Evaluation of Influenza A/Hong Kong/123/77 (H1N1) ts -1A2 and Cold-Adapted Recombinant Viruses in Seronegative Adult Volunteers

1980 ◽  
Vol 29 (2) ◽  
pp. 348-355 ◽  
Author(s):  
Brian R. Murphy ◽  
Margret B. Rennels ◽  
R. Gordon Douglas ◽  
Robert F. Betts ◽  
Robert B. Couch ◽  
...  
Keyword(s):  
Hemagglutination Inhibition ◽  
Influenza A ◽  
Wild Type Virus ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serological Response ◽  
Type Virus ◽  
Wild Type ◽  
Cold Adapted ◽  
Recombinant Viruses ◽  
Inhibition Response

Two attenuated influenza A donor viruses, the A/Udorn/72 ts -1A2 and the A/Ann Arbor/6/60 cold-adapted ( ca ) viruses, are being evaluated for their ability to reproducibly attenuate each new variant of influenza A virus to a specific and desired level by the transfer of one or more attenuating genes. Each of these donor viruses has been able to attenuate influenza A viruses belonging to the H3N2 subtype by the transfer of one or more attenuating genes. To determine whether these two donor viruses could attenuate a wild-type virus that belonged to a different influenza A subtype, ts -1A2 and ca recombinants of a wild-type virus representative of the A/USSR/77 (H1N1) Russian influenza strain were prepared and evaluated in adult doubly seronegative volunteers at several doses. The recombinants derived from both donor viruses were attenuated for the doubly seronegative adults. Less than 5% of infected vaccinees developed a febrile or systemic reaction, whereas five of six recipients of wild-type virus developed such a response. The 50% human infectious dose (HID 50 ) for each recombinant was approximately 10 5.0 50% tissue culture infective doses. The virus shed by the ts -1A2 and ca vaccinees retained the ts or ca phenotype, or both. This occurred despite replication of the recombinant viruses for up to 9 days. No evidence for transmission of the ca or ts -1A2 recombinant virus to controls was observed. A serum hemagglutination inhibition response was detected in less than 50% of the infected vaccinees. However, with the more sensitive enzyme-linked immunosorbent assay, a serological response was detected in 100% of the ca vaccinees given 300 HID 50 and approximately 70% of ca or ts vaccinees who received 10 to 32 HID 50 of virus. These results indicate that the recombinants derived from both donor viruses were satisfactorily attenuated and were stable genetically after replication in doubly seronegative adults although they induced a lower serum hemagglutination inhibition response than that found previously for H3N2 ts and ca recombinants.

scholarly journals In Vivo Dynamics of Reporter Flaviviridae Viruses

2019 ◽  
Vol 93 (22) ◽  
Author(s):  
Tomokazu Tamura ◽  
Manabu Igarashi ◽  
Bazarragchaa Enkhbold ◽  
Tatsuya Suzuki ◽  
Masatoshi Okamatsu ◽  
...  
Keyword(s):  
Luciferase Activity ◽  
Viral Proteins ◽  
Solvent Accessible Surface Area ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Recombinant Viruses ◽  
Diarrhea Virus ◽  
Foreign Genes

ABSTRACT Recombinant viruses possessing reporter proteins have been generated for virus research. In the case of the family Flaviviridae, we recently generated recombinant viruses, including the hepatitis C virus of the genus Hepacivirus, Japanese encephalitis virus (JEV) of the genus Flavivirus, and bovine viral diarrhea virus of the genus Pestivirus; all three viruses possess an 11-amino-acid subunit derived from NanoLuc luciferase (HiBiT). Here, we further developed the recombinant viruses and investigated their utility in vivo. Recombinant viruses harboring HiBiT in the E, NS1, or NS3 protein constructed based on the predicted secondary structure, solvent-accessible surface area, and root mean square fluctuation of the proteins exhibited comparable replication to that of the wild-type virus in vitro. The recombinant JEV carrying HiBiT in the NS1 protein exhibited propagation in mice comparable to that of the parental virus, and propagation of the recombinant was monitored by the luciferase activity. In addition, the recombinants of classical swine fever virus (CSFV) possessing HiBiT in the Erns or E2 protein also showed propagation comparable to that of the wild-type virus. The recombinant CSFV carrying HiBiT in Erns exhibited similar replication to the parental CSFV in pigs, and detection of viral propagation of this recombinant by luciferase activity was higher than that by quantitative PCR (qPCR). Taken together, these results demonstrated that the reporter Flaviviridae viruses generated herein are powerful tools for elucidating the viral life cycle and pathogeneses and provide a robust platform for the development of novel antivirals. IMPORTANCE In vivo applications of reporter viruses are necessary to understand viral pathogenesis and provide a robust platform for antiviral development. In developing such applications, determination of an ideal locus to accommodate foreign genes is important, because insertion of foreign genes into irrelevant loci can disrupt the protein functions required for viral replication. Here, we investigated the criteria to determine ideal insertion sites of foreign genes from the protein structure of viral proteins. The recombinant viruses generated by our criteria exhibited propagation comparable to that of parental viruses in vivo. Our proteomic approach based on the flexibility profile of viral proteins may provide a useful tool for constructing reporter viruses, including Flaviviridae viruses.

scholarly journals The Influenza A Virus M2 Cytoplasmic Tail Is Required for Infectious Virus Production and Efficient Genome Packaging

2005 ◽  
Vol 79 (6) ◽  
pp. 3595-3605 ◽  
Author(s):  
Matthew F. McCown ◽  
Andrew Pekosz
Keyword(s):  
Influenza A Virus ◽  
Influenza A ◽  
Infectious Virus ◽  
Virus Production ◽  
Cytoplasmic Tail ◽  
Host Cells ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Virus Particles

ABSTRACT The M2 integral membrane protein encoded by influenza A virus possesses an ion channel activity that is required for efficient virus entry into host cells. The role of the M2 protein cytoplasmic tail in virus replication was examined by generating influenza A viruses encoding M2 proteins with truncated C termini. Deletion of 28 amino acids (M2Stop70) resulted in a virus that produced fourfold-fewer particles but >1,000-fold-fewer infectious particles than wild-type virus. Expression of the full-length M2 protein in trans restored the replication of the M2 truncated virus. Although the M2Stop70 virus particles were similar to wild-type virus in morphology, the M2Stop70 virions contained reduced amounts of viral nucleoprotein and genomic RNA, indicating a defect in vRNP packaging. The data presented indicate the M2 cytoplasmic tail plays a role in infectious virus production by coordinating the efficient packaging of genome segments into influenza virus particles.

scholarly journals Ubiquitination of the Cytoplasmic Domain of Influenza A Virus M2 Protein is Crucial for Production of Infectious Virus Particles

2017 ◽  
pp. JVI.01972-17 ◽  
Author(s):  
Wen-Chi Su ◽  
Wen-Ya Yu ◽  
Shih-Han Huang ◽  
Michael M.C. Lai
Keyword(s):  
Virus Replication ◽  
Influenza A ◽  
Functional Role ◽  
Infectious Virus ◽  
Virus Production ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Virus Particles

Virus replication is mediated by interactions between virus and host. Here, we demonstrate that influenza A virus membrane protein 2 (M2) can be ubiquitinated. The lysine residue at position 78, which is located in the cytoplasmic domain of M2, is essential for M2 ubiquitination. An M2-K78R (Lys78→Arg78) mutant, which produces ubiquitination-deficient M2, showed a severe defect in production of infectious virus particles. M2-K78R mutant progeny contained more HA proteins, less viral RNAs and less internal viral proteins, including M1 and NP, than the wild-type virus. Furthermore, most of the M2-K78R mutant viral particles lacked viral ribonucleoproteins upon examination under electron microscopy and exhibited slightly lower densities. We also found that mutant M2 colocalized with M1 protein to a lesser extent than for wild-type virus. These findings may account for the reduced incorporation of viral ribonucleoprotein into virions. By blocking the second round of virus infection, we showed that the M2 ubiquitination-defective mutant exhibited normal level of virus replication during the first round of infection, thereby proving that M2 ubiquitination is involved in the virus production step. Finally, we found that M2-K78R mutant virus induced autophagy and apoptosis earlier than wild-type virus. Collectively, these results suggest that M2 ubiquitination plays an important role in infectious virus production by coordinating efficient packaging of the viral genome into virus particles and timing of viral-induced cell death.IMPORTANCEAnnual epidemics and recurring pandemics of influenza viruses represent a very high global health and economic burden. Influenza virus M2 protein has been extensively studied for its important roles in virus replication, particularly in viral entry and release. Rimantadine, one of the most commonly used antiviral drugs, binds to the channel lumen near the N-terminus of M2 proteins. However, viruses resistant to Rimantadine have emerged. M2 undergoes several posttranslational modifications, such as phosphorylation and palmitoylation. Here, we reveal that ubiquitination mediates the functional role of M2. A ubiquitination-deficient M2 mutant predominately produced virus particles either lacking viral ribonucleoproteins or containing smaller amounts of internal viral components, resulting in lower infectivity. Our findings offer insights into the mechanism of influenza virus morphogenesis, particularly the functional role of M1-M2 interactions in viral particle assembly, and can be applied to the development of new influenza therapies.

scholarly journals Activity of the Oral Neuraminidase Inhibitor A-322278 against the Oseltamivir-Resistant H274Y (A/H1N1) Influenza Virus Mutant in Mice

2008 ◽  
Vol 53 (2) ◽  
pp. 791-793 ◽  
Author(s):  
Mariana Baz ◽  
Yacine Abed ◽  
Benjamin Nehmé ◽  
Guy Boivin
Keyword(s):  
Influenza A ◽  
Neuraminidase Inhibitor ◽  
H1n1 Influenza ◽  
Mortality Rates ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
H1n1 Influenza Virus ◽  
Influenza A H1n1 ◽  
Virus Mutant

ABSTRACT The new oral neuraminidase (NA) inhibitor A-322278 was evaluated in mice infected with influenza A/H1N1 wild-type virus or the oseltamivir-resistant (H274Y mutant) virus. A-322278 decreased mortality rates and lung virus titers significantly more than oseltamivir in mice infected with the NA H274Y mutant when therapy was started 4 h before or even 48 h after infection.

scholarly journals Plasminogen-Binding Activity of Neuraminidase Determines the Pathogenicity of Influenza A Virus

2001 ◽  
Vol 75 (19) ◽  
pp. 9297-9301 ◽  
Author(s):  
Hideo Goto ◽  
Krisna Wells ◽  
Ayato Takada ◽  
Yoshihiro Kawaoka
Keyword(s):  
Cell Culture ◽  
Influenza A ◽  
Binding Activity ◽  
Glycosylation Site ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
C Terminus ◽  
Viral Hemagglutinin

ABSTRACT When expressed in vitro, the neuraminidase (NA) of A/WSN/33 (WSN) virus binds and sequesters plasminogen on the cell surface, leading to enhanced cleavage of the viral hemagglutinin. To obtain direct evidence that the plasminogen-binding activity of the NA enhances the pathogenicity of WSN virus, we generated mutant viruses whose NAs lacked plasminogen-binding activity because of a mutation at the C terminus, from Lys to Arg or Leu. In the presence of trypsin, these mutant viruses replicated similarly to wild-type virus in cell culture. By contrast, in the presence of plasminogen, the mutant viruses failed to undergo multiple cycles of replication while the wild-type virus grew normally. The mutant viruses showed attenuated growth in mice and failed to grow at all in the brain. Furthermore, another mutant WSN virus, possessing an NA with a glycosylation site at position 130 (146 in N2 numbering), leading to the loss of neurovirulence, failed to grow in cell culture in the presence of plasminogen. We conclude that the plasminogen-binding activity of the WSN NA determines its pathogenicity in mice.

Sign in / Sign up

Export Citation Format

Share Document