Phylodynamics of Highly Pathogenic Avian Influenza A(H5N1) Virus Circulating in Indonesian Poultry.

After its first detection in 1996, the highly pathogenic avian influenza A(H5Nx) virus has spread extensively worldwide. HPAIv A(H5N1) was first detected in Indonesia in 2003 and has been endemic in poultry in this country ever since. However, Indonesia has limited information related to the phylodynamics of HPAIv A(H5N1) in poultry. The present study aimed to increase the understanding of the evolution and temporal dynamics of HPAIv H5N1 in Indonesian poultry between 2003 and 2016. To this end, HPAIv A(H5N1) hemagglutinin sequences of viruses collected from 2003 to 2016 were analyzed using Bayesian evolutionary analysis sampling trees. Results indicated that the common ancestor of Indonesian poultry HPAIv H5N1 arose approximately five years after the common ancestor worldwide of HPAI A(H5Nx). In addition, this study indicated that only two introductions of HPAIv A(H5N1) occurred, after which these viruses continued to evolve due to extensive spread among poultry. Furthermore, this study revealed the divergence of H5N1 clade 2.3.2.1c from H5N1 clade 2.3.2.1b. Both clades 2.3.2.1c and 2.3.2.1b share a common ancestor, clade 1, suggesting that clade 2.3.2.1 originated and diverged from China and other Asian countries. Since there was limited sequence and surveillance data for the HPAIv A(H5N1) from wild birds in Indonesia, the exact role of wild birds in the spread of HPAIv in Indonesia is currently unknown. The evolutionary dynamics of the Indonesian HPAIv A(H5N1) highlight the importance of continuing and improved genomic surveillance and adequate control measures in the different regions of both the poultry and wild birds. Spatial genomic surveillance is useful to take adequate control measures. Therefore, it will help to prevent the future evolution of HPAI A(H5N1) and pandemic threats.

Estimating the introduction time of highly pathogenic avian influenza into poultry flocks.

The estimation of farm-specific time windows for the introduction of highly-pathogenic avian influenza (HPAI) virus can be used to increase the efficiency of disease control measures such as contact tracing and may help to identify risk factors for virus introduction. The aims of this research are to (1) develop and test an accurate approach for estimating farm-specific virus introduction windows and (2) evaluate this approach by applying it to 11 outbreaks of HPAI (H5N8) on Dutch commercial poultry farms during the years 2014 and 2016. We used a stochastic simulation model with susceptible, infectious and recovered/removed disease stages to generate distributions for the period from virus introduction to detection. The model was parameterized using data from the literature, except for the within-flock transmission rate, which was estimated from disease-induced mortality data using two newly developed methods that describe HPAI outbreaks using either a deterministic model (A) or a stochastic approach (B). Model testing using simulated outbreaks showed that both method A and B performed well. Application to field data showed that method A could be successfully applied to 8 out of 11 HPAI H5N8 outbreaks and is the most generally applicable one, when data on disease-induced mortality is scarce.

Emergence and Selection of a Highly Pathogenic Avian Influenza H7N3 Virus.

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.

Highly Pathogenic Avian Influenza A(H5N1) Outbreaks in West Java Indonesia 2015-2016: Clinical Manifestation and Associated Risk Factors.

Knowledge of outbreaks and associated risk factors is helpful to improve control of the Highly Pathogenic Avian Influenza A(H5N1) virus (HPAI) in Indonesia. This study was conducted to detect outbreaks of HPAI H5N1 in endemically infected regions by enhanced passive surveillance, to describe the clinical manifestation of these outbreaks and identify associated risk factors. From November 2015 to November 2016, HPAI outbreak investigations were conducted in seven districts of West Java. In total 64 outbreaks were confirmed out of 75 reported suspicions and outbreak characteristics were recorded. The highest mortality was reported in backyard chickens (average 59%, CI95%: 49-69%). Dermal apoptosis and lesions (64%, CI95%: 52-76%) and respiratory signs (39%, CI95%: 27-51%) were the clinical signs observed overall most frequently, while neurological signs were most frequently observed in ducks (68%, CI95%: 47-90%). In comparison with 60 non-infected control farms, the rate of visitor contacts onto a farm was associated with the odds of HPAI infection. Moreover, duck farms had higher odds of being infected than backyard farms, and larger farms had lower odds than small farms. Results indicate that better external biosecurity is needed to reduce transmission of HPAI A(H5N1) in Indonesia.

Reassortments among Avian Influenza A(H5N1) Viruses Circulating in Indonesia, 2015-2016.

Highly pathogenic avian influenza (HPAI) A(H5N1) viruses have been circulating since 2003 in Indonesia, with major impacts on poultry health, severe economic losses, and 168 fatal laboratory-confirmed human cases. We performed phylogenetic analysis on 39 full-genome H5N1 virus samples collected during outbreaks among poultry in 2015-2016 in West Java and compared them with recently published sequences from Indonesia. Phylogenetic analysis revealed that the hemagglutinin gene of all samples belonged to 2 genetic groups in clade 2.3.2.1c. We also observed these groups for the neuraminidase, nucleoprotein, polymerase, and polymerase basic 1 genes. Matrix, nonstructural protein, and polymerase basic 2 genes of some HPAI were most closely related to clade 2.1.3 instead of clade 2.3.2.1c, and a polymerase basic 2 gene was most closely related to Eurasian low pathogenicity avian influenza. Our results detected a total of 13 reassortment types among HPAI in Indonesia, mostly in backyard chickens in Indramayu.

The development of a multiplex serological assay for avian influenza based on Luminex technology.

Avian influenza (AI) is an infectious disease in birds with enormous impact on the poultry sector. AI viruses are divided into different subtypes based on the antigenicity of their surface proteins haemagglutinin (HA) and neuraminidases (NA). In birds, 16 HA subtypes and 9 NA subtypes are detected in different combinations. Traditional serological methods for the subtyping of AI antibodies are labour-intensive and have to be performed for each HA and NA subtype separately. This study describes the development of a multiplex serological assay for subtyping AI antibodies in poultry sera using Luminex xMAP technology. This multiplex assay allows the detection of all AI serotypes in one single assay. For all HA and NA subtypes, recombinant proteins were purified and coupled to colour-coded magnetic bead sets. Using the Luminex MAGPIX device, binding of serum antibodies to the antigens on the bead sets is detected by fluorescent secondary antibodies, and the different bead sets are identified. The results of the multiplex assay were compared with that of the traditional singleplex assays. We show that serotyping using the novel multiplex serological assay is consistent with the results of the traditional assays in 97.8% of the reference sera and in 90.8% of the field sera. The assay has a higher sensitivity than the traditional assays, and requires a smaller sample volume. Therefore, the assay will allow complete AI-serotyping in small volumes of field sera, which will improve the monitoring of AI subtypes circulating in poultry significantly.

Exploring contacts facilitating transmission of influenza A(H5N1) virus between poultry farms in West Java, Indonesia: A major role for backyard farms?

Highly pathogenic avian influenza virus (HPAIV) H5N1 has been reported in Asia, including Indonesia since 2003. Although several risk factors related to the HPAIV outbreaks in poultry in Indonesia have been identified, little is known of the contact structure of farms of different poultry production types (backyard chickens, broilers, layers, and ducks). This study aims to quantify the contact rates associated with the movement of people, and movements of live birds and products and equipment that affect the risk of HPAIV H5N1 transmission between poultry farms in Indonesia. On 124 poultry farms in 6 districts in West Java, logbooks were distributed to record the movements of farmers/staff and visitors and their poultry contacts. Most movements in backyard chicken, commercial native chicken, broiler and duck farms were visits to and from other poultry farms, whilst in layer farms visits to and from poultry companies, visits to egg collection houses and visit from other poultry farms were most frequent. Over 75% of persons visiting backyard chicken and duck farms had previously visited other poultry farms on the same day. Visitors of backyard chicken farms had the highest average contact rate, either direct contact with poultry on other farms before the visits (1.35 contact/day) or contact during their visits in the farms (10.03 contact/day). These results suggest that backyard chicken farms are most at risk for transmission of HPAIV compared to farms of the other poultry production types. Since visits of farm-to-farm were high, backyard farms could also a potential source for HPAIV transmission to commercial poultry farms.

Similar transmissibility of the Italian H7N1 highly pathogenic avian influenza virus and its low pathogenic avian influenza virus predecessor.

The transmissibility of the H7N1 highly pathogenic avian influenza virus (HPAIV), which caused a large epidemic in commercial poultry in Italy in 1999-2000, was studied in chickens and compared with that of the low pathogenic precursor virus (LPAIV). Group transmission experiments using the HPAIV were executed to estimate the infectious period (IP), the transmission parameter (ß) and the basic reproduction number (R0). These estimates were then compared with those reported for the LPAIV. The estimated ß and R0 were similar for both viruses, whilst the IP of the LPAIV was longer than that of the HPAIV. These findings indicate that transmissibility from chicken-to-chicken alone does not appear to confer an advantage for this LPAIV to evolve to a HPAIV.

Novel Highly Pathogenic Avian Influenza A(H5N6) Virus in the Netherlands, December 2017.

A novel highly pathogenic avian influenza A(H5N6) virus affecting wild birds and commercial poultry was detected in the Netherlands in December 2017. Phylogenetic analysis demonstrated that the virus is a reassortant of H5N8 clade 2.3.4.4 viruses and not related to the Asian H5N6 viruses that caused human infections.

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.

Different cross protection scopes of two avian influenza H5N1 vaccines against infection of layer chickens with a heterologous highly pathogenic virus.

Avian influenza (AI) virus strains vary in antigenicity, and antigenic differences between circulating field virus and vaccine virus will affect the effectiveness of vaccination of poultry. Antigenic relatedness can be assessed by measuring serological cross-reactivity using haemagglutination inhibition (HI) tests. Our study aims to determine the relation between antigenic relatedness expressed by the Archetti-Horsfall ratio, and reduction of virus transmission of highly pathogenic H5N1 AI strains among vaccinated layers. Two vaccines were examined, derived from H5N1 AI virus strains A/Ck/WJava/Sukabumi/006/2008 and A/Ck/CJava/Karanganyar/051/2009. Transmission experiments were carried out in four vaccine and two control groups, with six sets of 16 specified pathogen free (SPF) layer chickens. Birds were vaccinated at 4weeks of age with one strain and challenge-infected with the homologous or heterologous strain at 8weeks of age. No transmission or virus shedding occurred in groups challenged with the homologous strain. In the group vaccinated with the Karanganyar strain, high cross-HI responses were observed, and no transmission of the Sukabumi strain occurred. However, in the group vaccinated with the Sukabumi strain, cross-HI titres were low, virus shedding was not reduced, and multiple transmissions to contact birds were observed. This study showed large differences in cross-protection of two vaccines based on two different highly pathogenic H5N1 virus strains. This implies that extrapolation of in vitro data to clinical protection and reduction of virus transmission might not be straightforward.

Discordant detection of avian influenza virus subtypes in time and space between poultry and wild birds; Towards improvement of surveillance programs.

Avian influenza viruses from wild birds can cause outbreaks in poultry, and occasionally infect humans upon exposure to infected poultry. Identification and characterization of viral reservoirs and transmission routes is important to develop strategies that prevent infection of poultry, and subsequently virus transmission between poultry holdings and to humans. Based on spatial, temporal and phylogenetic analyses of data generated as part of intense and large-scale influenza surveillance programs in wild birds and poultry in the Netherlands from 2006 to 2011, we demonstrate that LPAIV subtype distribution differed between wild birds and poultry, suggestive of host-range restrictions. LPAIV isolated from Dutch poultry were genetically most closely related to LPAIV isolated from wild birds in the Netherlands or occasionally elsewhere in Western Europe. However, a relatively long time interval was observed between the isolations of related viruses from wild birds and poultry. Spatial analyses provided evidence for mallards (Anas platyrhynchos) being more abundant near primary infected poultry farms. Detailed year-round investigation of virus prevalence and wild bird species distribution and behavior near poultry farms should be used to improve risk assessment in relation to avian influenza virus introduction and retarget avian influenza surveillance programs.

Wind-Mediated Spread of Low-Pathogenic Avian Influenza Virus into the Environment during Outbreaks at Commercial Poultry Farms.

Avian influenza virus-infected poultry can release a large amount of virus-contaminated droppings that serve as sources of infection for susceptible birds. Much research so far has focused on virus spread within flocks. However, as fecal material or manure is a major constituent of airborne poultry dust, virus-contaminated particulate matter from infected flocks may be dispersed into the environment. We collected samples of suspended particulate matter, or the inhalable dust fraction, inside, upwind and downwind of buildings holding poultry infected with low-pathogenic avian influenza virus, and tested them for the presence of endotoxins and influenza virus to characterize the potential impact of airborne influenza virus transmission during outbreaks at commercial poultry farms. Influenza viruses were detected by RT-PCR in filter-rinse fluids collected up to 60 meters downwind from the barns, but virus isolation did not yield any isolates. Viral loads in the air samples were low and beyond the limit of RT-PCR quantification except for one in-barn measurement showing a virus concentration of 8.48 x 10(4) genome copies/m(3). Air samples taken outside poultry barns had endotoxin concentrations of ~50 EU/m(3) that declined with increasing distance from the barn. Atmospheric dispersion modeling of particulate matter, using location-specific meteorological data for the sampling days, demonstrated a positive correlation between endotoxin measurements and modeled particulate matter concentrations, with an R(2) varying from 0.59 to 0.88. Our data suggest that areas at high risk for human or animal exposure to airborne influenza viruses can be modeled during an outbreak to allow directed interventions following targeted surveillance.

Full-Genome Sequence of Influenza A(H5N8) Virus in Poultry Linked to Sequences of Strains from Asia, the Netherlands, 2014.

Genetic analyses of highly pathogenic avian influenza A(H5N8) virus from the Netherlands, and comparison with strains from Europe, South Korea, and Japan, showed a close relation. Data suggest the strains were probably carried to the Netherlands by migratory wild birds from Asia, possibly through overlapping flyways and common breeding sites in Siberia.

Silent spread of highly pathogenic Avian Influenza H5N1 virus amongst vaccinated commercial layers.

The aim of this study was to determine whether a single vaccination of commercial layer type chickens with an inactivated vaccine containing highly pathogenic avian influenza virus strain H5N1 A/chicken/Legok/2003, carried out on the farm, was sufficient to protect against infection with the homologous virus strain. A transmission experiment was carried out with pairs of chicken of which one was inoculated with H5N1 virus and the other contact-exposed. Results showed that the majority of the vaccinated birds developed haemagglutination inhibition (HI) titres below 4log2. No clinical signs were observed in the vaccinated birds and virus shedding was limited. However, nearly all vaccinated birds showed a four-fold or higher increase of HI titres after challenge or contact-exposure, which is an indication of infection. This implies that virus transmission most likely has occurred. This study showed that a single vaccination applied under field conditions induced clinical protection, but was insufficient to induce protection against virus transmission, suggesting that silent spread of virus in vaccinated commercial flocks may occur.

Pulmonary immunization of chickens using non-adjuvanted spray-freeze dried whole inactivated virus vaccine completely protects against highly pathogenic H5N1 avian influenza virus.

Highly pathogenic avian influenza (HPAI) H5N1 virus is a major threat to public health as well as to the global poultry industry. Most fatal human infections are caused by contact with infected poultry. Therefore, preventing the virus from entering the poultry population is a priority. This is, however, problematic in emergency situations, e.g. during outbreaks in poultry, as there are currently no mass application methods to effectively vaccinate large numbers of birds within a short period of time. To evaluate the suitability of needle-free pulmonary immunization for mass vaccination of poultry against HPAI H5N1, we performed a proof-of-concept study in which we investigated whether non-adjuvanted spray-freeze-dried (SFD) whole inactivated virus (WIV) can be used as a dry powder aerosol vaccine to immunize chickens. Our results show that chickens that received SFD-WIV vaccine as aerosolized powder directly at the syrinx (the site of the tracheal bifurcation), mounted a protective antibody response after two vaccinations and survived a lethal challenge with HPAI H5N1. Furthermore, both the number of animals that shed challenge virus, as well as the level of virus shedding, were significantly reduced. Based on antibody levels and reduction of virus shedding, pulmonary vaccination with non-adjuvanted vaccine was at least as efficient as intratracheal vaccination using live virus. Animals that received aerosolized SFD-WIV vaccine by temporary passive inhalation showed partial protection (22% survival) and a delay in time-to-death, thereby demonstrating the feasibility of the method, but indicating that the efficiency of vaccination by passive inhalation needs further improvement. Altogether our results provide a proof-of-concept that pulmonary vaccination using an SFD-WIV powder vaccine is able to protect chickens from lethal HPAI challenge. If the efficacy of pulmonary vaccination by passive inhalation can be improved, this method might be suitable for mass application.

Getting more out of less--a quantitative serological screening tool for simultaneous detection of multiple influenza A hemagglutinin-types in chickens.

Current avian influenza surveillance in poultry primarily targets subtypes of interest for the veterinary sector (H5, H7). However, as virological and serological evidence suggest, surveillance of additional subtypes is important for public health as well as for the poultry industry. Therefore, we developed a protein microarray enabling simultaneous identification of antibodies directed against different HA-types of influenza A viruses in chickens. The assay successfully discriminated negative from experimentally and naturally infected, seropositive chickens. Sensitivity and specificity depended on the cut-off level used but ranged from 84.4% to 100% and 100%, respectively, for a cut off level of ≥ 1:40, showing minimal cross reactivity. As this testing platform is also validated for the use in humans, it constitutes a surveillance tool that can be applied in human-animal interface studies.

Quantification of dust-borne transmission of highly pathogenic avian influenza virus between chickens.

BACKGROUND: Understanding the transmission of highly pathogenic avian influenza virus (HPAIv) between poultry flocks is essential to prevent and control epidemics. Dust, produced in infected chicken flocks, has been hypothesized to be an important mechanical vector for between-flock transmission of HPAIv. OBJECTIVES: The aim of our study was to quantify the amount of virus shed by infected birds and its relation to deposition of virus in the environment and the rate of dust-borne transmission between groups of chickens. METHODS: Four replicate experiments were performed, each replicate with two groups of 14 chickens housed in two separate rooms. In one group, chickens were inoculated with HPAIv. Ventilation forced the air from that room to the second (recipient) group through a tube. Deceased birds in the inoculated group were replaced with new susceptible birds up to day 10 p.i. Dust samples were collected daily. Trachea and cloaca swabs were collected daily to determine virus shedding and virus spread to the recipient group. RESULTS: The amount of virus detected in dust samples in the recipient room was, on average, 10(3·7) EID(50) /m(3) . Virus transmission from the inoculated to the recipient group occurred in two experiments. The transmission rate parameter for dust-borne transmission was estimated at 0·08 new infections/infectious chicken/day. CONCLUSIONS: The results of this study are a first step to elucidate the importance of dust-borne transmission of HPAIv between flocks and help interpret environmental samples.