Re-emergence of Avian leukosis virus subgroup J in the rooster of Hy-line brown layers and its transmission pattern in ocks

Avian leukosis virus subgroup J (ALV-J) is the most prevalent subgroup in chickens and exhibits increasing pathogenicity and stronger horizontal and vertical transmission ability among different kinds of chickens. Although vertical transmission of ALV-J from hens infected through articial insemination was reported before by the detection of swabs and serum, but there was no further research on the transmission pattern of ALVs in the roosters. In the present study, the introduction of Hy-line brown roosters signicantly increased the p27 positive rate of ALV in an indigenous ock detected by ELISA and virus isolation. Sequence analysis and IFA showed that it is classied into ALV-J subgroup, locating in a new branch compared with the domestic and foreign referential sequences. Meanwhile, the gp85 gene of the ALV-J isolated in the hens and its albumens had a homology of 94.1–99.7% with that in the roosters, which means that the strain is quite likely transmitted to the hens and their offspring through insemination of the roosters. In addition, there are four ALV-J infection status in plasma and semen of rooster (V + S+, V-S+, V + S-, V-S-), so the eradication of ALV in rooster requires simultaneous virus isolation of semen and plasma. Additionally, compared with ALV detection in samples by DF-1 cells, directly detecting ALV in semen by ELISA exists some false positives. Collectively, our results suggested that the incomplete eradication process of roosters leads to the sporadic ndings of ALV-J in laying hens


Introduction
Avian leukosis (AL), inducing malignant or benign tumorigenic diseases by avian leukosis virus (ALV), has caused immense economic losses all over the world in the poultry industry since its emergence [1,2]. ALVs were divided into 11 different subgroups (designated A to K) based on cross neutralization and gp85 sequences [3]. Among those, avian leukosis virus subgroup J (ALV-J) is the most prevalent subgroup in chickens and exhibits increasing pathogenicity and stronger vertical transmission ability in recent years [4][5][6]. ALV-J infection used to be a common problem in poultry industry during 2003-2010, causing myelocytoma of broilers and haemangioma of layers when its widespread [7][8][9][10][11]. Because of the widespread distribution of the ALV-J strains, together with the less organized character of the poultry industry especially among the local breeds of chickens, control and eradication of ALV in China remain a major challenge. Since 2018, a more tumorigenic ALV-J strain has appeared in the imported broiler breeders in China, inducing severe osteomas in keel and ribs, causing a massive pandemic, and hence becoming a major concern of avian health [3,11]. Therefore, many domestic farms have started selfchecking for ALV infection [3].
Rubin and his colleagues de ned four serological classes of susceptible birds: viraemia, no antibody (V + A−); no viraemia, with antibody (V − A+); viraemia, with antibody (V + A+); and no viraemia, no antibody (V − A−) [2,12]. Another study showed that roosters could spread ALV-J to hens by insemination and cause vertical transmission [13]. Yet the researchers did not elaborate on the ALV infection status of semen from male or the role of males in the transmission of ALV. In the present, roosters may be selected for freedom from ALV by ELISA tests of cloacal swabs or semen sampling, as well as virus isolation. However, the accuracy of these detection methods remains to be de ned. Page 3/17 In this study, it was discovered that ALV-J re-emerged in Hy-line brown roosters in recent years. Moreover, the hens were infected by insemination, and virus can be transmitted vertically to the breeding eggs.
Several infection states of ALV-J in roosters were clari ed and the reliability of different methods for detecting samples of males were compared. All those data may provide more detailed basis for the eradication of ALV.

Sample origin
The positive rates of virus isolation in ocks and p27 antigen in breeding eggs of an indigenous chicken were both within 1% for two consecutive generations. In order to introduce new genes, the introduction of 46 Hy-line brown fast feathering roosters and arti cial insemination were performed. Ever since, about 7.61% (7 from 92 samples) were found to be p27 positive in the albumen samples in this generation.
Therefore, semen and plasma samples from the 46 roosters, plasmas from 92 hens and 10 albumen samples of breeding eggs from the ve ALV positive hens ( 2 samples/hens) were collected for virus isolation and identi cation.

virus isolation and identi cation
Blood and semen were collected aseptically from the Hy-Line brown roosters and indigenous hens. The semen samples were diluted 5 times with PBS containing 2% penicillin and streptomycin. After mixing upside down gently, the supernatant was centrifuged to precipitate cells at 3000×g for 2 min. Albumens of 10 breeding eggs were collected aseptically from 5 ALV positive chickens, and diluted 4 times with DMEM in 5ml syringes. Virus isolation from plasma, semen and albumens were performed by inoculating DF1 cells as previously described [9]. The ALV group-speci c antigen p27 in the culture supernatant was detected by enzyme-linked immunosorbent assay (ELISA, IDEXX, USA(#99-0925)).

Indirect immuno uorescence assay (IFA)
The cell were washed with PBS and xed with cold acetone-alcohol mixture (3:2) for 5 min. Then, the cells were incubated with mouse anti-ALV-J monoclonal antibody JE9 [24] at 37°C for 60 min, following by incubation with goat anti-mouse IgG antibody conjugated with uorescein isothiocyanate (Sigma, California, USA) at 37°C for another 60 min. Finally, the cells were observed under uorescence microscope.

Primers and PCR Ampli cation
A pair of universal primers (The length of the ampli cation is about 2200bp), targeting env and LTR, was adopted to detect exogenous ALVs as previously reported (ALV-F:GATGAGGCGAGCCCTCTCTTTG/ALV-R: TGTGGTGGGAGGTAAAATGGCGT) [11]. DNA was extracted from the DF-1 cells. The conditions for PCR with primers ALV-F/R were as follows: 95°C for 5 min; followed by 31 cycles of 95°C for 50 s, 55°C for 40 s, and 72°C for 140 s; with a nal elongation step of 10 min at 72°C. The PCR product was analyzed by electrophoresis in 0.8% agarose in Tris-acetate-EDTA buffer. Molecular cloning of positive amplicons was performed and positive clones were con rmed and subjected to Sanger sequencing.

Sequence Analysis
Sequence alignments were assembled with other ALV-J referential sequences retrieved from the National Center for Biotechnology Information database (Table 1). Phylogenetic analysis was based on the neighbor-joining method with 1000 bootstrap replicates by MEGA ver. 6.0. The GenBank accession numbers of the strains used in this study are listed in Table 1. Table 1 The referential strains for the comparison of gp85 gene.

Strain
Year 3 Results

The introduction of Hy-line brown roosters with ALV-J increased the positive rate of ALV in the ock
Before the introduction of Hy-line brown roosters, the positive rate of virus isolation and p27 in the albumens in the ock was within 1% for two consecutive generations, and the peak egg production was about 70%. However, after the introduction of the roosters, the positive rate of virus isolation and p27 in the albumens increased to 5.4% (5/92) and 7.6% (7/92), respectively. The peak egg production dropped to about 65% from 70%. The albumen of two breeding eggs from each positive hen were collected for virus isolation, and only one albumen showed positive result. Collectively, our results indicated that the introduction of Hy-line brown roosters with ALV-J increased the positive rate of ALV in the ock.

The infection status of ALV in semen and plasma of a rooster
To determine whether the rooster infected the ALV, 46 semen and plasma samples from the 46 roosters were collected and detected by different methods. In 46 roosters, 14 roosters were tested to be positive for ALV in plasma or semen (30.43%). Among those, 12 roosters were positive in plasma (26.09%) and 8 roosters were positive in semen (17.39%). According to the results of virus isolation from semen and plasma of the 46 roosters, there are 4 infection status of Avian leukosis virus (Table 2). 6 roosters showed viraemia, with semen positive (V + S+, 13.04%); 6 roosters showed viraemia, with semen negative (V + S-, 13.04%); 2 roosters showed no viraemia, with semen positive (V-S+, 4.35%), while the remaining 32 roosters were double-negative (V-S-, 69.56%).

High proportion of false positives in semen directly detected by ELISA
In addition, the semen were detected directly by ELISA, and the results showed that 11 semen samples were positive, but only 6 corresponding semen samples showed positive from virus isolation by DF1 cells in plasmas or semen. while 5 semen samples showed positive in the semen by ELISA, which were negative ,due to no virus isolated by DF-1 cells. Therefore, Our data suggested that the ALV was directly detected in semen by ELISA is not inaccurate, which due to unknown material in the semen binding the p27 antigen.

Identi cation of ALV-J isolated from roosters, hens and albumens
14 positive samples from roosters, hens, and albumens were went through virus isolation and sequence analysis.Phylogenetic analysis of the gp85 gene, suggested that all of the isolated strains belonged to ALV-J. However, these strains are located in a new branch of the subgroup J, and do not show obvious homology through the comparison with domestic and foreign referential sequences (Fig. 1). In addition, we performed the IFA experiments, and consistent with phylogenetic analysis, the obvious green uorescence could been observed using the monoclonal antibody JE9 (Fig. 2).

The homology analysis of ALV-J strains in roosters, hens and albumens
The viruses isolated from roosters, hens, as well as albumens, show a homology between 94.1-99.7%, and are located in the same branch of the phylogenetic tree (Fig. 1), which demonstrate that they may be derived from the same source. The comparison of the gp85 sequence between semen and plasma sample of the same rooster shows that the nucleotide homology in most roosters (5/6) is more than 98.6%, but the other one (1/6) only has a homology of 96%, and its homology with that of the other 5 chicken is as low as 94.1%.

Discussion
Since the rst strain of ALV-J, HPRS-103, was isolated from meat-type breeder chickens in the UK in 1988, it has rapidly spread around the world, causing severe economic losses to the poultry industry [4,14].
Being the most pathogenic subgroup of ALV, ALV-J could induce malignant or benign tumorigenic diseases and immunosuppression in chicken, including hemangiomas, myelomas and brosarcomas [2,[15][16][17]. The outbreak of hemangioma associated with ALV-J was reported between 2006 and 2010 in China in commercial layer chickens [7,18]. However, with the implementation of the ALV puri cation project by a number of major breeder companies, successful eradication has been achieved at pedigree and multiplier levels. Therefore, there is few reports of ALV-J infection in layers in recent years. Zhao discovered that co-infection with avian hepatitis E virus and avian leukosis virus subgroup J is the cause of an outbreak of hepatitis and liver hemorrhagic syndromes in a Hy-line brown layer chicken ock in China recently [19]. This study also found subclinical infection of ALV-J in the roosters of Hy-line layers, which reminded us that the puri cation of ALV can not be overlooked, and needs to be continued through monitoring by proportional sampling.
Different methods were used to detect various samples of roosters and hens in the same ock. The ELISA method is used to detect semen directly, which showed that only 50% samples are consistent with the results of virus isolation. Virus isolation is used as the gold standard for detecting ALV, so the results achieved by ELISA has false positives and false negatives, resulting in missed and false mistaken of chickens. At the same time, different infection status in semen and plasma of the same rooster were clari ed, namely, viraemia, with semen positive (V + S+, 13.04%), no viraemia, but semen positive (V-S+, 4.35%), viraemia, but semen negative (V + S-, 13.04%), double-negative (V-S-, 69.56%). The positive rate of virus isolation from plasma is higher than that of semen, but the two are not one-to-one. Therefore, this suggests that we need to choose both semen and plasma for virus isolation when performing ALV puri cation of roosters in order to obtain a better puri cation effect.
Li previously found that ALV-J in semen could be transmitted to hens by insemination through animal experiments in SPF chicken [13], but they only discovered the antibody of ALV-J, without viraemia in the hens and their offspring. This study is based on the infected clinical ocks. The ALV-J was isolated not only in the hens, but also in its breeding eggs. Although the positive rate of virus isolation in albumens was low, one strain of ALV-J was found and sequenced. The homology analysis showed that the strain had a homology of 94.1-99.7% with that in the roosters. Up to 100% similarity means that the strain is quite likely transmitted to the hens and their offspring through insemination of the roosters. This study comprehensively described that ALV-J from roosters can be transmitted to hens and their offspring through insemination.
Previous studies have demonstrated that ALV-J displays a high level of genetic variation and recombination [20,21], which allows the development of new variants with changes in antigenicity, tissue tropism, host range and pathopoiesis [22]. Gao found that ALV-J is subject to greater selective pressure in the hen's follicles, which can promote the evolution of the virus [23]. In this study, we found that the ALV-J in the semen of the same rooster had only 96% homology with that in the plasma, which indicated that semen of roosters may suffer heavy selective pressure that promoted the evolution of ALV-J;and that is why the isolated strains located in a new branch in the phylogenetic tree compared with the referential stains.
In summary, we isolated ALV-J from Hy-line brown layers, and discovered the complete chain of the transmission of ALV-J from roosters to hens and then to the offspring through insemination and vertical transmission. Semen are detected by ELISA method is not completely accurate. There are four ALV-J infection status in plasma and semen of rooster, so the puri cation of ALV in rooster requires simultaneous virus isolation of semen and plasma. Therefore, we speculate that the reason why there are still some sporadic ndings of ALV-J in laying hens is probably due to the incomplete puri cation process of roosters.

Declarations Date Availability
The data used to support the ndings of this study are included within the article. Compliance with ethical standards

Con ict of Interest
The authors declare that the research was conducted in the absence of any commercial or nancial relationships that could be construed as a potential con ict of interest.

Ethical approval
This article does not include experiments with human participants or animals performed by any of the authors.

Figure 1
The phylogenetic tree of gp85 gene in the isolates and the referential strains. The phylogenetic tree is generated using the neighbor-joining method using MEGA5.1 software (bootstrap method with 1000 replicates). ALV-A to E, K and J were divided and marked on the right. The source of ALV-J reference strains is in parentheses. Purple rhombus represent the strains were isolated in the plasmas of the rooster. Green triangles represent the strains were isolated in the semens of the rooster. The other strains in this study are also highlighted by the red gure. The sources of reference and isolated strains are marked after each strain. The following indicates the abbreviations used for country names: USA=United States, UK=United Kingdom, CHN=China, RUS=Russia, EGY=Egypt, NGA=Nigeria.