Immunization of Chickens with Newcastle Disease Virus Expressing H5 Hemagglutinin Protects against Highly Pathogenic H5N1 Avian Influenza Viruses

Background

Highly-pathogenic avian influenza virus (HPAIV) and Newcastle disease virus (NDV) are the two most important poultry viruses in the world. Natural low-virulence NDV strains have been used as vaccines over the past 70 years with proven track records. We have previously developed a reverse genetics system to produce low-virulent NDV vaccine strain LaSota from cloned cDNA. This system allows us to use NDV as a vaccine vector for other avian pathogens.

Methodology/Principal Finding

Here, we constructed two recombinant NDVs (rNDVs) each of which expresses the hemagglutinin (HA) gene of HPAIV H5N1strain A/Vietnam/1203/2004 from an added gene. In one, rNDV (rNDV-HA), the open reading frame (ORF) of HA gene was expressed without modification. In the second, rNDV (rNDV-HAF), the ORF was modified so that the transmembrane and cytoplasmic domains of the encoded HA gene were replaced with those of the NDV F protein. The insertion of either version of the HA ORF did not increase the virulence of the rNDV vector. The HA protein was found to be incorporated into the envelopes of both rNDV-HA and rNDV-HAF. However, there was an enhanced incorporation of the HA protein in rNDV-HAF. Chickens immunized with a single dose of either rNDV-HA or rNDV-HAF induced a high titer of HPAIV H5-specific antibodies and were completely protected against challenge with NDV as well as lethal challenges of both homologous and heterologous HPAIV H5N1.

Conclusion and Significance

Our results suggest that these chimeric viruses have potential as safe and effective bivalent vaccines against NDV and. HPAIV. These vaccines will be convenient and affordable, which will be highly beneficial to the poultry industry. Furthermore, immunization with these vaccines will permit serological differentiation of vaccinated and avian influenza field virus infected animals.     

Recombinant Parainfluenza Virus 5 Vaccine Encoding the Influenza Virus Hemagglutinin Protects against H5N1 Highly Pathogenic Avian Influenza Virus Infection following Intranasal or Intramuscular Vaccination of BALB/c Mice

New approaches for vaccination to prevent influenza virus infection are needed. Emerging viruses, such as the H5N1 highly pathogenic avian influenza (HPAI) virus, pose not only pandemic threats but also challenges in vaccine development and production. Parainfluenza virus 5 (PIV5) is an appealing vector for vaccine development, and we have previously shown that intranasal immunization with PIV5 expressing the hemagglutinin from influenza virus was protective against influenza virus challenge (S. M. Tompkins, Y. Lin, G. P. Leser, K. A. Kramer, D. L. Haas, E. W. Howerth, J. Xu, M. J. Kennett, J. E. Durbin, R. A. Tripp, R. A. Lamb, and B. He, Virology 362:139–150, 2007). While intranasal immunization is an appealing approach, PIV5 may have the potential to be utilized in other formats, prompting us to test the efficacy of rPIV5-H5, which encodes the HA from H5N1 HPAI virus, in different vaccination schemes. In the BALB/c mouse model, a single intramuscular or intranasal immunization with a live rPIV5-H5 (ZL46) rapidly induced robust neutralizing serum antibody responses and protected against HPAI challenge, although mucosal IgA responses primed by intranasal immunization more effectively controlled virus replication in the lung. The rPIV5-H5 vaccine incorporated the H5 HA into the virion, so we tested the efficacy of an inactivated form of the vaccine. Inactivated rPIV5-H5 primed neutralizing serum antibody responses and controlled H5N1 virus replication; however, similar to other H5 antigen vaccines, it required a booster immunization to prime protective immune responses. Taken together, these results suggest that rPIV5-HA vaccines and H5-specific vaccines in particular can be utilized in multiple formats and by multiple routes of administration. This could avoid potential contraindications based on intranasal administration alone and provide opportunities for broader applications with the use of a single vaccine vector.     

Recombinant Parainfluenza Virus 5 Expressing Hemagglutinin of Influenza A Virus H5N1 Protected Mice against Lethal Highly Pathogenic Avian Influenza Virus H5N1 Challenge

A safe and effective vaccine is the best way to prevent large-scale highly pathogenic avian influenza virus (HPAI) H5N1 outbreaks in the human population. The current FDA-approved H5N1 vaccine has serious limitations. A more efficacious H5N1 vaccine is urgently needed. Parainfluenza virus 5 (PIV5), a paramyxovirus, is not known to cause any illness in humans. PIV5 is an attractive vaccine vector. In our studies, a single dose of a live recombinant PIV5 expressing a hemagglutinin (HA) gene of H5N1 (rPIV5-H5) from the H5N1 subtype provided sterilizing immunity against lethal doses of HPAI H5N1 infection in mice. Furthermore, we have examined the effect of insertion of H5N1 HA at different locations within the PIV5 genome on the efficacy of a PIV5-based vaccine. Interestingly, insertion of H5N1 HA between the leader sequence, the de facto promoter of PIV5, and the first viral gene, nucleoprotein (NP), did not lead to a viable virus. Insertion of H5N1 HA between NP and the next gene, V/phosphorprotein (V/P), led to a virus that was defective in growth. We have found that insertion of H5N1 HA at the junction between the small hydrophobic (SH) gene and the hemagglutinin-neuraminidase (HN) gene gave the best immunity against HPAI H5N1 challenge: a dose as low as 1,000 PFU was sufficient to protect against lethal HPAI H5N1 challenge in mice. The work suggests that recombinant PIV5 expressing H5N1 HA has great potential as an HPAI H5N1 vaccine.     

Persistence of Highly Pathogenic Avian Influenza H5N1 Virus Defined by Agro-Ecological Niche

The highly pathogenic avian influenza (HPAI) H5N1 virus has spread across Eurasia and into Africa. Its persistence in a number of countries continues to disrupt poultry production, impairs smallholder livelihoods, and raises the risk a genotype adapted to human-to-human transmission may emerge. While previous studies identified domestic duck reservoirs as a primary risk factor associated with HPAI H5N1 persistence in poultry in Southeast Asia, little is known of such factors in countries with different agro-ecological conditions, and no study has investigated the impact of such conditions on HPAI H5N1 epidemiology at the global scale. This study explores the patterns of HPAI H5N1 persistence worldwide, and for China, Indonesia, and India includes individual provinces that have reported HPAI H5N1 presence during the 2004–2008 period. Multivariate analysis of a set of 14 agricultural, environmental, climatic, and socio-economic factors demonstrates in quantitative terms that a combination of six variables discriminates the areas with human cases and persistence: agricultural population density, duck density, duck by chicken density, chicken density, the product of agricultural population density and chicken output/input ratio, and purchasing power per capita. The analysis identifies five agro-ecological clusters, or niches, representing varying degrees of disease persistence. The agro-ecological distances of all study areas to the medoid of the niche with the greatest number of human cases are used to map HPAI H5N1 risk globally. The results indicate that few countries remain where HPAI H5N1 would likely persist should it be introduced.     

人源中和性抗高致病性禽流感病毒H5N1基因工程全抗体的研制

运用噬菌体表面呈现技术,从禽流感病人恢复期血中获得淋巴细胞,通过基因工程手段,构建了人源抗H5NI禽流感病毒基因工程抗体文库.用纯化的人源H5N1禽流感病毒颗粒(A/Anhui/1/2005)及重组血凝素蛋白HA(A/Viet Nam/1203/2004)对Fab噬菌体抗体库进行富集筛选,成功地获得了抗禽流感病毒H5N1血凝素蛋白HA的人源单抗Fab段基因,并在大肠杆菌中获得有效表达.通过序列测定确定抗体轻重链型别,然后将阳性克隆的轻链和重链Fd段基因分别克隆入全抗体表达载体pAC-L-Fc后转染昆虫Sf9细胞,利用杆状病毒/昆虫细胞系统实现全抗体的分泌型表达.用ELISA、IFA和流式细胞术对所获人源单抗的功能特性进行鉴定.结果表明,我们获得了2株特异性针对H5N1禽流感病毒血凝素蛋白HA而与甲1型和甲3型人流感病毒无交叉反应的人源单抗(AVFlulgG01、AVFlulgG03).微量中和试验结果表明,除A/Guangdong/1/2006外,AVFlu-IgG01能够广泛地中和HA基因进化上属于Clade 2的中国南方、北方及中部地区的H5N1禽流感病毒分离株,同时还对属于Clade Ⅰ的越南H5N1分离株A/Viet Nam/1203/2004具有中和活性;AVFluIgG03虽然不能中和A/Viet Nam/1203/2004,但是对属于Clade 2的所有中国H5N1分离株均具有中和作用.人源中和性抗禽流感病毒H5N1基因工程全抗体的获得不仅为高致病性禽流感病毒H5N1的预防和治疗带来了希望,同时也为其疫苗研制提供了新的思路.     

Prior Infection of Chickens with H1N1 or H1N2 Avian Influenza Elicits Partial Heterologous Protection against Highly Pathogenic H5N1

There is a critical need to have vaccines that can protect against emerging pandemic influenza viruses. Commonly used influenza vaccines are killed whole virus that protect against homologous and not heterologous virus. Using chickens we have explored the possibility of using live low pathogenic avian influenza (LPAI) A/goose/AB/223/2005 H1N1 or A/WBS/MB/325/2006 H1N2 to induce immunity against heterologous highly pathogenic avian influenza (HPAI) A/chicken/Vietnam/14/2005 H5N1. H1N1 and H1N2 replicated in chickens but did not cause clinical disease. Following infection, chickens developed nucleoprotein and H1 specific antibodies, and reduced H5N1 plaque size in vitro in the absence of H5 neutralizing antibodies at 21 days post infection (DPI). In addition, heterologous cell mediated immunity (CMI) was demonstrated by antigen-specific proliferation and IFN-γ secretion in PBMCs re-stimulated with H5N1 antigen. Following H5N1 challenge of both pre-infected and naïve controls chickens housed together, all naïve chickens developed acute disease and died while H1N1 or H1N2 pre-infected chickens had reduced clinical disease and 70–80% survived. H1N1 or H1N2 pre-infected chickens were also challenged with H5N1 and naïve chickens placed in the same room one day later. All pre-infected birds were protected from H5N1 challenge but shed infectious virus to naïve contact chickens. However, disease onset, severity and mortality was reduced and delayed in the naïve contacts compared to directly inoculated naïve controls. These results indicate that prior infection with LPAI virus can generate heterologous protection against HPAI H5N1 in the absence of specific H5 antibody.     

Characterization of the H5N1 Highly Pathogenic Avian Influenza Virus Derived from Wild Pikas in China

The highly pathogenic H5N1 avian influenza virus emerged from China in 1996 and has spread across Eurasia and Africa, with a continuous stream of new cases of human infection appearing since the first large-scale outbreak among migratory birds at Qinghai Lake. The role of wild birds, which are the natural reservoirs for the virus, in the epidemiology of the H5N1 virus has raised great public health concern, but their role in the spread of the virus within the natural ecosystem of free-ranging terrestrial wild mammals remains unclear. In this study, we investigated H5N1 virus infection in wild pikas in an attempt to trace the circulation of the virus. Seroepidemiological surveys confirmed a natural H5N1 virus infection of wild pikas in their native environment. The hemagglutination gene of the H5N1 virus isolated from pikas reveals two distinct evolutionary clades, a mixed/Vietnam H5N1 virus sublineage (MV-like pika virus) and a wild bird Qinghai (QH)-like H5N1 virus sublineage (QH-like pika virus). The amino acid residue (glutamic acid) at position 627 encoded by the PB2 gene of the MV-like pika virus was different from that of the QH-like pika virus; the residue of the MV-like pika virus was the same as that of the goose H5N1 virus (A/GS/Guangdong [GD]/1/96). Further, we discovered that in contrast to the MV-like pika virus, which is nonpathogenic to mice, the QH-like pika virus is highly pathogenic. To mimic the virus infection of pikas, we intranasally inoculated rabbits, a species closely related to pikas, with the H5N1 virus of pika origin. Our findings first demonstrate that wild pikas are mammalian hosts exposed to H5N1 subtype avian influenza viruses in the natural ecosystem and also imply a potential transmission of highly pathogenic avian influenza virus from wild mammals into domestic mammalian hosts and humans.Highly pathogenic avian influenza (HPAI) is an extremely infectious, systemic viral disease that causes a high rate of mortality in birds. HPAI H5N1 viruses are now endemic in avian populations in Southeast Asia and have repeatedly been transmitted to humans (9, 14, 27). Since 2003, the H5N1 subtype has been reported in 391 human cases of influenza and has caused 247 human deaths in 15 countries, leading to greater than 60% mortality among infected individuals (38). Although currently incapable of sustained human-to-human transmission, H5N1 viruses undoubtedly pose a serious threat to public health, as well as to the global economy. Hence, preparedness for such a threat is a global priority (36).Wild birds are considered to be natural reservoirs for influenza A viruses (6, 18, 21, 35, 37). Of the 144 type A influenza virus hemagglutinin-neuraminidase (HA-NA) combinations, 103 have been found in wild birds (5, 7, 17, 37). Since the first HPAI outbreak among migratory wild birds appeared at Qinghai Lake in western China in May 2005 (3, 16, 25, 34, 41), HPAI viruses of the H5N1 subtype have been isolated from poultry throughout Eurasia and Africa. The continued occurrence of human cases has created a situation that could facilitate a pandemic emergence. There is heightened concern that wild birds are a reservoir for influenza A viruses that switch hosts and stably adapt to mammals, including horses, swine, and humans (11, 19, 22, 37).Despite the recent expansion of avian influenza virus (AIV) surveillance and genomic data (5, 17, 20, 21, 33, 40), fundamental questions remain concerning the ecology and evolution of these viruses. Little is known about how terrestrial wild mammals within their natural ecological systems affect HPAI H5N1 epidemiology or about the virus''s effects on public health, though there are many reports of natural and experimental H5N1 virus infection in animals belonging to the taxonomic orders Carnivora (12, 13, 15, 28, 29) and Artiodactyla (15). Herein, we provide the results of our investigation into H5N1 virus infection in wild pikas (Ochotona curzoniae of the order Lagomorpha) within their natural ecological setting. We describe our attempt to trace the circulation of H5N1 viruses and to characterize pika H5N1 influenza virus (PK virus).     

Highly Pathogenic H5N1 Influenza Virus Infection in Migratory Birds

H5N1avianinfluenza virus(AIV)has emerged as a pathogenic entityfor a variety of species,including humans,inre-cent years.Here we report an outbreak among migratory birds on Lake Qinghaihu,China,in May and June2005,inwhich more than a thousand birds were affected.Pancreatic necrosis and abnormal neurological symptoms were the majorclinical features.Sequencing of the complete genomes of four H5N1AIVstrains revealedthemto be reassortants relatedto a peregrine falconisolate from Hong Kong an…     

Genetic Characterization of a Novel Recombinant H5N2 Avian Influenza Virus Isolated from Chickens in Tibet

In this report, a novel H5N2 avian influenza virus (AIV) was isolated from chickens in Tibet in 2010, western China. Phylogenetic analysis demonstrated that it was a natural reassortant between H9N2 and H5N1 subtypes. It is of note that this virus has an HP genotype with HA, PB2, M, and NS genes homologous to those of A/peregrine falcon/Hong Kong/2142/2008(H5N1)-like HPAIV isolated from dead wild birds. Publishing this genome information will contribute to the investigation of avian influenza epidemiology and to further research of AIV''s biological properties.     

Estimation of Transmission Parameters of H5N1 Avian Influenza Virus in Chickens

Despite considerable research efforts, little is yet known about key epidemiological parameters of H5N1 highly pathogenic influenza viruses in their avian hosts. Here we show how these parameters can be estimated using a limited number of birds in experimental transmission studies. Our quantitative estimates, based on Bayesian methods of inference, reveal that (i) the period of latency of H5N1 influenza virus in unvaccinated chickens is short (mean: 0.24 days; 95% credible interval: 0.099–0.48 days); (ii) the infectious period of H5N1 virus in unvaccinated chickens is approximately 2 days (mean: 2.1 days; 95%CI: 1.8–2.3 days); (iii) the reproduction number of H5N1 virus in unvaccinated chickens need not be high (mean: 1.6; 95%CI: 0.90–2.5), although the virus is expected to spread rapidly because it has a short generation interval in unvaccinated chickens (mean: 1.3 days; 95%CI: 1.0–1.5 days); and (iv) vaccination with genetically and antigenically distant H5N2 vaccines can effectively halt transmission. Simulations based on the estimated parameters indicate that herd immunity may be obtained if at least 80% of chickens in a flock are vaccinated. We discuss the implications for the control of H5N1 avian influenza virus in areas where it is endemic.     

Geographical Spread of Highly Pathogenic Avian Influenza Virus H5N1 during the 2006 Outbreak in Austria

In spring 2006, highly pathogenic avian influenza virus (HPAIV) of subtype H5N1 was detected in Austria in 119 dead wild birds. The hemagglutinin cleavage site showed that the amino acid sequence motif was identical to that of the Qinghai lineage. For detailed analysis, the hemagglutinin (HA) and neuraminidase (NA) genes of 27 selected Austrian H5N1 viruses originating from different regions and wild bird species were analyzed phylogenetically, which revealed two clearly separated Austrian subclusters, both belonging to European cluster EMA-1. Subcluster South (SCS) contains virus isolates from the south of Austria as well as from Slovenia, Turkey, Egypt, and Nigeria. The second subcluster, Northwest (SCN), covered a larger group of viruses originating from different locations and wild bird species in the northern and very western parts of Austria, as well as from Bavaria and Switzerland. Surprisingly, virus isolates originating from two mute swans and one wild duck found on the north side of the Alps did not cluster with SCN but with SCS. Together with isolates from Bavarian, the Czech Republic, Italy, and Slovakia, they form a genuine subgroup, named subgroup Bavaria (SGB). This subgroup forms a link to SCN, indicating a spread of the virus from south to north. There has been a general assumption that the generic HPAI introduction route into Europe was from Russia to north Germany, introducing cluster EMA-2 into Europe. Interestingly, our findings support the assumption of an alternative introduction of the HPAI H5N1 virus from Turkey to central Europe, where it spread as cluster EMA-1 during the outbreak of 2006.Highly pathogenic H5N1 viruses have been recognized in Asia since 1996, when the first Asian H5N1 virus (A/Goose/Guandgdong/1/96) was isolated from sick geese in southern China (25). Since then, this virus has caused endemic infections in poultry in many southeast Asian countries (13, 18). Although influenza viruses in wild aquatic birds occasionally are transmitted to chickens and turkeys, where they may produce outbreaks of severe disease, they do not appear to have entered the wild bird populations to a substantial extent until late April to June 2005, when a large outbreak of H5N1 infection occurred at Qinghai Lake in western China, a major breeding site of migratory birds (2). Subsequently to the outbreak at Qinghai Lake from April to June 2005, H5N1 viruses have continued to cause outbreaks in Asia and Europe (http://www.who.int).A major molecular determinant for the pathogenicity of H5 and H7 viruses is the amino acid sequence specifying the proteolytic cleavage site of hemagglutinin (HA). In lowly pathogenic avian influenza virus (LPAIV), single basic residues at the cleavage site restrict the proteolytic activation of HA to the respiratory and intestinal tracts. In contrast, insertional mutations at the genomic locus encoding the endoproteolytic cleavage site resulting in the presence of a polybasic site render it accessible for ubiquitous protease, resulting in severe, systemic infections (17). All analyzed viruses originating from Qinghai Lake showed the series of basic amino acids at the HA cleavage site PQGERRRKKRGLF, which is characteristic of high pathogenicity in chickens. They also exhibited a 20-amino-acid deletion of the neuraminidase (NA) stalk (residues 49 to 68) that is characteristic of the NA of the A/Goose/Guandgdong/1/96 virus (2).Salzberg et al. analyzed 36 isolates of highly pathogenic avian influenza (HPAI) H5N1 viruses collected from Europe, northern Africa, the Middle East, and Asia and described the genetic relationships among these isolates, which affect birds and humans (16). He grouped the isolates into three distinct lineages, one encompassing all known non-Asian isolates, and hypothesized that this Europe-African lineage has been introduced into the European-African region at least three times and has split into three distinct, independently evolving sublineages: EMA-1, EMA-2, and EMA-3. These three clades possibly represent either separate introductions or a single introduction from Asia via Russia into Europe or any other western site, which then has subsequently evolved into three sublineages, EMA-1, EMA-2, and EMA-3 (16). EMA-2 contains the first German H5N1-positive swan found at the beginning of February 2006 on the Baltic island Ruegen (A/Cygnus cygnus/Germany/R65/06). This suggests a single introduction route for this cluster, because a phylogenetic analysis of the HA and the NA nucleotide sequences revealed that the closest genetic relative was an isolate from Astrakhan (A/Cygnus olor/Astrakhan/Ast05-2-3/2005). From Astrakhan, located in southern Russia, a westward movement of wild birds to central Europe in late January/early February 2006 is suggested (24).The aim of this study was to perform a phylogenetic analysis of Austrian HPAI H5N1 isolates from the outbreak of 2006 to determine their linkage to the European clusters EMA-1, EMA-2, and EMA-3 and to identify possible implications for H5N1 introduction routes into Austria.     

The Short Stalk Length of Highly Pathogenic Avian Influenza H5N1 Virus Neuraminidase Limits Transmission of Pandemic H1N1 Virus in Ferrets

H5N1 influenza viruses pose a pandemic threat but have not acquired the ability to support sustained transmission between mammals in nature. The restrictions to transmissibility of avian influenza viruses in mammals are multigenic, and overcoming them requires adaptations in hemagglutinin (HA) and PB2 genes. Here we propose that a further restriction to mammalian transmission of the majority of highly pathogenic avian influenza (HPAI) H5N1 viruses may be the short stalk length of the neuraminidase (NA) protein. This genetic feature is selected for when influenza viruses adapt to chickens. In our study, a recombinant virus with seven gene segments from a human isolate of the 2009 H1N1 pandemic combined with the NA gene from a typical chicken-adapted H5N1 virus with a short stalk did not support transmission by respiratory droplet between ferrets. This virus was also compromised in multicycle replication in cultures of human airway epithelial cells at 32°C. These defects correlated with a reduction in the ability of virus with a short-stalk NA to penetrate mucus and deaggregate virions. The deficiency in transmission and in cleavage of tethered substrates was overcome by increasing the stalk length of the NA protein. These observations suggest that H5N1 viruses that acquire a long-stalk NA through reassortment might be more likely to support transmission between humans. Phylogenetic analysis showed that reassortment with long-stalk NA occurred sporadically and as recently as 2011. However, all identified H5N1 viruses with a long-stalk NA lacked other mammalian adapting features and were thus several genetic steps away from becoming transmissible between humans.     

A Novel Bivalent Vaccine Based on a PB2-Knockout Influenza Virus Protects Mice from Pandemic H1N1 and Highly Pathogenic H5N1 Virus Challenges

Vaccination is an effective means to protect against influenza virus. Although inactivated and live-attenuated vaccines are currently available, each vaccine has disadvantages (e.g., immunogenicity and safety issues). To overcome these problems, we previously developed a replication-incompetent PB2-knockout (PB2-KO) influenza virus that replicates only in PB2 protein-expressing cells. Here, we generated two PB2-KO viruses whose PB2-coding regions were replaced with the HA genes of either A/California/04/2009 (H1N1pdm09) or A/Vietnam/1203/2004 (H5N1). The resultant viruses comparably, or in some cases more efficiently, induced virus-specific antibodies in the serum, nasal wash, and bronchoalveolar lavage fluid of mice relative to a conventional formalin-inactivated vaccine. Furthermore, mice immunized with these PB2-KO viruses were protected from lethal challenges with not only the backbone virus strain but also strains from which their foreign HAs originated, indicating that PB2-KO viruses with antigenically different HAs could serve as bivalent influenza vaccines.     

Experimental Infection of Macaques with a Wild Water Bird-Derived Highly Pathogenic Avian Influenza Virus (H5N1)

Highly pathogenic avian influenza virus (HPAIV) continues to threaten human health. Non-human primate infection models of human influenza are desired. To establish an animal infection model with more natural transmission and to determine the pathogenicity of HPAIV isolated from a wild water bird in primates, we administered a Japanese isolate of HPAIV (A/whooper swan/Hokkaido/1/2008, H5N1 clade 2.3.2.1) to rhesus and cynomolgus monkeys, in droplet form, via the intratracheal route. Infection of the lower and upper respiratory tracts and viral shedding were observed in both macaques. Inoculation of rhesus monkeys with higher doses of the isolate resulted in stronger clinical symptoms of influenza. Our results demonstrate that HPAIV isolated from a water bird in Japan is pathogenic in monkeys by experimental inoculation, and provide a new method for HPAIV infection of non-human primate hosts, a good animal model for investigation of HPAIV pathogenicity.     

Virus Shedding and Potential for Interspecies Waterborne Transmission of Highly Pathogenic H5N1 Influenza Virus in Sparrows and Chickens

To elucidate the role of sparrows as intermediate hosts of highly pathogenic avian influenza H5N1 viruses, we assessed shedding and interspecies waterborne transmission of A/duck/Laos/25/06 in sparrows and chickens. Inoculated birds shed virus at high titers from the oropharynx and cloaca, and infection was fatal. Waterborne transmission from inoculated sparrows to contact chickens was absent, while 25% of sparrows were infected via waterborne transmission from chickens. The viral shedding and susceptibility to infection we observed in sparrows, coupled with their presence in poultry houses, could facilitate virus spread among poultry and wild birds in the face of an H5N1 influenza virus outbreak.The H5N1 influenza A viruses remain a major global concern because of their rapid evolution, genetic diversity, broad host range, and ongoing circulation in wild and domestic birds. H5N1 influenza viruses have swept through poultry flocks across Asia and have spread westward through Eastern Europe to India and Africa since 2003 (1). Sixty-two countries have reported H5N1 influenza virus in domestic poultry/wild birds during the time period 2003 to 2009 (http://www.oie.int/eng/info_ev/en_AI_factoids_2.htm), and to date, more than 400 human infections have been documented in 16 countries, with a mortality rate of ∼61% (http://www.who.int/csr/disease/avian_influenza/country/cases_table_2009_05_22/en/index.html). Most human cases of H5N1 influenza have occurred after contact with infected poultry (13).Some of the more recent isolates of H5N1 highly pathogenic avian influenza (HPAI) virus do not cause overt disease in certain species of domestic and wild ducks; however, these viruses are 100% lethal to chickens and gallinaceous poultry. Because of ducks’ ability to “silently” spread H5N1 HPAI virus and their unresolved role as a reservoir, they are the focus of much research (5, 6, 11). In contrast, the possible role of passerine birds has received little attention, despite their widespread interaction with poultry at many sites worldwide (http://www.searo.who.int/LinkFiles/Publication_PHI-prevention-control-AI.pdf). The order Passeriformes includes more than half of all bird species, including sparrows. Since 2001, several outbreaks of H5N1 influenza virus infection have been reported in passerine birds in eastern Asia, often near infected poultry farms (15). Interestingly, the only confirmed presence of asymptomatic infection with HPAI H5N1 in wild birds was in tree sparrows in Henan Province, China. Both tree and house sparrows (Passer montanus and Passer domesticus, respectively) are members of the Old World sparrow family Passeridae, and in fact, the tree sparrow was not recognized as a species separate from that of the house sparrow until 1713 (http://www.arkive.org/tree-sparrow/passer-montanus/info.html?displayMode=factsheet). The four avian influenza virus isolates obtained from these asymptomatic infections were of the A/Goose/Guangdong/1/96 lineage and were highly pathogenic to experimentally infected chickens (4, 8).Under experimental conditions, passerine species have shown varied susceptibility to HPAI H5N1 viruses. Among sparrows, starlings, and pigeons inoculated with HPAI H5N1 virus isolates, only sparrows experienced lethal infection, and transmission to contact birds was extremely rare (2). Similarly, in sparrows and starlings inoculated with the H5N1 HPAI A/chicken/Hong Kong/220/97 virus, clinical signs were observed only in sparrows, and no deaths occurred (9).To assess the duration and routes of virus shedding and the waterborne virus transmission of HPAI H5N1 virus between sparrows and chickens, we inoculated groups of birds with A/duck/Laos/25/06, which had caused extremely high morbidity and mortality in domestic ducks (7) and was highly pathogenic to chickens, geese, and quail (J.-K. Kim and R. G. Webster, unpublished data). The virus was obtained from our collaborators in Lao People''s Democratic Republic and was grown in the allantoic cavities of 10-day-old embryonated chicken eggs (eggs) for 36 to 48 h at 35°C. The allantoic fluid was harvested, titrated (50% egg infective dose [EID₅₀] per milliliter), and stored at −80°C. All experiments were approved by the U.S. Department of Agriculture and the U.S. Centers for Disease Control and Prevention and were performed in biosafety level 3+ facilities at St. Jude Children''s Research Hospital. Wild house sparrows (Passer domesticus) were captured locally (Memphis, TN), and specific-pathogen-free outbred White Leghorn chickens (Gallus domesticus) were purchased from Charles River Laboratories (North Franklin, CT). All animal experiments were approved by the St. Jude Animal Care and Use Committee and complied with the policies of the National Institutes of Health and the Animal Welfare Act.Before inoculation, oropharyngeal and cloacal swabs were collected from sparrows, and baseline blood samples were collected from chickens to exclude preexisting H5N1 influenza virus infection. Eight sparrows were inoculated intranasally with 10⁶ EID₅₀ of virus in a volume of 100 μl, and five chickens were inoculated with 10² EID₅₀ of virus in a volume of 1 ml (0.5 ml intranasally, 0.5 ml intratracheally, and 1 drop per eye). All birds in each experimental group were housed in a single cage. Inoculated sparrows were provided with 1 liter of water in a shallow stainless steel pan at the bottom of the cage, and chickens were given 3 liters of water in a trough inside the cage. Twenty-four hours after inoculation, 1 liter of water was removed from the inoculated chickens’ cage and placed undiluted in a cage housing 8 contact sparrows; similarly, 1 liter of water was taken from the inoculated sparrows’ cage, mixed with 2 liters of fresh water, and placed in a cage housing 5 contact chickens. Clinical disease signs, including depression, huddling at the cage bottom, and ruffled feathers, were monitored through daily observation, and oropharyngeal and cloacal swabs obtained from all birds were collected daily for 14 days. Swab samples were titrated in eggs and expressed as log₁₀ EID₅₀/ml (10). The lower limit of detection was 0.75 log₁₀ EID₅₀/ml.Blood samples were taken from all surviving contact birds on day 14 of the study. Sera were treated with a receptor-destroying enzyme (Denka Seiken, Campbell, CA), as instructed by the manufacturer, and heat inactivated at 56°C for 30 min. Hemagglutination inhibition (HI; using 0.5% packed chicken red blood cells) titers were determined as the reciprocal of the highest serum dilution that inhibited 4 hemagglutinating units of virus. HI titers of ≥10 were considered suggestive of recent influenza virus infection.Inoculation with A/duck/Laos/25/06 was lethal to all birds (Table ​(Table1).1). While chickens succumbed to infection within 2 days postinoculation (p.i.), the mean time until death for sparrows was 4.1 days; mortality occurred rapidly (overnight) without prior observation of clinical signs. Expected clinical signs, should they have occurred, included moderate to severe depression, huddling at the cage bottom, and ruffled feathers (9). All inoculated birds shed virus from the oropharynx and, to a lesser extent, from the cloaca (Fig. 1A and B). The mean virus titers of inoculated chickens and sparrows were comparable on day 1 p.i.; however, on day 2 p.i., the mean oropharyngeal and cloacal viral titers of chickens were approximately 2 and 2.5 times greater, respectively, than those of sparrows (Fig. 1A and B). The virus titer in water used by inoculated sparrows was 10^0.75 EID₅₀/ml at 1 day p.i. and peaked at 10^1.75 EID₅₀/ml on days 2 and 4 p.i. (Fig. ​(Fig.1C).1C). No virus was detected in water from the inoculated chickens’ cage.Open in a separate windowFIG. 1.Mean oropharyngeal and cloacal virus titers in sparrows (A) and chickens (B) inoculated with a lethal dose of A/duck/Laos/25/06 (H5N1) virus. (C) Virus titers in the drinking water of inoculated sparrows. Sparrows were inoculated with 10⁶ EID₅₀/ml of virus, and chickens were inoculated with 10² EID₅₀/ml of virus. The lower limit of detection was 0.75 log₁₀ EID₅₀/ml.

TABLE 1.

Transmission rates, mortality rates, and mean peak titers of A/duck/Laos/25/06 (H5N1) virus in inoculated and contact birds