Evolution of high pathogenicity of H5 avian influenza virus: haemagglutinin cleavage site selection of reverse-genetics mutants during passage in chickens

ABSTRACT Low-pathogenicity avian influenza (LPAI) viruses of subtypes H5 and H7 have the ability to spontaneously mutate to highly pathogenic (HPAI) virus variants, causing high mortality in poultry. The highly pathogenic phenotype is caused by mutation of the hemagglutinin (HA) cleavage site, but additional mutations may play a role. Evidence from the field for the switch to high pathogenicity remains scarce. This study provides direct evidence for LPAI-to-HPAI virus mutation during H7N3 infection of a turkey farm in the Netherlands. No severe clinical symptoms were reported at the farm, but deep sequencing of isolates from the infected turkeys revealed a minority of HPAI virus sequences (0.06%) in the virus population. The HPAI virus contained a 12-nucleotide insertion in the HA cleavage site that was likely introduced by a single event as no intermediates with shorter inserts were identified. This suggests nonhomologous recombination as the mechanism of insertion. Analysis of different organs of the infected turkeys showed the largest amount of HPAI virus in the lung (4.4%). The HPAI virus was rapidly selected in experimentally infected chickens after both intravenous and intranasal/intratracheal inoculation with a mixed virus preparation. Full-genome sequencing revealed that both pathotypes contained a deletion in the stalk region of the neuraminidase protein. We identified additional mutations in HA and polymerase basic protein 1 (PB1) in the HPAI virus, which were already present as minority variants in the LPAI virus population. Our findings provide more insight into the molecular changes and mechanisms involved in the emergence and selection of HPAI viruses. IMPORTANCE Low-pathogenicity avian influenza (LPAI) viruses circulate in wild birds and can be transmitted to poultry. LPAI viruses can mutate to become highly pathogenic avian influenza (HPAI) viruses causing severe disease and death in poultry. Little is known about this switch to high pathogenicity. We isolated an LPAI H7N3 virus from an infected turkey farm and showed that this contains small amounts of HPAI virus. The HPAI virus rapidly outcompeted the LPAI virus in chickens that were experimentally infected with this mixture of viruses. We analyzed the genome sequences of the LPAI and HPAI viruses and identified several changes that may be important for a virus to become highly pathogenic. This knowledge may be used for timely identification of LPAI viruses that pose a risk of becoming highly pathogenic in the field.

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The PA Subunit of the Influenza Virus Polymerase Complex Affects Replication and Airborne Transmission of the H9N2 Subtype Avian Influenza Virus

Viruses ◽

10.3390/v11010040 ◽

2019 ◽

Vol 11 (1) ◽

pp. 40 ◽

Cited By ~ 1

Author(s):

Mengchan Hao ◽

Shaojie Han ◽

Dan Meng ◽

Rong Li ◽

Jing Lin ◽

...

Keyword(s):

Influenza Virus ◽

Avian Influenza ◽

Avian Influenza Virus ◽

Guinea Pigs ◽

Influenza A ◽

Reverse Genetics ◽

Species Barrier ◽

Airborne Transmission ◽

H9n2 Subtype ◽

Influenza A H1n1

The polymerase acidic (PA) protein is the third subunit of the influenza A virus polymerase. In recent years, studies have shown that PA plays an important role in overcoming the host species barrier and host adaptation of the avian influenza virus (AIV). The objective of this study was to elucidate the role of the PA subunit on the replication and airborne transmission of the H9N2 subtype AIV. By reverse genetics, a reassortant rSD01-PA was derived from the H9N2 subtype AIV A/Chicken/Shandong/01/2008 (SD01) by introducing the PA gene from the pandemic influenza A H1N1 virus A/swine/Shandong/07/2011 (SD07). Specific pathogen-free (SPF) chickens and guinea pigs were selected as the animal models for replication and aerosol transmission studies. Results show that rSD01-PA lost the ability of airborne transmission among SPF chickens because of the single substitution of the PA gene. However, rSD01-PA could infect guinea pigs through direct contact, while the parental strain SD01 could not, even though the infection of rSD01-PA could not be achieved through aerosol. In summary, our results indicate that the protein encoded by the PA gene plays a key role in replication and airborne transmission of the H9N2 subtype AIV.

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High-Pathogenicity Avian Influenza Virus in the Reproductive Tract of Chickens

Veterinary Pathology ◽

10.1177/0300985813490755 ◽

2013 ◽

Vol 50 (6) ◽

pp. 956-960 ◽

Cited By ~ 15

Author(s):

M.Sá e Silva ◽

D. R. Rissi ◽

M. Pantin-Jackwood ◽

D. E. Swayne

Keyword(s):

Influenza Virus ◽

Avian Influenza ◽

Avian Influenza Virus ◽

Reproductive Tract ◽

High Pathogenicity

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Contributions of the Avian Influenza Virus HA, NA, and M2 Surface Proteins to the Induction of Neutralizing Antibodies and Protective Immunity

Journal of Virology ◽

10.1128/jvi.02135-09 ◽

2009 ◽

Vol 84 (5) ◽

pp. 2408-2420 ◽

Cited By ~ 46

Author(s):

Baibaswata Nayak ◽

Sachin Kumar ◽

Joshua M. DiNapoli ◽

Anandan Paldurai ◽

Daniel R. Perez ◽

...

Keyword(s):

Influenza Virus ◽

Avian Influenza ◽

Avian Influenza Virus ◽

Neutralizing Antibodies ◽

Protective Immunity ◽

Reverse Genetics ◽

Severe Disease ◽

Serum Antibody ◽

Surface Proteins ◽

Serum Antibodies

ABSTRACT Highly pathogenic avian influenza virus (HPAIV) subtype H5N1 causes severe disease and mortality in poultry. Increased transmission of H5N1 HPAIV from birds to humans is a serious threat to public health. We evaluated the individual contributions of each of the three HPAIV surface proteins, namely, the hemagglutinin (HA), the neuraminidase (NA), and the M2 proteins, to the induction of HPAIV-neutralizing serum antibodies and protective immunity in chickens. Using reverse genetics, three recombinant Newcastle disease viruses (rNDVs) were engineered, each expressing the HA, NA, or M2 protein of H5N1 HPAIV. Chickens were immunized with NDVs expressing a single antigen (HA, NA, and M2), two antigens (HA+NA, HA+M2, and NA+M2), or three antigens (HA+NA+M2). Immunization with HA or NA induced high titers of HPAIV-neutralizing serum antibodies, with the response to HA being greater, thus identifying HA and NA as independent neutralization antigens. M2 did not induce a detectable neutralizing serum antibody response, and inclusion of M2 with HA or NA reduced the magnitude of the response. Immunization with HA alone or in combination with NA induced complete protection against HPAIV challenge. Immunization with NA alone or in combination with M2 did not prevent death following challenge, but extended the time period before death. Immunization with M2 alone had no effect on morbidity or mortality. Thus, there was no indication that M2 is immunogenic or protective. Furthermore, inclusion of NA in addition to HA in a vaccine preparation for chickens may not enhance the high level of protection provided by HA.

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