SRSF5-Mediated Alternative Splicing of M Gene is Essential for Influenza A Virus Replication: A Host-Directed Target Against Influenza Virus.

Splicing of influenza A virus (IAV) RNA is an essential process in the viral life cycle that involves the co-opting of host factors. Here, it is demonstrated that induction of host serine and arginine-rich splicing factor 5 (SRSF5) by IAV facilitated viral replication by enhancing viral M mRNA splicing. Mechanistically, SRSF5 with its RRM2 domain directly bounds M mRNA at conserved sites (M mRNA position 163, 709, and 712), and interacts with U1 small nuclear ribonucleoprotein (snRNP) to promote M mRNA splicing and M2 production. Mutations introduced to the three binding sites, without changing amino acid code, significantly attenuates virus replication and pathogenesis in vivo. Likewise, SRSF5 conditional knockout in the lung protects mice against lethal IAV challenge. Furthermore, anidulafungin, an approved antifungal drug, is identified as an inhibitor of SRSF5 that effectively blocks IAV replication in vitro and in vivo. In conclusion, SRSF5 as an activator of M mRNA splicing promotes IAV replication and is a host-derived antiviral target.

A study on the infection status and transmission of avian leukosis virus subgroup J in Hy-line brown roosters.

Avian leukosis virus subgroup J (ALV-J) is the most prevalent subgroup in chickens and exhibits increased pathogenicity and stronger horizontal and vertical transmission ability among different breeds. Although vertical transmission of ALV-J from infected hens through artificial insemination has been inferred from the detection of the p27 antigen in swabs and serum, there has been no further research on the transmission pattern of ALVs in roosters. In the present study, the positive rate of ALV increased significantly in an indigenous flock after detecting the p27 antigen via enzyme-linked immunosorbent assay (ELISA) and virus isolation in DF-1 cells. Viral sequence comparisons and an indirect fluorescent antibody assay showed that these isolates belonged to the ALV-J subgroup but formed a new branch in a phylogenetic tree when compared to domestic and foreign referential strains. The gp85 gene of the ALV-J isolated from hens and albumen was 94.1-99.7% identical to that in roosters, revealing that these isolates were quite likely transmitted to the hens and their offspring through the semen of ALV-infected roosters by artificial insemination from the Hy-line brown roosters. In addition, we defined four ALV-J infection states in plasma and semen of roosters (P+S+, P-S+, P+S-, and P-S-), which suggests that, in order to eradicate ALV in roosters, it is necessary to perform virus isolation using both semen and plasma. Additionally, ALV detection in semen by ELISA produced false-positive and false-negative results when compared to virus isolation in DF-1 cells. Collectively, our results suggested that an incomplete process of eradication of ALV from ALV-positive roosters led to the sporadic presence of ALV-J in laying hens.

The follicle promotes the evolution of variants of avian leukosis virus subgroup J in vertical transmission.

Avian leukosis virus (ALV) is one of the main causative agent of tumor development, which brings enormous economic losses to the poultry industry worldwide. ALV can be transmitted horizontally and vertically, and the latter often give rise to more adverse pathogenicity. However, the propagation and evolution of ALV underlying vertical transmission remain not-well understood. Herein, an animal model for the evolution of variants of ALV subgroup J (ALV-J) in the vertical transmission was built and different organs from infected hens and plasma from their ALV-positive progenies were collected, and then three segments in the hypervariable regions of ALV (gp85-A, gp85-B, LTR-C) were amplified and sequenced using conventional Sanger sequencing and MiSeq high-throughput sequencing, respectively. The results showed that the genomic diversity of ALV-J occurred in different organs from ALV-J infected hen, and that the dominant variants in different organs of parental hens, especially in follicle, changed significantly compared with original inoculum strain. Notably, the dominant variants in progenies exhibited higher homologies with variants in parental hens' follicle (88.9%-98.9%) than other organs (85.6%-91.1%), and most consistent mutations in the variants were observed between the progenies and parental hen's follicle. Furthermore, HyPhy analysis indicated that the global selection pressure value (ω) in the follicle is significantly higher than those in other organs. In summary, an animal model for vertical transmission was built and our findings revealed the evolution of variants of ALV in the process of vertical transmission, moreover, the variants were most likely to be taken to the next generation via follicle, which may be related to the higher selection pressure follicle underwent.

A New Strategy for the Detection of Chicken Infectious Anemia Virus Contamination in Attenuated Live Vaccine by Droplet Digital PCR.

Chicken infectious anemia virus (CIAV) causes the atrophy of bone marrow hematopoietic and lymphoid tissues in chicks, leading to huge economic losses all over the world. The using of attenuated vaccine contaminated with CIAV increased the mortality and the pathogenicity of other diseases in many farms. However, it is difficult to detect the CIAV contamination by general detection technology due to the extremely low dose of CIAV in vaccines. In this study, we established a new method called droplet digital Polymerase Chain Reaction (ddPCR) to detect CIAV contamination of vaccines more sensitively and accurately. The lowest detection limitation of this method is 2.4 copies of CIAV plasmid or CIAV contamination at 0.1 EID50/1000 feathers in vaccines without any positive signals of other viruses. Besides, the sensitivity of ddPCR is 100 times greater than that of conventional PCR and 10 times greater than that of real-time PCR. The ddPCR technique is more sensitive and more intuitive. Therefore, it could be valuable for the detection of CIAV contamination in vaccines.

Development and evaluation of a droplet digital PCR assay for the detection of fowl adenovirus serotypes 4 and 10 in attenuated vaccines.

In recent years, there has been an increase in reported cases of fowl adenovirus serotype 4 (FAdV-4) in chickens in China. The use of live attenuated vaccines contaminated with FAdV-4 has been proved to be one of the important causes of massive outbreaks of hydropericardium syndrome. To detect the contamination with FAdV-4 in attenuated vaccines more promptly and accurately, a droplet digital PCR (ddPCR) assay was developed for the rapid detection of FAdV-4 and FAdV-10. The ability of this assay to detect FAdV-4 contamination in attenuated Newcastle disease virus vaccines was assessed in comparison to a quantitative real-time PCR (qPCR) and a conventional PCR assay. The findings indicated that the ddPCR assay could detect FAdV-4 contamination at 0.1 EID50/1,000 feathers, while the qPCR could detect FAdV-4 contamination at 1 EID50/1,000 feathers with identical genomic targets, which was 1,000-fold more sensitive than conventional PCR detection with a sensitivity of 102 EID50/1,000 feathers. The ddPCR assay also showed high specificity for FAdV-4/10 and no positive signals were detected for other FAdVs. Consequently, the intuitive and rapid results were especially suitable for the detection of FAdV-4 contamination in vaccines. In this study, a ddPCR assay was developed to effectively detect and quantify low-dose FAdV-4 contamination, providing a new method for rapid detection of FAdV-4 contamination in various samples, especially vaccines.

The intracorporal interaction of fowl adenovirus type 4 and LaSota strain significantly aggravates the pathogenicity of one another after using contaminated Newcastle disease virus-attenuated vaccine.

In the past five years, inclusion body hepatitis-hydropericardium syndrome caused by fowl adenovirus type 4 (FAdV-4) has been rampant in China and is still an epidemic at present. We had found the contamination of FAdV-4 in Newcastle disease virus (NDV)-attenuated vaccine from a large farm with aforementioned disease, and then conjectured that the use of this contaminated vaccine might be an important reason for the outbreak of the FAdV-4 infection. To assess the pathogenicity of this contaminated vaccine and the interaction between FAdV and LaSota, systemic animal experiments were launched with FAdV infection via the contaminated vaccine, as well as FAdV direct infection. Results showed that no chicks died after FAdV direct infection, while a 16.7% mortality rate appeared after FAdV infection through the contaminated vaccine; the synergistic reaction of FAdV and LaSota aggravated their inhibitory action for weight growth of SPF chickens, enhanced one another's capacity for immune system destruction, promoted their multiplication in vivo, and restrained antibody production. This study demonstrated the intracorporal interaction and enhanced pathogenicity of FAdV-4 and LaSota strain in contaminated NDV-attenuated vaccine, illustrating how the exogenous virus causes infection and induces severe diseases, which reminds us that the damage of attenuated vaccine contaminated with FAdV-4 even in extremely low dose is not insignificant.

Synergetic pathogenicity of Newcastle disease vaccines LaSota strain and contaminated chicken infectious anemia virus.

Newcastle disease virus (NDV)-attenuated vaccine has been widely used to prevent ND in poultry flocks, while many reports also mentioned the exogenous virus contamination in attenuated vaccines, which might be the reason for the widespread of some contagious diseases. Recently, the chicken infectious anemia virus (CIAV) contamination in the NDV-attenuated vaccine was also found in China, though no systemic study has studied the pathogenicity or infection mechanism of this special transmission route. Accordingly, simulation experiments were launched using CIAV isolated from a contaminated NDV-attenuated vaccine. Results showed that using NDV-attenuated vaccine contaminated with CIAV could cause CIA in chickens with obvious symptoms, including anemia, hemorrhage, lymphoatrophy, and growth retardation, while the synergistic reaction of CIAV and LaSota prompted their multiplication in vivo and disturbed the production of antibodies against each other. And CIAV could significantly reduce the NDV antibody titers and decrease the protective effectiveness. This study showed the synergetic pathogenicity of CIAV and LaSota strain after using contaminated NDV-attenuated vaccine, helping us to understand how the CIAV causes infection and induces severe diseases with a relatively low dose through the mouth, as well as reminding us that the damage of an attenuated vaccine contaminated with CIAV even in extremely low dose is not insignificant.

A pilot study of acupuncture at pain acupoints for cervical cancer pain.

This retrospective study aimed to investigate the feasible effectiveness of acupuncture at pain acupoints for the treatment of patients with cervical cancer pain (CCP). A total of 64 cases were analyzed. All these cases were assigned to an acupuncture group or a control group according to the different therapies they received. The cases in the acupuncture group received acupuncture treatment at pain acupoints, while the subjects in the control group underwent acupuncture at regular acupoints. The primary endpoint was CCP, assessed by numeric rating scale (NRS). The secondary endpoints were evaluated by the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire-Core 30 (EORTC QLQ-C30), and Karnofsky Performance Status (KPS). In addition, adverse events were also recorded during the treatment period. After treatment, patients in the acupuncture group exerted greater outcomes in CCP reduction when compared with patients in the control group (P < .01). In addition, no adverse events were recorded in either group. The results of this study showed that acupuncture at pain acupoints might be efficacious in patients with CCP after 14-day treatment.

Newcastle disease virus-attenuated vaccine LaSota played a key role in the pathogenicity of contaminated exogenous virus.

Newcastle disease virus (NDV)-attenuated vaccine has been widely used since the 1950s and made great progress in preventing and controlling Newcastle disease. However, many reports mention exogenous virus contamination in attenuated vaccines, while co-contamination with fowl adenovirus (FAdV) and chicken infectious anaemia virus (CIAV) in the NDV-attenuated vaccine also emerged in China recently, which proved to be an important reason for the outbreaks of inclusion body hepatitis-hydropericardium syndrome in some flocks. It is amazing that exogenous virus contamination at extremely low doses still infected chickens and induced severe disease; thus, we speculated that there must be some interaction between the NDV-attenuated vaccine and the contaminated exogenous viruses within. Accordingly, simulation experiments were launched using FAdV and CIAV isolated from the abovementioned vaccine. The results showed that the pathogenicity of FAdV and CIAV co-infection through the contaminated vaccine was significantly higher than that of direct oral infection, while the synergistic reaction of these viruses and LaSota prompted their multiplication in vivo and disturbed the production of antibodies against each other. This study showed the interactions of FAdV, CIAV and LaSota after using contaminated NDV-attenuated vaccine, helping us to understand how the contaminated exogenous viruses cause infection and induce severe disease at a relatively low dose through the oral route.

Characterization of the novel genotype avian hepatitis E viruses from outbreaks of hepatic rupture haemorrhage syndrome in different geographical regions of China.

Since 2016, hepatic rupture haemorrhage syndrome (HRHS) has emerged in layer and broiler breeder hens in several provinces of China, and novel genotype avian hepatitis E viruses were detected from these chickens. To gain a better understanding of the genetic properties of the novel avian HEV strain, the capsid gene of four isolates from birds at four farms experiencing HRHS in different geographical regions were determined and compared with those of reported pathogenic and nonpathogenic avian HEV isolates as well as mammalian HEVs. Results showed that all those isolates share 80.1%-88.2% nucleotide sequence identity and 89.3%-91.9% amino acid sequence identity with other published avian HEV strains, while phylogenetic analysis further demonstrate that a novel genotype avian HEV was epidemic in China. Meanwhile, sequence analysis revealed that those novel isolates contain various amino acid mutations and even a hypervariable region in their major antigenic domains, which might be the critical factors for the pathogenicity elevation and even change their antigenicity. The data presented in this report will enhance the current understanding of the epidemiology and genetic diversity of the novel genotype avian HEV in China and provide additional insight into the critical factors that determine the pathogenicity of it.

Hepatic rupture hemorrhage syndrome in chickens caused by a novel genotype avian hepatitis E virus.

Since 2016, severe outbreaks of hepatic rupture hemorrhage syndrome (HRHS) have emerged in chickens in several Chinese provinces and caused huge economic losses to the poultry industry, but the etiological characteristics and pathogenic potential of it has remained unclear. This study sequenced the partial helicase and capsid gene of the potentially novel avian hepatitis E virus (HEV) isolated from chickens with HRHS and tested the pathogenicity of it on SPF chicks, while the appearance of clinical signs, histopathological changes, viral distribution, viremia and viral shedding were monitored for 14 days post-infection (dpi). Analysis revealed that the HRHS related avian HEV belongs to a novel genotype, and infected chicks developed the typical symptoms of HRHS. Thus, this study successfully developed an experimental infection model for studying the pathogenicity and role of the novel avian HEV in HRHS. Meanwhile, the novel avian HEV mainly existed in the liver and spleen, inducing a rapid viremia and chronic viral shedding in infected chicks, and could cause 40% mortality before 14 dpi. In conclusion, this study found the novel genotype avian HEV and confirmed its role in HRHS.

Characterization of avian leukosis virus subgroup J isolated between 1999 and 2013 in China.

Avian leukosis virus subgroup J (ALV-J) has successively infected white feather chickens, layer hens, cultivated yellow chickens, and indigenous chickens; infection rates and tumorigenicity have attracted increasingly extensive attention in China. To clarify the correlation of the epidemiological phenomenon of ALV-J with the evolution of envelope protein gp85, 140 strains of ALV-J isolated from chickens with different genetic backgrounds from 1999 to 2013 were compared. The homology of the gp85 protein and genetic genealogical relationships between 140 strains of ALV-J and the prototype strain HPRS-103, as well as between the same ALV-J strains and 8 American isolates, were analyzed and compared. The results showed that there was no significant difference in the variation range of homology of the gp85 protein between the prototype HPRS-103 and ALV-J isolates from different genetic backgrounds and different years. However, genetic pedigree analysis showed that virus strains that isolated from the same type of chickens remained close to each other on the phylogenetic tree, which means that there was a correlation between the genetic background of infected chickens and virus strains. Further analysis of amino acid sequences also found similar results and revealed that unique amino acid sites were formed in chickens with different genetic backgrounds, which proved that ALV-J could adapt to the new host through amino acid variation. Genetic sequence phylogenetic tree analysis was more representative than sequence homology comparisons for assessing ALV-J correlations. These conclusions contributed to the control and prevention of ALV infection. ALV-J is still prevalent in Chinese indigenous chickens, more attentions should be given to fulfill the purification.

Isolation and characterization of subgroup J Avian Leukosis virus associated with hemangioma in commercial Hy-Line chickens.

There was an outbreak of hemangioma associated with avian leukosis virus subgroup J (ALV-J) between 2006 and 2010 in China in commercial layer chickens. Recently, severe hemangiomas broke out in Hy-Line layer chickens on a poultry farm in 2017 where ALV was eradicated earlier. Six isolates of ALV-J, named SDAU1701-SDAU1706, were characterized by virus isolation and sequence analysis of the complete proviral genomes. Avian leukosis virus subgroup J was identified by an immunofluorescence assay with monoclonal antibody JE9, whereas Marek's disease virus or reticuloendotheliosis virus was not detected. Sequence analysis of the complete proviral genome revealed that there was 96.0-99.6% identity between each other and had a homology of 94.6-96.0% when compared with the reference strain. The six isolates formed one distinct lineage separate from the reference sequences in a phylogenetic-tree, which suggested that there were several genetic differences between these groups. Homology analysis of the env, pol, and gag genes of the six isolates showed that the env gene was more variable, especially the gp85 protein, which shared only 88.2-91.9% identity with the reference strains. Sequence comparisons of the gp85 protein indicated that 19 sites were different from those in the NX0101 and HPRS-103 strains inducing myeloid leukosis; among our strains, five mutations were identical to those in the viruses causing hemangioma. Four other distinctive mutations were detected in our six isolates. This study reminds us that the surveillance of viral eradication should be conducted continuously on a farm where ALVs were eradicated. To prevent the prevalence of ALVs, more attention should be paid to daily monitoring.

Newcastle disease virus-attenuated vaccine co-contaminated with fowl adenovirus and chicken infectious anemia virus results in inclusion body hepatitis-hydropericardium syndrome in poultry.

Inclusion body hepatitis-hydropericardium syndrome (IBH-HPS) induced by fowl adenovirus type 4 (FAdV-4) has caused huge economic losses to the poultry industry of China, but the source of infection for different flocks, especially flocks with high biological safety conditions, has remained unclear. This study tested the pathogenicity of Newcastle disease virus (NDV)-attenuated vaccine from a large-scale poultry farm in China where IBH-HPS had appeared with high mortality. Analysis revealed that the NDV-attenuated vaccine in use from the abovementioned poultry farm was simultaneously contaminated with FAdV-4 and chicken infectious anemia virus (CIAV). The FAdV and CIAV isolated from the vaccine were purified for the artificial preparation of an NDV-attenuated vaccine singly contaminated with FAdV or CIAV, or simultaneously contaminated with both of them. Seven-day-old specific pathogen-free chicks were inoculated with the artificially prepared contaminated vaccines and tested for corresponding indices. The experiments showed that no hydropericardium syndrome (HPS) and corresponding death occurred after administering the NDV-attenuated vaccine singly contaminated with FAdV or CIAV, but a mortality of 75% with IBH-HPS was commonly found in birds after administering the NDV-attenuated vaccine co-contaminated with FAdV and CIAV. In conclusion, this study found the co-contamination of FAdV-4 and CIAV in the same attenuated vaccine and confirmed that such a contaminated attenuated vaccine was a significant source of infection for outbreaks of IBH-HPS in some flocks.

Protection induced by a gp90 protein-based vaccine derived from a Reticuloendotheliosis virus strain isolated from a contaminated IBD vaccine.

BACKGROUND: Reticuloendotheliosis is an immunosuppressive disease caused by avian reticuloendotheliosis virus (REV). It is commonly found in poultry farms and has caused a notable economic loss worldwide. Despite this, there is currently no effective vaccine available to protect against REV infection. METHOD: In this study, gp90 protein derived from an REV isolated from a contaminated vaccine was co-administered with cytosine-phosphate-guanine oligodeoxynucleotide (CpG-ODN) adjuvant to hens to determine if it protects their chicks against REV infection. To synthesize the gp90 protein, the gp90 gene was amplified using polymerase chain reaction, expressed in Escherichia coli, and purified. The resulting recombinant protein was injected intramuscularly into breeder hens along with CpG-ODN adjuvant and then serum antibody levels were regularly evaluated. After the fertilized eggs from these vaccinated hens had hatched, the resulting chicks were challenged with a 102.7 50% tissue culture infectious dose (TCID50) of REV at 1 day old and the REV antibody levels in these hatched chickens were evaluated before and after the challenge. Viremia and growth rate were measured weekly and statistically analyzed. RESULTS: The results suggest that the gp90 recombinant protein was successfully prepared and, when used with CpG-ODN adjuvant to immunize breeder hens, induced serological antibody production against REV in both hens and their hatched chicks. In addition, the maternal antibodies induced by the gp90 protein vaccine effectively protected majority of the chicks from REV infection. CONCLUSIONS: Overall, we found the gp90 protein obtained in this study may be a potential vaccine candidate that had good immunogenicity and could be an auxiliary measure to accelerate the eradication of REV.

Co-infection of fowl adenovirus with different immunosuppressive viruses in a chicken flock.

In poultry, fowl adenovirus (FAdV) and immunosuppressive virus co-infection is likely to cause decreased egg production, inclusion body hepatitis, and pericardial effusion syndrome. In this study, fowl adenovirus infection was found in parental and descendent generations of chickens. We used quantitative polymerase chain reaction (PCR) and dot blot hybridization to detect the infection of reticuloendotheliosis (REV), avian leukosis virus (ALV), and chicken infectious anemia virus (CIAV) in 480 plasma samples. The test samples were 34.58% FADV-positive, 22.29% REV-positive, 7.5% CAV-positive, and 0.63% ALV-positive. Sequence analysis showed that FADV belonged to serotype 7, which can cause inclusion body hepatitis. The ALV strain was ALV-A, in which the homology of gp85 gene and SDAU09C1 was 97.3%. The positive rate was lower because of the purification of avian leukemia, whereas the phylogenetic tree analysis of REV showed that the highest homology was with IBD-C1605, which was derived from a vaccine isolate. Through pathogen detection in poultry we present, to our knowledge, the first discovery of fowl adenovirus type 7 infection in parental chickens and found that there was co-infection of FAdV and several immunosuppressive viruses, such as the purified ALV and CIAV. This indicates that multiple infection of different viruses is ever-present, and more attention should be given in the diagnosis process.

Characterization of subgroup J avian Leukosis virus isolated from Chinese indigenous chickens.

BACKGROUND: In spite of the purification of the laying hens and broilers of avian leukosis virus (ALV) has made remarkable achievements, the infection of ALV was still serious in Chinese indigenous chickens. METHODS: In order to assess the epidemic state of avian leukosis virus in indigenous chickens in China, 10 novel strains of ALV subgroup J (ALV-J), named JS16JH01 to JS16JH10, were isolated and identified by virus isolation and immunofluorescence antibody assays from a Chinese local breed farm with a sporadic incidence of tumors. To understand their virological characteristics further, the proviral genome of ENV-LTR was sequenced and compared with the reference strains. RESULTS: The homology of the gp85 gene between the ten ALV-J strains and NX0101 was in the range from 89.7-94.8% at the nuclear acid level. In addition, their gp85 genes were quite varied, with identities of 92-98% with themselves at the nuclear acid level. There were several snp and indel sites in the amino acid sequence of gp85 genes after comparison with other reference strains of ALV. Interestingly, a novel insertion in the gp85 region was found in two strains, JS16JH01 and JS16JH07, compared with NX0101 and HPRS-103. DISCUSSION: At present, owing to the large-scale purification of ALV in China, laying hens and broiler chickens with ALV infection are rarely detected, but ALVs are still frequently detected in the local chickens, which suggests that more efforts should be applied to the purification of ALV from indigenous chickens.

Dynamic Co-evolution and Interaction of Avian Leukosis Virus Genetic Variants and Host Immune Responses.

Subgroup J avian leukosis virus (ALV-J), a typical retrovirus, is characterized of existence of a cloud of diverse variants and considerable genetic diversity. Previous studies describing the evolutionary dynamics of ALV-J genetic variants mainly focused on the early infection period or few randomly selected clones. Here, we inoculated 30 specific-pathogen-free chickens with the same founder ALV-J stock of known genetic background. Six (three antibody positive and three antibody negative) chickens were selected among 15 chickens with viremia. Viruses were serially isolated in 36 weeks and then sequenced using MiSeq high-throughput sequencing platform. This produced the largest ALV-J dataset to date, composed of more than three million clean reads. Our results showed that host humoral immunity could greatly enhance the genetic diversity of ALV-J genetic variants. In particular, selection pressures promoted a dynamic proportional changes in ALV-J genetic variants frequency. Cross-neutralization experiment showed that along with the change of the dominant variant, the antibody titers specific to infectious clones corresponding to the most dominant variants in weeks 12 and 28 have also changed significantly in sera collected in weeks 16 and 32. In contrast, no shift of dominant variant was observed in antibody-negative chickens. Moreover, we identified a novel hypervariable region in the gp85 gene. Our study reveals the interaction between ALV-J and the host, which could facilitate the development of vaccines and antiviral drugs.

Cooperative effects of immune enhancer TPPPS and different adjuvants on antibody responses induced by recombinant ALV-J gp85 subunit vaccines in SPF chickens.

To explore the antibody responses and protective effects induced by subgroup J avian leukosis virus (ALV-J) gp85 protein vaccine plus different adjuvants (CpG and white oil adjuvant YF01) combined with the immune enhancer Taishan Pinus massoniana pollen polysaccharide (TPPPS), we immunized SPF chickens with the recombinant ALV-J gp85 protein, along with different adjuvants and immune enhancer, which protected the chickens by inducing different levels of protective antibodies. Results showed that a single injection of gp85 recombinant protein could only produce low-titre antibodies that were maintained over a short time in few chickens. When combined with YF01 or CpG adjuvants, the recombinant protein could induce high-titre antibodies in most of the immunized chickens. Moreover, when the immune enhancer TPPPS was used with the two adjuvants, it further elevated the antibody levels for a longer duration. The eggs from four groups with the highest levels of ALV-J antibodies were collected, hatched, and examined for maternal antibodies. The protection by the maternal antibodies against ALV-J infection in the TPPPS-immunized group was higher than that in the group without TPPPS, which was consistent with the observations in the parents. This study shows that the immune enhancer TPPPS, combined with YF01 or CpG adjuvants, can enhance the immunogenicity of gp85 recombinant proteins, and provide a better immuno-protection. It provides a powerful experimental basis for the development of ALV-J subunit vaccine. Efficient subunit vaccine development will also accelerate the process of purification of ALV-J.

Gp85 genetic diversity of avian leukosis virus subgroup J among different individual chickens from a native flock.

To compare the genetic diversity and quasispecies evolution of avian leukosis virus (ALV) among different individuals, 5 chickens, raised in Shandong Provice of China, were randomly selected from a local chicken flock associated with serious tumor cases. Blood samples were collected and inoculated into chicken embryo fibroblast and DF-1 cell lines for virus isolation and identification, respectively, of Marek's disease virus (MDV), reticuloendotheliosis virus (REV), and ALV. Five strains of ALV subgroup J (ALV-J) were identified, and the gp85 gene from each strain was amplified and cloned. For each strain, about 20 positive clones of gp85 gene were selected for sequence analyses and the variability of the quasispecies of the 5 strains was compared. The results showed that the nuclear acid length of gp85 gene of 5 ALV-J isolates is 921 bp, 921 bp, 924 bp, 918 bp, and 912 bp respectively, and amino acid homologies of different gp85 clones from the 5 ALV-J strains were 99.3 to 100%, 99.3 to 100%, 99.4 to 100%, 98.4 to 100%, 99.0 to 100%, respectively. The proportions of dominant quasispecies were 65.0%, 85.0%, 85.0%, 50.0%, 84.2%, respectively, and homology of the gp85 among these dominant quasispecies was 89.2 to 92.5%. These data demonstrated the composition of the ALV-J quasispecies varied among infected individuals even within the same flock, and the dominant quasispecies continued to evolve both for their proportion and gene mutation.