Chemical crosslinking of proteins of the influenza virion

Summary Purified influenza virus (A/FPV/Rostock/34; H7N1) was reacted with one of three chemical crosslinking reagents [dimethylsuberimidate (DMS), tartryl diazide (TDA) and formaldehyde] under conditions designed to give a ladder of crosslinked polypeptides (putative homo- and heteropolymers) when analysed by SDS-polyacrylamide gel electrophoresis under reducing conditions. The different virion polypeptides were identified by Western blotting with monospecific antisera against HA1, HA2, NP, and M1. When reacted with any crosslinker NP preferentially formed 2mer and 4mer homopolymers while M1 formed 2mers, 4mers, 6mers, and 8mers. 2mers and 3mers of HA1 were detected after crosslinking with TDA and DMS but homopolymers of HA2 could not be identified with certainty due to comigrating M1. One heteropolymer was clearly identified as 1NP: 1M1 (with DMS and TDA) and others, as expected, as components of the haemagglutinin spike 1HA1:1HA2, 2HA1: 2HA2, and 3HA1:3HA2. Formaldehyde gave rise only to HA1:HA2 polymers. The presence of other heteropolymers containing NP in conjunction with HA2 and HA1 seemed likely. Whenever HA2 ran with an M_r of about 50k it comigrated with M1 suggesting it may have formed (with DMS or TDA) a 1HA2:1M1 heterodimer. However it is possible that this band consisted of HA2 homodimers comigrating with M1 homodimers. Patterns of crosslinking with DMS and TDA were similar although not identical, but those obtained with formaldehyde were markedly different. All patterns were highly reproducible.     

Preparation of cross-linked hyaluronic acid film using 2-chloro-1-methylpyridinium iodide or water-soluble 1-ethyl-(3,3-dimethylaminopropyl)carbodiimide

In order to obtain much slower biodegradable films, which are often required for biomedical applications, we have developed a series of studies on heterogeneous cross-linking of hyaluronic acid (HA) films by using 2-chloro-1-methylpyridinium iodide (CMPI) or 1-ethyl-(3,3- dimethylaminopropyl)carbodiimide (EDC) as cross-linking reagents. From the in vitro degradation rate, we found that EDC cross-linked HA films completely dissolved in PBS at 37 °C during the period of 4-6 days. However, CMPI cross-linked HA films showed only a low percentage of weight loss over 30 days. This phenomenon could be explained from the mechanism of reaction between carboxyl group of HA and EDC. The latter reacted with carboxyl group to form an unstable intermediate O-acylurea, which showed a relatively low reactivity and quickly rearranged to form a stable N-acylurea. Thus, most of the EDC-activated carboxyl groups in HA were chemically transferred into N-acylurea or left as unreactive O-acylurea, and only a few of cross-linking bonds were formed between HA. On the other hand, the intermediate obtained from the reaction between carboxyl group and CMPI showed a relatively high reactivity and reacted with the hydroxyl group of the same and/or different molecules of HA to form an inter- and intramolecular esterification. Apparently, CMPI cross-linked HA films have a much higher cross-linking density and constructed a more rigid three-dimensional network. Therefore, it produced HA films, which dramatically increased its enzymatic stability in aqueous solution of hyaluronidase. The obtained results from elemental analyses, FT-IR spectra and NMR spectra also indicate that acylurea groups were introduced into EDC-cross-linked HA films.     

Preparation of cross-linked hyaluronic acid film using 2-chloro-1-methylpyridinium iodide or water-soluble 1-ethyl-(3,3-dimethylaminopropyl)carbodiimide

In order to obtain much slower biodegradable films, which are often required for biomedical applications, we have developed a series of studies on heterogeneous cross-linking of hyaluronic acid (HA) films by using 2-chloro-1-methylpyridinium iodide (CMPI) or 1-ethyl-(3,3-dimethylaminopropyl)carbodiimide (EDC) as cross-linking reagents. From the in vitro degradation rate, we found that EDC cross-linked HA films completely dissolved in PBS at 37 degrees C during the period of 4-6 days. However, CMPI cross-linked HA films showed only a low percentage of weight loss over 30 days. This phenomenon could be explained from the mechanism of reaction between carboxyl group of HA and EDC. The latter reacted with carboxyl group to form an unstable intermediate O-acylurea, which showed a relatively low reactivity and quickly rearranged to form a stable N-acylurea. Thus, most of the EDC-activated carboxyl groups in HA were chemically transferred into N-acylurea or left as unreactive O-acylurea, and only a few of cross-linking bonds were formed between HA. On the other hand, the intermediate obtained from the reaction between carboxyl group and CMPI showed a relatively high reactivity and reacted with the hydroxyl group of the same and/or different molecules of HA to form an inter- and intramolecular esterification. Apparently, CMPI cross-linked HA films have a much higher cross-linking density and constructed a more rigid three-dimensional network. Therefore, it produced HA films, which dramatically increased its enzymatic stability in aqueous solution of hyaluronidase. The obtained results from elemental analyses, FT-IR spectra and NMR spectra also indicate that acylurea groups were introduced into EDC-cross-linked HA films.     

In situ cross-linking of vesicular stomatitis virus proteins with reversible agents.

Several reversible cross-linking agents were tested for their ability to cross-link the proteins of intact vesicular stomatitis virus (VSV). Results were analyzed by two-dimensional acrylamide gel electrophoresis in the sodium dodecyl sulfate-discontinuous buffer system. Formaldehyde cross-linking was found to be reversible by heat, and gave results similar to the reagent dimethyl-3,3′-dithio-bisproprionimidate (DTBP), cleavable by reduction of its internal disulfide bond. These two reagents caused the formation of cross-linked polypeptides with molecular weights consistent with the species N:M, G:M, and G:N, where N, M, and G stand for the VSV nucleoprotein, membrane protein, and glycoprotein, respectively. Other agents tested included o-phenanthroline in the presence of Cu²⁺ and O₂, and the heavy metal ions Cu²⁺ and Ni²⁺, all reversible by β-mercaptoethanol. These agents gave similar results: No cross-linking of G proteins was observed, and of the possible heterodimers, only N:M was found. NS, the phosphopolypeptide of VSV, was found in complexes corresponding in molecular weight to homodimer and homotetramer species, possibly indicating a tetrameric configuration of the native NS protein. The apparent molecular weights of cross-linked polypeptides were found to be about 20% greater than for linear polypeptides of the same molecular weight, when the mobility in acrylamide gels of a number of cross-linked species from native proteins, enzymes, and viral nucleocapsids was compared to the mobility of linear polypeptides. The accessibility of sulfhydryl groups in native and SDS-disupted VSV was assayed by reaction with radioactive iodoacetic acid; it was found that the G protein contained no free sulfhydryl groups, explaining its failure to react with o-phenanthroline/Cu²⁺ and heavy metal ions. Removal of the G proteins from the virus with nonionic detergent prior to cross-linking with DTBP showed the coordinate loss of both the G and the N spots corresponding in apparent molecular weight to the G:N heterodimer. This result provides evidence that the glycoprotein of VSV may be capable of interacting in some manner with the core nucleoprotein.     

Use of cross-linking in studying the structure of RNA tumour viruses.

Treatment of intact avian myeloblastosis virus (AMV) with dimethyl suberimidate dihydrochloride (DMS), a cross-linking agent specific for amino groups, was found to result in progressive cross-linking among viral proteins, as revealed by polyacrylamide gel electrophoresis (PAGE) in the presence of sodium dodecyl sulphate (SDS). Free viral proteins were not cross-linked. The cross-linked protein complex with an apparent molecular weight of 50,000 daltons was studied in detail.     

Human influenza A virus: comparative analysis of the structural polypeptides by two-dimensional polyacrylamide gel electrophoresis.

The biochemical properties of the major virion polypeptides (HA₁, HA₂, M, NP, NA) of 19 influenza A virus strains have been compared by two-dimensional polyacrylamide gel electrophoresis using nonequilibrium pH gradient electrophoresis in the first dimension. The highly variable surface antigen of the virus, the hemagglutinin (HA), exhibited multiple polypeptide subspecies varying extensively in charge. Comparison of the HA of the different influenza A strains demonstrated that most strains exhibit a unique hemagglutinin with distinguishable electrophoresic properties. Differences in charge and/or molecular weight of at least one of the two HA subunits (HA₁ or HA₂) were found for strains within the same subtype and for serologically indistinguishable strains such as A/USSR/90/77 and A/FW/l/50. Differences in the matrix (M) and neuraminidase (NA) proteins also were observed between strains. The results of this study indicate that the comparative examination of the two-dimensional polypeptide patterns of a particular virus isolate may be useful for the purposes of strain identification, determination of strain purity, homogeneity, and determination of gene origin following experimental or natural recombination events.     

Study on Chemical Cross-linking Modification of Hyaluronan and the Biocompatibility of its Derivatives

Objective: Prepare cross-linked HA gels with higher mechanical stability,lower degradation velocity and desirable biocompatibility,so as to extend the usage of HA.Method: 1.Test molecular weight of HA (MrHA) by viscosimetry;2.Prepare cross-linked HA gels by DVS,GTA,DEC;3.Discuss the cross-linking and degradation procedure;4,evaluate the biocompatibility of the best HA gels.Results: The mechanical stability and durability to degradation of HA-DVS gels are superior to those of other gels,and when HA :DVS = 40:1 (g/g),at 35℃ and in 0.2M NaOH solution,the HA-DVS gel shows the best mechanical stability,and its cytotoxicity reaches class I,hemolysis ratio is lower than 5 %.Conclusion:Our HADVS gel can be used to prepare biologic scaffolds.     

Synthesis and cellular location of the ten influenza polypeptides individually expressed by recombinant vaccinia viruses

A complete set of recombinant vaccinia viruses that express each of the influenza virus polypeptides has been constructed. PB1, PB2, PA, HA, NP, M1, and NS1 genes were derived from influenza virus A/PR/8/34, NA from influenza virus A/Cam/46, and M2 and NS2 genes from influenza virus A/Udorn/72. Cells infected with these recombinant viruses synthesize influenza polypeptides that are precipitable with specific antisera and that have electrophoretic mobilities similar to the corresponding influenza virus polypeptides. Indirect immunofluorescence studies have shown that HA, NA, and MS2 proteins migrate to the cell surface; PB2, PB1, PA, NP, and NS1 proteins migrate to the cell nucleus; and M1 and NS2 are distributed throughout the cell, although NS2 accumulates preferentially in nuclei. These transport processes occurred independently of other influenza polypeptides and are therefore attributable to the intrinsic properties of the influenza polypeptides themselves.     

Entire genome characterization of human papillomavirus type 16 from infected Thai women with different cytological findings

Avian influenza has been regarded as a human health threat. A major measure to prevent its outbreak is vaccination. In this study, a series of expression plasmids carrying the hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), matrix protein 1 (M1), and matrix protein 2 (M2) genes, respectively, of the avian influenza virus (AIV) A/Chicken/Henan/12/2004(H5N1) strain were constructed. These plasmids were administered to mice by electroporation (50 μg for each per administration, 1–5 times, at an interval of 2 weeks), and the mice were challenged with the homologous virus later. The mice immunized with HA plasmid once and the NA plasmid twice survived 100%, and those with NP plasmid showed 60–80% survival rate with at least three immunizations. The mice immunized with M1 plasmid survived 25% with five immunizations, while M2 plasmid had no protection even with five immunizations. The mixture of M1 and NP plasmids protected 95% of the mice against the homologous virus, and 80% of the mice against a challenge with heterologous H1N1 (PR8) virus. Moreover, the homologous protection lasted at least 6 months. Our data provided a basis for selecting multiple-component AIV vaccines.     

Influenza virus proteins. Incorporation of newly synthesized viral proteins into virions

Incorporation of individual viral proteins (P1, P2, P3, NP, NA, HA, and M) into virions was studied by the "pulse-chase" method followed by electrophoretic analysis of the proteins in chick fibroblast cultures infected with influenza WSN virus. Viral proteins were found to incorporate into virions rather rapidly and for a long time after synthesis, with the exception of M protein incorporation of which into the virus markedly decreased by 4 hours after synthesis. Nucleocapsid proteins (NP, P1, P2, and P3) incorporated into the virus asynchronously: by 17 hours postinoculation, in NP protein prevailed the molecules synthesized after 6 hours of infection, in P2 protein the molecules synthesized in the first hours of infection, and P1 and P3 proteins were represented by a uniform set of molecules synthesized at various periods after inoculation. The evidence on heterogeneity of P2 and M proteins in the virus has been obtained. Possible mechanisms regulating the selection of molecules of individual viral proteins in formation of virus particles are discussed.     

A comparison of polypeptides in measles and SSPE virus strains.

The polypeptide patterns of twelve subacute sclerosing panencephalitis (SSPE) and measles viruses have been compared by slab gel electrophoresis. The polypeptide patterns of nine strains of SSPE and measles virus were identical. Differences in the NP protein and the HA protein of the Oddo strain, the P protein of the large plaque variant of the Lec strain of SSPE and in the M protein of the Hu 2 measles strain could not be correlated with biological characteristics such as plaque morphology, origin or haemagglutination properties.     

Emergence of a new swine H3N2 and pandemic (H1N1) 2009 influenza A virus reassortant in two Canadian animal populations, mink and swine

A swine H3N2 (swH3N2) and pandemic (H1N1) 2009 (pH1N1) influenza A virus reassortant (swH3N2/pH1N1) was detected in Canadian swine at the end of 2010. Simultaneously, a similar virus was also detected in Canadian mink based on partial viral genome sequencing. The origin of the new swH3N2/pH1N1 viral genes was related to the North American swH3N2 triple-reassortant cluster IV (for hemagglutinin [HA] and neuraminidase [NA] genes) and to pH1N1 for all the other genes (M, NP, NS, PB1, PB2, and PA). Data indicate that the swH3N2/pH1N1 virus can be found in several pigs that are housed at different locations.     

Preparation and characterization of cross-linked collagen-phospholipid polymer hybrid gels

2-methacryloyloxyethyl phosphorylcholine (MPC)-immobilized collagen gel was developed. Using 1-ethyl-3-(3-dimethyl aminopropyl)-1-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), we cross-linked a collagen film in 2-morpholinoethane sulfonic acid (MES) buffer (EN gel). EN gel was prepared under both pH 4.5 and pH 9.0 in order to observe changes in cross-linking ability. To cross-link MPC to collagen gel, poly(MPC-co-methacrylic acid) (PMA) having a carboxyl group side chain was chosen. E/N gel was added to the MES buffer having pre-NHS activated PMA to make MPC-immobilized collagen gel (MiC gel). MiC gel was prepared under both acidic and alkaline conditions to observe the changes in the cross-linking ability of PMA. X-ray photoelectron spectroscopy showed that the PMA was cross-linked with collagen under both acidic and alkaline conditions. Differential scanning calorimetry (DSC) results showed that the shrinkage temperature increased for the MiC gels and that the increase would be greater for the MiC gel prepared under alkaline conditions. The data showed that swelling would be less when the MiC gel was prepared under alkaline conditions. The biodegradation caused by collagenase was suppressed for the MiC gel prepared under alkaline conditions due to stable inter- and intrahelical networks.     

北京市甲型H1N1型流感病毒基因检测与流行趋势分析

目的 通过对甲H1N1型流感病毒基因的检测分析,揭示其基因变异情况及探讨甲型流感病毒的流行趋势,为甲型流感的防控提供理论依据.方法 样本为患者鼻咽部的粘膜上皮细胞和分泌物.取200μL标本置于核酸分离纯化系统中提取核酸,然后以LightCycler 480 PCR仪进行核酸扩增,最后分析曲线判断结果.每份标本均对甲型H1N1流感病毒基质蛋白基因（M基因）、核蛋白基因（NP基因）、血凝素基因（HA基因）、人类的RNA酶P基因（RNase P基因）进行检测和分析.结果判定：M基因、NP基因、HA基因、RNase P基因均阳性,为真阳性;HA基因阴性,其它三种基因阳性,为可疑阳性.结果 共检测3673份样本,其中,阴性2404份,甲型流感阳性样本共1269份,阳性率34.5％;阳性样本中季节性甲型流感641份,占50.5％;甲型H1N1流感阳性628份,占49.5％（真阳性433份,占68.9％,可疑阳性195份,占31.1％）.在整个流行季,真阳性与可疑阳性在相同时间的检出率比值基本不变.甲型H1N1检出高峰在10月,季节性甲流则分别在9月和12月.中青年人群的阳性检出率为57.5％,儿童为39.9％,而60岁以上的老年人仅占2.6％.结论 甲型H1N1流感以M、HA、NP、RNaesP四种基因阳性毒株为主,其流行高峰出现在10月份,感染人群以中青年和儿童为主.     

Sequence comparison between two quasi strains of H6N1 with different pathogenicity from a single parental isolate.

BACKGROUND AND PURPOSE: Relationships between gene change and virulence for hemagglutinin (HA) subtypes of avian influenza virus remain inconclusive. In this study, sequences of these nearly identical virus strains were obtained in order to elucidate the relationship between molecular determinants and virulence. METHODS: Two strains, with different virulence, of an H6N1 avian influenza virus were isolated from an infected chicken flock. Complete 8-gene fragments from the 2 strains were cloned and sequenced. Putative amino acid sequences were compared. RESULTS: Comparisons of the sequences from the 2 strains showed 0.65%, 0.79%, 0.28%, 1.23%, 0.80%, 0.20%, 0.43%, and 0.83% differences in PB2, PB1, PA, HA, NP, neuraminidase (NA), NS1 and NS2 proteins, respectively. The M1, M2, and PB1-F2 protein sequences from the strains were identical. The HA cleavage site of both strains contained a single R, despite their difference in virulence. Thus, the difference in virulence might be due to sequences other than the HA cleavage site. Most of the changes were in the HA2 part. The sequence immediately after the HA cleavage site was GILG in the non-virulent strain and GIFG in the virulent strain. The change from E to G at position 106 in the HA, near the receptor binding site, might influence the virulence. Other sequence changes likely to influence virulence were from K to R at position 291 (K291R) in NP protein and from P to T at position 101 (P101T) in NA protein. CONCLUSION: The amino acid changes identified in this study may be important in the virulence of influenza viruses.     

Recognition by major histocompatibility complex class I-restricted cytolytic T lymphocytes of cells expressing vaccinia-encoded viral and class I proteins

Target cells expressing influenza virus hemagglutinin (HA) could be recognized by cytotoxic T lymphocytes (CTL) in conjunction with the murine major histocompatibility complex class I antigen, H-2Kd, when both antigens were encoded by recombinant vaccinia virus. This recognition occurred if HA and H-2Kd were encoded by separate vaccinia viruses following dual infection of target cells or if HA and H-2Kd were encoded by a single recombinant virus. In contrast, target cells expressing nucleoprotein (NP) were only recognized by H-2Kd-restricted CTL if both NP and H-2Kd were encoded by the same vaccinia virus. These results show that the requirements for association of H-2Kd with different viral antigens derived from HA or NP can vary. Possible factors contributing to this difference are discussed.     

Synthesis and characterization of a novel hyaluronic acid hydrogel

Hyaluronic acid (hyaluronan, HA) has many medical applications as a biomaterial. To enhance its biostability, a novel hydrogel of cross-linked hyaluronic acid was prepared using a double cross-linking process, which involves building cross-linkages between hydroxyl group pairs and carboxyl group pairs. The present study explored a number of cross-linking processes in order to obtain different degrees of cross-linking, which were evaluated by the measurement of water absorption capacity as an index of the gel network density. To gain a better understanding of the stability of the gel, the chemical structure and particularly the rheological behaviour of the cross-linked HA, which included the influences of factors, such as degree of cross-linking, HA concentration and gel particle size, were investigated. The in vitro biostability against hyaluronidase and free radical degradation was tested to show that the cross-linked hydrogel had improved resistance to in vitro hyaluronidase and free radical degradation.     

Protective Efficacy of the Conserved NP,PB1, and M1 Proteins as Immunogens in DNA- and Vaccinia Virus-Based Universal Influenza A Virus Vaccines in Mice

The conventional hemagglutinin (HA)- and neuraminidase (NA)-based influenza vaccines need to be updated most years and are ineffective if the glycoprotein HA of the vaccine strains is a mismatch with that of the epidemic strain. Universal vaccines targeting conserved viral components might provide cross-protection and thus complement and improve conventional vaccines. In this study, we generated DNA plasmids and recombinant vaccinia viruses expressing the conserved proteins nucleoprotein (NP), polymerase basic 1 (PB1), and matrix 1 (M1) from influenza virus strain A/Beijing/30/95 (H3N2). BALB/c mice were immunized intramuscularly with a single vaccine based on NP, PB1, or M1 alone or a combination vaccine based on all three antigens and were then challenged with lethal doses of the heterologous influenza virus strain A/PR/8/34 (H1N1). Vaccines based on NP, PB1, and M1 provided complete or partial protection against challenge with 1.7 50% lethal dose (LD₅₀) of PR8 in mice. Of the three antigens, NP-based vaccines induced protection against 5 LD₅₀ and 10 LD₅₀ and thus exhibited the greatest protective effect. Universal influenza vaccines based on the combination of NP, PB1, and M1 induced a strong immune response and thus might be an alternative approach to addressing future influenza virus pandemics.     

Polypeptides specified by the influenza virus genome: I. Evidence for eight distinct gene products specified by fowl plague virus

The structural polypeptides of fowl plague virus (influenza A) and those synthesized in fowl plague virus-infected chick embryo fibroblasts have been analyzed by high resolution polyacrylamide gel electrophoresis. We detected eight distinct virus gene products: three polymerase-associated polypeptides (P₁, P₂, P₃), hemagglutinin (HA), nucleoprotein (NP), neuraminidase (NA), membrane polypeptide (M), and a nonstructural polypeptide (NS). The molecular weights of these polypeptides correlate closely with the molecular weights of the eight genome RNA species found in fowl plague virus.The three high molecular weight polypeptides, P₁, P₂, and P₃, were detected both in virions and infected cells, and their separate identity established by a two-dimensional tryptic peptide mapping procedure. An active RNA polymerase enzyme complex isolated from virions and virus-infected cells contained all three P proteins together with the NP protein. The nonstructural polypeptide (NS), together with the P proteins and the NP, appeared early in the infectious cycle, while the M protein and HA protein appeared later in infection. The NS and M polypeptides, which have similar molecular weights, were separated on SDS-polyacrylamide gels and shown to be distinct by tryptic peptide mapping.