The Emergence, Diversification, and Transmission of Subgroup J Avian Leukosis Virus Reveals that the Live Chicken Trade Plays a Critical Role in the Adaption and Endemicity of Viruses to the Yellow-Chickens.

The geographical spread and inter-host transmission of the subgroup J avian leukosis virus (ALV-J) may be the most important issues for epidemiology. An integrated analysis, including phylogenetic trees, homology modeling, evolutionary dynamics, selection analysis and viral transmission, based on the gp85 gene sequences of the 665 worldwide ALV-J isolates during 1988-2020, was performed. A new Clade 3 has been emerging and was evolved from the dominating Clade 1.3 of the Chinese Yellow-chicken, and the loss of a α-helix or ß-sheet of the gp85 protein monomer was found by the homology modeling. The rapid evolution found in Clades 1.3 and 3 may be closely associated with the adaption and endemicity of viruses to the Yellow-chickens. The early U.S. strains from Clade 1.1 acted as an important source for the global spread of ALV-J and the earliest introduction into China was closely associated with the imported chicken breeders in the 1990s. The dominant outward migrations of Clades 1.1 and 1.2, respectively, from the Chinese northern White-chickens and layers to the Chinese southern Yellow-chickens, and the dominating migration of Clade 1.3 from the Chinese southern Yellow-chickens to other regions and hosts, indicated that the long-distance movement of these viruses between regions in China was associated with the live chicken trade. Furthermore, Yellow-chickens have been facing the risk of infections of the emerging Clades 2 and 3. Our findings provide new insights for the epidemiology and help to understand the critical factors involved in ALV-J dissemination. IMPORTANCE Although the general epidemiology of ALV-J is well studied, the ongoing evolutionary and transmission dynamics of the virus remain poorly investigated. The phylogenetic differences and relationship of the clades and subclades were characterized, and the epidemics and factors driving the geographical spread and inter-host transmission of different ALV-J clades were explored for the first time. The results indicated that the earliest ALV-J (Clade 1.1) from the United States, acted as the source for global spreads, and Clades 1.2, 1.3 and 3 were all subsequently evolved. Also the epidemiological investigation showed that the early imported breeders and the inter-region movements of live chickens facilitated the ALV-J dispersal throughout China and highlighted the needs to implement more effective containment measures.

Transcription analysis of chicken embryo fibroblast cells infected with the recombinant avian leukosis virus isolate GX14FF03.

Infection with recombinant avian leukosis virus (ALV) has previously been linked to malignancies and immunosuppression. However, the processes behind the unique pathophysiology of recombinant ALV are poorly understood. In this study, we analyzed gene expression patterns in chicken fibroblast cells (CEFs) infected with the recombinant ALV isolate GX14FF03 and used the RNA-seq technique to perform a complete analysis of the transcribed mRNAs. A total of 907 significant differentially expressed genes (SDEGs) were identified. Among these SDEGs, the most significantly upregulated gene was interleukin 8-like 1 (IL8L1), while the most significantly downregulated gene was fibroblast growth factor 16 (FGF16). The 907 SDGEs were highly enriched (p < 0.05) for 252 Gene Ontology (GO) terms, including 197 BP, 3 CC, and 52 MF. According to KEGG data analysis, SDEGs are implicated in eight significant pathways (p < 0.05). Furthermore, protein-protein interaction (PPI) network analysis revealed that IL8L1 interacts with 17 genes. These findings shed light on the molecular mechanisms involved in recombinant ALV infection by showing the mRNA expression profile in CEFs infected with GX14FF03 virus.

Molecular characterization of two novel sub-sublineages of pigeon paramyxovirus type 1 in China.

Pigeon paramyxovirus type 1 (PPMV-1) infection is enzootic in pigeon flocks and poses a potential risk to the poultry industry in China. To gain insight into the biological characteristics and transmission routes of circulating PPMV-1 in pigeons, 13 PPMV-1 isolates from domestic pigeons isolated during 2011-2015 in Guangxi province, China, were characterized using a pathogenicity assessment and phylogenetic analysis. All PPMV-1 isolates were mesogenic or lentogenic strains and had a mean death time (MDT) in 9-day-old SPF chicken embryos and a intracerebral pathogenicity index (ICPI) values of 54-154 h and 0.00-0.90, respectively. Analysis of the F and HN gene sequences of the PPMV-1 isolates and the Newcastle Disease (ND) vaccine strain La Sota, revealed that the nucleotide sequence similarity of the F and HN genes were all < 85% between the PPMV-1 isolates and La Sota, significantly lower than those > 98% among the PPMV-1 isolates. The amino acids sequence of the F protein at the cleavage site of the 13 PPMV-1 isolates was 112RRQKR↓F117, characteristic of virulent Newcastle disease virus (NDV). All 13 isolates were classified as sublineage 4b by phylogenetic analysis and evolutionary distances, based on the F gene sequences. It was also found that the 13 isolates were divided into two novel sub-groups of sublineage 4b, sub-sublineages 4biig and 4biih. Since these two novel sub-sublineages had two different geographic sources, we speculated that they represent two different transmission routes of PPMV-1 in China. Phylogenetic analysis of these isolates will help to elucidate the sources of the transmission and evolution of PPMV-1 and may help to control PPMV-1 infection in the pigeon industry in China.

The phylogenetic analysis of the new emerging ALV-K revealing the co-prevailing of multiple clades in chickens and a proposal for the classification of ALV-K.

Subgroup K avian leukosis virus (ALV-K) is a new subgroup of avian leukosis virus (ALV) that was first defined in 2012 and has been become prevalent in Chinese native chickens in recent years. An in-depth analysis of the genetic diversity of ALV-K was performed in the study. By Blast analysis, the env gene and the sequences of the 25 ALV-K isolates we isolated were found to be closely related to the isolates from Guangdong, Hebei, Jiangsu, and Hubei provinces, China. Further eighty-nine sequences of the gp85 gene of ALV-K strains available were used in the phylogenetic and genetic distance analyses for the classification. ALV-K was divided into two second-order clades (Clades 1.1 and 1.2) and three third-order clades (Clades 1.2.1, 1.2.2, and 1.2.3), indicating that not only 1.1 and 1.2.3, the two old clades which are prevalent in Japan, but also two new clades (1.2.1, 1.2.2), are co-prevalent in China. The representative strains of each clade were defined for the first time. Notably, Clade 1.2.2 was found to have a deletion of an amino acid residue in the gp85 gene, which was obviously different from Clades 1.1, 1.2.1, and 1.2.3. The proposed classification method will facilitate future studies of ALV-K epidemiology and the comparison of sequences obtained across the world. The first global comprehensive molecular epidemiological analysis was accomplished on the emerging ALV-K.

Emergence and Molecular Characterization of an Avian Hepatitis E Virus From Donglan Black Chicken in Southern China.

Avian hepatitis E virus (HEV) is a major pathogen associated with hepatitis splenomegaly syndrome in chickens and has been reported in China. Phylogenetic trees, Bayesian analysis, positive selection sites screening, and recombination analysis were first used to comprehend the global avian HEVs. All the avian HEV strains, including a new isolate named GX20A1 got from Donglan Black chicken in Guangxi, China, were uniformly defined into four genotypes, and GX20A1, belongs to Genotype 3. The topology of the phylogenetic tree based on the sequences of a 339-bp fragment (coding the helicase) in open reading frame (ORF) 1 of the avian HEVs was consistent with that based on the full-genome sequence. The estimated evolution rate of avian HEVs is 2.73 × 10-3 substitution/site/year (95% confidence interval (CI): 8.01 × 10-4-4.91 × 10-3), and the estimated genetic diversity of the strains experienced a declining phase from 2010 to 2017 and stabilized after 2017. It was further found that the Genotype 3 HEVs, including isolates from Hungary and China, likely originated in the 1930s. Notably, GX20A1 was gathered in the same branch with a Genotype 3 Guangdong isolate CaHEV-GDSZ01, which appeared earlier than GX20A1. In addition, two positive selection sites were identified, one for each of ORF1 and ORF2. Overall, the study revealed that avian HEVs were uniformly defined into four genotypes, and a 339-bp fragment in ORF1 of the viral genome could be used for the classification. A Genotype 3 isolate GX20A1 was first found from Donglan Black chicken and most likely originated from Guangdong.

The emerging naturally reassortant strain of IBDV (genotype A2dB3) having segment A from Chinese novel variant strain and segment B from HLJ 0504-like very virulent strain showed enhanced pathogenicity to three-yellow chickens.

Novel variant infectious bursal disease virus (nvIBDV) is an emerging pathotype that can cause sub-clinical disease with severe, prolonged immunosuppression in young chickens. At present, two major pathotypes, including vvIBDV and nvIBDV, are prevailing in China. In this study, we propose that the nvIBDV is a new genotype (A2dB1b) and also first isolated and characterized a nvIBDV reassortant strain YL160304 (A2dB3) with segments A and B derived, respectively, from the nvIBDV and the HLJ-0504-like vvIBDV from yellow chickens in southern China. The YL160304 causes more extensive cytotropism and can infect specific-pathogen-free chicken embryos with severe subcutaneous hemorrhage. The pathogenicity of YL160304 to 4-week-old three-yellow chickens was determined and compared with those of the nvIBDV QZ191002 and the HLJ-0504-like vvIBDV NN1172. Weight gain was significantly reduced in all the challenged birds. No clinical signs and associated mortality were observed in the birds challenged with QZ191002, while the mortalities in the birds challenged with NN1172 and YL160304 were 30% (3/10) and 10% (1/10), respectively. At 7 days postchallenge, the bursa was severely damaged and the percentage of peripheral blood B lymphocyte (PBBL) decreased significantly in all the challenged birds and the quantity of the viral RNA detected in the bursa was in accordance with the results of the histomorphometry and the depletion of PBBL. This study not only confirmed the emerging epidemic of the novel variant and its reassortant strains, but also discovered that the naturally reassortant nvIBDV strain with the segment B of HLJ 0504-like vvIBDV can significantly enhance the pathogenicity to chickens.

Phylogenetic and Spatiotemporal Analyses of the Complete Genome Sequences of Avian Coronavirus Infectious Bronchitis Virus in China During 1985-2020: Revealing Coexistence of Multiple Transmission Chains and the Origin of LX4-Type Virus.

Infectious bronchitis (IB) virus (IBV) causes considerable economic losses to poultry production. The data on transmission dynamics of IBV in China are limited. The complete genome sequences of 212 IBV isolates in China during 1985-2020 were analyzed as well as the characteristics of the phylogenetic tree, recombination events, dN/dS ratios, temporal dynamics, and phylogeographic relationships. The LX4 type (GI-19) was found to have the highest dN/dS ratios and has been the most dominant genotype since 1999, and the Taiwan-I type (GI-7) and New type (GVI-1) showed an increasing trend. A total of 59 recombinants were identified, multiple recombination events between the field and vaccine strains were found in 24 isolates, and the 4/91-type (GI-13) isolates were found to be more prone to being involved in the recombination. Bayesian phylogeographic analyses indicated that the Chinese IBVs originated from Liaoning province in the early 1900s. The LX4-type viruses were traced back to Liaoning province in the late 1950s and had multiple transmission routes in China and two major transmission routes in the world. Viral phylogeography identified three spread regions for IBVs (including LX4 type) in China: Northeastern China (Heilongjiang, Liaoning, and Jilin), north and central China (Beijing, Hebei, Shanxi, Shandong, and Jiangsu), and Southern China (Guangxi and Guangdong). Shandong has been the epidemiological center of IBVs (including LX4 type) in China. Overall, our study highlighted the reasons why the LX4-type viruses had become the dominant genotype and its origin and transmission routes, providing more targeted strategies for the prevention and control of IB in China.

Genetic diversity of avian leukosis virus subgroup J (ALV-J): toward a unified phylogenetic classification and nomenclature system.

Avian leukosis virus subgroup J (ALV-J) has infected a variety of birds, causing major economic losses in China. Understanding the comprehensive criteria of classification and nomenclature of ALV-J would be useful for the investigation of the viral evolution and also for the prevention and control of this infection. An in-depth analysis of the genetic diversity of ALV-J was performed in the present study. Four hundred and seventy-five sequences of the gp85 gene, including thirteen of avian endogenous retrovirus designated ev/J and 462 of ALV-J, were used in the phylogenetic and the evolutionary distance analysis for this classification. The study identified that the current ALV-J strains were divided into two first-order clades (Clades 1 and 2) and three second-order clades (Clades 1.1, 1.2 and 1.3). The current Chinese ALV-J strains are predominantly in Clade 1.3, and the Chinese and Egyptian chicken flocks have been facing the emerging Clade 2 viruses. This system pioneers the classification efforts for ALV-J, which uses Pilot tree for rapid classification of the new isolates and also the addition of possible new clades. The proposed unified classification system will facilitate future studies of ALV-J epidemiology and genetic evolution and of the comparison of sequences obtained across the world.

Reemergence of reticuloendotheliosis virus and Marek's disease virus co-infection in Yellow-Chickens in Southern China.

The reticuloendotheliosis virus (REV) and the Marek's disease virus (MDV) cause reticuloendotheliosis (RE) and Marek's disease (MD) in poultry, respectively. According to epidemiological results obtained in our laboratory from 2010 to 2017, the positive rates of REV and MDV co-infection remained at low levels. In the present study, during the period of October 2018 to July 2020, 4 clinical cases with high morbidity (5%-20%) and mortality (2%-10%), caused by the co-infection of REV and vv+ MDV-like strains, were diagnosed and analyzed by histopathological observation, cell cultures and detection with ELISA and IFA, and the PCR and by sequencing of the isolates' genes. Sequencing and the sequence analysis on the complete genomes of the REV strains and the meq genes of the MDV strains were performed. The results, based on the complete genome, LTR, gag, pol, and env genes' nucleotide sequences of the REV strains, showed that the REV isolates and 68.0 % (17/25) of the reference strains were in a same branch, and all had a high sequence similarity (>99.0%). The similarities between the four isolates and a vv+MDV strain GX18NNM4 were very high, up to 99.3-99.8%. Also, the amino acid residuals at locations 71, 77, 80, 115, 139, 176, and 217 were all the same as A, E, Y, A, A, R, and A, respectively, in the meq gene of the four MDV isolates. In addition, the substitutes at P176R and P217A interrupted the stretches of the proline-rich repeat PPPP, indicating that these strains belonged to the vv+ MDV-like category. Our findings indicated that the more recent and frequent reemergence of REV and the subsequent co-infection with vv+ MDV-like strain has become one of the causes of the clinical outbreaks of tumors and is undoubtedly a threat to the poultry industry in southern China.

Analysis of the evolution and transmission dynamics of the field MDV in China during the years 1995-2020, indicating the emergence of a unique cluster with the molecular characteristics of vv+ MDV that has become endemic in southern China.

Marek's disease (MD) continues to threaten the sustainability of the world poultry industry. In this study, the sequences of the meq gene of 220 MDV strains isolated during the years 1964-2020 were analysed, including 50 from our group plus 170 isolates from the GenBank. Analyses, using phylogenetic trees, amino acid (aa)-mutation screening, evolutionary studies and transmission dynamics were all performed. All strains were divided into two clusters (Clusters 1 and 2), and Cluster 1 includes the mild strains, the vaccine strains and the foreign virulent strains, while Cluster 2 was dominated by the Chinese field strains. Our study identified that the Chinese field strains in Cluster 2 during the years 1995-2020 likely originated in the 1980s from abroad, and the estimated genetic diversity of these strains experienced two growth phases in the years 2005-2007.5 and 2015-2017. Viral phylogeography identified 3 major geographic provincial regions for the Chinese field strains of Cluster 2: the Northeastern Region (Jilin, Liaoning and Heilongjiang), the East-central Region (Henan, Shandong and Jiangsu) and the Southern Region (Guangxi, Guangdong and Yunnan). The spread of Northeastern strains to East-central chicken flocks and the further spread from Guangxi to Guangdong are strongly indicated. The emergence of the mutations A88T and Q93R together in the Southern strains during the years 2017-2020 with molecular characteristics of vv+ MDV were also found later than those in the Northern strains. Overall, the Chinese field strains in Cluster 2 in southern China in recent years have been rapidly evolving. Guangxi Province has become an epicentre for these viruses and the chicken flocks in the Southern region have been facing the adverse effects of the emerging vv+ MDV.

Recombinant subgroup B avian leukosis virus combined with the subgroup J env gene significantly increases its pathogenicity.

The differences among different sub-groups of the avian leukosis virus (ALV) genome are mainly concentrated in the env gene, which binds to cell-specific receptors and determines the characteristics of viral tropism and pathogenicity. In this study, two rescued viruses rGX15MM6-2 (ALV of subgroup J, ALV-J) and rGX14FF03 (ALV of subgroup B, ALV-B) and a recombinant virus rALV-B-Jenv (ALV-B's backbone with ALV-J's env) were generated and tested utilizing both in vitro and in vivo experiments. The results showed that the replication ability of the viruses released in DF-1 cell cultures was listed in order as rGX15MM6-2 > rALV-B-Jenv > rGX14FF03. rGX15MM6-2 caused the most serious suppression of body weight gain, exhibited a significant negative effect on the development of immune organs (P < 0.05) and lower antibody responses to vaccinations with the commercial oil-emulsion vaccines (OEVs) (P<0.05) in the challenged chickens. The viral detection showed that the positive rate in blood from the birds infected with rALV-B-Jenv were respectively higher than those from the birds infected with rGX14FF03 (P < 0.05). At 25 wpi, similar tumors were found in the abdominal cavity of the birds in rGX15MM6-2 and rALV-B-Jenv groups. The results demonstrated that the ALV-J env gene significantly increases the pathogenicity of the recombinant ALV-B. With the increasing incidence of co-infections of different subgroups of ALV in the field, the possibility of viral recombination is increasing and demands further study.

The Emergence of a vv + MDV Can Break through the Protections Provided by the Current Vaccines.

Marek's disease (MD) is an infectious malignant T-cell lymphoma proliferative disease caused by Marek's disease virus (MDV). In recent years, the emergence of very virulent (vv) and/or very virulent plus (vv +) strains of MDV in the field has been suggested as one of the causes of vaccination failure. The pathogenicity of the MDV strain GX18NNM4, isolated from a clinical outbreak in a broiler breeder flock that was vaccinated with CVI988/Rispens, was investigated. In the vaccination-challenge test, GX18NNM4 was able to break through the protections provided by the vaccines CVI988 and 814. It also significantly reduced body weight gain and caused marked gross lesions and a large area of infiltration of neoplastic lymphocyte cells in the heart, liver, pancreas, etc. of the infected birds. In addition, the expressions of programmed death 1 (PD-1) and its ligand, programmed death ligand 1 (PD-L1), in the spleens and cecal tonsils (CTs) of the unvaccinated challenged birds were significantly increased compared to those in the vaccinated challenged birds, indicating that the PD-1/PD-L1 pathway is related to immune evasion mechanisms. The results showed that the GX18NNM4 strain could cause severe immunosuppression and significantly decrease the protections provided by the current commercial vaccines, thus showing GX18NNM4 to be a vv + MDV strain.

Re-emergence of a genotype VIII virulent Newcastle disease virus isolated from Chinese game fowl after 13 years.

Circulation of dominant genotypes VI and VII of Newcastle disease virus (NDV) is causing significant economic losses to the poultry industry in China. However, reports of Newcastle disease (ND) outbreaks caused by genotype VIII strains of NDV are rare. In this study, a virulent genotype VIII strain of NDV, designated GXGB2011, was isolated from a vaccinated game fowl flock showing clinic signs of infection in Pinxiang county, Guangxi, China. The whole genome of the isolate was completely sequenced and was found to be comprised of 15,192 nucleotides (nt), encoding the six structural proteins in the order of 3'-NP-P-M-F-HN-L-5'. The pattern of cleavage site 112 RRQKR↓F117 in the fusion (F) protein and the intracerebral pathogenicity index (ICPI) value of 1.5 showed that the strain GXGB2011 was a velogenic NDV. The results of the challenge experiment with the 5-week-old SPF chickens showed that the strain was highly pathogenic with 100% morbidity and mortality of the challenged birds. Based on the detection of virus in different organs of the infected birds, the highest viral load in caecal tonsils was observed and viral levels in immune organs were higher than those in the respiratory organs. Bayesian reconstruction of complete genomes based on the sequences of 66 NDV reference strains showed that the strain belonged to the genotype VIII of NDV. Phylogenetic analysis showed that the strain was more closely related to the foreign strains gamefowl/U.S.(CA)/24225/98, 1ITTY94060 and IT-147/94 rather than to the first domestic strains of the emergence genotype VIII in Qinghai, China during 1979-1985. In summary, the results of the study demonstrated the re-emergence of a highly pathogenic virulent isolate of genotype VIII of NDV. These results indicate the risk that this genotype VIII of NDV may spread to commercial chickens from game fowl.