Emergence and Selection of a Highly Pathogenic Avian Influenza H7N3 Virus

Low pathogenic avian influenza (LPAI) viruses of subtypes H5 and H7 have the ability to spontaneously mutate into highly pathogenic (HPAI) variants, causing high mortality in poultry. The switch to high pathogenicity is poorly understood, and evidence from the field is scarce. This study provides direct evidence for LPAI to HPAI mutation from a turkey farm during an H7N3 outbreak in the Netherlands. At the farm, only mild clinical symptoms were reported, but the intravenous pathogenicity index measured for the virus isolated from the infected turkeys was consistent with a highly pathogenic virus. Using deep-sequencing, we showed that a minority of HPAI virus (0.06%) was present in the virus preparation. Analysis of different organs of the infected turkeys showed the highest percentage of HPAI virus was present in the lung (4.4%). The HPAI virus contained a 12-nucleotide insertion in the hemagglutinin (HA) cleavage site that was introduced by a single event, as no intermediates with shorter inserts were identified. The HPAI virus was rapidly selected in chickens, after both intravenous and intranasal/intratracheal inoculation with the mixed virus preparation. Full-genome sequencing revealed that both pathotypes contained a deletion in the stalk region of the neuraminidase protein. We identified mutations in HA and polymerase basic protein 1 (PB1) in the HPAI virus, which were already present as minority variants in the LPAI virus. Our findings provide more insight into the molecular changes and mechanisms involved in the emergence of HPAI viruses. This knowledge may be used for the timely identification of LPAI viruses that pose a risk of becoming highly pathogenic in the field.

Vaccination with Consensus H7 Elicits Broadly Reactive and Protective Antibodies against Eurasian and North American Lineage H7 Viruses

H7 subtype avian influenza viruses have caused outbreaks in poultry, and even human infection, for decades in both Eurasia and North America. Although effective vaccines offer the best protection against avian influenza viruses, antigenically distinct Eurasian and North American lineage subtype H7 viruses require the development of cross-protective vaccine candidates. In this study, a methodology called computationally optimized broadly reactive antigen (COBRA) was used to develop four consensus H7 antigens (CH7-22, CH7-24, CH7-26, and CH7-28). In vitro experiments confirmed the binding of monoclonal antibodies to the head and stem domains of cell surface-expressed consensus HAs, indicating display of their antigenicity. Immunization with DNA vaccines encoding the four antigens was evaluated in a mouse model. Broadly reactive antibodies against H7 viruses from Eurasian and North American lineages were elicited and detected by binding, inhibition, and neutralizing analyses. Further infection with Eurasian H7N9 and North American H7N3 virus strains confirmed that CH7-22 and CH7-24 conferred the most effective protection against hetero-lethal challenge. Our data showed that the consensus H7 vaccines elicit a broadly reactive, protective response against Eurasian and North American lineage H7 viruses, which are suitable for development against other zoonotic influenza viruses.

Epizootic of highly pathogenic H7N3 Avian Influenza in an ecologic reserve in Mexico

SummaryThis report includes a 2015 epizootic of highly pathogenic H7N3 avian influenza virus among captive and wild birds at “El Zapotal” ecologic reserve, located in the state of Chiapas, Mexico. Epidemiological control measures were implemented to prevent virus dissemination. The infection with the highly pathogenic H7N3 virus was detected predominantly among Plain Chachalaca (Ortalis vetula), with occasional detections in a White-fronted Parrot (Amazona albifrons) and a single Clay-colored Thrush (Turdus grayi). Here, we describe the characteristics of the outbreak environment, the surveillance strategy, the biosecurity measures, and the evaluation of the site, including external farms. These actions, timely implemented by the veterinary authorities, helped to contain the outbreak beyond the ecologic reserve. This contingency showed the importance of developing a more complete analysis of the existing risks and the challenges to implement minimal biosecurity measures in these facilities.

Emergence and Selection of a Highly Pathogenic Avian Influenza H7N3 Virus

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.

A North American H7N3 Influenza Virus Supports Reassortment with 2009 Pandemic H1N1 and Induces Disease in Mice without Prior Adaptation

ABSTRACTReassortment between H5 or H9 subtype avian and mammalian influenza A viruses (IAV) can generate a novel virus that causes disease and transmits between mammals. Such information is currently not available for H7 subtype viruses. We evaluated the ability of a low-pathogenicity North American avian H7N3 virus (A/shorebird/Delaware/22/2006) to reassort with mammalian or avian viruses using a plasmid-based competition assay. In addition to genome segments derived from an avian H7N9 virus, the H7N3 virus reassorted efficiently with the PB2, NA, and M segments from the 2009 pandemic H1N1 (PH1N1) virus.In vitroandin vivoevaluation of the H7N3:PH1N1 (7 + 1) reassortant viruses revealed that the PB2, NA, or M segments fromPH1N1 largely do not attenuate the H7N3 virus, whereas the PB1, PA, NP, or NS genome segments fromPH1N1 do. Additionally, we assessed the functionality of the H7N3:PH1N1 7 + 1 reassortant viruses by measuring the inflammatory responsein vivo. We found that infection with wild-type H7N3 resulted in increased inflammatory cytokine production relative to that seen with thePH1N1 strain and that the increase was further exacerbated by substitution ofPH1N1 PB2 but not NA or M. Finally, we assessed if any adaptations occurred in the individually substituted segments afterin vivoinoculation and found no mutations, suggesting thatPH1N1 PB2, NA, and M are genetically stable in the background of this H7N3 virus. Taking the data together, we demonstrate that a North American avian H7N3 IAV is genetically and functionally compatible with multiple gene segments from the 2009 pandemic influenza virus strain without prior adaptation.IMPORTANCEThe 2009 pandemic H1N1 virus continues to circulate and reassort with other influenza viruses, creating novel viruses with increased replication and transmission potential in humans. Previous studies have found that this virus can also reassort with H5N1 and H9N2 avian influenza viruses. We now show that several genome segments of the 2009 H1N1 virus are also highly compatible with a low-pathogenicity avian H7N3 virus and that these reassortant viruses are stable and not attenuated in an animal model. These results highlight the potential for reassortment of H1N1 viruses with avian influenza virus and emphasize the need for continued surveillance of influenza viruses in areas of cocirculation between avian, human, and swine viruses.

Serological comparison of antibodies to avian influenza viruses, subtypes H5N2, H6N1, H7N3 and H7N9 between poultry workers and non-poultry workers in Taiwan in 2012

SUMMARYIn Taiwan, avian influenza virus (AIV) subtypes H5N2, H6N1 and H7N3 have been identified in domestic poultry, and several strains of these subtypes have become endemic in poultry. To evaluate the potential of avian-to-human transmission due to occupational exposure, an exploratory analysis of AIV antibody status in poultry workers was conducted. We enrolled 670 poultry workers, including 335 live poultry vendors (LPVs), 335 poultry farmers (PFs), and 577 non-poultry workers (NPWs). Serum antibody titres against various subtypes of viruses were analysed and compared. The overall seropositivity rates in LPVs and PFs were 2·99% (10/335) and 1·79% (6/335), respectively, against H5N2; and 0·6% (2/335) and 1·19% (4/335), respectively, for H7N3 virus. Of NPWs, 0·35% (2/577) and 0·17% (1/577) were seropositive for H5N2 and H7N3, respectively. Geographical analysis revealed that poultry workers whose workplaces were near locations where H5N2 outbreaks in poultry have been reported face greater risks of being exposed to viruses that result in elevated H5N2 antibody titres. H6N1 antibodies were detected in only one PF, and no H7N9 antibodies were found in the study subjects. Subclinical infections caused by H5N2, H6N1 and H7N3 viruses were thus identified in poultry workers in Taiwan. Occupational exposure is associated with a high risk of AIV infection, and the seroprevalence of particular avian influenza strains in humans reflects the endemic strains in poultry in this region.