Genomic characteristics of a chicken infectious anemia virus strain isolated from contaminated chicken pox attenuated vaccine

Chicken infectious anemia virus (CIAV) can be transmitted by contaminated live vaccines, and causes huge economic losses. This study evaluated the contamination status of CIAV in 24 batches of vaccines by recombinase-aided amplication assay (RAA), uorescence quantitative PCR and dot blot assay, and then determined a contaminated avian pox attenuated vaccine. The whole genome of the CIAV contaminant was then sequenced and named as JS2020-PFV (Genbank accession number: MW234428, 2296bp). It showed 94.5–99.9% identities with reference strains, and shared the closest evolution relationship with AB1K which was isolated from a chicken farm in Turkey. All these suggested that the use of CIAV contaminated live vaccine may be the reason for its epidemic in poultry.


Introduction
Chicken infectious anemis virus (CIAV) induces severe anemia and immunosuppression in poultry (Rosenberger et al.,1998). After its rst report in 1979 (Yuasa et al., 1979), CIAV has been detected worldwide and has caused huge economic losses (Fatoba et al.,2019). Recently, CIAV infections have also frequently appeared in different chicken farms in China (Zhang et al., 2013;Fang et al., 2017;Tan et al., 2020). The pathogenesis of CIAV has been well understood that it mainly causes the atrophy of bone marrow, hematopoietic tissue, and lymphatic tissues (e.g., thymus) in young chickens (Adair et al.,2000). CIAV can be transmitted both vertically and horizontally via respiratory and digestive tracts (ref), but how it spreads across different regions and even countries remains unclear.
It is worth noting that the rst strain of CIAV was isolated from the contaminated attenuated vaccine (Yuasa et al., 1979). And, after that, similar reports emerged in a lot of countries (Marin et al., 2013;Varela et al., 2014;, revealing that the use of contaminated live vaccines is an important way for the transmission of CIAV. Through interactions with attenuated vaccine strains or co-infected viruses, CIAV triggers more serious clinical symptoms with strong immunosuppressive ability (Su et al., 2018 a,b;. More importantly, the attenuated vaccines are usually inoculated at a low age, the CIAV infection caused by it will greatly interfere with the immune responses against vaccines, resulting in secondary infection (Zhang et al., 2017;. Therefore, monitoring the vaccine contaminations is of great signi cance for preventing CIAV infection in poultry. As a relatively conservative DNA virus, the detection of CIAV is relatively easy, and there are a large number of published methods (Han et al., 2019;Techera et al., 2019;Li et al., 2019). Although the sensitivity and accuracy of these methods are very high, they are not suitable for farms to detect the contamination in vaccines due to long operation time, complex experiments or high price. Recombinaseaided ampli cation (RAA) is a new isothermal ampli cation technology, requiring no classical thermostable enzyme. RAA has been widely used to detect multiple viruses, even the SARS-CoV-19 (Wu et al.,2020). The reaction, containing a recombinase UvsX obtained from Escherichia coli and the single strand DNA-binding protein (SSB) used in a polymerase chain reaction (PCR), can form single-stranded Page 3/15 DNA without heating and can be nished within 20 min at a constant temperature of 39°C (Rohrman et al.,2012).
This study aims to use RAA to moniter the CIAV contamination status of live vaccines, and verify its e cacy by comparing to uorescence quantitative PCR assay (qPCR) and dot blot. The whole genome of contaminated CIAV strain was sequenced for further analysis.

Materials And Methods
Vaccine collection 24 batchs of live vaccines against Newcastle disease virus, infectious bronchitis, infectious laryngotracheitis encephalomyelitis, avian pox, Marek disease virus, mycoplasma gallisepticum, viral arthritis, were collected from chicken farms. All these vaccines were diluted with normal saline according to the instructions, and 200 µl of the dilute was used for DNA extraction.

DNA Extraction
DNA was extracted using the DNA extraction kit (Omega, Bio-Tek, USA) according to the manufacturer's instructions as the template for RAA, qPCR and dot blot.

RAA
The primers and probe were designed according to the published CIAV genome (Table 1; Table2). The RAA uorescence kit and RAA ampli cation instrument were provided by Hangzhou ZC Bio-Sci & Tech Co., Ltd (Hangzhou, China). ddH 2 O was used as the negative control and standard plasmid contianing full-length CIAV genomes was contructed by our lab and used as positive control . RAA detection methods and primer probes are shown in Table 1.

Sample detection with dot blot hybridization
The samples were tested by PCR combined with dot blot hybridization , and the nitrocellulose membrane was purchased from Boehringer Millipore (Merck, Germany).
Ampli cation and sequencing of whole genome of samples According to the sequence published in the GenBank, three primers were designed to amplify the sample DNA using DNAStar 6.0 application. The primers are shown in Table 1. The ampli cation procedure was as follows: 94℃ for 5 min; 94℃ for 30 s, 55℃ for 30 s, and 72℃ for 30 s (34 cycles); 72℃ for 10 min (35 cycles); and 4℃. The PCR products were identi ed using 1% agarose gel electrophoresis and stained with ethidium bromide. The bands were puri ed with Omega gel recovery and puri cation kit (Omega Bio-Tek, Norcross, GA, USA), and the recovered target fragments were attached to the pMD18-T vector (Takara, Dalian, Chian). After overnight ligation at 16℃, the ligation products were transformed into competent cells of E. coli DH5α(Takara, Japan), and the positive clones were screened. Finally, the bacterial liquid identi ed as positive by PCR was sent to Shanghai Bioengineering Co., Ltd. for sequencing.

Sequence analysis
The DNA sequences were assembled using DNAStar (version 6.0). Multiple sequence alignment was performed using the Clustal W (BioEdit version 7.0) program, and the comparison of sequence identity was performed using MegAlign software (DNAStar). Phylogenetic analysis was performed using the maximum likeihood (ML) method on RAxML.

Results of CIAV nucleic acid test in vaccines
RAA, qPCR assay, and dot blot were employed to detect CIAV contamination in live vaccines. Results showed that an avian pox attenuated vaccine sample were determined as CIAV positive in all three methods (Figure 1). These results also suggested that the sensitivity and accuracy of RAA is similar to that of qPCR and dot blot, hence it can be used in daily moniter of vaccine contamination.

Whole genome sequencing
In order to further reveal the molecular characteristics of the CIAV contaminants, the whole genome of this strain was sequenced using three pairs of primers. Electrophoretic analysis con rmed the positive amplication by PCR, and the three segments were 842bp, 990bp and 802bp, respectively (Figure 2), which was consistent with the expected band size. Furthermore, the whole genome of this isolate was 2296 bp, covering three open-reading-frame (ORF), namely VP1, VP2, and VP3.
The lengths of VP1, VP2 and VP3 were 1350 bp, 651 bp and 366 bp, respectively. The isolate was named JS2020-PFV and its sequence was uploaded to GenBank gene library with the accession number of MW234428.

Genome analysis of JS2020-PFV
The whole genome of JS2020-PFV strain was compared with that of 56 other CIAV strains. Results showed that JS2020-PFV shared 94.5-99.9% identities with other reference strains. Among them, JS2020-PFV had the highest identity (99%) with the AB1K strain (MT259319) isolated from a chicken farm in Turkey. A phylogenetic tree was constructed based on the whole genome sequence of JS2020-PFV and reference strains, and the results further con rmed the closest evolution relationship between JS2020-PFV and AB1K (Figure 3).

Analysis of VP1 gene variation in JS2020-PFV strain
The VP1 gene of JS2020-PFV strain shared 94.0-99.9% identities with the reference strains, with the highest identity (99.9%) with AB1K. The genetic evolution relationship between the VP1 gene of JS2020-PFV strain and reference strains was consistent with the whole genome sequence (Figure 3). Further analysis of the VP1 gene of JS2020-PFV strain revealed that there are two amino acid mutations within which had never been found before, namely threonine at position 89 of VP1 protein changed to lysine and glutamine at position 448 changed to lysine (Table 2).
Difference analysis of transcriptional regulatory elements in non-coding regions Clustal W method was used for multiple alignment analysis of the non-coding regions of JS2020-PFV with 9 reference strains. Compared with the reference strains, the the motif in non-coding regions of CIAV was highly conserved (Figure 4). Softberry's Nsite online service analysis showed that four CREB sites associated with viral apoptotic capacity were also conserved in the new isolate. The JS2020-PFV and most of the reference strains all had four DR regions in the non-coding region, except for one additional DR region in the CUX-1 strain.

Discussion
Over the past two decades, many live vaccines have been proved to be contaminated with exogenous viruses, including avian leukosis virus (Mao et al., 2020), fowl adenovirus , reticuloendotheliosis virus (Li et al., 2015), CIAV  and others. This phenomenon is mainly caused by the use of SPF chicken embryos infected with exogenous viruses (Li et al., 2016). The use of contaminated vaccines will not only cause the spread of the virus, but also cause serious clinical symptoms, especially vaccines contaminated with CIAV that induces strong immunosuppression. Previsous studies demonstrated the the co-infection with CIAV can signi cantly improve the pathogenicity of the LaSota strain in NDV vaccines, and decrease the antibody titer, which makes the vaccinated chickens vulnerable to wild NDV strains . Therefore, monitering the exogenous viruses contamination in live vaccination is of great signi cance for poultry farms.
Currently, the standard routine for detecting exogenous virus contamination in vaccines mainly depend on cell culture and SPF chicken inspections, but the working cycle of the above methods is too long, so it is not conducive to the vaccine work in farms. Alternatively, several molecular detection methods were established for detecting exogenous viruses, such as CIAV and FAdV Dong et al., 2019). However, none of them can be directly used in farms because of complex equipment or di cult operations.
In this study, RAA assay, which can be nished with 15 minutes in a hand-held device, was employed to detect the contaminated CIAV in live vaccines. We designed the primers and probe based on the published CIAV genomes, and optimized the methods using the constructed plasmid containing the whole genome of CIAV . After that, this method was used to analyze 24 batchs of live vaccine, and the traditional qPCR and dot blot were used at the same time to verify the results of RAA tests. Results found that one avain pox vaccine was positive for CIAV in all these three methods indicating that the detection results were true and reliable. It should be noted that RAA completed the whole detection process in only 15 minutes, while qPCR and dot blot took 1.5 and 14 hours respectively. Therefore, the RAA method is more suitable for monitoring vaccine contamination in farms because of its exibility and convenience.
Furthermore, the whole genome sequence of CIAV was sequenced and named as JS2020-PFV. It was found that JS2020-PFV had extremely high identities (99%) and the closest evolution relationship with the AB1K strain (MT259319) that isolated from a chicken farm in Turkey. Besides, JS2020-PFV shared the same batch with Dutch isolate (D10068), American isolate (DI072479), American isolate (L14767) and Bangladesh isolate BD-3 (AF395114), while it was relatively unrelated to chinese isolates, including SDLY18 (FJ172347), GD-K-12 (KF224935) and SD15 (KX811526). These results are consistent with the fact that the vaccine was imported from a foreign company, suggesting that the transnational trade of vaccines may be an important way for some strains to spread across countries and regions.
The amino acid positions of VP1 protein are closely related to CIAV pathogenicity and cell proliferation (Fang et al. 2019). Previous studies have shown that amino acid positions 139 and 144 on VP1 were associated with the e ciency of virus proliferation and transmission in MSB1 cells (Yamaguchi et al. 2001). The amino acid positions 139 and 144 on the VP1 protein of JS2020-PFV are lysine (K) and glutamic acid (E), which are consistent with the strain with high replication ability (Yamaguchi et al. 2001). Besides, glutamine (Q) at position 394 was associated with higher pathogenicity, while histidine (H) at position 394 was related with lower pathogenicity (Yamaguchi et al. 2001). In this study, the amino acid at position 394 of VP1 protein of JS2020-PFV is glutamine (Q), suggesting that this strain may also be highly pathogenic. There are more than ten motifs related to transcriptional regulation in the noncoding region of CAV, which are closely related to viral replication and transcriptional regulation. After comparing and analyzing the non-coding regions of the selected strains in this paper, it was found that these motifs were very conservative. We speculate that these related motifs may be necessary for the replication of the virus itself.
Overall, CIAV contamination was detected from a FPV vaccine by RAA, and genome analysis suggested that it may be a highly pathogenic strain, which reminded us to pay close attention to the possible contamination of exogenous virus in live poultry vaccine. Detection of CIAV nucleic acid in attenuated chicken pox vaccine RAA rapid assay (A), nucleic acid dot hybridization (B), and uorescence quantitative PCR assay (C) were simultaneously adopted to detect CIAV contamination in vaccine samples, and all the methods showed positive results. Structural analysis of the non-coding region of JS2020-PFV strain The sequences in black frames are the motifs of transcriptional regulatory elements in this study