Experimental infection of H5N1 and H5N8 highly pathogenic avian influenza viruses in Northern Pintail (Anas acuta)

The wide geographic spread of Eurasian Goose/Guangdong lineage highly pathogenic avian influenza (HPAI) clade 2.3.4.4 viruses by wild birds is of great concern. In December 2014, an H5N8 HPAI clade 2.3.4.4 Group A (2.3.4.4A) virus was introduced to North America. Long‐distance migratory wild aquatic birds between East Asia and North America, such as Northern Pintail (Anas acuta ), were strongly suspected of being a source of intercontinental transmission. In this study, we evaluated the pathogenicity, infectivity and transmissibility of an H5N8 HPAI clade 2.3.4.4A virus in Northern Pintails and compared the results to that of an H5N1 HPAI clade 2.3.2.1 virus. All of Northern Pintails infected with either H5N1 or H5N8 virus lacked clinical signs and mortality, but the H5N8 clade 2.3.4.4 virus was more efficient at replicating within and transmitting between Northern Pintails than the H5N1 clade 2.3.2.1 virus. The H5N8‐infected birds shed high titre of viruses from oropharynx and cloaca, which in the field supported virus transmission and spread. This study highlights the role of wild waterfowl in the intercontinental spread of some HPAI viruses. Migratory aquatic birds should be carefully monitored for the early detection of H5 clade 2.3.4.4 and other HPAI viruses.     

Evolutionary and Ecological Dynamics of Transboundary Disease Caused by H5N1 Virus in Southeast Asia

Southeast Asia has been the breeding ground for many emerging diseases in the past decade, and it is in this region that new genetic variants of HPAI H5N1 viruses have been emerging. Cross‐border movement of animals accelerates the spread of H5N1, and the changing environmental conditions also exert strong selective pressure on the viruses. The transboundary zoonotic diseases caused by H5N1 pose a serious and continual threat to global economy and public health. Here, we divided the H5N1 viruses isolated in Southeast Asia during 2003–2009 into four groups according to their phylogenetic relationships among HA gene sequences. Molecular evolution analysis suggests populations in expansion rather than a positive selection for group 2 and group 3, yet group 4 is under strong positive selection. Site 193 was found to be a potential glycosylation site and located in receptor‐binding domain. Note that site 193 tends to appear in avian isolates instead of human strains. Population dynamics analysis reveals that the effective population size of infections in Southeast Asia has undergone three obvious increases, and the results are consistent with the epidemiological analysis. Ecological and phylogeographical analyses show that agro‐ecological environments, migratory birds, domestic waterfowl, especially free‐ranging ducks, are crucial in the occurrence, maintenance and spread of H5N1 virus. The epidemiological links between Indonesia and Suphanburi observed suggest that viruses in Indonesia were originated from multiple introductions.     

Co‐circulation and characterization of HPAI‐H5N1 and LPAI‐H9N2 recovered from a duck farm,Yogyakarta, Indonesia

In July 2016, an avian influenza outbreak in duck farms in Yogyakarta province was reported to Disease Investigation Center (DIC), Wates, Indonesia, with approximately 1,000 ducks died or culled. In this study, two avian influenza (AI) virus subtypes, A/duck/Bantul/04161291‐OR/2016 (H5N1) and A/duck/Bantul/04161291‐OP/2016 (H9N2) isolated from ducks in the same farm during an AI outbreak in Bantul district, Yogyakarta province, were sequenced and characterized. Our results showed that H5N1 virus was closely related to the highly pathogenic AI (HPAI) H5N1 of clade 2.3.2.1c, while the H9N2 virus was clustered with LPAI viruses from China, Vietnam and Indonesia H9N2 (CVI lineage). Genetic analysis revealed virulence characteristics for both in avian and in mammalian species. In summary, co‐circulation of HPAI‐H5N1 of clade 2.3.2.1c and LPAI‐H9N2 was identified in a duck farm during an AI outbreak in Yogyakarta province, Indonesia. Our findings raise a concern of the potential risk of the viruses, which could increase viral transmission and/or threat to human health. Routine surveillance of avian influenza viruses should be continuously conducted to understand the dynamic and diversity of the viruses for influenza prevention and control in Indonesia and SEA region.     

Identification of Potential Risk Factors Associated with Highly Pathogenic Avian Influenza Subtype H5N1 Outbreak Occurrence in Lagos and Kano States,Nigeria, During the 2006–2007 Epidemics

Highly pathogenic avian influenza HPAI H5N1 was first reported in Africa in 2006, in Nigeria. The country experienced severe outbreaks in 2006 and 2007, strongly affecting the poultry population. Current knowledge on potential risk factors for HPAI H5N1 occurrence in poultry farms in Nigeria is limited. Therefore, we conducted a case–control study to identify potential farm‐level risk factors for HPAI H5N1 occurrence in two areas of the country that were affected by the disease in 2006 and 2007, namely the States of Lagos and Kano. A case–control study was conducted at the farm level. A convenience sample of 110 farms was surveyed. Data on farm characteristics, farm management and trade practices were collected. Logistic regression was used to identify factors associated with farms that confirmed positive for HPAI. Having a neighbouring poultry farm was identified as a potential risk factor for disease occurrence [OR, 5.23; 95% CI, (0.88–30.97); P‐value = 0.048]. Farm staff washing their hands before handling birds was a protective factor [OR, 0.14; 95% CI, (0.05–0.37); P‐value <0.001], as well as not allowing traders to enter the farm [OR, 0.23; 95% CI, (0.08–0.70); P‐value = 0.008]. Our study highlighted the importance of trade and proximity between poultry farms in the epidemiology of HPAI H5N1 and the role of biosecurity in disease prevention in Kano and Lagos States. Despite the limitations owing to the sampling strategy, these results are consistent with other risk factor studies previously conducted on HPAI H5N1 in both Africa and other regions, suggesting similar risk factor patterns for HPAI H5N1 virus spread and substantiating current knowledge regarding the epidemiology of the disease. Finally, this study generated information from areas where data are difficult to obtain.     

Spatio‐temporal distribution of outbreaks of highly pathogenic avian influenza virus subtype H5N1 in Vietnam, 2015– 2018

Despite strong commitments of the central and provincial veterinary authorities in Vietnam to control highly pathogenic avian influenza H5N1 (HPAI‐H5N1) in poultry and to diminish the risk of human infection, outbreaks continue to occur in poultry. This study describes the spatio‐temporal distribution of HPAI‐H5N1 outbreaks in Vietnam for the period December 2014–April 2018 using the space‐time K‐function and the space‐time scan statistic. The space‐time K‐function analyses showed statistically significant spatio‐temporal clustering of HPAI‐H5N1 outbreaks in poultry during the study period of up to 50 days and 60 kilometres. The space‐time scan statistic test identified three statistically significant space‐time clusters of HPAI‐H5N1 in the south of the country where the incidence of HPAI‐H5N1 outbreaks was greater than that expected if outbreaks were randomly distributed in space and time. The analyses indicated shortfalls in the effectiveness of control measures used to control HPAI‐H5N1 throughout the study period. Development of a better understanding of the relative impact of HPAI‐H5N1 control measures (depopulation of infected flocks, vaccination, movement restrictions) on space‐time interaction would allow animal health authorities to focus their efforts on control measures shown to have the greatest relative effect.     

Risk of Introduction of H5N1 HPAI from Europe to Spain by Wild Water Birds in Autumn

Highly pathogenic avian influenza (HPAI) subtype H5N1 continues to circulate across Eurasia and Africa since its unprecedented rapid spread in 2005. Diffusion by wild bird movements has been evidenced in the European Union in 2006 and 2007. Spain is an important wintering quarter for aquatic birds from northern latitudes, so identifying the critical areas and species where an outbreak is prone to happen is necessary. This work presents an assessment of the risk of introduction of H5N1 HPAI in Spain by aquatic wild birds estimating a relative risk value per province. For this purpose, an assessment of the release and exposure to the risk of infection with H5N1 HPAI of 25 selected water bird species has been carried out. Parameters considered in the assessment include H5N1 HPAI notifications from 2006 to 2008 and factors that favour the occurrence or persistence of H5N1 HPAI (wetlands’ surface, low temperatures), together with aquatic wild birds’ movements parameters (departure, destination, stop‐overs, abundance) and parameters relative to the susceptible population in Spain: poultry density and wild aquatic abundance. Results show the relative risk for each Spanish province of experiencing H5N1 HPAI introduced by wild aquatic birds helping to identify higher risk areas.     

Experimental infection of clade 1.1.2 (H5N1), clade 2.3.2.1c (H5N1) and clade 2.3.4.4 (H5N6) highly pathogenic avian influenza viruses in dogs

Since the emergence of highly pathogenic avian influenza (HPAI) H5N1 in Asia, the haemagglutinin (HA) gene of this virus lineage has continued to evolve in avian populations, and H5N1 lineage viruses now circulate concurrently worldwide. Dogs may act as an intermediate host, increasing the potential for zoonotic transmission of influenza viruses. Virus transmission and pathologic changes in HPAI clade 1.1.2 (H5N1)‐, 2.3.2.1c (H5N1)‐ and 2.3.4.4 (H5N6)‐infected dogs were investigated. Mild respiratory signs and antibody response were shown in dogs intranasally infected with the viruses. Lung histopathology showed lesions that were associated with moderate interstitial pneumonia in the infected dogs. In this study, HPAI H5N6 virus replication in dogs was demonstrated for the first time. Dogs have been suspected as a “mixing vessel” for reassortments between avian and human influenza viruses to occur. The replication of these three subtypes of the H5 lineage of HPAI viruses in dogs suggests that dogs could serve as intermediate hosts for avian–human influenza virus reassortment if they are also co‐infected with human influenza viruses.     

The Effectiveness of Preventative Mass Vaccination Regimes Against the Incidence of Highly Pathogenic Avian Influenza on Java Island,Indonesia

We conducted an operational research study involving backyard and semicommercial farms on Java Island, Indonesia, between April 2008 and September 2009 to evaluate the effectiveness of two preventive mass vaccination strategies against highly pathogenic avian influenza (HPAI). One regimen used Legok 2003 H5N1 vaccine, while the other used both Legok 2003 H5N1 and HB1 Newcastle disease (ND) vaccine. A total of 16 districts were involved in the study. The sample size was estimated using a formal power calculation technique that assumed a detectable effect of treatment as a 50% reduction in the baseline number of HPAI‐compatible outbreaks. Within each district, candidate treatment blocks with village poultry populations ranging from 80 000 to 120 000 were created along subdistrict boundary lines. Subsequently, four of these blocks were randomly selected and assigned one treatment from a list that comprised control, vaccination against HPAI, vaccination against HPAI + ND. Four rounds of vaccination were administered at quarterly intervals beginning in July 2008. A vaccination campaign involved vaccinating 100 000 birds in a treatment block, followed by another 100 000 vaccinations 3 weeks later as a booster dose. Data on disease incidence and vaccination coverage were also collected at quarterly intervals using participatory epidemiological techniques. Compared with the unvaccinated (control) group, the incidence of HPAI‐compatible events declined by 32% (P = 0.24) in the HPAI‐vaccinated group and by 73% (P = 0.00) in the HPAI‐ and ND‐vaccinated group. The effect of treatment did not vary with time or district. Similarly, an analysis of secondary data from the participatory disease and response (PDSR) database revealed that the incidence of HPAI declined by 12% in the HPAI‐vaccinated group and by 24% in the HPAI + ND‐vaccinated group. The results suggest that the HPAI + ND vaccination significantly reduced the incidence of HPAI‐compatible events in mixed populations of semicommercial and backyard poultry.     

Avian Influenza H5N1 Surveillance and its Dynamics in Poultry in Live Bird Markets,Egypt

H5N1, a highly pathogenic avian influenza (H5N1 HPAI), is an endemic disease that is significant for public health in Egypt. Live bird markets (LBMs) are widespread in Egypt and play an important role in HPAI disease dynamics. The aim of the study was to evaluate the H5N1 HPAI prevalence in representative LBMs from 2009 to 2014, assess the effects of other variables and evaluate past outbreaks and human cases. It was found that ducks and geese are high‐risk species and that the prevalence of H5N1 HPAI was higher immediately after the political crises of 2011. The end of a calendar year (June to December) was a high‐risk period for positive samples, and the risk in urban LBMs was twice the risk in rural LBMs. Winter and political unrest was associated with higher H5N1 HPAI prevalence. Both human and poultry populations will continue to rise in Egypt, so continued poultry outbreaks are likely to be linked to more human cases. LBMs will continue to play a role in the dynamics of poultry disease in Egypt, and there is a need to reorganize markets in terms of biosecurity and traceability. It may also be beneficial to reduce inter‐governorate inter‐regional movements associated with poultry trade through promotion of regional trade or in the alternative provide sanitary features along the poultry market chain to reduce the speed of H5N1 HPAI infections. Policy formulation, design and enforcement must be pro‐poor, and consideration of the sociocultural and economic realities in Egypt is important. The LBMs provide ideal platforms to carry out sound surveillance plans and mitigate zoonotic risks of H5N1 HPAI to humans.     

Genetic Diversity and Phylogenetic Analysis of Highly Pathogenic Avian Influenza (HPAI) H5N1 Viruses Circulating in Bangladesh from 2007–2011

Highly pathogenic avian influenza (HPAI) H5N1 virus has been endemic in Bangladesh since its first isolation in February 2007. Phylogenetic analysis of the haemagglutinin (HA) gene of HPAI H5N1 viruses demonstrated that 25 Bangladeshi isolates including two human isolates from 2007–2011 along with some isolates from neighbouring Asian countries (India, Bhutan, Myanmar, Nepal, China and Vietnam) segregate into two distinct clades (2.2 and 2.3). There was clear evidence of introduction of clade 2.3.2 and 2.3.4 viruses in 2011 in addition to clade 2.2 viruses that had been in circulation in Bangladesh since 2007. The data clearly demonstrated the movement of H5N1 strains between Asian countries included in this study due to migration of wild birds and/or illegal movement of poultry across borders. Interestingly, the two human isolates were closely related to the clade 2.2 Bangladeshi chicken isolates indicating that they have originated from chickens. Furthermore, comparative amino acid sequence analysis revealed several substitutions (including 189R>K and 282I>V) in HA protein of some clade 2.2 Bangladeshi viruses including the human isolates, suggesting there was antigenic drift in clade 2.2.3 viruses that were circulating between 2008 and 2011. Overall, the data imply genetic diversity among circulating viruses and multiple introductions of H5N1 viruses with an increased risk of human infections in Bangladesh, and establishment of H5N1 virus in wild and domestic bird populations, which demands active surveillance.     

Emergence and spread of highly pathogenic avian influenza A(H5N8) in Europe in 2016‐2017

Circulation of highly pathogenic avian influenza (HPAI ) viruses poses a continuous threat to animal and public health. After the 2005–2006 H5N1 and the 2014–2015 H5N8 epidemics, another H5N8 is currently affecting Europe. Up to August 2017, 1,112 outbreaks in domestic and 955 in wild birds in 30 European countries have been reported, the largest epidemic by a HPAI virus in the continent. Here, the main epidemiological findings are described. While some similarities with previous HPAI virus epidemics were observed, for example in the pattern of emergence, significant differences were also patent, in particular the size and extent of the epidemic. Even though no human infections have been reported to date, the fact that A/H5N8 has affected so far 1,112 domestic holdings, increases the risk of exposure of humans and therefore represents a concern. Understanding the epidemiology of HPAI viruses is essential for the planning future surveillance and control activities.     

Highly Pathogenic Avian Influenza Virus A/H5N1 Infection in Vaccinated Meat Duck Flocks in the Mekong Delta of Vietnam

We investigated episodes of suspected highly pathogenic avian influenza (HPAI)‐like illness among 12 meat duck flocks in two districts in Tien Giang province (Mekong Delta, Vietnam) in November 2013. In total, duck samples from 8 of 12 farms tested positive for HPAI virus subtype A/haemagglutinin 5 and neuraminidase 1 (H5N1) by real‐time RT‐PCR. Sequencing results confirmed clade of 2.3.2.1.c as the cause of the outbreaks. Most (7/8) laboratory‐confirmed positive flocks had been vaccinated with inactivated HPAI H5N1 clade 2.3.4 vaccines <6 days prior to onset of clinical signs. A review of vaccination data in relation to estimated production in the area suggested that vaccination efforts were biased towards larger flocks and that vaccination coverage was low [21.2% ducks vaccinated with two shots (range by district 7.4–34.9%)]. The low‐coverage data, the experimental evidence of lack of cross‐protection conferred by the currently used vaccines based on clade 2.3.4 together with the short lifespan of meat duck flocks (60–70 days), suggest that vaccination is not likely to be effective as a tool for control of H5N1 infection in meat duck flocks in the area.     

Highly pathogenic avian influenza subtype H5Nx clade 2.3.4.4 outbreaks in Dutch poultry farms, 2014–2018: Clinical signs and mortality

In recent years, different subtypes of highly pathogenic avian influenza (HPAI) viruses caused outbreaks in several poultry types worldwide. Early detection of HPAI virus infection is crucial to reduce virus spread. Previously, the use of a mortality ratio threshold to expedite notification of suspicion in layer farms was proposed. The purpose of this study was to describe the clinical signs reported in the early stages of HPAI H5N8 and H5N6 outbreaks on chicken and Pekin duck farms between 2014 and 2018 in the Netherlands and compare them with the onset of an increased mortality ratio (MR). Data on daily mortality and clinical signs from nine egg‐producing chicken farms and seven Pekin duck farms infected with HPAI H5N8 (2014 and 2016) and H5N6 (2017–2018) in the Netherlands were analysed. In 12 out of 15 outbreaks for which a MR was available, MR increase preceded or coincided with the first observation of clinical signs by the farmer. In one chicken and two Pekin duck outbreaks, clinical signs were observed prior to MR increase. On all farms, veterinarians observed clinical signs of general disease. Nervous or locomotor signs were reported in all Pekin duck outbreaks, but only in two chicken outbreaks. Other clinical signs were observed less frequently in both chickens and Pekin ducks. Compared to veterinarians, farmers observed and reported clinical signs, especially respiratory and gastrointestinal signs, less frequently. This case series suggests that a MR with a set threshold could be an objective parameter to detect HPAI infection on chicken and Pekin duck farms at an early stage. Observation of clinical signs may provide additional indication for farmers and veterinarians for notifying a clinical suspicion of HPAI infection. Further assessment and validation of a MR threshold in Pekin ducks are important as it could serve as an important tool in HPAI surveillance programs.     

Risk factors associated with highly pathogenic avian influenza subtype H5N8 outbreaks on broiler duck farms in South Korea

Highly Pathogenic Avian Influenza (HPAI ) subtype H5N8 outbreaks occurred in poultry farms in South Korea in 2014 resulting in significant damage to the poultry industry. Between 2014 and 2016, the pandemic disease caused significant economic loss and social disruption. To evaluate the risk factors for HPAI infection in broiler duck farms, we conducted a retrospective case–control study on broiler duck farms. Forty‐three farms with confirmed laboratories on premises were selected as the case group, and 43 HPAI ‐negative farms were designated as the control group. Control farms were matched based on farm location and were within a 3‐km radius from the case premises. Spatial and environmental factors were characterized by site visit and plotted through a geographic information system (GIS ). Univariable and multivariable logistic regression models were developed to assess possible risk factors associated with HPAI broiler duck farm infection. Four final variables were identified as risk factors in a final multivariable logistic model: “Farms with ≥7 flocks” (odds ratio [OR ] = 6.99, 95% confidence interval [CI ] 1.34–37.04), “Farm owner with ≥15 years of raising poultry career” (OR  = 7.91, 95% CI 1.69–37.14), “Presence of any poultry farms located within 500 m of the farm” (OR  = 6.30, 95% CI 1.08–36.93) and “Not using a faecal removal service” (OR  = 27.78, 95% CI 3.89–198.80). This highlights that the HPAI H5N8 outbreaks in South Korea were associated with farm owner education, number of flocks and facilities and farm biosecurity. Awareness of these factors may help to reduce the spread of HPAI H5N8 across broiler duck farms in Korea during epidemics. Greater understanding of the risk factors for H5N8 may improve farm vulnerability to HPAI and other subtypes and help to establish policies to prevent re‐occurrence. These findings are relevant to global prevention recommendations and intervention protocols.     

Genetic and pathogenic characteristics of clade 2.3.2.1c H5N1 highly pathogenic avian influenza viruses isolated from poultry outbreaks in Laos during 2015–2018

Since 2004, there have been multiple outbreaks of H5 highly pathogenic avian influenza (HPAI) viruses in Laos. Here, we isolated H5N1 HPAI viruses from poultry outbreaks in Laos during 2015–2018 and investigated their genetic characteristics and pathogenicity in chickens. Phylogenetic analysis revealed that the isolates belonged to clade 2.3.2.1c and that they differed from previous Laos viruses with respect to genetic composition. In particular, the isolates were divided into two genotypes, each of which had a different NS segments. The results of possible migration analysis suggested a high likelihood that the Laos isolates were introduced from neighbouring countries, particularly Vietnam. The recent Laos isolate, A/Duck/Laos/NL‐1504599/2018, had an intravenous pathogenicity index score of 3.0 and showed a 50% chicken lethal dose of 10^2.5 EID₅₀/0.1 ml, indicating high pathogenicity. The isolated viruses exhibited no critical substitution in the markers associated with mammalian adaptation, but possess markers related to neuraminidase inhibitor resistance. These results emphasize the need for ongoing surveillance of circulating influenza virus in South‐East Asia, including Laos, to better prepare for and mitigate global spread of H5 HPAI.     

Survivability of highly pathogenic avian influenza virus (H5N1) in naturally preened duck feathers at different temperatures

Ducks are the “Trojan Horses” for Asian H5N1 avian influenza viruses (AIV) and attain carrier status without displaying overt infection. These birds help in the spread of the virus among the poultry and human population through direct or indirect contact. Preen oil is the secretion of preen gland of water birds such as ducks. In a process called preening, the water birds spread preen oil across their feather and body. Preen oil has been known to play a significant role in the accumulation of various pathogens including Highly Pathogenic Avian Influenza (HPAI) from water onto feathers. However, the studies are scarce on the role of preen oil in the survivability of HPAIV. We conducted a simulative study to analyse the effect of preen oil on the survivability of the HPAI virus (H5N1) on duck feathers. Duck feather samples along with relevant controls were spiked with the H5N1 virus at two different initial concentrations (10⁴ EID₅₀ and 10⁶ EID₅₀), stored at 37°C, 25°C and 10°C temperatures and tested at regular intervals for percent infectivity by egg culture method and qRT‐PCR. The infectivity and viral load were significantly higher in naturally preened duck feathers in comparison to the three preen oil deficit controls at both low and high initial concentrations of virus (10⁴ EID₅₀ and 10⁶ EID₅₀). Maximum persistence was seen at 10°C in naturally preened duck feathers spiked with 10⁶ EID₅₀ concentration of viruses. It was also seen that depletion of preen oil from duck feathers reduced the persistence of the virus. These results demonstrate that preen oil plays a significant role in survivability and protection of HPAIV on duck feathers. This study herein will present new avenues in understanding one of the epidemiological niches of HPAIV.     

Experimental infection of highly pathogenic avian influenza viruses,Clade 2.3.4.4 H5N6 and H5N8, in Mandarin ducks from South Korea

Outbreaks of highly pathogenic avian influenza (HPAI ) have been reported worldwide. Wild waterfowl play a major role in the maintenance and transmission of HPAI . Highly pathogenic avian influenza subtype H5N6 and H5N8 viruses simultaneously emerged in South Korea. In this study, the comparative pathogenicity and infectivity of Clade 2.3.4.4 Group B H5N8 and Group C H5N6 viruses were evaluated in Mandarin duck (Aix galericulata ). None of the ducks infected with H5N6 or H5N8 viruses showed clinical signs or mortality. Serological assays revealed that the HA antigenicity of H5N8 and H5N6 viruses was similar to each other. Moreover, both the viruses did not replicate after cross‐challenging with H5N8 and H5N6 viruses, respectively, as the second infection. Although both the viruses replicated in most of the internal organs of the ducks, viral replication and shedding through cloaca were higher in H5N8‐infected ducks than in H5N6‐infected ducks. The findings of this study provide preliminary information to help estimate the risks involved in further evolution and dissemination of Clade 2.3.4.4 HPAI viruses among wild birds.