WSN Influenza Virus Research Articles

Sustained generation of peroxide from the air by carbon nano onion under visible light to combat RNA virus.

Graphical abstractCarbon nano onion (CNO) from dried grass has been synthesized by carbonization in the size range, 20 to 100 nm. This shows catalytic property to transform aerial oxygen under visible light to generate reactive oxygen species (ROS). A concept has been presented herein to show that this CNO even under room light generates hydrogen peroxide which inhibits WSN influenza virus (H1N1). The advantage of introducing CNO, synthesized from a cheap source to cater to the global need, is to sterilize infected hospitals indoor and outdoor, aircraft carriers, air conditioner vents due to its sustained conversion of air to ROS. Thus, CNO use could prevent frequent evacuation as used by conventional sanitisers to sterilize infected places from other RNA virus and hospital pathogens under COVID-19 pandemic.Carbon nano onion (CNO) under aerial oxygen on exposure with visible light generates ROS which is capable to rupture the lipid envelope of SARS-CoV-2 followed by disintegrating its RNA.

ChemInform Abstract: Spiromastilactones: A New Class of Influenza Virus Inhibitors from Deep‐Sea Fungus.

AbstractMost of the novel spiromastilactones A—M (13 examples) shows inhibitory activities against WSN influenza virus with low cytotoxicity.

Spiromastilactones: A new class of influenza virus inhibitors from deep-sea fungus

A new class of phenolic lactones with trivial names of spiromastilactones A-M (1–13) was isolated from a deep-sea derived Spiromastix sp. fungus. Their structures featured by various chlorination at aromatic rings were determined on the basis of extensive spectroscopic analyses. An antiviral assay revealed that most of the tested compounds exert inhibitory activity against WSN influenza virus with low cytotoxicity, while the structure–activity relationships were discussed. Spiromastilactone D (4), a 5′-chloro-2′-methoxy substituted analogue, displayed the most potent to inhibit a panel of influenza A and B viruses in addition to drug-resistant clinical isolates. Mechanistic investigation resulted in that compound 4 bonded to hemagglutinin protein (HA), potentially at the spherical head, and disrupted the HA-sialic acid receptor interaction, that is essential for the attachment and entry of all influenza viruses. In addition, compound 4 also showed inhibitory effect toward viral genome replication via targeting viral RNP complex. The synergistic effects of 4 on both viral entry and replication assumed it to be a promising lead for the development of a new influenza inhibitor.

Influenza virus infection in multinucleated skeletal myofibers

We examined the progression of the WSN influenza virus infection in isolated, multinucleated rat skeletal myofibers. Contrary to mononucleated cells, the adsorbed virions showed markedly delayed entry kinetics. Viral budding occurred on the sarcolemma, but the hemagglutinin envelope glycoprotein matured inefficiently and was poorly cleaved. Compatible with this, plaque assays indicated that infective viral particles were not formed. In situ hybridization studies showed that at low-dose infection, viral RNA production was restricted to one or a few nuclei within a myofiber. Dual in situ hybridization indicated that two different viral RNAs usually co-localized in the same nucleus or nuclei, suggesting that different viral genome segments replicated in the same nucleus. Newly synthesized viral ribonucleoprotein particles (vRNPs) did not re-enter virgin nuclei. Therefore, a single infected nucleus was able to support viral protein production, and notably, these proteins could reach hundreds of micrometers from the nucleus of origin. These results suggest that after viral disassembly in the endosome, the genome segments remained glued together and entered a myonucleus as a package. Spreading of the infection into virgin nuclei either by vRNPs or newly made virions did not occur, and thus the infection was abortive.

Plasmid expressing the influenza HA gene protects old mice from lethal challenge with influenza virus.

Virus-based influenza vaccines induce less protection in old compared to young subjects due, in part, to age-associated alterations in the immune response. This study shows that old mice produce a less diverse HI antibody response after immunization than adult mice. However, immunization of old and young mice with plasmids expressing the HA gene induced comparable clearance of influenza virus from the lungs and the same level of protection from a lethal challenge with live WSN influenza virus. Thus, genetic immunization may offer advantages for the elderly over virus-base vaccines.

Influenza virus RNA replication in vitro: synthesis of viral template RNAs and virion RNAs in the absence of an added primer

The two steps in influenza virus RNA replication are (i) the synthesis of template RNAs, i.e., full-length copies of the virion RNAs, and (ii) the copying of these template RNAs into new virion RNAs. We prepared nuclear extracts from infected HeLa cells that catalyzed both template RNA and virion RNA synthesis in vitro in the absence of an added primer. Antibody depletion experiments implicated nucleocapsid protein molecules not associated with nucleocapsids in template RNA synthesis for antitermination at the polyadenylation site used during viral mRNA synthesis. Experiments with the WSN influenza virus temperature-sensitive mutant ts56 containing a defect in the nucleocapsid protein proved that the nucleocapsid protein was indeed required for template RNA synthesis both in vivo and in vitro. Nuclear extracts prepared from mutant virus-infected cells synthesized template RNA at the permissive temperature but not at the nonpermissive temperature, whereas the synthesis of mRNA-size transcripts was not decreased at the nonpermissive temperature. Antibody depletion experiments showed that nucleocapsid protein molecules not associated with nucleocapsids were also required for the copying of template RNA into virion RNA. In contrast to the situation with the synthesis of transcripts complementary to virion RNA, no discrete termination product(s) were made during virion RNA synthesis in vitro in the absence of nucleocapsid protein molecules.

Identification of the defects in the hemagglutinin gene of two temperature-sensitive mutants of A/WSN/33 influenza virus

Two temperature-sensitive mutants of WSN influenza virus, ts-61S and ts-134, possess defects in the hemagglutinin (HA) gene. These defects are characterized as a defective intracellular transport of the HA at the nonpermissive temperature and a marked thermolability. The nucleic acid sequences of the HA gene of these two viruses, as well as a series of revertant viruses, were determined. The deduced amino acid sequences demonstrate that the HA of ts-61S varied from the wild type protein by three amino acids while that of ts-134 differed by two residues. For both mutants, analysis of revertant viruses indicated that the phenotype of transport inhibition at the nonpermissive temperature and heat lability were associated with a single amino acid change in the globular portion of the molecule. In the case of ts-61S, the critical change in the HA was the replacement of a serine residue at position 110 with that of a proline. The mutational defect in the HA of ts-134 was due to the substitution of a tyrosine residue at position 159 with that of a histidine residue. Four of five revertants of ts-134 were suppressor revertants, of which some of the compensatory changes did not restore thermostability to the HA.

Influenza viral (A/WSN/33) hemagglutinin is expressed and glycosylated in the yeast Saccharomyces cerevisiae.

Recombinant plasmids were constructed in which genes coding for either the entire or the signal-minus (amino acid residues 2-17 deleted) hemagglutinin (HA) of WSN influenza virus were placed under the control of the alcohol dehydrogenase I gene promoter of Saccharomyces cerevisiae. Both recombinant plasmids were shown to direct the synthesis of HA-specific polypeptides that were detected by immunoprecipitation with antiviral antibodies. The complete HA produced in yeast had an approximate Mr of 70,000 and was glycosylated, as determined by the endoglycosidase H sensitivity, and was bound to membrane. Therefore, the complete HA polypeptide possessing the signal sequence probably traversed the yeast secretory pathways. Signal-minus HA, on the other hand, had a lower molecular weight and was nonglycosylated. The specific binding of yeast HA with antiviral antibodies could be competitively inhibited by influenza viral HA, demonstrating that the HA produced in yeast contained antigenic determinants of the native viral HA.

Complete sequence analyses show that two defective interfering influenza viral RNAs contain a single internal deletion of a polymerase gene.

Defective interfering (DI) influenza viral RNAs arise by internal deletion of progenitor RNAs. By using recombinant DNA cloning and DNA sequence analysis techniques, we have deduced the complete sequence of two such RNAs (L2b and L3), both arising from the same polymerase (P1) gene of WSN influenza virus. We have also partially determined the sequence of the P1 polymerase gene, including the sequence at the point of deletion and the flanking regions. Our sequence study shows the following. (i) Both L2b and L3 arise by a simple deletion in the P1 gene. (ii) L2b and L3 are 683 and 441 nucleotides long, respectively. (iii) The first 413 and 244 nucleotides of the 5' ends of L2b and L3, respectively, are identical to those of the 5' end of the P1 gene. (iv) The last 270 nucleotides of L2b and 197 nucleotides of L3 are the same as those of the 3' end of the P1 gene. (v) The entire sequence of L3 is present in the sequence of L2b. (vi) Both the 5' and the 3' termini, including the transcription stop and poly(A) addition signals of the progenitor P1 gene, are present in both L2b and L3. (vii) The sequences at the deletion point and the flanking region of the P1 gene do not resemble the consensus splicing sequence of spliced mRNA suggesting that a replicational event rather than splicing is involved in the formation of influenza defective interfering RNAs.

Activation of mouse lymphocytes by vesicular stomatitis virus.

Vesicular stomatitis virus (VSV) is a mitogen for mouse spleen cells, and infectious virus is not required for mitogenesis. At concentrations between 10 and 100 microgram per culture, VSV stimulated DNA synthesis and blast transformation. Maximal activation by VSV occurred 48 h after culture initiation. Spleen cells depleted of T-lymphocytes by treatment with anti-Thy 1.2 and complement and those obtained from congenitally athymic BALB/c nu/nu mice were activated by VSV, suggesting that VSV is a B-cell mitogen. Activation of spleen cells was independent of the host in which the virus was grown, since VSV grown in BHK-21, HKCC, or MDBK cells was mitogenic. The mitogenesis was specific for VSV, since MDBK cell-grown WSN influenza virus was not a mitogen in this in vitro activation system, VSV-specific antibody prevented VSV mitogenesis, and VSV was mitogenic for spleen cells from C3H/HeJ mice which were resistant to mitogenesis by endotoxin.

Sequence relationships among defective interfering influenza viral RNAs.

Each clone of ts-52 and ts+ WSN influenza virus, when serially passaged at high multiplicity, gives rise to defective interfering (DI) virus with a unique set of new RNA species. The new RNAs (DI RNA) from several DI viruses were examined by the technique of RNase T1 oligonucleotide two-dimensional electrophoresis. It was found that each DI RNA arises from a specific segment of standard viral RNA. All DI RNA studied arose from the viral polymerase genes (P1, P2, and P3). DI RNAs originating from the same polymerase gene were interrelated. Certain of these DI RNAs appeared to contain completely overlapping nucleotide sequences. Others contained both overlapping and nonoverlapping nucleotide sequences. The latter DI RNAs may be formed from the progenitor viral RNA segment by a mechanism other than a common initiation (or termination) point and a simple deletion from one end.

Diversity and generation of defective interfering influenza virus particles

A number of individually isolated, plaque-purified clones of ts-52 WSN influenza virus were analyzed for their ability to generate defective interfering (DI) virus particles. Clonal stocks, when passaged four or five times serially in MDBK cells at high multiplicity produced DI viruses each with a characteristic set of DI RNA segments. Aliquots of different viral passages from a given clone (VP1 through VP3), when passaged independently produced the same DI virus suggesting that DI viruses are already present in VP1. Furthermore, additional experiments suggested that DI viruses characteristic of a given clone were even present in the clonal stock virus and became amplified during undiluted passages. When aliquots of a virus clone (directly isolated from a plaque) were passaged independently some clones produced identical DI viruses at VP4 suggesting that DI viruses were already present in the plaque whereas aliquots of another clone produced different DI viruses suggesting that DI viruses were absent in the clone and were generated subsequently during passages. Thus DI virus can be generated even in the plaque produced by a single infectious virus. Large quantities of a particular DI virus could be produced by coinfection with a helper infectious virus. However, continued amplification of DI viruses for a large number of passages by a helper infectious virus resulted in disappearance of some DI RNA segments with the appearance of possible new DI RNA segments. Mixed infections of two different DI viruses in the presence of helper virus resulted in the production of both types of major DI viruses. Furthermore, we have developed an assay for quantitating DI virus directly using an infectious center reduction method. Our data suggest that a single DI virus particle can inhibit infectious center formation by standard virus and that standard virus is replaced almost entirely by DI virus after four undiluted passages.

P1 is required for initiation of cRNA synthesis in WSN influenza virus

Ts101, a mutant of WSN influenza virus, has a temperature-sensitive lesion in P1, a structural protein of the virion. P1 is required for cRNA synthesis, and no detectable cRNA is synthesized by ts101 in infected cells at the nonpermissive temperature. In the present study, the virion transcriptase activity of ts101 is shown to be temperature-sensitive in vitro, but only at low concentrations of the dinucleotide primer ApG, which is required specifically for initiation. At saturating concentrations of ApG, ts101 was not distinguishable from ts+. It is suggested that P1 is required for initiation of transcription, and normally functions in vivo in the recognition of the putative physiological primer. Inasmuch as P1 and P3 are known to be the only gene products which are required for the synthesis of viral cRNA, it seems likely that P3 functions in the elongation process.

Temperature-sensitive virion transcriptase activity in mutants of WSN influenza virus.

Temperature-sensitive mutants of WSN influenza virus (10, 11, 12) were tested in vitro for activity of the virion RNA-dependent RNA transcriptase at various temperatures. Temperature-sensitivity was found for virion transcriptase activity of mutants belonging to complementation/recombination group I, but not groups II, IV and V. It was not possible on the basis of the results to specify the precise biochemical lesion of mutants from group III.