scholarly journals Direct RNA Sequencing of the Complete Influenza A Virus Genome

2018 ◽  
Author(s):  
Matthew W. Keller ◽  
Benjamin L. Rambo-Martin ◽  
Malania M. Wilson ◽  
Callie A. Ridenour ◽  
Samuel S. Shepard ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Influenza A ◽  
Splice Variants ◽  
Rna Virus ◽  
Allantoic Fluid ◽  
Virus Genome ◽  
Chemical Degradation ◽  
Original Form ◽  
Sequencing Platform ◽  
Oxford Nanopore

ABSTRACTFor the first time, a complete genome of an RNA virus has been sequenced in its original form. Previously, RNA was sequenced by the chemical degradation of radiolabelled RNA, a difficult method that produced only short sequences. Instead, RNA has usually been sequenced indirectly by copying it into cDNA, which is often amplified to dsDNA by PCR and subsequently analyzed using a variety of DNA sequencing methods. We designed an adapter to short highly conserved termini of the influenza virus genome to target the (-) sense RNA into a protein nanopore on the Oxford Nanopore MinION sequencing platform. Utilizing this method and total RNA extracted from the allantoic fluid of infected chicken eggs, we demonstrate successful sequencing of the complete influenza virus genome with 100% nucleotide coverage, 99% consensus identity, and 99% of reads mapped to influenza. By utilizing the same methodology we can redesign the adapter in order to expand the targets to include viral mRNA and (+) sense cRNA, which are essential to the viral life cycle. This has the potential to identify and quantify splice variants and base modifications, which are not practically measurable with current methods.

scholarly journals REACTIONS BETWEEN INFLUENZA VIRUS AND A COMPONENT OF ALLANTOIC FLUID

1948 ◽  
Vol 88 (4) ◽  
pp. 463-484 ◽  
Author(s):  
Paul H. Hardy ◽  
Frank L. Horsfall
Keyword(s):  
Influenza Virus ◽  
Influenza A Virus ◽  
Influenza A ◽  
Allantoic Fluid ◽  
Partial Dissociation

Evidence is presented which shows that there is a component present in normal allantoic fluid, probably mucoprotein in nature, capable of combining with influenza A virus (PR8), and that following combination between this component and the virus only partial dissociation of the complex occurs. Evidence is also presented which strongly suggests that the component is present in virus-infected allantoic fluid in which it is in part combined with the virus and in part free although altered by viral action. The probability that the component is present as well in highly purified preparations of influenza virus, and its effect upon various reactions obtained with this agent are discussed.

scholarly journals N2Gas Plasma Inactivates Influenza Virus by Inducing Changes in Viral Surface Morphology, Protein, and Genomic RNA

2013 ◽  
Vol 2013 ◽  
pp. 1-7 ◽  
Author(s):  
Akikazu Sakudo ◽  
Naohiro Shimizu ◽  
Yuichiro Imanishi ◽  
Kazuyoshi Ikuta
Keyword(s):  
Influenza Virus ◽  
Plasma Treatment ◽  
Influenza A ◽  
Allantoic Fluid ◽  
High Voltage Pulse ◽  
Influenza Viruses ◽  
Blue Staining ◽  
Enzyme Linked Immunosorbent Assay ◽  
Gas Plasma ◽  
Electron Microscopy Analysis

We have recently treated with N2gas plasma and achieved inactivation of bacteria. However, the effect of N2gas plasma on viruses remains unclear. With the aim of developing this technique, we analyzed the virucidal effect of N2gas plasma on influenza virus and its influence on the viral components. We treated influenza virus particles with inert N2gas plasma (1.5 kpps; kilo pulses per second) produced by a short high-voltage pulse generated from a static induction thyristor power supply. A bioassay using chicken embryonated eggs demonstrated that N2gas plasma inactivated influenza virus in allantoic fluid within 5 min. Immunochromatography, enzyme-linked immunosorbent assay, and Coomassie brilliant blue staining showed that N2gas plasma treatment of influenza A and B viruses in nasal aspirates and allantoic fluids as well as purified influenza A and B viruses induced degradation of viral proteins including nucleoprotein. Analysis using the polymerase chain reaction suggested that N2gas plasma treatment induced changes in the viral RNA genome. Scanning electron microscopy analysis showed that aggregation and fusion of influenza viruses were induced by N2gas plasma treatment. We believe these biochemical changes may contribute to the inactivation of influenza viruses by N2gas plasma.

scholarly journals THE QUANTITATIVE DETERMINATION OF INFLUENZA VIRUS AND ANTIBODIES BY MEANS OF RED CELL AGGLUTINATION

1942 ◽  
Vol 75 (1) ◽  
pp. 49-64 ◽  
Author(s):  
George K. Hirst
Keyword(s):  
Influenza Virus ◽  
Influenza A ◽  
Allantoic Fluid ◽  
Red Cells ◽  
Virus Neutralization ◽  
Influenza B ◽  
Logarithmic Scale ◽  
Neutralization Titer ◽  
Cell Agglutination ◽  
Agglutination Titer

1. The agglutination titer for chicken red cells of freshly prepared or carefully stored suspensions of PR8 influenza virus, that is to say virus of maximum pathogenicity, was found to be proportional to the mouse lethal titer of the same preparations. 2. The agglutination titer of infected allantoic fluid procured in a standard way is relatively constant, regardless of the influenza strain used and its pathogenicity for mice. 3. Virus preparations inactivated by heat or storage may retain their agglutinating power. 4. Certain animal sera contain a partially heat-labile factor which, in low dilution, inhibits the agglutination of chicken red cells by influenza A and influenza B viruses. 5. The agglutination inhibition test, using ferret and human sera, gives qualitative data regarding influenza antibodies which are similar to the information obtained on the same sera by means of the virus neutralization test. 6. There is a definite relationship between the agglutination inhibition titer and the virus neutralization titer of a serum. On a logarithmic scale of both variables, this relationship is essentially linear within the range investigated. 7. The agglutination inhibition titer of immune ferret serum is inversely proportional to the amount of virus used in the test.

scholarly journals Incorporation of influenza A virus genome segments does not absolutely require wild-type sequences

2009 ◽  
Vol 90 (7) ◽  
pp. 1734-1740 ◽  
Author(s):  
Ken Fujii ◽  
Makoto Ozawa ◽  
Kiyoko Iwatsuki-Horimoto ◽  
Taisuke Horimoto ◽  
Yoshihiro Kawaoka
Keyword(s):  
Influenza Virus ◽  
Influenza A Virus ◽  
Influenza A ◽  
Virus Genome ◽  
Wild Type ◽  
Genome Packaging ◽  
Viral Rnas ◽  
Virion Incorporation ◽  
Specific Sequences ◽  
Type Sequence

The efficient incorporation of influenza virus genome segments into virions is mediated by cis-acting regions at both ends of the viral RNAs. It was shown previously that nt 16–26 at the 3′ end of the non-structural (NS) viral RNA of influenza A virus are important for efficient virion incorporation and that nt 27–56 also contribute to this process. To understand further the signalling requirements for genome packaging, this study performed linker-scanning mutagenesis in the latter region and found that nt 27–35 made an appreciable contribution to the efficient incorporation of the NS segment. An NS vRNA library was then generated composed of an RNA population with randomized nucleotides at positions 16–35 such that the virus could select the sequences it required for virion incorporation. The sequences selected differed from the wild-type sequence and no conserved nucleotides were selected. The ability of non-wild-type sequences to function in this manner indicates that the incorporation of influenza A virus genome segments does not absolutely require specific sequences.

ADSORPTION OF INFLUENZA VIRUS

1947 ◽  
Vol 25e (2) ◽  
pp. 43-52 ◽  
Author(s):  
Ronald Hare ◽  
Marjorie Curl
Keyword(s):  
Influenza Virus ◽  
Flow Rate ◽  
Influenza A ◽  
Allantoic Fluid ◽  
Fuller’S Earth ◽  
Filter Aids

Influenza A and B viruses in allantoic fluid can be adsorbed by certain grades of diatomaceous earths usually employed as filter aids, there being a marked degree of correlation between the 'flow rate' of the earths and their ability to adsorb the virus. Of the clays, kaolin and fuller's earth are similarly able to adsorb the virus as are certain varieties of charcoal. Elution of the adsorbed virus is also possible provided a protein-containing solution such as broth, serum, or isinglass be employed, the highest yield being obtained with virus adsorbed on silica earths and eluted with isinglass solution.

scholarly journals The effects of a deleterious mutation load on patterns of influenza A/H3N2's antigenic evolution in humans

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Katia Koelle ◽  
David A Rasmussen
Keyword(s):  
Influenza Virus ◽  
Genetic Linkage ◽  
Influenza A ◽  
Deleterious Mutation ◽  
Rna Virus ◽  
Phylogenetic Analyses ◽  
Molecular Pathways ◽  
Deleterious Mutations ◽  
Mutation Load ◽  
Antigenic Evolution

Recent phylogenetic analyses indicate that RNA virus populations carry a significant deleterious mutation load. This mutation load has the potential to shape patterns of adaptive evolution via genetic linkage to beneficial mutations. Here, we examine the effect of deleterious mutations on patterns of influenza A subtype H3N2's antigenic evolution in humans. By first analyzing simple models of influenza that incorporate a mutation load, we show that deleterious mutations, as expected, act to slow the virus's rate of antigenic evolution, while making it more punctuated in nature. These models further predict three distinct molecular pathways by which antigenic cluster transitions occur, and we find phylogenetic patterns consistent with each of these pathways in influenza virus sequences. Simulations of a more complex phylodynamic model further indicate that antigenic mutations act in concert with deleterious mutations to reproduce influenza's spindly hemagglutinin phylogeny, co-circulation of antigenic variants, and high annual attack rates.

Influenza

2021 ◽  
Author(s):  
Leong Hoe Nam
Keyword(s):  
Influenza Virus ◽  
Pandemic Influenza ◽  
Influenza A ◽  
Respiratory Infections ◽  
Rna Virus ◽  
Treatment Options ◽  
Antigenic Drift ◽  
New Techniques ◽  
Influenza A H1n1 ◽  
Viral Respiratory Infections

The influenza virus is a segmented RNA virus with different mechanisms for mutations, and hence for minor (antigenic drift) and major (antigenic shift) changes. Influenza virus A was responsible for pandemics on average every 30 years in the past, with the most recent being the 2009 swine-origin influenza A H1N1 (SO-H1N1). The clinical picture is unspecific: seasonal or pandemic influenza cannot be differentiated from other viral respiratory infections on clinical grounds. PCR has become the standard for microbiological confirmation of the diagnosis. Treatment options remain limited with neuraminidase inhibitors (oseltamivir; zanamivir). Resistance may occur under treatment or under prophylaxis; however, it is still rare overall. Vaccination is still the preferred method for prevention. However, the long lead time for production (at least 6 months) poses a challenge. Innovative new techniques like cell culture or recombinant productions are urgently needed. Pandemic influenza vaccines for SO-H1N1 were shown to be effective and safe in children, pregnant women, adults, and also in elderly. Pre-pandemic vaccines (H5N1) are also available.

Madin Darby canine kidney cell-lines used in influenza virus production has resulted in canine 28s being integrated in the influenza virus genome - evidence from Influenza A patients in Hong Kong

2020 ◽  
Author(s):  
Sandeep Chakraborty
Keyword(s):  
Influenza Virus ◽  
Hong Kong ◽  
Influenza Vaccine ◽  
Mdck Cell ◽  
Influenza A ◽  
Mdck Cells ◽  
Virus Genome ◽  
Kidney Cells ◽  
Darby Canine Kidney ◽  
Canine Kidney

In 1989, 54 nucleotides from chicken 18s were seen to be inserted into the haemagglutinin gene of an influenza virus increasing viral pathogenicity [1]. Previously, I have reported human 18s sequences (from sequence vectors) appended to the influenza virus genome in Covid19 patients from Wuhan and Hong Kong [2]. These human ribosomal sequences are supposed to increase the transcription of the virus in the human cell, and thus will be more pathogenic. Here, I report the circulation of Influenza A genomes with 28s from canine integrated, showing that the escape of lab-made viruses is quite prevalent.Canine 28s sequence appended to flu genomesA recent (2020,Accid:PRJNA605947) study from the University of Hong Kong that did Nanopore sequencing to find novel targets for detection and surveillance of Influenza A viruses [3] shows the integration of canine 28s (Fig 1) sequence to the flu genome. The full read (SRR11067307.3179,SI:fullread.fa) splits into the flu virus (1-1605, SI:flu.baltimore.fa,(Baltimore/R0197/2017(H1N1)) nucleocapsid protein (NP) gene) and canine 28 (1606-1973, SI:canine.28s.fa Accid:XR 004817748.1, Canis lupus dingo 28S)). There is no reason to find canine 28s in clinical samples, barring the fact that canine kidney cells are used to manufacture the virus for several applications, including vaccines.Madin Darby canine kidney cells (MDCK) - why MDCK?The advantages of using MDCK influenza production is well known [4]. MDCK is better in the replication of live attenuated influenza viruses than most other cell lines (like Vero), thus yielding more virus in large-scale production of influenza virus [5]. The virus replicates rapidly in MDCK to ‘produce high titers in MDCK cells in as few as 3 to 10 passages, i.e., in 10–30 days’ [4]. Also, MDCK cells are also good for the production of certain influenza B virus vaccines, and MDCK cell-derived components are not allergenic [6]. Vaccines made using MDCK cellsInfluvac, a split virus vaccine produced in adherent MDCK cells, in the Netherlands in 1999 [7]. The use of MDCK cells (MedImmune) for production of live attenuated influenza vaccine in both serum containing and serum-free media was found to be more efficient [8]. Another trivalent MDCK cell culture-derived influenza vaccine is Optaflu [9]. ”Flucelvax Quadrivalent is the only cell-based inactivated flu vaccine that has been licensed by the FDA for use in the United States.” (https://www.cdc.gov/flu/prevent/cell-based.htm)

scholarly journals ANTIBODY RESPONSE OF HUMAN BEINGS FOLLOWING VACCINATION WITH INFLUENZA VIRUSES

1942 ◽  
Vol 75 (5) ◽  
pp. 495-511 ◽  
Author(s):  
G. K. Hirst ◽  
E. R. Rickard ◽  
Loring Whitman ◽  
F. L. Horsfall
Keyword(s):  
Influenza Virus ◽  
Antibody Response ◽  
Influenza A ◽  
Geometric Mean ◽  
Allantoic Fluid ◽  
Influenza Viruses ◽  
Human Beings ◽  
Significant Difference ◽  
Antibody Levels

Eleven different preparations of influenza virus were used to vaccinate large groups of human beings. The antibody response to these vaccines was measured by means of the in vitro agglutination inhibition test, and the geometric mean titers of sera taken 2 weeks after vaccination were compared. From these comparisons the following conclusions were drawn: 1. There was a wide individual variation in the antibody response of human beings to the same preparation of influenza virus administrated subcutaneously. The amount of antibody produced by a group with a low prevaccination antibody level was very nearly the same as the amount produced by groups that had higher initial levels. 2. The use of the X strain of distemper virus in the preparation of an influenza vaccine did not enhance the antigenicity of the influenza virus present. 3. Within certain limits the mean antibody response of human beings increased as the amount of virus injected was increased. When large amounts of influenza A virus were given, the antibody response was of the same order of magnitude as that which occurred following actual infection by this virus. 4. When the vaccine was prepared from allantoic fluid, there was no significant difference in the antibody response of human beings given active virus, formalin-inactivated virus, heat-inactivated virus, or virus inactivated by the drying process. 5. Ground infected chick embryos, when diluted with infected allantoic fluid, gave a greater antibody response than allantoic fluid alone (when the virus remained active). The antigenicity of such a preparation was diminished when the virus was inactivated by formalin. 6. Antibody levels 6 and 9 weeks after vaccination showed a marked drop from the 2-week postvaccination levels. In a small group the antibody levels at 5 months were still further reduced. Those individuals who possessed the higher titers tended to lose their antibodies faster than did those at a lower level.

scholarly journals STUDIES IN HUMAN IMMUNIZATION AGAINST INFLUENZA

1944 ◽  
Vol 80 (4) ◽  
pp. 265-273 ◽  
Author(s):  
G. K. Hirst ◽  
E. R. Rickard ◽  
W. F. Friedewald
Keyword(s):  
Influenza Virus ◽  
Antibody Response ◽  
Attack Rate ◽  
Influenza A ◽  
Allantoic Fluid ◽  
Great Majority ◽  
High Order ◽  
Human Beings ◽  
Antibody Levels ◽  
A Current

The administration to human beings of formalin-killed influenza virus, concentrated from allantoic fluid, resulted in a high order of antibody response within 2 weeks after injection. Even after 1 year the great majority of individuals vaccinated had antibody levels considerably above their prevaccination titer for the PR8, Lee, and a current 1943 strain. An investigation of the occurrence of epidemic influenza A in seven widely separated populations, 1 year after vaccination of part of these groups, showed that the attack rate among vaccinated persons was consistently lower than that of control individuals. The average reduction in attack rate was of the order of 35 per cent.

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