Isolation and genetic characteristics of Novel H4N1 Avian Influenza viruses in ChongQing, China

Background Avian influenza viruses (AIVs) constitute significant zoonotic pathogens encompassing a broad spectrum of subtypes. Notably, the H4 subtype of AIVs has a pronounced ability to shift hosts. The escalating prevalence of the H4 subtype heightens the concern for its zoonotic potential, signaling an urgent need for vigilance. Methods During the period from December 2021 to November 2023, we collected AIV-related environmental samples and assessed them using a comprehensive protocol that included nucleic acid testing, gene sequencing, isolation culture, and resequencing. Results In this study, a total of 934 environmental samples were assessed, revealing a remarkably high detection rate (43.66%, 289/662) of AIV in the live poultry market. Notably, the H4N1 subtype AIV (cs2301) was isolated from the live poultry market and its complete genome sequence was successfully determined. Subsequent analysis revealed that cs2301, resulting from a reassortment event between wild and domesticated waterfowl, exhibits multiple mutations and demonstrates potential for host transfer. Conclusions Our research once again demonstrates the significant role of wild and domesticated waterfowl in the reassortment process of avian influenza virus, enriching the research on the H4 subtype of AIV, and emphasizing the importance of proactive monitoring the environment related to avian influenza virus. Supplementary Information The online version contains supplementary material available at 10.1186/s12985-024-02352-8.

on algorithms such as BWA and self-developed virus identification method.For influenza positive sample, reads are assembled using custom software IAP and assembled contigs of influenza will be used for phylogenetic analysis.

Applications
This software is only applicable for high-throughput sequencing data analysis of this kit: MGIEasy Respiratory Microorganisms Genome Amplification Kit. 2) This manual and the information contained within are proprietary to MGI Tech Co., Ltd.

Compatibility
(hereinafter called MGI), and ate intended solely for the contractual use of its customer in connection with the use of the product described herein and for no other purpose.
Any person or organization can not entirely or partially reprint, copy, revise, distribute or disclose to others the manual without the prior written consent of MGI.Any unauthorized person should not use this manual.
3) MGI does not make any promise of this manual, including (but not limited to) any commercial of special purpose and any reasonable implied guarantee.MGI has taken measures to guarantee the correctness of this manual.However, MGI is not responsible for missing parts in the manual and reserves the right to revise the manual and the software, so as to improve the reliability, performance or design.

Assembly of influenza genome
If the input sample is identified as influenza positive, MGI FluTrack will complete influenza assembly use self-developed program IAP and output filtered contigs of each influenza genome segment.

Phylogenetic analysis
MGI FluTrack will complete phylogenetic analysis of positive samples with assembled influenza genome contigs.

Report generation
Generate HTML report of input sample with Python script.
Chapter 3. User manual MGI FluTrack manages the entire process of sample input and output through the ZLIMS-MGI lab information management system.The following introduces the operation guide for using the MGI FluTrack analysis software based on the ZLIMS system.

Introduction
This chapter describes how to start MGI FluTrack analysis based on the ZLIMS-MGI system.
Please read this manual carefully before using ZLIMS-MGI to ensure correct analysis.

Applications
MGIEasy Respiratory Microorganisms Genome Amplification Kit.

Operational environment
The server has been configured with the required system environment such as Linux.
If using Chrome browser to login ZLIMS, the browser version should be between 63.0 and 92.0, which ensures FTP folder can be browsed correctly.
3.2 Scenario 1: Sequencing platform + Analysis server (Sequencing + ZLIMS Lite automatic analysis system) The operation consists of five steps: Login to ZLIMS system, Download the sample template, Fill in and import the sample template, Sequencing on the platform and Task status monitoring and viewing.The sequencing task will be started when the operation of sequencing on the platform is completed, and the bioinformatics analysis will be triggered automatically after the sequencing is completed.When the analysis task status icon turns as , it indicates that the task is complete and users can check the report (See section 3.4 for details).[1] Chinese template corresponds to Chinese system environment, English template corresponds to English system environment.
[2] The sample type of the imported data must already exist in the technical route under the MGI FluTrack product.
[3] In the template, the input fields with * are required, and the fields without * are optional.In the imported data, the required field must not be empty.
[4] One sample can only correspond to one barcode, the "Sample ID" in excel must be unique and the "Sample Name" in excel must be unique or empty (Figure 3-5).
[5] Cells in the excel cannot be merged, and spaces or special characters are not allowed in both ends of the string in the cells.

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The sample information filled in can be identical with some sample information already entered in the system.

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Library ID: Library's product number.After sequencing and analysis successful, the result report can be viewed by clicking the icon in the report management , see section 3.4 for detailed operations.Note for Excel: [1] Chinese template corresponds to Chinese system environment, English template corresponds to English system environment.
[2] The sample type of the imported data must already exist in the technical route under the MGI FluTrack product.
[3] In the template, the input fields with * are required, and the fields without * are optional.In the imported data, the required field must not be empty.
[4] One sample can only correspond to one FASTQ file or one pair of FASTQ files, the "Sample ID" in excel must be unique and the "Sample Name" in excel must be unique or empty (Figure 3-25).
[5] Cells cannot be merged in Excel, and spaces or special characters are not allowed in both ends of the string in the cells.

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The sample information filled in can be identical with some sample information already entered in the system.

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Split Data : The downsampling number of reads, the unit is K/M/G, for example: 1K/1M/1G, which means that the corresponding number of reads will be used for analysis, if not set, the 5M data will be used for analysis by default.If DNB loads two chips at the same time, that is, chip 2 is on the machine while the task of chip 1 is still running, then the two chips will enter the same load DNB task and be analyzed together; but if chip 2 is on the machine after chip 1 finished, then chip 2 will create a new task, and two analysis tasks would be launched separately.

Time of sample entry and impact
Sample entry is divided into DNB Sample Entry and Analysis Sample Entry.
1) The DNB Sample Entry time needs to be before the DNB sample is loaded on the sequencer, if the DNB sample is not recorded in the ZLIMS system in advance, but loads on the sequencer directly, the system will automatically create a sample with the sample ID equal to the DNB ID.When you load the DNB directly on the sequencer next time, the system will no longer recognize the DNB ID, because the DNB ID already exists in the system.
2) The Analysis Sample Entry time needs to be after the sequencing is completed, obtain the FASTQ path and fill it in the Analysis Sample Entry.The running time of 5M reads sample will be less than 1 hour.
E. What is the basis for PCT threshold in influenza identification?

7 ) 4 Chapter
are all illustrations.The contents might be slightly different from the software, please refer to the software purchased.5) Intel® is trademark of Intel Corporation or its subsidiaries in the U.S. and/ or other countries.Other names and brands mentioned in this manual may be claimed as the property of others.The operating examples in this user manual do not apply to the ZTRON Pro Appliance product.For getting information on the operation of the software product on the ZTRON Pro, please refer to the user manual for ZTRON Pro Appliance.an automatic analysis software based on Linux operating system, which includes data filtering and quality control, identification of influenza A/B, assembly of influenza genome, phylogenetic analysis and report generation, the figure below is the overall workflow of MGI FluTrack:

Figure 2 - 1
Figure 2-1 Workflow of MGI FluTrack 2.1.1Data filtering and quality control Raw data is the regular FASTQ format.If you set the [Split Data] value in the input form, the software will perform the downsampling process on the raw FASTQ data, otherwise it will use

3.2. 1
Step 1: Login to ZLIMS system Double-click the MGI ZLIMS shortcut on the desktop to enter the ZLIMS system login interface, enter the account ID and password (Figure 3-1), and click Login to enter the main interface (Figure 3-2).

Figure 3
Figure 3-3 MGI FluTrack sample template download Open the MGI FluTrack sample template Excel, and you can see two worksheets, which are

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Pooled Library ID: Pooling homogenized product number.SplitData : The downsampling number of reads, the unit is K/M/G, for example: 1K/1M/1G, which means that the corresponding number of reads will be used for analysis, if not set, the 5M data will be used for analysis by default. Primer file: Primer file of Multi-PCR. DNB ID: It cannot be the same as the DNB ID entered in the system, each lane can only allow one DNB ID, and the DNB ID must be consistent with the DNB ID entered by the corresponding lane during sequencing (see 3.2.4step 4-c).

Figure 3 -
Figure 3-5 example for samples from the sample template (DNB Sample Entry)Remarks:

Figure 3
Figure 3-8-a Sample information import interface

Figure 3 - 9
Figure 3-9 Sample status 3.2.4Step 4: Operation of Sequencing on the platform Use the DNB ID in Step 3 of 3.2.2 to sequence on the platform.Take MGISEQ-200RS as an

Figure 3 -
Figure 3-11-f Sequencing settings 7) After entering the interface, fill in [DNB ID] and [Recipe] select the [Customize], click to enter the Customize parameter settings (Figure 3-12).Note: The DNB ID entered here must be consistent with the DNB ID in the [DNB Sample Entry] form of the analysis system.

Figure 3 -
Figure 3-12 Sequencing Barcode scheme and sequencing scheme settings

Figure 3 -
Figure 3-13 Fill in the sequencing cartridge ID 10) Go to the next step and fill in the sequencing slide ID in the [Flow cell ID] window, click to enter the next step (Figure 3-14).

Figure 3 -
Figure 3-14 Fill in the Flow cell ID 11) Enter the last step, confirm the sequencing information on the interface, and click the

3. 3
Figure 3-17 Step 1 for data uploading 2) Use the left mouse button, select the folder containing the data in the [Mounted mobile storage device directory], and drag it to the [rawdata] directory (Figure 3-18):

Figure 3 -
Figure 3-20 Step two for folder permission modification 5) Ensure that all user permissions are as shown in the figure below, click [Change] (Figure 3-21):

Figure 3 -
Figure 3-23 MGI FluTrack sample template download Open the MGI FluTrack sample template Excel, and you can see two worksheets: [DNB

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Primer file: Primer file of Multi-PCR. If FASTQ data uploaded are in SE (single-end) type, only ' Read1 files path(*)'field needs to be filled in, or if in PE (paired-end) type,'Read2 files path ' field also needs to be filled in at the same time, If the sequencing type is incorrectly filled, the analysis can't be completed normally.And the data path must be filled in full path and correctly (see 3.3.1),the path can't contain special characters such as spaces.

Figure 3 -
Figure 3-25 Fill in the sample template (Analysis Sample Entry)

Figure 3 -
Figure 3-26 Sample template import interface 3.3.4Step 4: Start analysis 1) Click [Laboratory]-[Workflow-Sample Lite], select [MGI FluTrack] as the product to be used for analysis, check the samples to be analyzed, click the [Create Task] button (Figure 3-27), and there will be a pop-up page for analysis details.

Figure 3 -
Figure 3-28 Task details page 3) Click [Save] button on the analysis parameter interface to start analysis (Figure 3-29), and close the opened analysis details page (Figure 3-29).

Figure 3 -
Figure 3-29 Analysis parameter interface 3.3.5Step 5: View sample task status The analysis progress can be viewed on the [Sample] interface (Figure 3-30).After the

Figure 3 -Figure 3 -
Figure 3-30 Sample analysis progress 1) If the analysis task is completed successfully, it will be automatically closed.2) If the analysis task fails, you can find the analysis task on the right interface [Production Management Lite], Click the icon and then operate as needed: a.If you want to restart the analysis, check the failed sample and click [Redo Analysis].b.If you do not need to redo the analysis, click the [Finish] button in the bottom right corner of the page to close the current analysis task.3.4 View report and download result files 1) [Analysis Report] in [Report] interface can preview the analysis report of single sample (Figure 3-31), after clicking a certain node in the phylogenetic analysis result, user can click 'Pin the code' to close the pop-up window.Furthermore, user can adjust the position of the pop-up window by dragging the tree diagram with the left mouse button (Figure 3-31-a).Note: In this preview mode, the link in the web report is invalid.If you need the complete report content, please download it according to the operations in this section 3) or 4).

Figure 3 -
Figure 3-32 Download analysis report in batches Note: In this preview mode, the link in the web report is invalid.And the downloaded files only include HTML files, so links to tables, views, etc. in HTML are invalid.To get the complete report content, please download it according to the operation in this section 3). 3) [Analysis Result File] in [Report] interface (Figure 3-33)corresponds to the result directory of analyzed samples, click the icon to enter the path where the compressed result package (Result.tar.gz) is located, and download the report and results of the corresponding sample.

Figure 3 -
Figure 3-33 Download result files in Report interface

Figure 3 -
Figure 3-34 Analysis status 3.5.2Load DNB on two slides at the same time