Development of a colloidal gold immunochromatographic strip for rapid detection of avian coronavirus infectious bronchitis virus.

Avian infectious bronchitis virus (IBV) still causes serious economic losses in the poultry industry. Currently, there are multiple prevalent genotypes and serotypes of IBVs. It is imperative to develop a new diagnosis method that is fast, sensitive, specific, simple, and broad-spectrum. A monoclonal hybridoma cell, N2D5, against the IBV N protein was obtained after fusion of myeloma SP2/0 cells with spleen cells isolated from the immunized Balb/c mice. The N2D5 monoclonal antibody (mAb) and the previously prepared mouse polyclonal antibody against the IBV N protein were used to target IBV as a colloidal gold-mAb conjugate and a captured antibody, respectively, in order to develop an immunochromatographic strip. The optimal pH and minimum antibody concentration in the reaction system for colloidal gold-mAb N2D5 conjugation were pH 6.5 and 30 µg/mL, respectively. Common avian pathogens were tested to evaluate the specificity of the strip and no cross-reaction was observed. The sensitivity of the strip for detecting IBV was 10-1.4522 EID50/mL. The strip showed a broad-spectrum cross-reactive capacity for detecting IBV antigens, including multiple IBV genotypes in China and all of the seven serotypes of IBV that are currently prevalent in southern China. Additionally, the result can be observed within 2 min without any equipment. The throat and cloacal swab samples of chickens that were artificially infected with three IBV strains were tested using the developed strip and the qPCR method; the strip test demonstrated a high consistency in detecting IBV via qPCR gene detection. In conclusion, the immunochromatographic strip that was established is rapid, sensitive, specific, simple, practical, and broad-spectrum; additionally, it has the potential to serve as an on-site rapid detection method of IBV and can facilitate the surveillance and control of the disease, especially in resource-limited areas.

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.

Lactobacillus plantarum GX17 benefits growth performance and improves functions of intestinal barrier/intestinal flora among yellow-feathered broilers.

Lactobacillus plantarum has recently been found to be a natural source feed additive bacteria with great advantages in food safety and animal welfare. Discovering novel strains with commercial application potentiation could benefit the local poultry industry, and in particular support Chinese farmers. In this study, we tested a recently isolated novel strain of Lactobacillus plantarum GX17 as a feed additive on the growth performance and intestinal barrier functions of 1-day-old Chinese yellow-feather chicks. As good as other commercial probiotics, feeding with Lactobacillus plantarum GX17 showed significant improvements in humoral immune responses and enhanced the immune effect after vaccination for either the Newcastle disease vaccine or the avian influenza vaccine. This study also found that feeding with Lactobacillus plantarum GX17 improved the feed-to-weight ratio and caused a significant increase of the villus length to crypt depth ratio. Furthermore, Lactobacillus plantarum GX17 significantly up-regulated the mRNA expression of CLDN, MUC2, and TLR2, all of which are jejunum-associated barrier genes, indicating an improvement of the intestinal barrier functions by enhancing the tight junction between epithelia cells. These results are comparable to the effects of feeding the commercial complex probiotics that improve the expression levels of CLDN, ocludin, MUC2, TLR2, and TLR4. In terms of maintaining intestinal health, commercial complex probiotics increased the relative abundance of Parabacteroides and Romboutsia, while Lactobacillus plantarum GX17 increased the relative abundance of Pseudoflavonifractor. Our data suggest that Lactobacillus plantarum GX17 could enhance the intestinal absorption of nutrients and therefore improve the growth performance of Chinese yellow-feather chicks. In conclusion, compared with the commercial complex probiotics, Lactobacillus plantarum GX17 has more positive effects on the growth performance and intestinal barrier function of yellow-feather chickens, and can be used as a feed additive.

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.

Analysis of the global origin, evolution and transmission dynamics of the emerging novel variant IBDV (A2dB1b): The accumulation of critical aa-residue mutations and commercial trade contributes to the emergence and transmission of novel variants.

The Chinese IBDV novel variant (nvIBDV), belonging to the genotype A2dB1b, an emerging pathotype that can cause subclinical disease with severe, prolonged immunosuppression, poses a new threat to the poultry industry. The process of the global origin, evolution and transmission dynamics of nvIBDV, however, is poorly understood. In this study, phylogenetic trees, site substitutions of amino acid (aa) and highly accurate protein structure modelling, selection pressure, evolutionary and transmission dynamics of nvIBDV were analysed. Interestingly, nvIBDV was classified into the same genogroup with the early US antigenic variants (avIBDV) but in a new lineage with a markedly different and specific pattern of 17 aa-residual substitutions: 13 in VP2 (77D, 213N, 221K, 222T, 249K, 252I, 253Q, 254N, 284A, 286I, 299S, 318D and 323E) and four in VP1 (141I, 163V, 240E and 508K). Importantly, the aa-residues 299S and 163V may play a key role in cell binding and polymerase activity, respectively. The effective population size of the circulating avIBDV experienced two growth phases, respectively, in the years 1999-2007 (in North America) and 2015-2021 (in Asia), which is consistent with the observed trend of the epidemic outbreaks. The most recent common ancestor (tMRCA) of avIBDV most first originated in the USA and was dated around the 1970s. After its emergence, the ancestor virus of this group probably spread to China around the 1990s and the variants experienced a long-term latent circulation with the accumulation of several critical aa-residue mutations in VP2 until re-emerging in 2016. At present, central China has become the epicentre of nvIBDV spread to other parts of China and Asian countries. Importantly, a strong correlation seems to exist between the transmission patterns of virus and the flow of commercial trade of live poultry and products. These findings provide important insights into the origin, evolution and transmission of the nvIBDV and will assist in the development of programs for control strategies for these emerging viruses.

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.

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.

Identification of a novel avian coronavirus infectious bronchitis virus variant with three-nucleotide-deletion in nucleocapsid gene in China.

A novel avian infectious bronchitis virus (IBV) variant, designated as GX-NN160421, was isolated from vaccinated chicken in Guangxi, China, in 2016. Based on analysis of the S1 gene sequence, GX-NN160421 belonged to the New-type 1 (GVI-1) strain. More importantly, three consecutive nucleotides (AAC) deletions were found in the highly conserved structure gene N. The serotype of GX-NN160421 was different from those of the commonly used vaccine strains. The mortality of the GX-NN160421 strain was 3.33%, which contrasted with 50% mortality in the clinical case, but high levels of virus shedding lasted at least 21 days. In conclusion, the first novel IBV variant with three-nucleotide-deletion in the N gene was identified, and this unique variant is low virulent but with a long time of virus shedding, indicating the continuing evolution of IBV and emphasizing the importance of limiting exposure to novel IBV strains as well as extensive monitoring of new IBVs.

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.

Construction and Immunogenicity Comparison of Three Virus-Like Particles Carrying Different Combinations of Structural Proteins of Avian Coronavirus Infectious Bronchitis Virus.

Infectious bronchitis virus (IBV) poses massive economic losses in the global poultry industry. Here, we firstly report the construction and immunogenicity comparison of virus-like particles (VLPs) carrying the S, M and E proteins (SME-VLPs); VLPs carrying the S and M proteins (SM-VLPs); and VLPs carrying the M and E proteins (ME-VLPs) from the dominant serotype representative strain GX-YL5 in China. The neutralizing antibody response induced by the SME-VLPs was similar to that induced by the inactivated oil vaccine (OEV) of GX-YL5, and higher than those induced by the SM-VLPs, ME-VLPs and commercial live vaccine H120. More importantly, the SME-VLPs elicited higher percentages of CD4+ and CD8+ T lymphocytes than the SM-VLPs, ME-VLPs and OEV of GX-YL5. Compared with the OEV of GX-YL5, higher levels of IL-4 and IFN-γ were also induced by the SME-VLPs. Moreover, the mucosal immune response (sIgA) induced by the SME-VLPs in the tear and oral swabs was comparable to that induced by the H120 vaccine and higher than that induced by the OEV of GX-YL5. In the challenge experiment, the SME-VLPs resulted in significantly lower viral RNA levels in the trachea and higher protection scores than the OEV of GX-YL5 and H120 vaccines, and induced comparable viral RNA levels in the kidneys, and tear and oral swabs to the OEV of GX-YL5. In summary, among the three VLPs, the SME-VLPs carrying the S, M and E proteins of IBV could stimulate the strongest humoral, cellular and mucosal immune responses and provide effective protection, indicating that it would be an attractive vaccine candidate for IB.

ALV-J-contaminated commercial live vaccines induced pathogenicity in Three-Yellow chickens: one of the transmission routes of ALV-J to commercial chickens.

One avian leukosis virus of subgroup J (ALV-J) strain GX14YYA1 was isolated from a commercial bivalent Newcastle disease (ND)-infectious bronchitis (IB) vaccine in our previous study. To evaluate the pathogenicity of the ALV-J-contaminated vaccine on commercial chickens, day-old Three-Yellow chicks in group I were vaccinated with ALV-J-contaminated bivalent ND-IB live vaccine by intranasal and eye drop at 1-day-old for the primary vaccination and at 7-day-old for the secondary vaccination. Groups II and III were kept as the normal vaccination group with the noncontaminated ND-IB vaccine and blank control groups, respectively. The birds of different groups were maintained separately in isolators for 175 d. The first viremia was detected at 4 wk of age and 20% (2/10) of the birds maintained viremia during 11 to 25 wk of age. At the same time, the birds in group I experienced a significant suppression of body weight gain when compared with those of groups II and III (P < 0.05). In addition, the birds in group I showed obvious ALV-J hemangioma-type anatomical lesions in the liver and tumors were observed in the abdominal cavity. The results demonstrated that the ALV-J contaminated commercial live vaccines can induce pathogenicity in commercial Three-Yellow chickens and indicate that ALV-J-contaminated commercial live vaccines could be one of the transmission routes of ALV-J to commercial chickens.

Rapid and visual detection of the emerging novel duck reovirus by using a specific and sensitive reverse transcription recombinase polymerase amplification method.

Duck spleen necrosis disease (DSND) caused by Novel Duck Reovirus (NDRV), is an emerging infectious disease that causes severely threaten to duck industry. Currently, the popular conventional RT-PCR technique for detecting NDRV is time consuming. So, it is essential to develop a rapid and accurate molecular diagnosis techniques of the pathogen for the purpose to effective control of the disease. In our study, a simple, rapid and reliable detection method was developed by an isothermal reverse transcription-recombinase polymerase amplification (RT-RPA). The RT-RPA primers were designed targeting the S3 gene of NDRV, and its specificity was verified by testing a series of other waterfowl pathogens. A total of 20 field and experimental samples from infected ducklings were tested by the RT-RPA and compared with the results of the conventional RT-PCR and the quantitative RT-PCR simultaneously. The RT-RPA method could detect as little as 4.14 × 102 copies/µl of the target gene in the sensitivity analysis, which was 10×higher sensitive than the conventional RT-PCR. The major advantage of the RT-RPA method is that it could be performed as an isothermal reaction at 37 ℃ and completed within 20 min. In addition, no cross-reactivity was detected with other waterfowl-origin viruses. Also, the amplified products could be visualized faster, without the gel electrophoresis, by adding the SYBR Green I and observing them under an ultraviolet light. The newly developed RT-RPA method offers a simple, rapid and accurate for rapid detection of NDRV, which especially useful in on-site facilities and resource-limited areas.

The Full Region of N-Terminal in Polymerase of IBDV Plays an Important Role in Viral Replication and Pathogenicity: Either Partial Region or Single Amino Acid V4I Substitution Does Not Completely Lead to the Virus Attenuation to Three-Yellow Chickens.

Infectious Bursal Disease Virus (IBDV) has haunted the poultry industry with severe, prolonged immunosuppression of chickens when infected at an early age and can easily lead to other secondary infections. Understanding the pathogenic mechanisms could lead to effective prevention and control of Infectious Bursal Disease (IBD). Evidence suggests that the N-terminal domain of polymerase in segment B plays an important role, but it is not clear which part or residual is crucial for the pathogenicity. Using a reverse genetics technique, a molecular clone (rNN1172) of the parental vvIBDV strain NN1172 was generated, and its pathogenicity was found to be the same as the parental virus. Then, three recombinant chimeric viruses were rescued based on the rNN1172 and substituted with the counterparts in the N-terminal domain of the attenuated vaccine strain B87: the rNN1172-B87VP1a (substituting the full region of the 1-167 aa residuals), the rNN1172-B87VP1a∆4 (substituting the region of the 5-167 aa residuals), and the rNN1172-VP1∆4 (one single aa residual substitution V4I), to better explore the role of the N-terminal domain of polymerase on the viral pathogenicity. Interestingly, all these substitutions played different roles in the viral pathogenicity: the mortality of the rNN1172-B87VP1a-challenged chickens was significantly reduced from 30% to 0%. No obvious lesion was found in the histopathological examination, and the lowest viral genome copy number was also detected in the bursa when compared to the parental and two other recombinant viruses. The mortalities caused by rNN1172-B87VP1a∆4 and rNN1172-B87VP1∆4, respectively, were all reduced to 10% and had a delayed onset of death. Our results also revealed that the pathogenicity of the IBDV was consistent with the viral replication efficiency in vivo (bursae). This study demonstrated that the full region of the N-terminal of polymerase plays an important role in viral replication and pathogenicity, but the substitutions of its partial region or a single residual do not completely lead to the virus attenuation to Three-Yellow chickens, although that significantly reduces its pathogenicity.

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 diagnosis and successful replication of a clinical case of Duck Spleen Necrosis Disease: An experimental co-infection of an emerging unique reovirus and Salmonella indiana reveals the roles of each of the pathogens.

Duck spleen necrosis disease (DSND) is an emerging infectious disease that causes significant economic loss in the duck industry. In 2018, a duck reovirus (named DRV/GX-Y7) and Salmonella indiana were both isolated from the spleens and livers of diseased ducks with DSND in China. The DRV/GX-Y7 strain could propagate in the Vero, LMH, DF-1 and DEF cells with obvious cytopathic effects. The genome of DRV/GX-Y7 was 23,418 bp in length, contained 10 dsRNA segments, ranging from 3959 nt (L1) to 1191 nt (S4). The phylogenetic analysis showed that the DRV/GX-Y7 strain was in the same branch with the new waterfowl-origin reovirus cluster, but was obviously far distant from the clusters of other previous waterfowl-origin reoviruses Muscovy duck reovirus (MDRV) and goose-origin reovirus (GRV), broiler/layer-origin reovirus (ARV) and turkey-origin reovirus (TRV). The RDP and SimPlot program analysis revealed that there were two potential genetic reassortment events in the M2 and S1 segments of the genome. In order to have a clear insight into the pathogenic mechanism of DRV/GX-Y7 and S. Indiana in clinical DSND, an infection experiment was further conducted by challenging commercial ducklings with the two isolates individually and with both. The results showed that DRV/GX-Y7 produced severe hemorrhagic and/or necrotic lesions in the immune organs (thymus, spleen, and bursae) of experimentally infected ducklings. And, that the co-infection of DRV/GX-Y7 and S. Indiana could greatly enhance the pathogenesis by increasing the morbidity and mortality in ducklings whose clinical symptoms and lesions were similar to the natural clinical DSND cases. In summary, the results suggested that the pathogen causing duck spleen necrosis was an emerging unique genetic reassortment strain of duck Orthoreovirus that was significantly different from any previously reported waterfowl-derived Orthoreovirus and the co-infection with the Salmonella isolate could increase the severity of the disease.

Full-length cDNA sequence analysis of 85 avian leukosis virus subgroup J strains isolated from chickens in China during the years 1988-2018: coexistence of 2 extremely different clusters that are highly dependent upon either the host genetic background or the geographic location.

During the process of transmission and spread of avian leukosis virus subgroup J (ALV-J) in chickens worldwide, the viral genome is constantly changing. A comprehensive and systematic study of the evolutionary process of ALV-J in China is needed. In this study, we amplified the full-length viral cDNA sequences of 16 ALV-J isolates of Yellow-chicken origin and analyzed and compared these sequences with another 69 ALV-J strains isolated during the years 1988-2018. These isolates were then sorted into 2 clusters: cluster I included isolates that mainly originated from the layers and White-feather broilers from northern China; cluster II included isolates mainly from the Yellow-chicken, most of them being from southern China. According to the sequence homologies of the whole genome and gag, pol, gp85, and gp37 genes, the ALV-J strains are more likely to randomly change in different directions from the original strain HPRS-103 as time passes. The results of entropy analysis of the sequences of gag, pol, and env revealed that the env gene had the largest variation, and the gag gene nonconserved sites are mainly concentrated in p19, p10, and p12. In addition, 84.71% (72/85) of the isolates had the 205-nucleotide (nt) deletion in the 3'UTR region, and 30.59% (26/85) of the isolates had the 125-nt to 127-nt deletion in the E element. Our study provides evidence for the coexistence of 2 extremely different clusters of ALV-J prevailing in China and in some other countries during the period of 1988-2018 and implies that the clusters are highly dependent on the host genetic background and the geographic location.

Vertical transmission of ALV from ALV-J positive parents caused severe immunosuppression and significantly reduced marek's disease vaccine efficacy in three-yellow chickens.

In order to evaluate the influence of the vertical transmission of avian leukosis virus (ALV) from J subgroup (ALV-J) positive parents on the vaccine efficacy of Marek's disease virus (MDV), ALV-J positive male breeders × female breeders of Three-yellow chickens and the ALV negative male breeder × the negative female breeders were used respectively for crossbreeding to produce eggs and the hatching offspring. The commercial CVI988/Rispens vaccine was used to vaccinate the crossbred offspring at 1-day-old. At 7-days-old, the birds were inoculated with the inactivated oil-emulsion vaccines (OEVs) AIV-H5 monovalent and NDV + AIV-H9 bivalent, respectively. Then the birds were challenged with a Chinese very virulent (vv) MDV field strain GXY2 at 14-day-old. The results showed that the viral load of the challenged GXY2 in the offspring from the ALV-J positive breeders was significantly higher than that from the ALV-negative breeders' (P < 0.05), and the mortality and tumor incidence of offspring from the ALV-J positive breeders were higher than those of the ALV-negative breeders. Also the offspring of the ALV-J positive breeders exhibited a significant negative effect on the development of the immune organs (P < 0.05) and lower antibody responses to the vaccinations with the commercial OEVs (P<0.05). The MD vaccine protective index in the offspring from the ALV-J positive breeders was lower than that from the ALV-negative breeders. The results of the study demonstrated that the vertical transmission of ALV from the ALV-J positive parents caused severe immunosuppression and significantly reduced the Marek's disease vaccine efficacy in Three-yellow chickens.

A novel recombinant avian leukosis virus isolated from gamecocks induced pathogenicity in Three-Yellow chickens: a potential infection source of avian leukosis virus to the commercial chickens.

One natural recombinant avian leukosis virus (ALV) strain GX14DJ3-18 was isolated from a native gamecock by DF-1 cell culture and identified with Polymerase Chain Reaction (PCR), immunofluorescence assay and the viral genome's nucleotide sequencing. This strain was revealed as a novel recombinant virus with nucleotide sequence similarities of 95.4% Long Terminal Repeated (LTR), 95.8% 5', UTR, 97.9% gag, and 92.9% 3'untranslated regions (UTR) in ALV-J. Also we found sequence similarities of 99.3% pol and 99.0% gp37 in ALV-E, and 89.9% gp85 in ALV-A. The simulated congenital infection with GX14DJ3-18 in Three-Yellow chickens exhibited a significant negative effect on the development of immune organs (P < 0.05). Also, lower antibody responses were found to vaccinations with the commercial vaccines of Newcastle disease virus and with subtypes H5 and H9 of avian influenza virus (P < 0.05). The incidence of tumor or tumor-like lesions in the challenged birds was 14.28% (5/35), while none were observed in the un-challenged control group (0/35). These results suggested that GX14DJ3-18 is a novel recombinant ALV that can induce pathogenicity in the commercial Three-Yellow chickens. We speculated that cross-provincial sales of gamecocks in which ALVs have not been eradicated thoroughly might be a potential route for the transmission of ALVs to commercial chickens.