克隆抗体进行免疫荧光检测，观察到很强的特异性荧光，证明ＡｇＭ基因ｃＤＮＡ能进行表达。     

Protective immunity of Hantaan virus nucleocapsid and envelope protein studied using baculovirus-expressed proteins

Summary Recombinant Hantaan virus nucleocapsid protein (rNP) and recombinant envelope (rEnv) proteins were prepared using a baculovirus expression system to examine the role of Hantaan virus structural proteins in protective immunity. Passive transfer of spleen cells from mice immunized with rNP conferred partial protection or prolongation of time to death from fatal Hantaan virus infection in suckling mice which were challenged with Hantaan virus at 40 LD₅₀ (survival rate: 43%) or 4 LD₅₀ (survival rate: 43%). The T cell-enriched fraction protected one mouse from lethal infection but the B cell-enriched fraction had no such effect on fatal HTN infection. The protective effects of the antibody against HTN challenge were examined by passive immunization. The monoclonal antibody ECO 2 directed to NP also conferred partial survival and significant difference in time to death. Although rEnv antigen failed to induce neutralizing antibody, both immune spleen cells and immune serum to rEnv conferred partial protection upon suckling mice. These results indicate that both nucleocapsid and envelope proteins of Hantaan virus were responsible for induction of cell mediated protective immunity. Vero E 6 cells infected with Hantaan virus expressed envelope protein on the surface, as determined by flow cytometry. However, there was only negligible expression of nucleocapsid protein.     

Maturation of Hantaan virus glycoproteins G1 and G2.

Hantaan virus-infected Vero E6 cell lysates were used for immunoprecipitation with monoclonal antibodies against glycoprotein G1 (MAbG1) or G2 (MAbG2). When cell lysates were prepared with buffer containing nonionic detergent, both G1 and G2 glycoproteins were precipitated with either MAbG1 or MAbG2. In contrast, when cell lysates were prepared with a buffer containing ionic detergents MAbG1 precipitated only glycoprotein G1 and MAbG2 precipitated only glycoprotein G2. Heterodimers and possibly higher oligomeric forms of the glycoproteins were detected on nonreducing SDS-polyacrylamide gels only after chemical cross-linking and immunoprecipitation with either MAbG1 or MAbG2. In order to determine the sites of Hantaan virus glycoproteins maturation and the G1-G2 complex formation, infected cells were treated with inhibitors that prevent specific steps of oligosaccharide processing. Furthermore, glycoproteins G1 and G2 immunoprecipitated from infected cell lysates or from isolated virus particles were tested for sensitivity to endoglycosidase H, endoglycosidase F, and endoglycosidase D. The results of these experiments show that maturation of both G1 and G2 takes place in the endoplasmic reticulum (ER). Furthermore, G1-G2 complex formation occurs in the ER as well, since the two glycoproteins co-precipitated with either MAbG1 or MAbG2 from infected cell lysates treated with brefeldin A and prepared with buffer containing nonionic detergent.     

Inhibiting effects of fucoidans on Hantaan virus adsorption on a model of peritoneal macrophages in vitro

A model of peritoneal macrophages was used to study the effect of fucoidans from brown seaweeds on the adsorption of Hantaan virus. Fucoidans were found to have antiviral activity, but to differ in the inhibition of hantavirus adsorption, which was associated with their structural features. The mechanism of their action is assumed to be shown via ligand-receptor interaction with certain cell membrane receptors and via blockage of hantavirus glycoproteins (G1 and G2), resulting in adsorption inhibition and preventing viral penetration into the macrophages.     

Antiviral neutralizing antibody to Hantaan virus as determined by plaque reduction technique

Summary The 76–118 strain of Hantaan virus was titrated in E6 (Vero) cells by the plaque method using agarose overlay medium. Visible plaques, formed 10 days post-infection, were uniformly 2–3 mm in diameter. Dose-response experiments showed that a single infectious particle initiated the formation of a plaque. Infectivity titers by the plaque method were equivalent to those obtained by the endpoint method (TCID₅₀) using the immunofluorescence antibody technique (IFA) for antigen detection. The single-cycle growth pattern of the virus showed an eclipse phase of 7 to 9 hours, with production of cell-free infectious virus 18 hours post-infection. Plaque reduction neutralization tests suggested that complement enhanced the neutralizing activity of sera; rat sera were particularly complement-dependent. The plaque reduction neutralization test was about 10 times more sensitive than the TCID₅₀ neutralization test. Convalescent phase sera from patients with hemorrhagic fever with renal syndrome (HFRS) having higher IF antibody titers to Hantaan virus than to nephropathia epidemica (NE) virus were capable of neutralizing Hantaan virus, while sera from patients with higher IF antibody titers to NE virus than Hantaan virus did not contain neutralizing antibody to Hantaan virus.With 3 FiguresOn leave from the Department of Virology, Kyushu University School of Medicine, Fukuoka 812, Japan.     

Identification of Hantaan virus-related structures in kidneys of cadavers with haemorrhagic fever with renal syndrome

Summary The etiologic agent of haemorrhagic fever with renal syndrome (HFRS), Hantaan virus, was first isolated in 1976. Since then numerous Hantaan-like viruses have been isolated and five serotypes of Hantavirus have been recognized. Serological studies indicate that these viruses are globally distributed, with each serotype occurring in specific areas. Hantaan virus has been intensively studied antigenically, biochemically, and genetically. However there is still a paucity of information on the pathogenesis of Hantaan virus in the human host.In this paper, we report the detection by thin section immune electron microscopy of the occurrence of numerous dense precipitates, typical inclusion bodies, a surface antigen layer, as well as Hantaan virion-like structures in the kidneys of patients that died during the acute phase of HFRS. These findings may shed some light on understanding the pathogenesis of HFRS in target organs most affected by the disease, such as the kidneys.     

Seroepidemiology of Hantaan virus infection in Taiwan

In order to investigate the infection rate of Hantaan virus in Taiwan, a total of 6,536 human serum samples were collected from residents, selected by stratified random sampling, from 19 townships covering four different ethnic groups: Aborigines, Fukien Taiwanese, Hakka Taiwanese, and Mainland Chinese. Serum samples were screened for Hantaan virus antibodies by indirect immunofluorescence. The prototype Hantaan virus (76/118)-infected Vero E6 cells were used as the viral antigen for the antibody detection. Among 6,536 human serum samples, 403 (6.2%) samples had Hantaan virus antibodies. The seropositive rates for males and females were 6.1% and 6.2%, respectively. A higher seropositive rate was found among Aborigines on the Orchid Islets (11.5%) and Fukien Taiwanese on the Penghu Islets (11.6%), while the lowest rate was observed among Hakka Taiwanese in the south of Taiwan (2.5%). In comparing with different ethnic groups, the highest prevalence was found among Fukien Taiwanese (8.1%) and the lowest among Mainland Chinese (4.9%). Among the different geographical areas, the highest positive rate was found in western Taiwan (7.1%) and the lowest in southern Taiwan (5.4%). Hantaan virus antibodies were also detected in 22 of 548 (4.0%) rat serum samples. The highest seropositive rate was found in rat sera collected from the Orchid Islets (21.4%). None of the rat sera collected from Hsinchu, Miaoli, Changhua, Nantu, Yunlin, Chi-ayi, Tainan, and Penghu Counties were positive. Hantaan virus antibodies were found in rats: Rattus rattus (20%), Bandicota indica (9.0%), Rattus norvegicus (8.3%), Bandicota nemorivaga (6.3%), Rattus losea (4.2%), and Apodemus agrarius (1.6%). Hantaan virus antibodies were not detected in rat sera collected from species of Rattus coxinga, Rattus culturatus, Mus musculus, Mus caroli, Suncus murinus, and Apodemus semotus. The results show that the Hantaan or Hantaan-related virus exists and is distributed widely in both human and rats in Taiwan. © 1996 Wiley-Liss, Inc.     

汉滩病毒G1S0.7嵌合基因重组腺病毒的构建及鉴定

目的在本室前期工作的基础上构建汉滩病毒M基因G1片段与S基因0.7kb片段嵌合基因的重组腺病毒。方法构建含有汉滩病毒G1S0.7嵌合基因的转移载体pShuttle-G1S0.7,然后通过特异性的酶切将嵌合基因与腺病毒DNA相连,电转化E.coli JM109,获得重组腺病毒Adeno-G1S0.7 DNA,转染HFX293细胞得到重组腺病毒。进一步对重组腺病毒的滴度和表达产物进行鉴定。结果构建的含G1S0.7嵌合基因的重组腺病毒,滴度可达10^13～10^15 PFU／L;该重组腺病毒感染Vero-E6细胞后,表达出可被抗汉滩病毒核蛋白及糖蛋白G1的特异性单抗（mAb）所识别的融合蛋白。结论利用腺病毒表达系统,成功地表达同时具有核蛋白及糖蛋白G1生物学活性的融合蛋白,为进一步研究其免疫学特性奠定了基础。