scholarly journals Insertion of Basic Amino Acids in the Hemagglutinin Cleavage Site of H4N2 Avian Influenza Virus (AIV)—Reduced Virus Fitness in Chickens is Restored by Reassortment with Highly Pathogenic H5N1 AIV

2020 ◽  
Vol 21 (7) ◽  
pp. 2353
Author(s):  
Marcel Gischke ◽  
Reiner Ulrich ◽  
Olanrewaju I. Fatola ◽  
David Scheibner ◽  
Ahmed H. Salaheldin ◽  
...  
Keyword(s):  
Amino Acids ◽  
Avian Influenza ◽  
Cleavage Site ◽  
Influenza Viruses ◽  
Viral Fitness ◽  
Highly Pathogenic ◽  
Basic Amino Acids ◽  
The Impact ◽  
Virus Fitness

Highly pathogenic (HP) avian influenza viruses (AIVs) are naturally restricted to H5 and H7 subtypes with a polybasic cleavage site (CS) in hemagglutinin (HA) and any AIV with an intravenous pathogenicity index (IVPI) ≥ 1.2. Although only a few non-H5/H7 viruses fulfill the criteria of HPAIV; it remains unclear why these viruses did not spread in domestic birds. In 2012, a unique H4N2 virus with a polybasic CS 322PEKRRTR/G329 was isolated from quails in California which, however, was avirulent in chickens. This is the only known non-H5/H7 virus with four basic amino acids in the HACS. Here, we investigated the virulence of this virus in chickens after expansion of the polybasic CS by substitution of T327R (322PEKRRRR/G329) or T327K (322PEKRRKR/G329) with or without reassortment with HPAIV H5N1 and H7N7. The impact of single mutations or reassortment on virus fitness in vitro and in vivo was studied. Efficient cell culture replication of T327R/K carrying H4N2 viruses increased by treatment with trypsin, particularly in MDCK cells, and reassortment with HPAIV H5N1. Replication, virus excretion and bird-to-bird transmission of H4N2 was remarkably compromised by the CS mutations, but restored after reassortment with HPAIV H5N1, although not with HPAIV H7N7. Viruses carrying the H4-HA with or without R327 or K327 mutations and the other seven gene segments from HPAIV H5N1 exhibited high virulence and efficient transmission in chickens. Together, increasing the number of basic amino acids in the H4N2 HACS was detrimental for viral fitness particularly in vivo but compensated by reassortment with HPAIV H5N1. This may explain the absence of non-H5/H7 HPAIV in poultry.

scholarly journals Insertion of Basic Amino Acids in the Hemagglutinin Cleavage Site of H4N2 Avian Influenza Virus (AIV) Reduced Virus Fitness in Chickens which is Restored by Reassortment with Highly Pathogenic H5N1 AIV

2020 ◽  
Author(s):  
Marcel Gischke ◽  
Reiner Ulrich ◽  
Olanrewaju I. Fatola ◽  
David Scheibner ◽  
Ahmed H. Salaheldin ◽  
...  
Keyword(s):  
Amino Acids ◽  
Avian Influenza ◽  
Cleavage Site ◽  
Influenza Viruses ◽  
Viral Fitness ◽  
Highly Pathogenic ◽  
Basic Amino Acids ◽  
The Impact ◽  
Virus Fitness

Highly pathogenic (HP) avian influenza viruses (AIVs) are naturally restricted to H5 and H7 subtypes with a polybasic cleavage site (CS) in the hemagglutinin (HA) and any AIV with an intravenous pathogenicity index (IVPI) ≥1.2. Only few non-H5/H7 viruses fulfill the criteria of HPAIVs; nevertheless, it remains unknown why these viruses did not spread in domestic birds. In 2012, a unique H4N2 virus with a polybasic CS 322PEKRRTR/G329 was isolated from quails in California which, however, was avirulent in chickens. This is the only known non-H5/H7 virus with four basic amino acids in the HACS. Here, we investigated the virulence of this virus in chickens after expansion of the polybasic CS by substitution of T327R (322PEKRRRR/G329) or T327K (322PEKRRKR/G329) with or without reassortment with HPAIVs H5N1 and H7N7. The impact of single mutations or reassortment on virus fitness in vitro and in vivo was studied. Efficient cell culture replication of T327R/K carrying H4N2 viruses increased by trypsin, particularly in MDCK cells, and reassortment with HPAIV H5N1. Likewise, replication, virus excretion and bird-to-bird transmission of H4N2 was remarkably compromised by the CS mutations, but restored after reassortment with HPAIV H5N1, although not with HPAIV H7N7. Viruses carrying the H4-HA with or without R327 or K327 mutations and the other gene segments from HPAIV H5N1 exhibited high virulence and efficient transmission in chickens. Together, increasing the number of basic amino acids in the H4N2 HACS was detrimental for viral fitness particularly in vivo but compensated by reassortment with HPAIV H5N1. This may explain the absence of non-H5/H7 HPAIVs in poultry.

scholarly journals Molecular Determinants within the Surface Proteins Involved in the Pathogenicity of H5N1 Influenza Viruses in Chickens

2004 ◽  
Vol 78 (18) ◽  
pp. 9954-9964 ◽  
Author(s):  
Diane J. Hulse ◽  
Robert G. Webster ◽  
Rupert J. Russell ◽  
Daniel R. Perez
Keyword(s):  
Amino Acids ◽  
Avian Influenza ◽  
Cleavage Site ◽  
Glycosylation Site ◽  
Influenza Viruses ◽  
Surface Proteins ◽  
Avian Influenza Viruses ◽  
Highly Pathogenic ◽  
H5n1 Influenza ◽  
High Pathogenicity

ABSTRACT Although it is established that the cleavage site and glycosylation patterns in the hemagglutinin (HA) play important roles in determining the pathogenicity of H5 avian influenza viruses, some viruses exist that are not highly pathogenic despite possessing the known characteristics of high pathogenicity (i.e., their HA contains multiple basic amino acids at the cleavage site and has glycosylation patterns similar to that of the highly pathogenic H5 viruses). Currently little is known about the H5N1 viruses that fall into this intermediate category of pathogenicity. We have identified strains of H5N1 avian influenza viruses that have markers typical of high pathogenicity but distinctly differ in their ability to cause disease and death in chickens. By analyzing viruses constructed by reverse-genetic methods and containing recombinant HAs, we established that amino acids 97, 108, 126, 138, 212, and 217 of HA, in addition to those within the cleavage site, affect pathogenicity. Further investigation revealed that an additional glycosylation site within the neuraminidase (NA) protein globular head contributed to the high virulence of the H5N1 virus. Our findings are in agreement with previous observations that suggest that the activities of the HA and NA proteins are functionally linked.

scholarly journals A Unique Multibasic Proteolytic Cleavage Site and Three Mutations in the HA2 Domain Confer High Virulence of H7N1 Avian Influenza Virus in Chickens

2015 ◽  
Vol 90 (1) ◽  
pp. 400-411 ◽  
Author(s):  
El-Sayed M. Abdelwhab ◽  
Jutta Veits ◽  
Kerstin Tauscher ◽  
Mario Ziller ◽  
Jens P. Teifke ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Avian Influenza ◽  
Avian Influenza Virus ◽  
Cleavage Site ◽  
Influenza Viruses ◽  
Highly Pathogenic ◽  
Systemic Spread ◽  
H7n1 Virus

ABSTRACT In 1999, after circulation for a few months in poultry in Italy, low-pathogenic (LP) avian influenza (AI) H7N1 virus mutated into a highly pathogenic (HP) form by acquisition of a unique multibasic cleavage site (mCS), PEIPKGSRVRR*GLF (asterisk indicates the cleavage site), in the hemagglutinin (HA) and additional alterations with hitherto unknown biological function. To elucidate these virulence-determining alterations, recombinant H7N1 viruses carrying specific mutations in the HA of LPAI A/chicken/Italy/473/1999 virus (Lp) and HPAI A/chicken/Italy/445/1999 virus (Hp) were generated. Hp with a monobasic CS or carrying the HA of Lp induced only mild or no disease in chickens, thus resembling Lp. Conversely, Lp with the HA of Hp was as virulent and transmissible as Hp. While Lp with a multibasic cleavage site (Lp_CS445) was less virulent than Hp, full virulence was exhibited when HA2 was replaced by that of Hp. In HA2, three amino acid differences consistently detected between LP and HP H7N1 viruses were successively introduced into Lp_CS445. Q450L in the HA2 stem domain increased virulence and transmission but was detrimental to replication in cell culture, probably due to low-pH activation of HA. A436T and/or K536R restored viral replication in vitro and in vivo . Viruses possessing A436T and K536R were observed early in the HPAI outbreak but were later superseded by viruses carrying all three mutations. Together, besides the mCS, stepwise mutations in HA2 increased the fitness of the Italian H7N1 virus in vivo . The shift toward higher virulence in the field was most likely gradual with rapid optimization. IMPORTANCE In 1999, after 9 months of circulation of low-pathogenic (LP) avian influenza virus (AIV), a devastating highly pathogenic (HP) H7N1 AIV emerged in poultry, marking the largest epidemic of AIV reported in a Western country. The HPAIV possessed a unique multibasic cleavage site (mCS) complying with the minimum motif for HPAIV. The main finding in this report is the identification of three mutations in the HA2 domain that are required for replication and stability, as well as for virulence, transmission, and tropism of H7N1 in chickens. In addition to the mCS, Q450L was required for full virulence and transmissibility of the virus. Nonetheless, it was detrimental to virus replication and required A436T and/or K536R to restore replication, systemic spread, and stability. These results are important for better understanding of the evolution of highly pathogenic avian influenza viruses from low-pathogenic precursors.

scholarly journals SequenceAnalysis of Recent H7 Avian Influenza Viruses Associated with ThreeDifferent Outbreaks in Commercial Poultry in the UnitedStates

2003 ◽  
Vol 77 (24) ◽  
pp. 13399-13402 ◽  
Author(s):  
Erica Spackman ◽  
Dennis A. Senne ◽  
Sherrill Davison ◽  
David L. Suarez
Keyword(s):  
Amino Acids ◽  
Avian Influenza ◽  
Cleavage Site ◽  
Influenza Viruses ◽  
Avian Influenza Viruses ◽  
Receptor Binding Site ◽  
Highly Pathogenic ◽  
Genetic Features ◽  
Commercial Poultry ◽  
Live Bird

ABSTRACT The hemagglutinin (HA) and neuraminidase (NA) genes of H7 avian influenza virus (AIV) isolated between 1994 and 2002 from live-bird markets (LBMs) in the northeastern United States and from three outbreaks in commercial poultry have been characterized. Phylogenetic analysis of the HA and NA genes demonstrates that the isolates from commercial poultry were closely related to the viruses circulating in the LBMs. Also, since 1994, two distinguishing genetic features have appeared in this AIV lineage: a deletion of 17 amino acids in the NA protein stalk region and a deletion of 8 amino acids in the HA1 protein which is putatively in part of the receptor binding site. Furthermore, analysis of the HA cleavage site amino acid sequence, a marker for pathogenicity in chickens and turkeys, shows a progression toward a cleavage site sequence that fulfills the molecular criteria for highly pathogenic AIV.

scholarly journals H5N2 Avian Influenza Outbreak in Texas in 2004: the First Highly Pathogenic Strain in the United States in 20 Years?

2005 ◽  
Vol 79 (17) ◽  
pp. 11412-11421 ◽  
Author(s):  
Chang-Won Lee ◽  
David E. Swayne ◽  
Jose A. Linares ◽  
Dennis A. Senne ◽  
David L. Suarez
Keyword(s):  
United States ◽  
Amino Acid ◽  
Avian Influenza ◽  
Cleavage Site ◽  
Basic Amino Acid ◽  
The United States ◽  
Influenza Viruses ◽  
Single Point Mutation ◽  
Highly Pathogenic

ABSTRACT In early 2004, an H5N2 avian influenza virus (AIV) that met the molecular criteria for classification as a highly pathogenic AIV was isolated from chickens in the state of Texas in the United States. However, clinical manifestations in the affected flock were consistent with avian influenza caused by a low-pathogenicity AIV and the representative virus (A/chicken/Texas/298313/04 [TX/04]) was not virulent for experimentally inoculated chickens. The hemagglutinin (HA) gene of the TX/04 isolate was similar in sequence to A/chicken/Texas/167280-4/02 (TX/02), a low-pathogenicity AIV isolate recovered from chickens in Texas in 2002. However, the TX/04 isolate had one additional basic amino acid at the HA cleavage site, which could be attributed to a single point mutation. The TX/04 isolate was similar in sequence to TX/02 isolate in several internal genes (NP, M, and NS), but some genes (PA, PB1, and PB2) had sequence of a clearly different origin. The TX/04 isolate also had a stalk deletion in the NA gene, characteristic of a chicken-adapted AIV. By analyzing viruses constructed by in vitro mutagenesis followed by reverse genetics, we found that the pathogenicity of the TX/04 virus could be increased in vitro and in vivo by the insertion of an additional basic amino acid at the HA cleavage site and not by the loss of a glycosylation site near the cleavage site. Our study provides the genetic and biologic characteristics of the TX/04 isolate, which highlight the complexity of the polygenic nature of the virulence of influenza viruses.

scholarly journals Novel Highly Pathogenic Avian A(H5N2) and A(H5N8) Influenza Viruses of Clade 2.3.4.4 from North America Have Limited Capacity for Replication and Transmission in Mammals

mSphere ◽  
2016 ◽  
Vol 1 (2) ◽  
Author(s):  
Bryan S. Kaplan ◽  
Marion Russier ◽  
Trushar Jeevan ◽  
Bindumadhav Marathe ◽  
Elena A. Govorkova ◽  
...  
Keyword(s):  
North America ◽  
Avian Influenza ◽  
North American ◽  
Influenza Viruses ◽  
Morbidity And Mortality ◽  
Avian Influenza Viruses ◽  
Highly Pathogenic ◽  
The North ◽  
Domestic Poultry ◽  
The Impact

ABSTRACT Highly pathogenic H5 influenza viruses have been introduced into North America from Asia, causing extensive morbidity and mortality in domestic poultry. The introduced viruses have reassorted with North American avian influenza viruses, generating viral genotypes not seen on other continents. The experiments and analyses presented here were designed to assess the impact of this genetic diversification on viral phenotypes, particularly as regards mammalian hosts, by comparing the North American viruses with their Eurasian precursor viruses. Highly pathogenic influenza A(H5N8) viruses from clade 2.3.4.4 were introduced to North America by migratory birds in the fall of 2014. Reassortment of A(H5N8) viruses with avian viruses of North American lineage resulted in the generation of novel A(H5N2) viruses with novel genotypes. Through sequencing of recent avian influenza viruses, we identified PB1 and NP gene segments very similar to those in the viruses isolated from North American waterfowl prior to the introduction of A(H5N8) to North America, highlighting these bird species in the origin of reassortant A(H5N2) viruses. While they were highly virulent and transmissible in poultry, we found A(H5N2) viruses to be low pathogenic in mice and ferrets, and replication was limited in both hosts compared with those of recent highly pathogenic avian influenza (HPAI) H5N1 viruses. Molecular characterization of the hemagglutinin protein from A(H5N2) viruses showed that the receptor binding preference, cleavage, and pH of activation were highly adapted for replication in avian species and similar to those of other 2.3.4.4 viruses. In addition, North American and Eurasian clade 2.3.4.4 H5NX viruses replicated to significantly lower titers in differentiated normal human bronchial epithelial cells than did seasonal human A(H1N1) and highly pathogenic A(H5N1) viruses isolated from a human case. Thus, despite their having a high impact on poultry, our findings suggest that the recently emerging North American A(H5N2) viruses are not expected to pose a substantial threat to humans and other mammals without further reassortment and/or adaptation and that reassortment with North American viruses has not had a major impact on viral phenotype. IMPORTANCE Highly pathogenic H5 influenza viruses have been introduced into North America from Asia, causing extensive morbidity and mortality in domestic poultry. The introduced viruses have reassorted with North American avian influenza viruses, generating viral genotypes not seen on other continents. The experiments and analyses presented here were designed to assess the impact of this genetic diversification on viral phenotypes, particularly as regards mammalian hosts, by comparing the North American viruses with their Eurasian precursor viruses.

scholarly journals Characterization of Low-Pathogenicity H5N1 Avian Influenza Viruses from North America

2007 ◽  
Vol 81 (21) ◽  
pp. 11612-11619 ◽  
Author(s):  
Erica Spackman ◽  
David E. Swayne ◽  
David L. Suarez ◽  
Dennis A. Senne ◽  
Janice C. Pedersen ◽  
...  
Keyword(s):  
North America ◽  
Avian Influenza ◽  
North American ◽  
Wild Bird ◽  
Geographic Origin ◽  
Influenza Viruses ◽  
Highly Pathogenic ◽  
Infectious Dose ◽  
Antigenic Relatedness

ABSTRACT Wild-bird surveillance in North America for avian influenza (AI) viruses with a goal of early identification of the Asian H5N1 highly pathogenic AI virus has identified at least six low-pathogenicity H5N1 AI viruses between 2004 and 2006. The hemagglutinin (HA) and neuraminidase (NA) genes from all 6 H5N1 viruses and an additional 38 North American wild-bird-origin H5 subtype and 28 N1 subtype viruses were sequenced and compared with sequences available in GenBank by phylogenetic analysis. Both HA and NA were phylogenetically distinct from those for viruses from outside of North America and from those for viruses recovered from mammals. Four of the H5N1 AI viruses were characterized as low pathogenicity by standard in vivo pathotyping tests. One of the H5N1 viruses, A/MuteSwan/MI/451072-2/06, was shown to replicate to low titers in chickens, turkeys, and ducks. However, transmission of A/MuteSwan/MI/451072-2/06 was more efficient among ducks than among chickens or turkeys based on virus shed. The 50% chicken infectious dose for A/MuteSwan/MI/451072-2/06 and three other wild-waterfowl-origin H5 viruses were also determined and were between 105.3 and 107.5 50% egg infective doses. Finally, seven H5 viruses representing different phylogenetic clades were evaluated for their antigenic relatedness by hemagglutination inhibition assay, showing that the antigenic relatedness was largely associated with geographic origin. Overall, the data support the conclusion that North American H5 wild-bird-origin AI viruses are low-pathogenicity wild-bird-adapted viruses and are antigenically and genetically distinct from the highly pathogenic Asian H5N1 virus lineage.

scholarly journals Pathogenesis and Transmission Assessments of Two H7N8 Influenza A Viruses Recently Isolated from Turkey Farms in Indiana Using Mouse and Ferret Models

2016 ◽  
Vol 90 (23) ◽  
pp. 10936-10944 ◽  
Author(s):  
Xiangjie Sun ◽  
Jessica A. Belser ◽  
Joanna A. Pulit-Penaloza ◽  
Hui Zeng ◽  
Amanda Lewis ◽  
...  
Keyword(s):  
Avian Influenza ◽  
Influenza A ◽  
Influenza Viruses ◽  
Avian Influenza Viruses ◽  
Highly Pathogenic ◽  
Pathogenic Avian Influenza ◽  
Domestic Poultry ◽  
Commercial Turkey

ABSTRACTAvian influenza A H7 viruses have caused multiple outbreaks in domestic poultry throughout North America, resulting in occasional infections of humans in close contact with affected birds. In early 2016, the presence of H7N8 highly pathogenic avian influenza (HPAI) viruses and closely related H7N8 low-pathogenic avian influenza (LPAI) viruses was confirmed in commercial turkey farms in Indiana. These H7N8 viruses represent the first isolation of this subtype in domestic poultry in North America, and their virulence in mammalian hosts and the potential risk for human infection are largely unknown. In this study, we assessed the ability of H7N8 HPAI and LPAI viruses to replicatein vitroin human airway cells andin vivoin mouse and ferret models. Both H7N8 viruses replicated efficientlyin vitroandin vivo, but they exhibited substantial differences in disease severity in mammals. In mice, while the H7N8 LPAI virus largely remained avirulent, the H7N8 HPAI virus exhibited greater infectivity, virulence, and lethality. Both H7N8 viruses replicated similarly in ferrets, but only the H7N8 HPAI virus caused moderate weight loss, lethargy, and mortality. The H7N8 LPAI virus displayed limited transmissibility in ferrets placed in direct contact with an inoculated animal, while no transmission of H7N8 HPAI virus was detected. Our results indicate that the H7N8 avian influenza viruses from Indiana are able to replicate in mammals and cause severe disease but with limited transmission. The recent appearance of H7N8 viruses in domestic poultry highlights the need for continued influenza surveillance in wild birds and close monitoring of the potential risk to human health.IMPORTANCEH7 influenza viruses circulate in wild birds in the United States, but when the virus emerges in domestic poultry populations, the frequency of human exposure and the potential for human infections increases. An H7N8 highly pathogenic avian influenza (HPAI) virus and an H7N8 low-pathogenic avian influenza (LPAI) virus were recently isolated from commercial turkey farms in Indiana. To determine the risk that these influenza viruses pose to humans, we assessed their pathogenesis and transmissionin vitroand in mammalian models. We found that the H7N8 HPAI virus exhibited enhanced virulence, and although transmission was only observed with the H7N8 LPAI virus, the ability of this H7 virus to transmit in a mammalian host and quickly evolve to a more virulent strain is cause for concern. Our findings offer important insight into the potential for emerging H7 avian influenza viruses to acquire the ability to cause disease and transmit among mammals.

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