Characterization of an H6N1 avian influenza virus from an illegally imported product

Background : H6N1 is low pathogenic virus commonly found in wild and domestic birds across many continents. To investigate the infective status of imported food, we detected avian influenza virus (AIV) in illegally imported frozen food from international flight. Methods : The virus was isolated, the complete genome was sequenced, and the pathogenicity was tested. PQIATR/G was found in the motif of the HA cleavage site of the isolated strain, which conforms to the molecular characteristics of a low pathogenic AIV. The amino acid residues at positions 186, 190, 226, and 228 of the HA protein receptor binding sites were proline (P), valine (V), glutamine (Q), serine (S), respectively, exhibiting the molecular characteristics required to bind to human-like receptors. The SPF chicken test showed that the IVPI was 0 and the virus was a low pathogenic AIV. The infectious results in BALB/c mice showed that the isolate could be replicated in the lower respiratory tract of mice. Mice infected with a high dose of the virus displayed moderate weight loss but no obvious clinical symptoms. Results :The results of the sequence analysis showed that the avian influenza virus was of the H6N1 subtype, which shared the highest nucleotide identity with the AIV subtypes prevalent in Taiwan province. Conclusions : It is important to intensily monitor the illegal carrying.


Background
Avian influenza viruses (AIVs) have caused huge economic losses to the global poultry industry and posed a great threat to global public health [1][2] . Currently, 16 HA subtypes (H1-H6) and 9 NA (N1-N9) of influenza viruses have been isolated from birds [3] . AIVs can be divided into highly pathogenic AIV (HPAIV) and low pathogenic AIV (LPAIV). HPAIV can directly infect waterfowl and poultry, and may occasionally infect humans. Moreover, LPAIV may lead to the generation of HPAIV through genetic mutations or assortment. Thus, LPAIV infections are becoming a major threat to the poultry industry and only limited protection is provided by the available inactivated vaccines due to an improper cold chain or administered dose [4] . Although migratory birds and commercial live poultry have been considered to be major factors associated with spread of HPAIVs, smuggled avian products are also 3 risk factors for AIV spread internationally. In 2005, a low pathogenic H10N7 AIV was isolated from one of the smuggled duck carcasses, which was later seized by official controls in a warehouse in Italy [5] .
From 2015 to 2016, highly (H5N1 and H5N6) and low (H9N2 and H1N2) pathogenic AIVs were isolated from raw chickens and duck products illegally imported to Japan by international passengers [6] .
On Oct 13 th , 2017, a frozen chicken illegally introduced into China by international passengers was seized. In this study, a low pathogenic H6N1 AIV was isolated from this chicken. The results clearly show that illegal raw products can contribute to the spread of AIV and we should intensely monitor such illegal animal transportation.

Poultry product confiscated at inspection and quarantine
One frozen uncooked chicken was confiscated at Pudong International Airport by a passenger from airline CI501 flying from Taiwan province to Shanghai. [7] The liver and lung tissues of the frozen uncooked chickens were collected and cut into small pieces and placed in a tube with magnetic beads followed by rubbing. The homogenate was then centrifuged at 10 000 rpm for 10 min, and the supernatants were used for RNA extraction. Viral RNA was extracted with the QIAamp viral RNA mini kit (Qiagne, Hilden, Germany) and maintained at -80℃ until use.

Real-time RT-PCR
The RNA were detected using an influenza A RT-PCR assay LE (Qiagen, Hilden, Germany) for all known influenza viruses. A H5/H7/H9 subtype (Qiagen, Hilden, Germany) kit was used for further analysis. All procedure was carried out according to the kit instructions.

Virus isolation and identification
The virus was isolated in specific pathogen-free (SPF) chicken embryonated eggs at 35℃ for 72 h.
Allantoic fluid was harvested and hemagglutination (HA) assay with 1% turkey red blood cells (TRBCs) was used to confirm the presence of influenza viruses. The HA positive samples were further identified by reverse transcription-polymerase chain reaction (RT-PCR) using primers published by Hoffmann [8 ] .

Viral RNA extraction and RT-PCR
Viral RNA was extracted from allantoic fluid using an RNeasy Mini kit (Qiagen, Hilden, Germany) and transcribed into complimentary DNA (cDNA). The PCR reaction contained 3.0 μL cDNA, 1 μL forward primer and reverse primer, 12.5 μL RT-PCR Buffer, 1.25 μL Enzyme Mix, and 6.25 μL RNasefree water with a final volume of 25 μL. A single PCR program was used at 50 ℃for 30 min;94℃ for 2 min, 35 cycles of denaturation at 94℃ for 30 s, annealing at 57℃ for 30 s, and extension at 72℃ for 3 min. The program ended with one cycle at 72℃ for 5 min. [7] The PCR products were visualized by agarose gel electrophoresis and purified using a QIA quick PCR purification kit (Qiagen, Hilden, Germany). The purified PCR products were sequenced and the full genome sequences of the viruses were deposited in the GenBank database (accession no. MP706030-MP706037). Phylogenetic trees were generated using the neighbor-joining method with MEGA software (version 5.01, Molecular Evolutionary Genetics Analysis (MEGA; http://megasoftware.net/), and the bootstrap value was tested with 1 000 replications for each gene.

Intravenous pathogenicity index (IVPI) and Mouse Experiments
The isolated viruses were inoculated intravenously in 10 six-week-old SPF chickens with 0.1 mL of a 1/10 dilution of a bacteria-free, infective allantoic fluid. The chickens were observed 10 days for sick and death. Stocks of the isolated virus were produced by passaging one time in 10-day-old SPF embryonated chicken eggs following a 10-fold serial dilution. Virus titers in virus stocks were determined with standard methods using 10-day-old SPF embryonated chicken eggs (ECEs). Virus suspensions were prepared in PBS (pH 7.2), and the allantoic cavities of five ECEs were infected with 0.1 mL of each dilution. The ECEs were incubated at 37℃ with a relative humidity of 60% for 72 h.
The presence of viruses was determined with the hemagglutination assay. Viral titers were expressed as log10 EID 50 /mL.

5
To investigate the pathogenicity in mammals, the virus (2×10 8 EID 50 /0.1mL) was 10-fold serially diluted and each dilution was inoculated into six-week-old BALB/c mice Beijing Vital River Laboratory Animal Technology Co., Ltd. at a dose of 50 μL per animal. The five mice were monitored and weighed daily at two-week intervals. The mice to be euthanized were placed in a sealed box, and carbon dioxide was released to suffocate the mice. The lungs, nasal turbinates, spleens, and brains of the mice were collected and the viral titers were determined.

Real-time RT-PCR
Real-time RT-PCR showed that influenza A virus was positive ( Figure 1A), and the H5, H7, and H9 subtypes were negative ( Figure 1B-D).

Virus isolation and genome sequencing
All samples were inoculated in 10-day-old SPF embryonated eggs, and the allantoic fluid was harvested after 72 h. The HA titer was approximately 2 8 -2 9 . The isolated virus was named A/chicken/Taiwan/6634/2017 (i.e., TW6634). After blasting with HA and NA sequences, TW6634 was found to be closely related to the H6N1 subtype.

Sequence analysis
The sequence analysis revealed that the TW6634 genes shared the highest nucleotide identity with the AIV subtypes prevalent in Taiwan province ( A bioinformatics analysis of the TW6634 viral genome revealed that the receptor binding sites of the HA gene of the TW6634 virus were P186, V190, Q226, and S228 (H3 numbering). This indicates that TW6634 can bind to the human α 2,6-sialic acid receptor. The NS1 gene had an Alanine mutation at locus 149, which suggested that it might improve the virulence of the virus in chickens [9][10] . The M2 gene had an Asparagine mutation at locus 31, suggesting that the virus might be resistant to 6 amantadine [11][12] .

Animal experiments
After inoculating the virus into SPF chickens, all 10 chickens survived for 10 days, did not display any clinical signs, and the IVPI was 0.0, and then humanely euthanized with cervical dislocation. Thus, TW6634 is a LPAIV strain. After inoculating the TW6634 virus into BALB/c mice, none of the mice displayed any clinical signs and survived the entire observation period. Weight monitoring revealed that only moderate transient weight loss occurred in the mice infected with a high dose of the virus (10 8 EID 50 and 10 7 EID 50 groups) (Fig. 2). Three days after infection, four of the infected mice were sacrificed, and the lungs, sinuses, spleen, and brain tissues were collected for viral isolation and titration. The virus was isolated only in the lungs of infected mice with a titer of 10 3.7 EID 50 /g ( Table   2).

Discussion
In the present study, we isolated a LPAIV H6N1 strain from illegal frozen poultry seized from an international airplane. To further characterize the isolated virus, we conducted genome sequencing, phylogenetic analysis, and a pathogenicity evaluation. The IVPI of TW6634 was found to be 0.0, which is consistent with the amino acid sequence (PQIATR/GLF) at its HA cleavage site. The BLAST sequence analysis revealed that TW6634 displayed the highest sequence homology with the endemic H6N1 subtype of AIVs in Taiwan, with which it had the closest genetic relationship. The analysis of the HA receptor binding site showed that TW6634 could bind to the human α 2, 6-sialic acid receptor. In TW6634-infected mice, the virus was found to replicate in the lungs with weak pathogenicity and the mice that received a high dose of infection only exhibited moderate temporary weight loss. Influenza infections in poultry are of great concern due to the impact on both bird and public health, agricultural trade, and the high cost of associated control [13] . In June 2013, the first case of human avian H6N1 infection was reported in a Taiwanese woman [14] .
Every year, a large number of illegally imported poultry products were confiscated in Shanghai. The illegally products include cooked eggs, chicken thigh meats, cooked chicken, cooked ducks and so on. 7 This is the first time a complete frozen chicken include visceral was illegally imported by international passenger. The LPAI virus caused localized virus infections in respiratory and gastrointestinal tracts [15] . In this experiment, we collected samples from lung tissues may also increases the chances of detecting the virus.
International trade and movement, especially the illegal movement and smuggling of foods, are risk factors for the spread of infectious disease. The results of the present study indicate that it is necessary to further monitor international imports and exports. Moreover, H6N1 is a LPAIV commonly found in poultry and wild birds. The viral sequences isolated from poultry product shows that it is the closest to the Taiwan H6N1 virus strain native to poultry. Recently, the isolation rate of the AIV H6 subtype in China has been increasing [16][17][18] , and the infection rate of waterfowl is relatively high. Therefore, such rates may be related to the gradual enhancement of the adaptive ability of the waterfowl AIV H6 subtype to infect poultry [18] . Receptor binding tests show that more than one-third of the AIV H6 subtypes can recognize human receptors [18] . In addition, animal experiments have shown that some AIV H6 subtypes can effectively replicate in mice and be transmitted by contact [18] .
Moreover, some studies have shown that the H6 virus can mutate into a HPAIV via mutations the sequence of the HA cleavage site, which results in sequences that are characteristic of HPAIVs [19] . Therefore, the AIV H6 subtype is a potential threat to both humans and poultry.

Conclusion
In this study, we could not confirm the origin of poultry products and only able to trace the flight routine. However, the products illegally imported could be the origin of spreading the viruses and it is essential to increase publicity and awareness and strengthen inspection.

Ethics Approval and Consent to Participate
In this research, all the studies using animals were submitted as protocols approved by the Animal Care and Ethics Committee of China Animal Health and Epidemiology Center (No.2017-25).All experiments were performed according to the guidelines of the committee. According to the Customs of PRC, the animal products were prohibited and we have the right to confiscate the product and 8 detect them.

Consent to publication
Not applicable.

Availability of data and materials
All data generated or analysed during this study are included in this published article.

Competing Interest
The author declared that they have no conflict of interest.

Author's contributions
WY designed the study and perfomed the experiments with JW and LJ, and analyzed the data together with ZL, ZQ and LC. WY wrote the initial draft of the manuscript and JW revised the manuscript. All authors have read and approved the manuscript.  Figure 1 Results of Real-time RT-PCR. A. Influenza A assay; B. H5 subtype assay; C. H7 subtype assay; D. H9 subtype assay.
12 Figure 2 Results of weight change rate in mice experiment.

Supplementary Files
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