scholarly journals Molecular Basis of Replication of Duck H5N1 Influenza Viruses in a Mammalian Mouse Model

2005 ◽  
Vol 79 (18) ◽  
pp. 12058-12064 ◽  
Author(s):  
Zejun Li ◽  
Hualan Chen ◽  
Peirong Jiao ◽  
Guohua Deng ◽  
Guobin Tian ◽  
...  
Keyword(s):  
Amino Acid ◽  
Host Range ◽  
Reverse Genetics ◽  
Southern China ◽  
Single Gene ◽  
Influenza Viruses ◽  
Single Amino Acid ◽  
Species Barrier ◽  
H5n1 Influenza ◽  
The One

ABSTRACT We recently analyzed a series of H5N1 viruses isolated from healthy ducks in southern China since 1999 and found that these viruses had progressively acquired the ability to replicate and cause disease in mice. In the present study, we explored the genetic basis of this change in host range by comparing two of the viruses that are genetically similar but differ in their ability to infect mice and have different pathogenicity in mice. A/duck/Guangxi/22/2001 (DKGX/22) is nonpathogenic in mice, whereas A/duck/Guangxi/35/2001 (DKGX/35) is highly pathogenic. We used reverse genetics to create a series of single-gene recombinants that contained one gene from DKGX/22 and the remaining seven gene segments from DKGX/35. We find that the PA, NA, and NS genes of DKGX/22 could attenuate DKGX/35 virus to some extent, but PB2 of DKGX/22 virus attenuated the DKGX/35 virus dramatically, and an Asn-to-Asp substitution at position 701 of PB2 plays a key role in this function. Conversely, of the recombinant viruses in the DKGX/22 background, only the one that contains the PB2 gene of DKGX/35 was able to replicate in mice. A single amino acid substitution (Asp to Asn) at position 701 of PB2 enabled DKGX/22 to infect and become lethal for mice. These results demonstrate that amino acid Asn 701 of PB2 is one of the important determinants for this avian influenza virus to cross the host species barrier and infect mice, though the replication and lethality of H5N1 influenza viruses involve multiple genes and may result from a constellation of genes. Our findings may help to explain the expansion of the host range and lethality of the H5N1 influenza viruses to humans.

scholarly journals A Single-Amino-Acid Substitution in a Polymerase Protein of an H5N1 Influenza Virus Is Associated with Systemic Infection and Impaired T-Cell Activation in Mice

2009 ◽  
Vol 83 (21) ◽  
pp. 11102-11115 ◽  
Author(s):  
Jamie L. Fornek ◽  
Laura Gillim-Ross ◽  
Celia Santos ◽  
Victoria Carter ◽  
Jerrold M. Ward ◽  
...  
Keyword(s):  
Immune Response ◽  
Influenza Virus ◽  
Amino Acid ◽  
T Cell ◽  
Amino Acid Substitution ◽  
Systemic Infection ◽  
Influenza Viruses ◽  
Single Amino Acid Substitution ◽  
Single Amino Acid ◽  
H5n1 Influenza

ABSTRACT The transmission of H5N1 influenza viruses from birds to humans poses a significant public health threat. A substitution of glutamic acid for lysine at position 627 of the PB2 protein of H5N1 viruses has been identified as a virulence determinant. We utilized the BALB/c mouse model of H5N1 infection to examine how this substitution affects virus-host interactions and leads to systemic infection. Mice infected with H5N1 viruses containing lysine at amino acid 627 in the PB2 protein exhibited an increased severity of lesions in the lung parenchyma and the spleen, increased apoptosis in the lungs, and a decrease in oxygen saturation. Gene expression profiling revealed that T-cell receptor activation was impaired at 2 days postinfection (dpi) in the lungs of mice infected with these viruses. The inflammatory response was highly activated in the lungs of mice infected with these viruses and was sustained at 4 dpi. In the spleen, immune-related processes including NK cell cytotoxicity and antigen presentation were highly activated by 2 dpi. These differences are not attributable solely to differences in viral replication in the lungs but to an inefficient immune response early in infection as well. The timing and magnitude of the immune response to highly pathogenic influenza viruses is critical in determining the outcome of infection. The disruption of these factors by a single-amino-acid substitution in a polymerase protein of an influenza virus is associated with severe disease and correlates with the spread of the virus to extrapulmonary sites.

scholarly journals A Single-Amino-Acid Substitution in the NS1 Protein Changes the Pathogenicity of H5N1 Avian Influenza Viruses in Mice

2007 ◽  
Vol 82 (3) ◽  
pp. 1146-1154 ◽  
Author(s):  
Peirong Jiao ◽  
Guobin Tian ◽  
Yanbing Li ◽  
Guohua Deng ◽  
Yongping Jiang ◽  
...  
Keyword(s):  
Amino Acid ◽  
Avian Influenza ◽  
Host Cell ◽  
Amino Acid Substitution ◽  
Influenza Viruses ◽  
Single Amino Acid ◽  
Ns1 Protein ◽  
Avian Influenza Viruses ◽  
H5n1 Influenza ◽  
H5n1 Avian Influenza

ABSTRACT In this study, we explored the molecular basis determining the virulence of H5N1 avian influenza viruses in mammalian hosts by comparing two viruses, A/Duck/Guangxi/12/03 (DK/12) and A/Duck/Guangxi/27/03 (DK/27), which are genetically similar but differ in their pathogenicities in mice. To assess the genetic basis for this difference in virulence, we used reverse genetics to generate a series of reassortants and mutants of these two viruses. We found that a single-amino-acid substitution of serine for proline at position 42 (P42S) in the NS1 protein dramatically increased the virulence of the DK/12 virus in mice, whereas the substitution of proline for serine at the same position (S42P) completely attenuated the DK/27 virus. We further demonstrated that the amino acid S42 of NS1 is critical for the H5N1 influenza virus to antagonize host cell interferon induction and for the NS1 protein to prevent the double-stranded RNA-mediated activation of the NF-κB pathway and the IRF-3 pathway. Our results indicate that the NS1 protein is critical for the pathogenicity of H5N1 influenza viruses in mammalian hosts and that the amino acid S42 of NS1 plays a key role in undermining the antiviral immune response of the host cell.

scholarly journals The polymerase complex genes contribute to the high virulence of the human H5N1 influenza virus isolate A/Vietnam/1203/04

2006 ◽  
Vol 203 (3) ◽  
pp. 689-697 ◽  
Author(s):  
Rachelle Salomon ◽  
John Franks ◽  
Elena A. Govorkova ◽  
Natalia A. Ilyushina ◽  
Hui-Ling Yen ◽  
...  
Keyword(s):  
Reverse Genetics ◽  
Mammalian Species ◽  
Polymerase Activity ◽  
Human Case ◽  
Influenza Viruses ◽  
Species Barrier ◽  
Ns Gene ◽  
H5n1 Influenza ◽  
High Virulence ◽  
Pandemic Threat

H5N1 influenza viruses transmitted from poultry to humans in Asia cause high mortality and pose a pandemic threat. Viral genes important for cell tropism and replication efficiency must be identified to elucidate and target virulence factors. We applied reverse genetics to generate H5N1 reassortants combining genes of lethal A/Vietnam/1203/04 (VN1203), a fatal human case isolate, and nonlethal A/chicken/Vietnam/C58/04 (CH58) and tested their pathogenicity in ferrets and mice. The viruses' hemagglutinins have six amino acids differences, identical cleavage sites, and avian-like α-(2,3)–linked receptor specificity. Surprisingly, exchanging hemagglutinin and neuraminidase genes did not alter pathogenicity, but substituting CH58 polymerase genes completely attenuated VN1203 virulence and reduced viral polymerase activity. CH58's NS gene partially attenuated VN1203 in ferrets but not in mice. Our findings suggest that for high virulence in mammalian species an avian H5N1 virus with a cleavable hemagglutinin requires adaptive changes in polymerase genes to overcome the species barrier. Thus, novel antivirals targeting polymerase proteins should be developed.

scholarly journals The R251K Substitution in Viral Protein PB2 Increases Viral Replication and Pathogenicity of Eurasian Avian-like H1N1 Swine Influenza Viruses

Viruses ◽  
2020 ◽  
Vol 12 (1) ◽  
pp. 52 ◽  
Author(s):  
Mengkai Cai ◽  
Ruting Zhong ◽  
Chenxiao Qin ◽  
Zhiqing Yu ◽  
Xiaoyan Wen ◽  
...  
Keyword(s):  
Amino Acid ◽  
Reverse Genetics ◽  
H1n1 Virus ◽  
Swine Influenza ◽  
Polymerase Activity ◽  
Human Infection ◽  
Influenza Viruses ◽  
Epidemiological Surveillance ◽  
Single Amino Acid ◽  
Swine Industry

The Eurasian avian-like swine (EA) H1N1 virus has affected the Chinese swine industry, and human infection cases have been reported occasionally. However, little is known about the pathogenic mechanism of EA H1N1 virus. In this study, we compared the mouse pathogenicity of A/swine/Guangdong/YJ4/2014 (YJ4) and A/swine/Guangdong/MS285/2017 (MS285) viruses, which had similar genotype to A/Hunan/42443/2015 (HuN-like). None of the mice inoculated with 106 TCID50 of YJ4 survived at 7 days post infection, while the survival rate of the MS285 group was 100%. Therefore, a series of single fragment reassortants in MS285 background and two rescued wild-type viruses were generated by using the reverse genetics method, and the pathogenicity analysis revealed that the PB2 gene contributed to the high virulence of YJ4 virus. Furthermore, there were 11 amino acid differences in PB2 between MS285 and YJ4 identified by sequence alignment, and 11 single amino acid mutant viruses were generated in the MS285 background. We found that the R251K mutation significantly increased the virulence of MS285 in mice, contributed to high polymerase activity and enhanced viral genome transcription and replication. These results indicate that PB2-R251K contributes to the virulence of the EA H1N1 virus and provide new insight into future molecular epidemiological surveillance strategies.

scholarly journals The NS1 Gene Contributes to the Virulence of H5N1 Avian Influenza Viruses

2006 ◽  
Vol 80 (22) ◽  
pp. 11115-11123 ◽  
Author(s):  
Zejun Li ◽  
Yongping Jiang ◽  
Peirong Jiao ◽  
Aiqin Wang ◽  
Fengju Zhao ◽  
...  
Keyword(s):  
Amino Acid ◽  
Avian Influenza ◽  
Recombinant Virus ◽  
Influenza Viruses ◽  
Single Amino Acid ◽  
Amino Acid Difference ◽  
Ns1 Protein ◽  
Highly Pathogenic ◽  
H5n1 Influenza ◽  
Ns1 Gene

ABSTRACT In the present study, we explored the genetic basis underlying the virulence and host range of two H5N1 influenza viruses in chickens. A/goose/Guangdong/1/96 (GS/GD/1/96) is a highly pathogenic virus for chickens, whereas A/goose/Guangdong/2/96 (GS/GD/2/96) is unable to replicate in chickens. These two H5N1 viruses differ in sequence by only five amino acids mapping to the PA, NP, M1, and NS1 genes. We used reverse genetics to create four single-gene recombinants that contained one of the sequence-differing genes from nonpathogenic GS/GD/2/96 and the remaining seven gene segments from highly pathogenic GS/GD/1/96. We determined that the NS1 gene of GS/GD/2/96 inhibited the replication of GS/GD/1/96 in chickens, while the substitution of the PA, NP, or M gene did not change the highly pathogenic properties of GS/GD/1/96. Conversely, of the recombinant viruses generated in the GS/GD/2/96 background, only the virus containing the NS1 gene of GS/GD/1/96 was able to replicate and cause disease and death in chickens. The single-amino-acid difference in the sequence of these two NS1 genes resides at position 149. We demonstrate that a recombinant virus expressing the GS/GD/1/96 NS1 protein with Ala149 is able to antagonize the induction of interferon protein levels in chicken embryo fibroblasts (CEFs), but a recombinant virus carrying a Val149 substitution is not capable of the same effect. These results indicate that the NS1 gene is critical for the pathogenicity of avian influenza virus in chickens and that the amino acid residue Ala149 correlates with the ability of these viruses to antagonize interferon induction in CEFs.

scholarly journals Lethality to Ferrets of H5N1 Influenza Viruses Isolated from Humans and Poultry in 2004

2005 ◽  
Vol 79 (4) ◽  
pp. 2191-2198 ◽  
Author(s):  
Elena A. Govorkova ◽  
Jerold E. Rehg ◽  
Scott Krauss ◽  
Hui-Ling Yen ◽  
Yi Guan ◽  
...  
Keyword(s):  
Amino Acid ◽  
Influenza A ◽  
Influenza Viruses ◽  
Avian Species ◽  
Single Amino Acid ◽  
Amino Acid Difference ◽  
Upper Respiratory Tract ◽  
Influenza A Viruses ◽  
H5n1 Influenza ◽  
High Virus

ABSTRACT The 2004 outbreaks of H5N1 influenza viruses in Vietnam and Thailand were highly lethal to humans and to poultry; therefore, newly emerging avian influenza A viruses pose a continued threat, not only to avian species but also to humans. We studied the pathogenicity of four human and nine avian H5N1/04 influenza viruses in ferrets (an excellent model for influenza studies). All four human isolates were fatal to intranasally inoculated ferrets. The human isolate A/Vietnam/1203/04 (H5N1) was the most pathogenic isolate; the severity of disease was associated with a broad tissue tropism and high virus titers in multiple organs, including the brain. High fever, weight loss, anorexia, extreme lethargy, and diarrhea were observed. Two avian H5N1/04 isolates were as pathogenic as the human viruses, causing lethal systemic infections in ferrets. Seven of nine H5N1/04 viruses isolated from avian species caused mild infections, with virus replication restricted to the upper respiratory tract. All chicken isolates were nonlethal to ferrets. A sequence analysis revealed polybasic amino acids in the hemagglutinin connecting peptides of all H5N1/04 viruses, indicating that multiple molecular differences in other genes are important for a high level of virulence. Interestingly, the human A/Vietnam/1203/04 isolate had a lysine substitution at position 627 of PB2 and had one to eight amino acid changes in all gene products except that of the M1 gene, unlike the A/chicken/Vietnam/C58/04 and A/quail/Vietnam/36/04 viruses. Our results indicate that viruses that are lethal to mammals are circulating among birds in Asia and suggest that pathogenicity in ferrets, and perhaps humans, reflects a complex combination of different residues rather than a single amino acid difference.

scholarly journals A Single Point Mutation, Asn16→Lys, Dictates the Temperature-Sensitivity of the Reovirus tsG453 Mutant

Viruses ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 289
Author(s):  
Kathleen K. M. Glover ◽  
Danica M. Sutherland ◽  
Terence S. Dermody ◽  
Kevin M. Coombs
Keyword(s):  
Amino Acid ◽  
Temperature Sensitivity ◽  
Reverse Genetics ◽  
Core Protein ◽  
Single Point ◽  
Single Amino Acid ◽  
Single Point Mutation ◽  
Temperature Sensitive ◽  
Amino Acid Substitutions ◽  
Gene Encoding

Studies of conditionally lethal mutants can help delineate the structure-function relationships of biomolecules. Temperature-sensitive (ts) mammalian reovirus (MRV) mutants were isolated and characterized many years ago. Two of the most well-defined MRV ts mutants are tsC447, which contains mutations in the S2 gene encoding viral core protein σ2, and tsG453, which contains mutations in the S4 gene encoding major outer-capsid protein σ3. Because many MRV ts mutants, including both tsC447 and tsG453, encode multiple amino acid substitutions, the specific amino acid substitutions responsible for the ts phenotype are unknown. We used reverse genetics to recover recombinant reoviruses containing the single amino acid polymorphisms present in ts mutants tsC447 and tsG453 and assessed the recombinant viruses for temperature-sensitivity by efficiency-of-plating assays. Of the three amino acid substitutions in the tsG453 S4 gene, Asn16-Lys was solely responsible for the tsG453ts phenotype. Additionally, the mutant tsC447 Ala188-Val mutation did not induce a temperature-sensitive phenotype. This study is the first to employ reverse genetics to identify the dominant amino acid substitutions responsible for the tsC447 and tsG453 mutations and relate these substitutions to respective phenotypes. Further studies of other MRV ts mutants are warranted to define the sequence polymorphisms responsible for temperature sensitivity.

scholarly journals Mutations in the PA Protein of Avian H5N1 Influenza Viruses Affect Polymerase Activity and Mouse Virulence

2017 ◽  
pp. JVI.01557-17 ◽  
Author(s):  
Gongxun Zhong ◽  
Mai Quynh Le ◽  
Tiago J.S. Lopes ◽  
Peter Halfmann ◽  
Masato Hatta ◽  
...  
Keyword(s):  
Amino Acid ◽  
Case Fatality ◽  
Polymerase Activity ◽  
Influenza Viruses ◽  
Viral Polymerase ◽  
Highly Pathogenic ◽  
H5n1 Influenza ◽  
Increased Risk ◽  
Severe Infections ◽  
Viral Determinants

To study the influenza viral determinants of pathogenicity, we characterized two highly pathogenic avian H5N1 influenza viruses isolated in Vietnam in 2012 (A/duck/Vietnam/QT1480/2012; QT1480) and 2013 (A/duck/Vietnam/QT1728/2013; QT1728) and found that the activity of their polymerase complexes differed significantly, even though both viruses were highly pathogenic in mice. Further studies revealed that the PA-S343A/E347D mutations reduced viral polymerase activity and mouse virulence when tested in the genetic background of QT1728 virus. In contrast, the PA-343S/347E mutations increased the polymerase activity of QT1480 and the virulence of a low pathogenic H5N1 influenza virus. The PA-343S residue (which alone increased viral polymerase activity and mouse virulence significantly relative to viral replication complexes encoding PA-343A) is frequently found in H5N1 influenza viruses of several subclades; infection with a virus possessing this amino acid may pose an increased risk to humans.IMPORTANCEH5N1 influenza viruses cause severe infections in humans with a case fatality rate that exceeds 50%. The factors that determine the high virulence of these viruses in humans are not fully understood. Here, we identified two amino acid changes in the viral polymerase PA protein that affect the activity of the viral polymerase complex and virulence in mice. Infection with viruses possessing these amino acid changes may pose an increased risk to humans.

The Single E627K Amino Acid Substitution in PB2 Enhances the Pathogenicity of Wild-Bird-Origin H6N6 Subtype Avian Influenza Virus in Mice

2020 ◽  
Vol 7 (1) ◽  
Author(s):  
Miura H ◽  
◽  
Ozeki Y ◽  
Omatsu T ◽  
Katayama Y ◽  
...  
Keyword(s):  
Amino Acid ◽  
Avian Influenza ◽  
Wild Bird ◽  
Virus Transmission ◽  
Influenza Viruses ◽  
Whole Genome Sequence ◽  
Species Barrier ◽  
The Impact ◽  
Original Virus ◽  
Wild Waterfowl

Avian Influenza Viruses (AIVs) are harbored by wild waterfowl as a natural host, and there is a species barrier restricting virus transmission from birds to mammals, including humans. However, it has been reported that, through genetic mutations, AIVs occasionally infect mammals and acquire high pathogenicity. The Amino Acid (aa) substitution of glutamic acid to lysine at position 627 (E627K) in polymerase basic protein 2 (PB2) is one of the wellknown factors underlying mammalian adaptation. Although this substitution was previously observed in mammalian-adapted H5, H7, and H9 AIV subtypes, the impact of this mutation on the mammalian adaptation of other AIV subtypes is not fully verified. Here, we isolated the low pathogenic AIV subtype H6N6 from a wild bird fecal sample in Tokachi Subprefecture, Hokkaido, Japan. We passaged this H6N6 subtype in BALB/c mice four times and acquired the mouse-adapted virus. Whole-genome sequence analysis showed that the adapted virus had only one aa substitution (E627K) in PB2. The adapted virus-inoculated mice tended to show increased weight loss and mortality compared with the original virus-inoculated mice. The viral titer in the lungs of the adapted virus-inoculated mice was significantly higher than that of the original virus-inoculated mice. Additionally, the virus isolated from the lung of the original virus-inoculated mice with serious symptoms harbored the E627K substitution. Our findings indicate the possibility that the PB2 E627K substitution in H6N6 subtype AIV rapidly appears in mammalian hosts and contributes to the enhanced pathogenicity of this virus.

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