Multiple Reassorted Viruses as Cause of Highly Pathogenic Avian Influenza A(H5N8) Virus Epidemic, the Netherlands, 2016.

In 2016, an epidemic of highly pathogenic avian influenza A virus subtype H5N8 in the Netherlands caused mass deaths among wild birds, and several commercial poultry farms and captive bird holdings were affected. We performed complete genome sequencing to study the relationship between the wild bird and poultry viruses. Phylogenetic analysis showed that the viruses are related to H5 clade 2.3.4.4 viruses detected in Russia in May 2016 but contained novel polymerase basic 2 and nucleoprotein gene segments and 2 different variants of the polymerase acidic segment. Molecular dating suggests that the reassortment events most likely occurred in wild birds in Russia or Mongolia. Furthermore, 2 genetically distinct H5N5 reassortant viruses were detected in wild birds in the Netherlands. Our study provides evidence for fast and continuing reassortment of H5 clade 2.3.4.4 viruses, which might lead to rapid changes in virus characteristics, such as pathogenicity, infectivity, transmission, and zoonotic potential.

Tissue tropism and pathology of highly pathogenic avian influenza H5N6 virus in chickens and Pekin ducks.

The highly pathogenic avian influenza (HPAI) H5N6 virus caused outbreaks on commercial poultry farms in the Netherlands in 2017-2018, holding chickens and Pekin ducks. Intravenous pathogenicity index (IVPI) tests confirmed the high pathogenicity of the virus. Tissues derived from birds from infected farms (natural infection) and IVPI tests (experimental infection) were used to compare histopathology and virus distribution in both poultry species. After natural infection in chickens, histopathologic changes were present in the respiratory tract and several internal organs in both chickens and Pekin ducks. Viral antigen expression in the tissues of chickens varied from that in ducks. Virus expression was found in epithelial, mononuclear and endothelial cells in chickens. In contrast to the major role infected endothelial cells seem to play in systemic infections of chickens, in ducks the number of infected endothelial cells was very limited. Therefore, endothelial cell infection likely does not play a major role in systemic infection and disease progression in HPAI H5N6 virus infected Pekin ducks.

Detection of Low Pathogenic Avian Influenza Virus Subtype H10N7 in Poultry and Environmental Water Samples During a Clinical Outbreak in Commercial Free-Range Layers, Netherlands 2017.

Wild birds are the natural reservoir of the avian influenza virus (AIV) and may transmit AIV to poultry via direct contact or indirectly through the environment. In the Netherlands, a clinically suspected free-range layer flock was reported to the veterinary authorities by the farmer. Increased mortality, a decreased feed intake, and a drop in egg production were observed. Subsequently, an infection with low pathogenic avian influenza virus was detected. This study describes the diagnostic procedures used for detection and subtyping of the virus. In addition to routine diagnostics, the potential of two different environmental diagnostic methods was investigated for detecting AIV in surface water. AIV was first detected using rRT-PCR and isolated from tracheal and cloacal swabs collected from the hens. The virus was subtyped as H10N7. Antibodies against the virus were detected in 28 of the 31 sera tested. An intravenous pathogenicity index (IVPI) experiment was performed, but no clinical signs (IVPI = 0) were observed. Post-mortem examination and histology confirmed the AIV infection. Multiple water samples were collected longitudinally from the free-range area and waterway near the farm. Both environmental diagnostic methods allowed the detection of the H10N7 virus, demonstrating the potential of these methods in detection of AIV. The described methods could be a useful additional procedure for AIV surveillance in water-rich areas with large concentrations of wild birds or in areas around poultry farms. In addition, these methods could be used as a tool to test if the environment or free-range area is virus-free again, at the end of an AIV epidemic.

Spread of Highly Pathogenic Avian Influenza (HPAI) H5N5 Viruses in Europe in 2016-2017 Appears Related to the Timing of Reassortment Events.

During the epizootic of highly pathogenic avian influenza (HPAI) H5N8 virus in Europe in 2016-2017, HPAI viruses of subtype H5N5 were also isolated. However, the detection of H5N5 viruses was limited compared to H5N8. In this study, we show that the genetic constellation of a newly isolated H5N5 virus is different from two genotypes previously identified in the Netherlands. The introduction and spread of the three H5N5 genotypes in Europe was studied using spatiotemporal and genetic analysis. This demonstrated that the genotypes were isolated in distinguishable phases of the epizootic, and suggested multiple introductions of H5N5 viruses into Europe followed by local spread. We estimated the timing of the reassortment events, which suggested that the genotypes emerged after the start of autumn migration. This may have prevented large-scale spread of the H5N5 viruses on wild bird breeding sites before introduction into Europe. Experiments in primary chicken and duck cells revealed only minor differences in cytopathogenicity and replication kinetics between H5N5 genotypes and H5N8. These results suggest that the limited spread of HPAI H5N5 viruses is related to the timing of the reassortment events rather than changes in virus pathogenicity or replication kinetics.

Genetic analysis identifies potential transmission of low pathogenic avian influenza viruses between poultry farms.

Poultry can become infected with low pathogenic avian influenza (LPAI) viruses via (in)direct contact with infected wild birds or by transmission of the virus between farms. This study combines routinely collected surveillance data with genetic analysis to assess the contribution of between-farm transmission to the overall incidence of LPAI virus infections in poultry. Over a 10-year surveillance period, we identified 35 potential cases of between-farm transmission in the Netherlands, of which 10 formed geographical clusters. A total of 21 LPAI viruses were isolated from nine potential between-farm transmission cases, which were further studied by genetic and epidemiological analysis. Whole genome sequence analysis identified close genetic links between infected farms in seven cases. The presence of identical deletions in the neuraminidase stalk region and minority variants provided additional indications of between-farm transmission. Spatiotemporal analysis demonstrated that genetically closely related viruses were detected within a median time interval of 8 days, and the median distance between the infected farms was significantly shorter compared to farms infected with genetically distinct viruses (6.3 versus 69.0 km; p < 0.05). The results further suggest that between-farm transmission was not restricted to holdings of the same poultry type and not related to the housing system. Although separate introductions from the wild bird reservoir cannot be excluded, our study indicates that between-farm transmission occurred in seven of nine virologically analysed cases. Based on these findings, it is likely that between-farm transmission contributes considerably to the incidence of LPAI virus infections in poultry.

Susceptibility of Chickens to Low Pathogenic Avian Influenza (LPAI) Viruses of Wild Bird- and Poultry-Associated Subtypes.

Analysis of low pathogenic avian influenza (LPAI) viruses circulating in the Netherlands in a previous study revealed associations of specific hemagglutinin (HA) and neuraminidase (NA) subtypes with wild bird or poultry hosts. In this study, we identified putative host associations in LPAI virus internal proteins. We show that LPAI viruses isolated from poultry more frequently carried the allele A variant of the nonstructural protein (NS) gene, compared to wild bird viruses. We determined the susceptibility of chickens to wild bird-associated subtypes H3N8 and H4N6 and poultry-associated subtypes H8N4 and H9N2, carrying either NS allele A or B, in an infection experiment. We observed variations in virus shedding and replication patterns, however, these did not correlate with the predicted wild bird- or poultry-associations of the viruses. The experiment demonstrated that LPAI viruses of wild bird-associated subtypes can replicate in chickens after experimental infection, despite their infrequent detection in poultry. Although the NS1 protein is known to play a role in immune modulation, no differences were detected in the limited innate immune response to LPAI virus infection. This study contributes to a better understanding of the infection dynamics of LPAI viruses in chickens.

Circulation of low pathogenic avian influenza (LPAI) viruses in wild birds and poultry in the Netherlands, 2006-2016.

In this study, we explore the circulation of low pathogenic avian influenza (LPAI) viruses in wild birds and poultry in the Netherlands. Surveillance data collected between 2006 and 2016 was used to evaluate subtype diversity, spatiotemporal distribution and genetic relationships between wild bird and poultry viruses. We observed close species-dependent associations among hemagglutinin and neuraminidase subtypes. Not all subtypes detected in wild birds were found in poultry, suggesting transmission to poultry is selective and likely depends on viral factors that determine host range restriction. Subtypes commonly detected in poultry were in wild birds most frequently detected in mallards and geese. Different temporal patterns in virus prevalence were observed between wild bird species. Virus detections in domestic ducks coincided with the prevalence peak in wild ducks, whereas virus detections in other poultry types were made throughout the year. Genetic analysis of the surface genes demonstrated that most poultry viruses were related to locally circulating wild bird viruses, but no direct spatiotemporal link was observed. Results indicate prolonged undetected virus circulation and frequent reassortment events with local and newly introduced viruses within the wild bird population. Increased knowledge on LPAI virus circulation can be used to improve surveillance strategies.