Research

11 pages, 1757 KiB

Open AccessArticle

Simultaneous Detection of Three Subgroups of Avian Leukosis Virus Using the Nanoparticle-Assisted PCR Assay

by Miaoli Wu, Shuaiqi Hu, Yujun Zhu, Feng Cong and Shengwang Liu

Viruses 2024, 16(1), 15; https://doi.org/10.3390/v16010015 - 21 Dec 2023

Cited by 1 | Viewed by 734

Nanoparticle-assisted polymerase chain reaction (nanoPCR) is a novel method for the rapid detection of pathogens. A sensitive and specific multiple nanoPCR assay was developed for simultaneous detection of avian leucosis virus (ALV) subgroups A, B and J. In this study, three pairs of [...] Read more.

Nanoparticle-assisted polymerase chain reaction (nanoPCR) is a novel method for the rapid detection of pathogens. A sensitive and specific multiple nanoPCR assay was developed for simultaneous detection of avian leucosis virus (ALV) subgroups A, B and J. In this study, three pairs of primers were designed, based on the conserved region of the gp85 gene. An exploration of the optimal primer concentration and annealing temperature were carried out, for better performance of the nanoPCR assay. According to the results, the multiple nanoPCR assay amplified 336 pb, 625 bp and 167 bp fragments of ALV-A, -B and -J, respectively, and showed no cross-reactivity with irrelevant pathogens, suggesting the excellent specificity of the assay. The constructed standard DNA templates were used to estimate the limit of detection. As shown by the results, the detection limit of the nanoPCR assay was nearly 10 copies/μL. To further evaluate the detection ability of the assay, 186 clinical samples were detected using the nanoPCR assay, among which, 14 samples were confirmed as ALV positive; the results were further confirmed by sequencing. In conclusion, a highly specific and sensitive nanoPCR assay was successfully developed, which could be a useful tool for clinical diagnosis as well as for the discrimination of ALV-A, -B and -J. Full article

(This article belongs to the Special Issue Avian Viral Disease Associated with Immunosuppression and Immune-Evasion)

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23 pages, 4388 KiB

Open AccessArticle

Whole Genomic Constellation of Avian Reovirus Strains Isolated from Broilers with Arthritis in North Carolina, USA

by Islam Nour, Sonsiray Alvarez-Narvaez, Telvin L. Harrell, Steven J. Conrad and Sujit K. Mohanty

Viruses 2023, 15(11), 2191; https://doi.org/10.3390/v15112191 - 31 Oct 2023

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Abstract

Avian reovirus (ARV) is an emerging pathogen which causes significant economic challenges to the chicken and turkey industry in the USA and globally, yet the molecular characterization of most ARV strains is restricted to a single particular gene, the sigma C gene. The [...] Read more.

Avian reovirus (ARV) is an emerging pathogen which causes significant economic challenges to the chicken and turkey industry in the USA and globally, yet the molecular characterization of most ARV strains is restricted to a single particular gene, the sigma C gene. The genome of arthrogenic reovirus field isolates (R18-37308 and R18-38167), isolated from broiler chickens in North Carolina (NC), USA in 2018, was sequenced using long-read next-generation sequencing (NGS). The isolates were genotyped based on the amino acid sequence of sigma C (σC) followed by phylogenetic and amino acid analyses of the other 11 genomically encoded proteins for whole genomic constellation and genetic variation detection. The genomic length of the NC field strains was 23,494 bp, with 10 dsRNA segments ranging from 3959 bp (L1) to 1192 bp (S4), and the 5′ and 3′ untranslated regions (UTRs) of all the segments were found to be conserved. R18-37308 and R18-38167 were found to belong to genotype (G) VI based on the σC analysis and showed nucleotide and amino acid sequence identity ranging from 84.91–98.47% and 83.43–98.46%, respectively, with G VI strains. Phylogenetic analyses of individual genes of the NC strains did not define a single common ancestor among the available completely sequenced ARV strains. Nevertheless, most sequences supported the Chinese strain LY383 as a probable ancestor of these isolates. Moreover, amino acid analysis revealed multiple amino acid substitution events along the entirety of the genes, some of which were unique to each strain, which suggests significant divergence owing to the accumulation of point mutations. All genes from R18-37308 and R18-38167 were found to be clustered within genotypic clusters that included only ARVs of chicken origin, which negates the possibility of genetic pooling or host variation. Collectively, this study revealed sequence divergence between the NC field strains and reference ARV strains, including the currently used vaccine strains could help updating the vaccination regime through the inclusion of these highly divergent circulating indigenous field isolates. Full article

(This article belongs to the Special Issue Avian Viral Disease Associated with Immunosuppression and Immune-Evasion)

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26 pages, 6902 KiB

Open AccessArticle

Whole Genome Sequencing of Infectious Bursal Disease Viruses Isolated from a Californian Outbreak Unravels the Underlying Virulence Markers and Highlights Positive Selection Incidence

by Islam Nour, Julia R. Blakey, Sonsiray Alvarez-Narvaez and Sujit K. Mohanty

Viruses 2023, 15(10), 2044; https://doi.org/10.3390/v15102044 - 03 Oct 2023

Viewed by 1116

Abstract

Outbreaks of the immunosuppressive infectious bursal disease (IBD) are frequently reported worldwide, despite the vaccination regimes. A 2009 Californian IBD outbreak caused by rA and rB isolates was described as very virulent (vv) IBD virus (IBDV); however, molecular factors beyond this virulence were [...] Read more.

Outbreaks of the immunosuppressive infectious bursal disease (IBD) are frequently reported worldwide, despite the vaccination regimes. A 2009 Californian IBD outbreak caused by rA and rB isolates was described as very virulent (vv) IBD virus (IBDV); however, molecular factors beyond this virulence were not fully uncovered. Therefore, segments of both isolates were amplified, successfully cloned, whole genome sequenced by Next Generation Sequencing, genotyped, and the leading virulence factors were entirely investigated in terms of phylogenetic and amino acid analysis and protein modeling for positive selection orientation and interaction analysis. rA and rB isolates displayed the highest amino acid identity (97.84–100%) with Genotype 3 strains. Interestingly, rA and rB contained all virulence hallmarks of hypervariable (HVR), including 222A, 242I, 249Q, 256I, 284A, 286T, 294I, 299S, and 318G, as well as the serine-rich heptapeptide sequence. Moreover, we pinpointed the A3B2 genotype of rA and rB, predominant in non-reassortants, and we highlighted the absence of recombination events. Furthermore, gene-wise phylogenetic analysis showed the entire genes of rA and rB clustered with the vvIBDVs and emphasized their share in IBDV virulence. VP5 showed a virulence marker, MLSL (amino acid sequence). VP2 encountered three significant novel mutations apart from the HVR, including G163E in rA and Y173C and V178A in rB, all residing within interacting motifs. VP4 contained 168Y, 173N, 203S, and 239D characteristic for the vv phenotype. A235V mutation was detected at the dsRNA binding domain of VP3. In VP1, the TDN triplet and the mutation (V4I) were detected, characteristic of hypervirulence occurring at the N-terminus responsible for protein priming. Although selection analysis revealed seven sites, codon 222 was the only statistically significant selection site. The VP2 modeling of rA and rB highlighted great structure fitness, with 96.14% Ramachandran favored positioning including the 222A, i.e., not influencing the structure stability. The 222A was found to be non-interface surface residue, associated with no interaction with the attachment-mediated ligand motif. Our findings provide pivotal insights into the evolution and underlying virulence factors and will assist in the development of control strategies via sequence-based continuous monitoring for the early detection of novel vv strains. Full article

(This article belongs to the Special Issue Avian Viral Disease Associated with Immunosuppression and Immune-Evasion)

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16 pages, 1749 KiB

Open AccessArticle

Emerging Hypervirulent Marek’s Disease Virus Variants Significantly Overcome Protection Conferred by Commercial Vaccines

by Jin-Ling Liu, Man Teng, Lu-Ping Zheng, Feng-Xia Zhu, Shu-Xue Ma, Lin-Yan Li, Zhi-Hui Zhang, Shu-Jun Chai, Yongxiu Yao and Jun Luo

Viruses 2023, 15(7), 1434; https://doi.org/10.3390/v15071434 - 25 Jun 2023

Cited by 3 | Viewed by 1558

Abstract

As one of the most important avian immunosuppressive and neoplastic diseases, Marek’s disease (MD), caused by oncogenic Marek’s disease virus (MDV), has caused huge economic losses worldwide over the past five decades. In recent years, MD outbreaks have occurred frequently in MD-vaccinated chicken [...] Read more.

As one of the most important avian immunosuppressive and neoplastic diseases, Marek’s disease (MD), caused by oncogenic Marek’s disease virus (MDV), has caused huge economic losses worldwide over the past five decades. In recent years, MD outbreaks have occurred frequently in MD-vaccinated chicken flocks, but the key pathogenic determinants and influencing factors remain unclear. Herein, we analyzed the pathogenicity of seven newly isolated MDV strains from tumor-bearing chickens in China and found that all of them were pathogenic to chicken hosts, among which four MDV isolates, SDCW01, HNXZ05, HNSQ05 and HNSQ01, were considered to be hypervirulent MDV (HV-MDV) strains. At 73 days of the virus infection experiment, the cumulative incidences of MD were 100%, 93.3%, 90% and 100%, with mortalities of 83.3%, 73.3%, 60% and 86.7%, respectively, for the four viruses. The gross occurrences of tumors were 50%, 33.3%, 30% and 63.3%, respectively, accompanied by significant hepatosplenomegaly and serious atrophy of the immune organs. Furthermore, the immune protection effects of four commercial MD vaccines against SDCW01, CVI988, HVT, CVI988+HVT, and 814 were explored. Unexpectedly, during the 67 days of post-virus challenge, the protection indices (PIs) of these four MD vaccines were only 46.2%, 38.5%, 50%, and 28%, respectively, and the birds that received the monovalent CVI988 or HVT still developed tumors with cumulative incidences of 7.7% and 11.5%, respectively. To our knowledge, this is the first demonstration of the simultaneous comparison of the immune protection efficacy of multiple commercial MD vaccines with different vaccine strains. Our study revealed that the HV-MDV variants circulating in China could significantly break through the immune protection of the classical MD vaccines currently widely used. For future work, there is an urgent need to develop novel, more effective MD vaccines for tackling the new challenge of emerging HV-MDV strains or variants for the sustainable control of MD. Full article

(This article belongs to the Special Issue Avian Viral Disease Associated with Immunosuppression and Immune-Evasion)

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14 pages, 8097 KiB

Open AccessArticle

Comparative Pathogenicity of Three Strains of Infectious Bursal Disease Virus Closely Related to Poultry Industry

by Kailin Li, Xinxin Niu, Nan Jiang, Wenying Zhang, Guodong Wang, Kai Li, Mengmeng Huang, Yulong Gao, Xiaole Qi and Xiaomei Wang

Viruses 2023, 15(6), 1257; https://doi.org/10.3390/v15061257 - 26 May 2023

Cited by 2 | Viewed by 1755

Abstract

Infectious bursal disease (IBD) is an acute, highly contagious, immunosuppressive, and fatal infectious disease of young chickens caused by infectious bursal disease virus (IBDV). Since 2017, a new trend has been discovered in the IBDV epidemic, with very virulent IBDV (vvIBDV) and novel [...] Read more.

Infectious bursal disease (IBD) is an acute, highly contagious, immunosuppressive, and fatal infectious disease of young chickens caused by infectious bursal disease virus (IBDV). Since 2017, a new trend has been discovered in the IBDV epidemic, with very virulent IBDV (vvIBDV) and novel variant IBDV (nVarIBDV) becoming the two current dominant strains in East Asia including China. In this study, we compared the biological characteristics of the vvIBDV (HLJ0504 strain), nVarIBDV (SHG19 strain), and attenuated IBDV (attIBDV, Gt strain) using specific-pathogen-free (SPF) chicken infection model. The results showed that vvIBDV distributed in multiple tissues, replicated the fastest in lymphoid organs such as bursa of Fabricius, induced significant viremia and virus excretion, and is the most pathogenic virus with a mortality of more than 80%. The nVarIBDV had a weaker replication capability and did not kill the chickens but caused severe damage to the central immune organ bursa of Fabricius and B lymphocytes and induced significant viremia and virus excretion. The attIBDV strain was found not to be pathogenic. Further studies preliminarily suggested that the expression level of inflammatory factors triggered by HLJ0504 was the highest, followed by the SHG19 group. This study is the first to systematically compare the pathogenic characteristics of three IBDVs closely related to poultry industry from the perspectives of clinical signs, micro-pathology, virus replication, and distribution. It is of great importance to obtain an extensive knowledge of epidemiology, pathogenicity, and comprehensive prevention, and control of various IBDV strains. Full article

(This article belongs to the Special Issue Avian Viral Disease Associated with Immunosuppression and Immune-Evasion)

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16 pages, 1552 KiB

Open AccessArticle

Differences in Pathogenicity and Vaccine Resistance Discovered between Two Epidemic Strains of Marek’s Disease Virus in China

by Zheng-Hao Yu, Yan-Ping Zhang, Xing-Ge Lan, Ya-Nan Wang, Rong-Rong Guo, Kai Li, Li Gao, Xiao-Le Qi, Hong-Yu Cui, Xiao-Mei Wang, Yu-Long Gao and Chang-Jun Liu

Viruses 2023, 15(4), 945; https://doi.org/10.3390/v15040945 - 11 Apr 2023

Cited by 1 | Viewed by 1584

Abstract

Despite highly effective vaccines, Marek’s disease (MD) causes great economic loss to the poultry industry annually, largely due to the continuous emergence of new MD virus (MDV) strains. To explore the pathogenic characteristics of newly emerged MDV strains, we selected two strains (AH/1807 [...] Read more.

Despite highly effective vaccines, Marek’s disease (MD) causes great economic loss to the poultry industry annually, largely due to the continuous emergence of new MD virus (MDV) strains. To explore the pathogenic characteristics of newly emerged MDV strains, we selected two strains (AH/1807 and DH/18) with clinically different pathotypes. We studied each strain’s infection process and pathogenicity and observed differences in immunosuppression and vaccine resistance. Specific pathogen-free chickens, unvaccinated or vaccinated with CVI988, were challenged with AH/1807 or DH/18. Both infections induced MD damage; however, differences were observed in terms of mortality (AH/1807: 77.8%, DH/18: 50%) and tumor rates (AH/1807: 50%, DH/18: 33.3%). The immune protection indices of the vaccine also differed (AH/1807: 94.1, DH/18: 61.1). Additionally, while both strains caused interferon-β and interferon-γ expression to decline, DH/18 infection caused stronger immunosuppression than AH/1807. This inhibition persisted even after vaccination, leading to increased replication of DH/18 that ultimately broke through vaccine immune protection. These results indicate that both strains have different characteristics, and that strains such as DH/18, which cause weaker pathogenic damage but can break through vaccine immune protection, require further attention. Our findings increase the understanding of the differences between epidemic strains and factors underlying MD vaccination failure in China. Full article

(This article belongs to the Special Issue Avian Viral Disease Associated with Immunosuppression and Immune-Evasion)

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13 pages, 3525 KiB

Open AccessArticle

J Subgroup Avian Leukosis Virus Strain Promotes Cell Proliferation by Negatively Regulating 14-3-3σ Expressions in Chicken Fibroblast Cells

by Moyu Wang, Hongmei Li, Xiyu Sun, Jianhua Qiu, Changhua Jing, Huiyue Jia, Yujie Guo and Huijun Guo

Viruses 2023, 15(2), 404; https://doi.org/10.3390/v15020404 - 31 Jan 2023

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Abstract

This study focuses on clarifying the regulation of chicken 14-3-3σ protein on the fibrous histiocyte proliferation caused by ALV-J-SD1005 strain infection. DF-1 cells were inoculated with 10² TCID₅₀ of ALV-J-SD1005 strain; the cell proliferation viability was dramatically increased and 14-3-3σ expressions [...] Read more.

This study focuses on clarifying the regulation of chicken 14-3-3σ protein on the fibrous histiocyte proliferation caused by ALV-J-SD1005 strain infection. DF-1 cells were inoculated with 10² TCID₅₀ of ALV-J-SD1005 strain; the cell proliferation viability was dramatically increased and 14-3-3σ expressions were dramatically decreased within 48 h after inoculation. Chicken 14-3-3σ over-expression could significantly decrease the cell proliferation and the ratio of S-phase cells, but increase the ratio of G2/M-phase cells in ALV-J-infected DF-1 cells; by contrast, chicken 14-3-3σ knockdown expression could cause the opposite effects. Additionally, chicken 14-3-3σ over-expression could also dramatically down-regulate the expressions of CDK2/CDC2, but up-regulate p53 expressions in the DF-1 cells; in contrast, the knockdown expression could significantly increase the expressions of CDK2/CDC2 and decrease p53 expressions. It can be concluded that chicken 14-3-3σ can inhibit cell proliferation and cell cycle by regulating CDK2/CDC2/p53 expressions in ALV-J-infected DF1 cells. ALV-J-SD1005 strain can promote cell proliferation by reducing 14-3-3σ expressions. This study helps to clarify the forming mechanism of acute fibrosarcoma induced by ALV-J infection. Full article

(This article belongs to the Special Issue Avian Viral Disease Associated with Immunosuppression and Immune-Evasion)

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15 pages, 2356 KiB

Open AccessArticle

Differential Replication and Cytokine Response between Vaccine and Very Virulent Marek’s Disease Viruses in Spleens and Bursas during Latency and Reactivation

by Bo Jiang, Jing Wang, Mengyao Cao, Huan Jin, Wenxiao Liu, Jing Cheng, Linyi Zhou, Jian Xu and Yongqing Li

Viruses 2023, 15(1), 6; https://doi.org/10.3390/v15010006 - 20 Dec 2022

Cited by 1 | Viewed by 1158

Abstract

Marek’s disease virus (MDV) infection results in Marek’s disease (MD) in chickens, a lymphoproliferative and oncogenic deadly disease, leading to severe economic losses. The spleen and bursa are the most important lymphoid and major target organs for MDV replication. The immune response elicited [...] Read more.

Marek’s disease virus (MDV) infection results in Marek’s disease (MD) in chickens, a lymphoproliferative and oncogenic deadly disease, leading to severe economic losses. The spleen and bursa are the most important lymphoid and major target organs for MDV replication. The immune response elicited by MDV replication in the spleen and bursa is critical for the formation of latent MDV infection and reactivation. However, the mechanism of the host immune response induced by MDV in these key lymphoid organs during the latent and reactivation infection phases is not well understood. In the study, we focused on the replication dynamics of a vaccine MDV strain MDV/CVI988 and a very virulent MDV strain MDV/RB1B in the spleen and bursa in the latent and reactivation infection phases (7–28 days post-inoculation [dpi]), as well as the expression of some previously characterized immune-related molecules. The results showed that the replication ability of MDV/RB1B was significantly stronger than that of MDV/CVI988 within 28 days post-infection, and the replication levels of both MDV strains in the spleen were significantly higher than those in the bursa. During the latent and reactivation phase of MDV infection (7–28 dpi), the transcriptional upregulation of chicken IL-1β, IL6, IL-8L1 IFN-γ and PML in the spleen and bursa induced by MDV/RB1B infection was overall stronger than that of MDV/CVI988. However, compared to MDV/RB1Binfection, MDV/CVI988 infection resulted in a more effective transcriptional activation of CCL4 in the latent infection phase (7–14 dpi), which may be a characteristic distinguishing MDV vaccine strain from the very virulent strain. Full article

(This article belongs to the Special Issue Avian Viral Disease Associated with Immunosuppression and Immune-Evasion)

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