Amino Acid Sequence Of Glycoprotein Research Articles

Molecular characterization of the 2022 Sudan virus disease outbreak in Uganda.

Ebola disease (EBOD) is a public health threat with a high case fatality rate. Most EBOD outbreaks have occurred in remote locations, but the 2013-2016 Western Africa outbreak demonstrated how devastating EBOD can be when it reaches an urban population. Here, the 2022 Sudan virus disease (SVD) outbreak in Mubende District, Uganda, is summarized, and the genetic relatedness of the new variant is evaluated. The Mubende variant exhibited 96% amino acid similarity with historic SUDV sequences from the 1970s and a high degree of conservation throughout the outbreak, which was important for ongoing diagnostics and highly promising for future therapy development. Genetic differences between viruses identified during the Mubende SVD outbreak were linked with epidemiological data to better interpret viral spread and contact tracing chains. This methodology should be used to better integrate discrete epidemiological and sequence data for future viral outbreaks.

Expanding Mouse-Adapted Yamagata-like Influenza B Viruses in Eggs Enhances In Vivo Lethality in BALB/c Mice.

Despite the yearly global impact of influenza B viruses (IBVs), limited host range has been a hurdle to developing a readily accessible small animal disease model for vaccine studies. Mouse-adapting IBV can produce highly pathogenic viruses through serial lung passaging in mice. Previous studies have highlighted amino acid changes throughout the viral genome correlating with increased pathogenicity, but no consensus mutations have been determined. We aimed to show that growth system can play a role in mouse-adapted IBV lethality. Two Yamagata-lineage IBVs were serially passaged 10 times in mouse lungs before expansion in embryonated eggs or Madin–Darby canine kidney cells (London line) for use in challenge studies. We observed that virus grown in embryonated eggs was significantly more lethal in mice than the same virus grown in cell culture. Ten additional serial lung passages of one strain again showed virus grown in eggs was more lethal than virus grown in cells. Additionally, no mutations in the surface glycoprotein amino acid sequences correlated to differences in lethality. Our results suggest growth system can influence lethality of mouse-adapted IBVs after serial lung passaging. Further research can highlight improved mechanisms for developing animal disease models for IBV vaccine research.

Infectious keratoconjunctivitis in free-ranging mule deer in Wyoming: a retrospective study and identification of a novel alphaherpesvirus.

We describe the clinicopathologic findings, relative prevalence, and pathogens associated with infectious keratoconjunctivitis in mule deer ( Odocoileus hemionus) in Wyoming. Seventeen cases with ocular lesions were identified among 1,036 mule deer postmortem submissions (1.6%) in an ~16 y period. Sixteen cases were observed in winter and most were in male (15 cases) and juvenile (13 cases) deer. Blindness was the most commonly reported clinical sign (10 cases). A herpesvirus was detected only in the 4 cases of bilateral necrotizing bulbar conjunctivitis. Phylogenetic analysis of glycoprotein amino acid sequences consistently identified this virus as a novel alphaherpesvirus. In 2 of these herpesvirus-positive cases, Actinomyces sp. and Moraxella ovis were also identified. Trueperella pyogenes was identified in 4 cases of unilateral ulcerative keratitis, keratoconjunctivitis, and panophthalmitis. M. ovis was cultured from 3 cases of bilateral conjunctivitis and keratoconjunctivitis. In the remaining cases, isolates included Moraxella bovis (1 case), Staphylococcus sp. and Streptococcus sp. (2), Flavobacterium sp. and Pseudomonas sp. (2), Escherichia coli and Enterobacter sp. (1), and bovine viral diarrhea virus 1 (1). No pathogens were identified in 2 cases. The relative prevalence of keratoconjunctivitis in mule deer in Wyoming appears to be low, and this disease is most commonly associated with infection by a novel alphaherpesvirus, T. pyogenes, and M. ovis.

Isolation and genotype analysis of spring viremia of carp virus strains from Heilongjiang Province in China

PDF HTML阅读 XML下载 导出引用 引用提醒 黑龙江地区鲤春病毒血症病毒的分离与基因型分析 DOI: 作者: 作者单位: 1. 中国水产科学研究院 黑龙江水产研究所, 黑龙江 哈尔滨 150070;2. 上海海洋大学 水产与生命学院, 上海 201306 作者简介: 纪锋(1990-),男,硕士研究生,从事鱼类病毒学研究.E-mail:fengjii@foxmail.com 通讯作者: 中图分类号: S941 基金项目: 黑龙江省应用技术研究与开发计划项目（GA13B401）. Isolation and genotype analysis of spring viremia of carp virus strains from Heilongjiang Province in China Author: Affiliation: 1. Heilongjiang Fishery Research Institute, Chinese Academy of Fishery Sciences, Harbin 150070, China;2. College of Fishery and Life Science, Shanghai Ocean University, Shanghai 201306, China Fund Project: 摘要 | 图/表 | 访问统计 | 参考文献 | 相似文献 | 引证文献 | 资源附件 | 文章评论 摘要:对2015-2016年黑龙江不同地区的40个养殖场送检的鲤（）进行鲤春病毒血症病毒（springviremia of carp virus，SVCV）的细胞培养分离、PCR鉴定、病毒滴度测定、病毒表面糖蛋白（glycoprotein，G）氨基酸序列聚类分析及基因分型研究。细胞培养结果显示，来自4个不同养殖场的鲤组织样本能够感染鲤上皮细胞（epitheliomapapulosum cyprini，EPC）产生典型细胞病变（cytopathic effect，CPE），收集病毒悬液分别称为Shlj1~Shlj4。PCR鉴定结果表明，该4株病毒均为SVCV。病毒滴度实验测算出SVCV Shlj 1~Shlj 4的滴度分别为106.28、106.88、107.57和106.38 TCID50/mL。Shlj的糖蛋白基因核苷酸序列的聚类分析和遗传进化分析结果显示，Shlj 1~Shlj 4与GenBank收录的中国参考株A2、BJ0505-2和美国参考株USA、212364聚为一簇，同源性为98.4%~99.8%；Shlj 1~Shlj 4毒株之间的糖蛋白核苷酸序列相似性在98.6%~99.8%，其中Shlj 3与美国SCVC毒株USA、212364具有最高的核苷酸相似性（99.8%），Shlj 2与英国参考毒株880163具有最低的相似性（88.0%）。糖蛋白氨基酸序列比对结果显示，Shlj4中氨基酸突变最多，与另3个毒株差异较大。基因型分析结果显示，Shlj 1~Shlj 4均为基因Ia型。本研究结果表明，黑龙江地区2015-2016年间的SVCV检出率约为10%，并且来源于不同养殖场的病毒分离株的核酸序列呈现不同程度的差异，该结果进一步证明SVCV毒株在中国不同的鲤养殖环境中正在不断地进化。 Abstract:Spring viremia of carp virus (SVCV) was routinely isolated and identified from tissues of carp () from 40 carp farms in Heilongjiang Province by separation in cell culture and identification using polymerase chain reaction. SVCV isolates were subjected to virus titer determination, phylogenetic reconstruction based on their glycoprotein amino acid sequences and genotyping. Cell culture results showed that the isolates from four different farms infected carp epithelial cells and caused a cytopathic effect. The isolated SVCV strains were designated as Shlj1-Shlj4 and were confirmed as SVCVs by polymerase chain reaction. The titers of SVCV Shlj1-Shlj 4 were 106.28, 106.88, 107.57, and 106.38×the 50% tissue culture infectious dose/mL, respectively. The results of the Shlj glycoprotein gene cluster analysis showed that Shlj1-Shlj4 clustered with China strains A2 and BJ0505-2 as well as America strains USA and 212364. The nucleotide sequence similarities of Shlj 1-Shlj 4 with those previously reported SVCV strains were 98.4%-99.8%, and the similarities among Shlj 1-Shlj 4 were 98.6%-99.8%. The highest nucleotide sequence similarity (99.8%) was observed between Shlj3 and USA/212364, and Shlj2 had the lowest similarity (88.0%) with England reference strain 880163. The amino acid sequence alignment results showed that, among the four Shlj strains, the glycoproteins of Shlj 4 contained the most mutations. Genotype analysis showed that Shlj 1-Shlj 4 all belonged to genotype Ia. The study showed that the SVCV detection rate was about 10% in Heilongjiang Province during 2015-2016, and the glycoprotein gene sequences of SVCV strains from different farms varied, indicating that SVCV strains are evolving in the Chinese fish farm environment. 参考文献 相似文献 引证文献

Molecular Characterization of a Novel Bovine Viral Diarrhea Virus Isolate SD-15.

As one of the major pathogens, bovine viral diarrhea virus caused a significant economic loss to the livestock industry worldwide. Although BVDV infections have increasingly been reported in China in recent years, the molecular aspects of those BVDV strains were barely characterized. In this study, we reported the identification and characterization of a novel BVDV isolate designated as SD-15 from cattle, which is associated with an outbreak characterized by severe hemorrhagic and mucous diarrhea with high morbidity and mortality in Shandong, China. SD-15 was revealed to be a noncytopathic BVDV, and has a complete genomic sequence of 12,285 nucleotides that contains a large open reading frame encoding 3900 amino acids. Alignment analysis showed that SD-15 has 93.8% nucleotide sequence identity with BVDV ZM-95 isolate, a previous BVDV strain isolated from pigs manifesting clinical signs and lesions resembling to classical swine fever. Phylogenetic analysis clustered SD-15 to a BVDV-1m subgenotype. Analysis of the deduced amino acid sequence of glycoproteins revealed that E2 has several highly conserved and variable regions within BVDV-1 genotypes. An additional N-glycosylation site (240NTT) was revealed exclusively in SD-15-encoded E2 in addition to four potential glycosylation sites (Asn-X-Ser/Thr) shared by all BVDV-1 genotypes. Furthermore, unique amino acid and linear epitope mutations were revealed in SD-15-encoded Erns glycoprotein compared with known BVDV-1 genotype. In conclusion, we have isolated a noncytopathic BVDV-1m strain that is associated with a disease characterized by high morbidity and mortality, revealed the complete genome sequence of the first BVDV-1m virus originated from cattle, and found a unique glycosylation site in E2 and a linear epitope mutation in Erns encoded by SD-15 strain. Those results will broaden the current understanding of BVDV infection and lay a basis for future investigation on SD-15-related pathogenesis.

Differentially expressed genes after viral haemorrhagic septicaemia virus infection in olive flounder (Paralichthys olivaceus)

A strain of viral haemorrhagic septicaemia virus (VHSV) was isolated from cultured olive flounder (Paralichthys olivaceus) during epizootics in South Korean. This strain showed high mortality to olive flounder in in vivo challenge experiment. The complete genomic RNA sequences were determined and phylogenetic analysis of the amino acid sequences of glycoprotein revealed that this isolate was grouped into genotype IVa of genus Novirhabdovirus. Expression profile of genes in olive flounder was analyzed at day 1 and day3 after infection with this VHSV isolate by using cDNA microarray containing olive flounder 13K cDNA clones. Microarray analysis revealed 785 up-regulated genes and 641 down-regulated genes by at least two-fold in virus-infected fish compared to healthy control groups. Among 785 up-regulated genes, we identified seven immune response-associated genes, including the interferon (IFN)-induced 56-kDa protein (IFI56), suppressor of cytokine signaling 1 (SOCS1), interleukin 8 (IL-8), cluster of differentiation 83 (CD83), α-globin (HBA), VHSV-induced protein-6 (VHSV6), and cluster of differentiation antigen 9 (CD9). Our results confirm previous reports that even virulent strain of VHSV induces expression of genes involved in protective immunity against VHSV.

Creation of DNA vaccine vector based on codon-optimized gene of rabies virus glycoprotein (G protein) with consensus amino acid sequence

An optimized design of the rabies virus glycoprotein (G protein) for use within DNA vaccines has been suggested. The design represents a territorially adapted antigen constructed taking into account glycoprotein amino acid sequences of the rabies viruses registered in the Russian Federation and the vaccine Vnukovo-32 strain. Based on the created consensus amino acid sequence, the nucleotide codon-optimized sequence of this modified glycoprotein was obtained and cloned into the pVAX1 plasmid (a vector of the last generation used in the creation of DNA vaccines). A twofold increase in this gene expression compared to the expression of the Vnukovo-32 strain viral glycoprotein gene in a similar vector was registered in the transfected cell culture. It has been demonstrated that the accumulation of modified G protein exceeds the number of the control protein synthesized using the plasmid with the Vnukovo-32 strain viral glycoprotein gene by 20 times. Thus, the obtained modified rabies virus glycoprotein can be considered to be a promising DNA vaccine antigen.

양식 넙치, Paralichthys olivaceus에서 분리한 viral hemorrhagic septicemia virus (VHSV)의 항원성 분석

The amino acid sequence of glycoprotein of Korean VHSV isolate (KR`01-1) was analyzed using the DNAStar Protean system. Based on the flexibility, hydrophilicity, antigenic index and surface probability, three regions (Gp1, Gp2 and Gp3) were selected as potential antigenic determinants. Three oligopeptides containing the amino acid sequences of the three regions were synthesized and polyclonal antibodies were raised against them. The activities of the antibodies were analyzed by Western blotting and virus neutralization test. The results showed that antibodies raised against oligopeptides Gp1 and Gp2 neutralized the infectivity of VHSV, suggesting that they can be possible candidates for subunit vaccines against VHS diseases in olive flounder.

Stable Isotope Tagging of Epitopes

Identification of peptides presented in major histocompatibility complex (MHC) class I molecules after viral infection is of strategic importance for vaccine development. Until recently, mass spectrometric identification of virus-induced peptides was based on comparative analysis of peptide pools isolated from uninfected and virus-infected cells. Here we report on a powerful strategy aiming at the rapid, unambiguous identification of naturally processed MHC class I-associated peptides, which are induced by viral infection. The methodology, stable isotope tagging of epitopes (SITE), is based on metabolic labeling of endogenously synthesized proteins during infection. This is accomplished by culturing virus-infected cells with stable isotope-labeled amino acids that are expected to be anchor residues (i.e. residues of the peptide that have amino acid side chains that bind into pockets lining the peptide-binding groove of the MHC class I molecule) for the human leukocyte antigen allele of interest. Subsequently these cells are mixed with an equal number of non-infected cells, which are cultured in normal medium. Finally peptides are acid-eluted from immunoprecipitated MHC molecules and subjected to two-dimensional nanoscale LC-MS analysis. Virus-induced peptides are identified through computer-assisted detection of characteristic, binomially distributed ratios of labeled and unlabeled molecules. Using this approach we identified novel measles virus and respiratory syncytial virus epitopes as well as infection-induced self-peptides in several cell types, showing that SITE is a unique and versatile method for unequivocal identification of disease-related MHC class I epitopes.

Characterization of T-cell reactive epitopes in glycoprotein G of herpes simplex virus type 2 using synthetic peptides

We have previously shown that the CD4+ T-cell response to herpes simplex virus type 2 glycoprotein G-2 is type-specific and can thus be used to evaluate herpes simplex virus type 2-specific T-cell responses in individuals with a concomitant herpes simplex virus type 1 infection. In this study we have followed the glycoprotein G-2-specific T-cell responses over time, and also tried to identify T-cell epitopes in the membrane bound portion and the secreted portion of glycoprotein G-2 using synthetic peptides spanning the whole amino acid sequence of glycoprotein G-2. We found that the magnitude of the glycoprotein G-2-specific response varied considerably in infected individuals over time, even though all patients responded to at least one of the two glycoproteins at all time-points examined. We could also document strong T-cell responses to synthetic peptides from the secreted glycoprotein G-2 but only low responses to synthetic peptides corresponding to sequences from the heavily glycosylated membrane-bound glycoprotein G-2. We were able to map an immunogenic region (amino acid 31-125) within the secreted glycoprotein G-2. This region of the glycoprotein induced proliferative responses in 47% of the herpes simplex virus type 2-infected individuals. However, we were not able to identify any universal T-cell epitope.

Heptad-repeat sequences in the glycoprotein of rhabdoviruses.

Two or three regions containing three or more successive newly defined heptads of a–d hydrophobic amino acid repeats have been located in the cDNA-derived amino acid sequences of glycoprotein G of all rhabdoviruses examined (rabies, vesicular stomatitis, fish, and plant rhabdoviruses) by computer search. These new heptad-repeats differ from those previously reported in other viruses because of the presence of all the hydrophobic amino acids in positions a or d, and because they are not predicted to form coiled coils by current methods and thus they have not been detected previously in any rhabdoviruses. The two or three heptad-repeat regions were the only parts of the glycoprotein with at least three successive heptad-repeats in all the rhabdoviral sequences studied and had low sequence variability among the members of each of the rhabdoviral genus but show no sequence similarity among the different genus. All these newly detected heptad repeats were in the vicinity of some of the higher hydrophobic regions in each of the rhabdovirus genera studied and were found mostly, but not always, outside the extra amino acid sequences that occur in the longer insect or plant rhabdovirus glycoprotein G. The correspondence of position and structure of these heptad-repeats among all the rhabdoviruses suggests its participation in common function(s), most probably related to viral fusion with cellular membranes.

Phylogenetic Relationships of Dengue-2 Viruses

RNA oligonucleotide fingerprinting studies on a large number of virus isolates previously demonstrated considerable genetic variation in isolates of dengue (DEN-)-2 serotype. We report the entire envelope (E) glycoprotein gene and deduced amino acid sequences of 16 DEN-2 viruses and the phylogenetic relationships of these, plus 17 additional published DEN E gene sequences. Comparison of DEN-2 E glycoprotein gene sequences revealed base substitutions scattered throughout the entire gene with as much as 22% sequence divergence. Aligned E glycoprotein amino acid sequences revealed the viruses differed by as much as 10%. There appeared to be constraints on the overall structure of the E protein to maintain biological function. Clusters of amino acid substitutions were present in the hydrophobic membrane anchor region at the carboxyl terminal end of the protein. Maximum parsimony analysis of the E gene sequences allowed construction of a phylogram indicating evolutionary relationships of the virus isolates within the DEN-2 serotype. Five genetic subtypes were identified. Phylogenetic relationships of the DEN-2 serotype and other flaviviruses based on E protein sequences reflected traditional antigenic and serologic classifications.

Conformational energy calculations on glycosylated turns in glycoproteins.

AbstractAnalysis of the amino acid sequence of glycoproteins has suggested the β‐turn as a likely site of glycosylation in glycoproteins. According to this model, the peptide chain traverses the interior of a globular protein, reversing its direction at the protein surface, a likely point for the attachment of hydrophilic carbohydrate residues. In order to search for plausible conformations of glycosylated β‐turns in asparagine‐linked glycoproteins, we have adapted the conformational energy calculation method of Scheraga and coworkers for use in carbohydrates. The parameters for nonbonded and hydrogen‐bonded interactions have been published, and electrostatic parameters are derived from a CNDO calculation on a model glycopeptide. Our results indicate that the orientation of the glycosyl amide bond having the amide proton nearly trans to the anomeric proton of the sugar has the lowest energy. Although CD and nmr experiments in our laboratory have consistently found this conformation, our calculations show the conformation having these two protons in a cis relationship to lie very close in energy. Calculations on the glycopeptide linkage model, α‐N‐acetyl, δ‐N(2‐acetamido‐1,2‐dideoxy‐β‐D‐glucopyranosyl)‐N′‐methyl‐L‐asparaginyl amide show that several distinct geometries are allowed for glycosylated β‐turns. For a type I β‐turn, three conformations of the glycosylated side chain are found within 4 kcal of the minimum, while two conformations of the glycosylated side chain are allowed for a type II turn. The hydrogen‐bonded C7 conformation is also allowed. Stereoviews of the low‐energy conformations reveal no major hydrogen‐bonding interaction between the peptide and sugar.