Effects of swab pool size and transport medium on the detection and isolation of avian influenza viruses in ostriches.

BACKGROUND: Rigorous testing is a prerequisite to prove freedom of notifiable influenza A virus infections in commercially farmed ostriches, as is the isolation and identification of circulating strains. Pooling 5 ostrich tracheal swabs in a 50 % v/v phosphate-buffered saline (PBS): glycerol transport medium (without antibiotics) is the current standard practice to increase reverse transcription real time PCR (RT-rtPCR) testing throughput and simultaneously reduce the test costs. In this study we investigated whether doubling ostrich tracheal swabs to 10 per pool would affect the sensitivity of detection of H5N8 high pathogenicity avian influenza virus (HPAIV) and H7N1 low pathogenicity avian influenza virus (LPAIV) by quantitative RT-rtPCR, and we also compared the effect of a protein-rich, brain heart infusion broth (BHI) virus transport media containing broad spectrum antimicrobials (VTM) on the efficacy of isolating the H5N8 and H7N1 viruses from ostrich tracheas, since the historical isolation success rate from these birds has been poor. RESULTS: Increasing the ostrich swabs from 5 to 10 per pool in 3 mls of transport medium had no detrimental effect on the sensitivity of the RT-rtPCR assay in detecting H5N8 HPAIV or H7N1 LPAIV; and doubling of the swab pool size even seemed to improve the sensitivity of virus detection at levels that were statistically significant (p less than or equal to 0.05) in medium and low doses of spiked H5N8 HPAIV and at high levels of spiked H7N1 LPAIV. On virus isolation, more samples were positive when swabs were stored in a protein-rich viral transport medium supplemented with antimicrobials in PBS: glycerol (10/18 vs. 7/18 for H5N8 HPAI); although the differences were not statistically significant, overall higher virus titres were detected (106.7 - 103.0 vs. 106.6 - 103.1 EID50 for H5N8 HPAIV and 105.5 - 101.4 vs. 105.1 - 101.3 EID50 for H7N1 LPAIV); and fewer passages were required with less filtration for both H5N8 HPAI and H7N1 LPAI strains. CONCLUSION: Ostrich tracheal swab pool size could be increased from 5 to 10 in 3mls of VTM with no loss in sensitivity of the RT-rtPCR assay in detecting HPAI or LPAI viruses, and HPAI virus could be isolated from a greater proportion of swabs stored in VTM compared to PBS: glycerol without antibiotics.

H7N1 Low Pathogenicity Avian Influenza Viruses in Poultry in the United States During 2018.

Here, we report three detections of H7N1 low pathogenicity avian influenza viruses (LPAIV) from poultry in Missouri (n = 2) and Texas (n = 1) during February and March 2018. Complete genome sequencing and comparative phylogenetic analysis suggest that the H7 LPAIV precursor viruses were circulating in wild birds in North America during the fall and winter of 2017 and spilled over into domestic poultry in Texas and Missouri independently during the spring of 2018.

Nota de investigación­Virus de la influenza aviar de baja patogenicidad H7N1 en avicultura, Estados Unidos, 2018. En este artículo se reportan tres detecciones del virus de influenza aviar de baja patogenicidad H7N1 (LPAIV) en avicultura en Missouri (n = 2) y Texas (n = 1) durante febrero y marzo del 2018. La secuenciación completa del genoma y el análisis filogenético comparativo sugieren que precursores de este virus de influenza de baja patogenicidad H7 circulaban en aves silvestres en América del Norte durante el otoño y el invierno de 2017 y se propagaron a las aves comerciales en Texas y Missouri de forma independiente durante la primavera del 2018.

Emergence of highly pathogenic H5N2 and H7N1 influenza A viruses from low pathogenic precursors by serial passage in ovo.

Highly pathogenic (HPAI) strains emerge from their low pathogenic (LPAI) precursors and cause severe disease in poultry with enormous economic losses, and zoonotic potential. Understanding the mechanisms involved in HPAI emergence is thus an important goal for risk assessments. In this study ostrich-origin H5N2 and H7N1 LPAI progenitor viruses were serially passaged seventeen times in 14-day old embryonated chicken eggs and Ion Torrent ultra-deep sequencing was used to monitor the incremental changes in the consensus genome sequences. Both virus strains increased in virulence with successive passages, but the H7N1 virus attained a virulent phenotype sooner. Mutations V63M, E228V and D272G in the HA protein, Q357K in the nucleoprotein (NP) and H155P in the neuraminidase protein correlated with the increased pathogenicity of the H5N2 virus; whereas R584H and L589I substitutions in the polymerase B2 protein, A146T and Q220E in HA plus D231N in the matrix 1 protein correlated with increased pathogenicity of the H7N1 virus in embryos. Enzymatic cleavage of HA protein is the critical virulence determinant, and HA cleavage site motifs containing multibasic amino acids were detected at the sub-consensus level. The motifs PQERRR/GLF and PQRERR/GLF were first detected in passages 11 and 15 respectively of the H5N2 virus, and in the H7N1 virus the motifs PELPKGKK/GLF and PELPKRR/GLF were detected as early as passage 7. Most significantly, a 13 nucleotide insert of unknown origin was identified at passage 6 of the H5N2 virus, and at passage 17 a 42 nucleotide insert derived from the influenza NP gene was identified. This is the first report of non-homologous recombination at the HA cleavage site in an H5 subtype virus. This study provides insights into how HPAI viruses emerge from low pathogenic precursors and demonstrated the pathogenic potential of H5N2 and H7N1 strains that have not yet been implicated in HPAI outbreaks.

Replication and pathogenic potential of influenza A virus subtypes H3, H7, and H15 from free-range ducks in Bangladesh in mammals.

Surveillance of wild aquatic birds and free-range domestic ducks in the Tanguar Haor wetlands in Bangladesh has identified influenza virus subtypes H3N6, H7N1, H7N5, H7N9, and H15N9. Molecular characterization of these viruses indicates their contribution to the genesis of new genotypes of H5N1 influenza viruses from clade 2.3.2.1a that are dominant in poultry markets in Bangladesh as well as to the genesis of the highly pathogenic H5N8 virus currently causing disease outbreaks in domestic poultry in Europe and the Middle East. Therefore, we studied the antigenicity, replication, and pathogenicity of influenza viruses isolated from Tanguar Haor in the DBA/2J mouse model. All viruses replicated in the lung without prior mammalian adaptation, and H7N1 and H7N9 viruses caused 100% and 60% mortality, respectively. H7N5 viruses replicated only in the lungs, whereas H7N1 and H7N9 viruses also replicated in the heart, liver, and brain. Replication and transmission studies in mallard ducks showed that H7N1 and H7N9 viruses replicated in ducks without clinical signs of disease and shed at high titers from the cloaca of infected and contact ducks, which could facilitate virus transmission and spread. Our results indicate that H7 avian influenza viruses from free-range ducks can replicate in mammals, cause severe disease, and be efficiently transmitted to contact ducks. Our study highlights the role of free-range ducks in the spread of influenza viruses to other species in live poultry markets and the potential for these viruses to infect and cause disease in mammals.

Risk of Human Infections With Highly Pathogenic H5N2 and Low Pathogenic H7N1 Avian Influenza Strains During Outbreaks in Ostriches in South Africa.

Background: Risk factors for human infection with highly pathogenic (HP) and low-pathogenic (LP) avian influenza (AI) H5N2 and H7N1 were investigated during outbreaks in ostriches in the Western Cape province, South Africa. Methods: Serum surveys were conducted for veterinarians, farmworkers, and laboratory and abattoir workers involved in 2 AI outbreaks in the Western Cape province: (1) controlling and culling of 42000 ostriches during (HPAI)H5N2 outbreaks in ostriches (2011) (n = 207); (2) movement control during (LPAI)H7N1 outbreaks in 2012 (n = 66). A third serosurvey was conducted on state veterinarians from across the country in 2012 tasked with disease control in general (n = 37). Antibodies to H5 and H7 were measured by means of hemagglutination inhibition and microneutralization assays, with microneutralization assay titers >40 considered positive. Results: Two of 207 (1%) participants were seropositive for H5 and 4 of 207 (2%) for H7 in 2011, compared with 1 of 66 (1.5%) and 8 of 66 (13%) in 2012. Although individuals in all professions tested seropositive, abattoir workers (10 of 97; 10.3%) were significantly more at risk of influenza A(H7N1) infection (P = .001) than those in other professions (2 of 171;1.2%). Among state veterinarians, 4 of 37(11%) were seropositive for H7 and 1 of 37 (2.7%) for H5. Investigations of (LP)H7N1-associated fatalities in wild birds and quarantined exotic birds in Gauteng, AI outbreaks in poultry in KwaZulu-Natal, and ostriches in Western Cape province provide possible exposure events. Conclusion: (LPAI)H7N1 strains pose a greater infection-risk than (HPAI)H5N2 strains to persons involved in control of outbreaks in infected birds, with ostrich abattoir workers at highest risk.

Risk for Low Pathogenicity Avian Influenza Virus on Poultry Farms, the Netherlands, 2007-2013.

Using annual serologic surveillance data from all poultry farms in the Netherlands during 2007-2013, we quantified the risk for the introduction of low pathogenicity avian influenza virus (LPAIV) in different types of poultry production farms and putative spatial-environmental risk factors: distance from poultry farms to clay soil, waterways, and wild waterfowl areas. Outdoor-layer, turkey (meat and breeder), and duck (meat and breeder) farms had a significantly higher risk for LPAIV introduction than did indoor-layer farms. Except for outdoor-layer, all poultry types (i.e., broilers, chicken breeders, ducks, and turkeys) are kept indoors. For all production types, LPAIV risk decreased significantly with increasing distance to medium-sized waterways and with increasing distance to areas with defined wild waterfowl, but only for outdoor-layer and turkey farms. Future research should focus not only on production types but also on distance to waterways and wild bird areas. In addition, settlement of new poultry farms in high-risk areas should be discouraged.

Rapid detection of avian influenza A virus by immunochromatographic test using a novel fluorescent dye.

Sensitive and rapid diagnostic systems for avian influenza (AI) virus are required to screen large numbers of samples during a disease outbreak and to prevent the spread of infection. In this study, we employed a novel fluorescent dye for the rapid and sensitive recognition of AI virus. The styrylpyridine phosphor derivative was synthesized by adding allyl bromide as a stable linker and covalently immobilizing it on latex beads with antibodies generating the unique Red dye 53-based fluorescent probe. The performance of the innovative rapid fluorescent immnunochromatographic test (FICT) employing Red dye 53 in detecting the AI virus (A/H5N3) was 4-fold and 16-fold higher than that of Europium-based FICT and the rapid diagnostic test (RDT), respectively. In clinical studies, the presence of human nasopharyngeal specimens did not alter the performance of Red dye 53-linked FICT for the detection of H7N1 virus. Furthermore, in influenza A virus-infected human nasopharyngeal specimens, the sensitivity of the Red dye 53-based assay and RDT was 88.89% (8/9) and 55.56% (5/9) relative to rRT-PCR, respectively. The photostability of Red dye 53 was higher than that of fluorescein isothiocyanate (FITC), showing a stronger fluorescent signal persisting up to 8min under UV. The Red dye 53 could therefore be a potential probe for rapid fluorescent diagnostic systems that can recognize AI virus in clinical specimens.

Risk factors for H7 and H9 infection in commercial poultry farm workers in provinces within Pakistan.

A cross sectional survey was conducted involving 354 farm poultry workers on 85 randomly selected commercial poultry farms in high density poultry farm areas in Pakistan to estimate the sero-prevalence of H5, H7 and H9 and to identify the potential risk factors for infection with the avian influenza virus. A haemagglutination inhibition test titre at 1:160 dilution was considered positive, based on WHO guidelines. The estimated sero-prevalence was 0% for H5, 21.2% for H7 and 47.8% for H9. Based on a generalized linear mixed model, the significant risk factors for H7 infection were area, type of farm and age of poultry worker. Risk of infection increased with the age of poultry workers. Compared with broiler farms, breeder farms presented a greater risk of infection (odds ratio [OR]=3.8, 95% confidence interval [CI]: 1.4, 10.1). Compared with the combined Khyber Pakhtunkhwa Province and Federal area, North Punjab had higher observed biosecurity measures and presented a lesser risk of infection (OR=0.3, 95% CI 0.1, 0.9). Biosecurity should therefore be enhanced (especially in breeder farms) to reduce the occupational risks in poultry farm workers and to decrease the risk of emergent human-adapted strains of AI H7 and H9 viruses.

Análisis de las coinfecciones detectadas entre los virus gripales A y B y otros virus respiratorios, 2012-2013 / Analysis of co-infections between influenza A and influenza B viruses and other respiratory viruses, 2012-2013

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Serological evidence of H7, H5 and H9 avian influenza virus co-infection among herons in a city park in Jiangxi, China.

Extensive surveillance of influenza A viruses in different avian species is critical for understanding its transmission. Here, a breeding colony of Little Egrets and Black-crowned Night Herons was monitored both serologically and virologically in a city park of Jiangxi in 2009. A portion of herons had antibodies against H7 (52%), H5 (55%) and H9 (6%) subtype avian influenza virus (AIV) in egg yolk samples, and 45% had antibodies against different AIV serotypes (H5, H7 or H9) simultaneously. Greater numbers of samples with anti-AIV H5N1 recombination-4 (Re-4, clade 7) antibodies were measured compared with those containing anti-H5N1 Re-1 (clade 0) and Re-5 (clade 2.3.4) antibodies. Eight strains of H5 and 9 strains of H9 were isolated from poultry of nearby markets. These results indicate wild birds are at risk from infection and co-infection with H7, H5, and H9 subtypes. Investigation of wild bird infection might provide an early warning sign of potential novel AIVs circulating in the nearby poultry industry and even in human society.

Genetic characterization of influenza A virus subtype H7N1 isolated from quail, Thailand.

In Thailand, surveillance for the highly pathogenic avian influenza virus H5N1 (HPAI-H5N1) has revealed high prevalence of the virus in quail in live-bird markets. This study monitored avian influenza viruses (AIVs) in quail farms in an area at high risk for HPAI-H5N1 over a 12-month period from 2009 to 2010. One-step real-time RT-PCR (rRT-PCR) results showed that 1.18 % of swab samples (24/2,040) were AIV positive. Among the rRT-PCR positive samples, three samples were identified as subtype H7N1. One Thai H7N1 virus designated "A/quail/Thailand/CU-J2882/2009 (H7N1)" was subjected to whole genome sequencing and genetic characterization. Phylogenetic analysis showed that the HA gene of the Thai H7N1 virus groups with those of the H7 Eurasian viruses. Interestingly, the NA gene of the virus was found to be closely related to those of the HPAI-H5N1 viruses from Vietnam and Thailand. This study constitutes the first report on AIV H7N1 in Thailand. Our results suggest the possibility of genetic reassortment between AIV-H7NX and HPAI-H5N1 in quail. The HA cleavage site of the Thai H7N1 virus contains no multiple amino acid insertions, suggesting low pathogenic characteristics for this virus.

Upconversion luminescence resonance energy transfer (LRET)-based biosensor for rapid and ultrasensitive detection of avian influenza virus H7 subtype.

Avian influenza viruses (AIV) with good adaptation and various mutations have threatened both human and animals' health. The H7 subtypes have the potential to cause pandemic threats to human health due to the highly pathogenic characteristics. Therefore, it is quite urgent to develop a novel biosensor for rapid and sensitive detection of H7 subtypes. In this work, a biosensor based on luminescence resonance energy transfer (LRET) from BaGdF5:Yb/Er upconversion nanoparticles (UCNPs) to gold nanoparticles (AuNPs) has been developed for rapid and sensitive H7 subtypes detection. The amino modified capture oligonucleotide probes are covalently linked to poly(ethylenimine) (PEI) modified BaGdF5:Yb/Er UCNPs. The thiol modified oligonucleotides with H7 hemagglutinin gene sequence are conjugated to surfaces of AuNPs. The hybridization process between complementary strands of H7 Hemagglutinin gene and its probe brings the energy donor and acceptor into close proximity, leading to the quenching of fluorescence of UCNPs. A linear response is obtained ranging from 10 pm to 10 nm and the limit of detection (LOD) is around 7 pm with detection time around 2 hours. This biosensor is expected to be a valuable diagnostic tool for rapid and sensitive detection of AIV.

Persistence of low-pathogenic H5N7 and H7N1 avian influenza subtypes in filtered natural waters.

Wild aquatic birds are the natural reservoir of avian influenza virus (AIV), and the virus is transmitted among birds through a fecal-oral route. Infected birds excrete significant amounts of AIV into the environment, and thereby sustain the circulation of AIV in the bird populations. Improved knowledge on the influence of environmental factors on the persistence of AIV in natural habitats would be valuable for risk assessments. The presented work investigated the persistence of two low-pathogenic AIV subtypes in natural water samples. The study included two AIVs formerly isolated from wild ducks, which were suspended in filtered natural fresh, brackish or sea water with salinity of 0, 8000 and 20,000 parts per million (ppm), respectively. Also sterilized brackish and sea waters were included in order to examine the influence of microbial flora on virus persistence. All water samples were incubated at temperatures representative for seasonal variation of ambient temperatures in Northern Europe (4, 17 and 25 °C). The results showed a clear correlation between persistence of viral infectivity and temperature, salinity and presence of microbial flora. While independent of virus subtype, the persistence of infectivity was negatively affected by increased temperature, salinity as well as presence of natural microbial flora. The study provides insight on impact of essential physical, chemical and biological parameters on persistence of AIV in aquatic environments. Studies determining the importance of additional environmental parameters and the detailed mechanisms of microbial inactivation of AIV should be encouraged.

Guiding outbreak management by the use of influenza A(H7Nx) virus sequence analysis.

The recently identified human infections with avian influenza A(H7N9) viruses in China raise important questions regarding possible source and risk to humans. Sequence comparison with an influenza A(H7N7) outbreak in the Netherlands in 2003 and an A(H7N1) epidemic in Italy in 1999­2000 suggests that widespread circulation of A(H7N9) viruses must have occurred in China. The emergence of human adaptation marker PB2 E627K in human A(H7N9) cases parallels that of the fatal A(H7N7) human case in the Netherlands.

Proteins of duck influenza virus responsible for acquisition of pathogenicity in chickens.

Influenza virus rgVac1sub-P0 (H5N1) (rgVac1-P0), in which a pair of dibasic amino acid residues was introduced at the cleavage site of the HA of a reassortant of H5N2 and H7N1 viruses of duck origin, was low pathogenic in chickens. Vac1sub-P3 (H5N1) (Vac1-P3) was selected as a highly pathogenic avian influenza virus by 3 consecutive passages in chickens from low pathogenic strain rgVac1-P0. Comparison of amino acid sequences of the virus proteins and experimental infection of chickens with a series of recombinant viruses demonstrated that in addition to the HA, each of the PA, NP, M1, and M2 of Vac1-P3 are responsible for the acquisition of pathogenicity in chickens. These 4 proteins of Vac1-P3 synergistically contributed to efficient virus replication in chickens.

Systemic distribution of different low pathogenic avian influenza (LPAI) viruses in chicken.

BACKGROUND: Since we were able to isolate viable virus from brain and lung of H7N1 low pathogenic avian influenza virus (LPAIV) infected chickens, we here examined the distribution of different LPAIV strains in chickens by measuring the viral AI RNA load in multiple organs. Subtypes of H5 (H5N1, H5N2), H7 (H7N1, H7N7) and H9 (H9N2), of chicken (H5N2, H7N1, H7N7, H9N2), or mallard (H5N1) origin were tested. The actual presence of viable virus was evaluated with virus isolation in organs of H7N7 inoculated chickens. FINDINGS: Viral RNA was found by PCR in lung, brain, intestine, peripheral blood mononuclear cells, heart, liver, kidney and spleen from chickens infected with chicken isolated LPAIV H5N2, H7N1, H7N7 or H9N2. H7N7 virus could be isolated from lung, ileum, heart, liver, kidney and spleen, but not from brain, which was in agreement with the data from the PCR. Infection with mallard isolated H5N1 LPAIV resulted in viral RNA detection in lung and peripheral blood mononuclear cells only. CONCLUSION: We speculate that chicken isolated LPAI viruses are spreading systemically in chicken, independently of the strain.

Selection of variant viruses during replication and transmission of H7N1 viruses in chickens and turkeys.

The influence of different glycosylation patterns of the haemagglutinin glycoprotein of H7N1 avian influenza viruses on virus replication in vivo was examined. Experimental infection of chickens and turkeys was carried out with H7N1 avian influenza viruses with alternative sites of glycosylation in the haemagglutinin and infected birds were sampled daily by swabbing the buccal and cloacal cavities. cDNAs of the HA1 coding region of the HA gene were prepared from the swabs and cloned into plasmids. Sequencing multiple plasmids made from individual swabs taken over the period of virus shedding showed that viruses with specific patterns of glycosylation near the receptor binding site were stable when birds were infected with a single variant, but when presented with a mixed population of viruses encoding differing patterns of glycosylation a specific variant was rapidly selected in the infected host.

Antibody response and viral shedding profile of Egyptian geese (Alopochen aegyptiacus) infected with low pathogenicity H7N1 and H6N8 avian influenza viruses.

Egyptian geese (Alopochen aegypticus), a duck species endemic to sub-Saharan Africa and occasionally implicated in the transmission of avian influenza viruses (AIV) to farmed ostriches, were experimentally infected with low pathogenicity H7N1 and H6N8 viruses to assess viral shedding and immune profiles. Following the first infection with H7N1 virus, high titers of virus were shed from both the tracheae and cloacae for at least 7 days postinfection, and tracheal shedding lasting until day 14. All detectable shedding from both tracheae and cloacae had ceased within 28 days of infection. Antibody titers peaked at day 7 postinfection, but the initial immune response was short-lived. Birds that received a second challenge with the homologous H7N1 virus mounted a more robust response that lasted beyond 66 days postchallenge, and H7N1 virus was detected, albeit at much lower levels, until day 28 post secondary infection (psi) in the cloaca and beyond day 28 psi in the trachea. Birds that received an initial infection with H7N1 virus were also challenged with H6N8 virus, and because a comparable shedding pattern to the H7N1 challenge group was observed, we concluded that the effect of any nonspecific immunity was negligible.

Systemic virus distribution and host responses in brain and intestine of chickens infected with low pathogenic or high pathogenic avian influenza virus.

BACKGROUND: Avian influenza virus (AIV) is classified into two pathotypes, low pathogenic (LP) and high pathogenic (HP), based on virulence in chickens.Differences in pathogenicity between HPAIV and LPAIV might eventually be related to specific characteristics of strains, tissue tropism and host responses. METHODS: To study differences in disease development between HPAIV and LPAIV, we examined the first appearance and eventual load of viral RNA in multiple organs as well as host responses in brain and intestine of chickens infected with two closely related H7N1 HPAIV or LPAIV strains. RESULTS: Both H7N1 HPAIV and LPAIV spread systemically in chickens after a combined intranasal/intratracheal inoculation. In brain, large differences in viral RNA load and host gene expression were found between H7N1 HPAIV and LPAIV infected chickens. Chicken embryo brain cell culture studies revealed that both HPAIV and LPAIV could infect cultivated embryonic brain cells, but in accordance with the absence of the necessary proteases, replication of LPAIV was limited. Furthermore, TUNEL assay indicated apoptosis in brain of HPAIV infected chickens only. In intestine, where endoproteases that cleave HA of LPAIV are available, we found minimal differences in the amount of viral RNA and a large overlap in the transcriptional responses between HPAIV and LPAIV infected chickens. Interestingly, brain and ileum differed clearly in the cellular pathways that were regulated upon an AI infection. CONCLUSIONS: Although both H7N1 HPAIV and LPAIV RNA was detected in a broad range of tissues beyond the respiratory and gastrointestinal tract, our observations indicate that differences in pathogenicity and mortality between HPAIV and LPAIV could originate from differences in virus replication and the resulting host responses in vital organs like the brain.

Persistence of low-pathogenic avian influenza H5N7 and H7N1 subtypes in house flies (Diptera: Muscidae).

Avian influenza caused by avian influenza virus (AIV) has a negative impact on poultry production. Low-pathogenic AIV (LPAIV) is naturally present in wild birds, and the introduction of the virus into domestic poultry is assumed to occur through contact with wild birds and by human activity, including the movement of live and dead poultry, and fomites such as clothing and vehicles. At present, the possible role of insects in the spread of AIV is dubious. The objective of the present work was to investigate the potential transmission of LPAIV by persistence of the virus in the alimentary tract of house flies, Musca domestica L. (Diptera: Muscidae). Flies were fed three virus concentrations of two AIV strains and then incubated at different temperatures for up to 24 h. The persistence of the two virus strains in the flies declined with increasing incubation temperatures and incubation periods. Similarly, increased virus uptake by the flies increased the persistence of virus. Persistence of infective AIV in flies differed significantly between the two virus strains. The laboratory experiments of the present study indicate that the house fly can be a potential carrier of AIV.