Newcastle disease virus activates diverse signaling pathways via Src to facilitate virus entry into host macrophages.

As an intrinsic cellular mechanism responsible for the internalization of extracellular ligands and membrane components, caveolae-mediated endocytosis (CavME) is also exploited by certain pathogens for endocytic entry [e.g., Newcastle disease virus (NDV) of paramyxovirus]. However, the molecular mechanisms of NDV-induced CavME remain poorly understood. Herein, we demonstrate that sialic acid-containing gangliosides, rather than glycoproteins, were utilized by NDV as receptors to initiate the endocytic entry of NDV into HD11 cells. The binding of NDV to gangliosides induced the activation of a non-receptor tyrosine kinase, Src, leading to the phosphorylation of caveolin-1 (Cav1) and dynamin-2 (Dyn2), which contributed to the endocytic entry of NDV. Moreover, an inoculation of cells with NDV-induced actin cytoskeletal rearrangement through Src to facilitate NDV entry via endocytosis and direct fusion with the plasma membrane. Subsequently, unique members of the Rho GTPases family, RhoA and Cdc42, were activated by NDV in a Src-dependent manner. Further analyses revealed that RhoA and Cdc42 regulated the activities of specific effectors, cofilin and myosin regulatory light chain 2, responsible for actin cytoskeleton rearrangement, through diverse intracellular signaling cascades. Taken together, our results suggest that an inoculation of NDV-induced Src-mediated cellular activation by binding to ganglioside receptors. This process orchestrated NDV endocytic entry by modulating the activities of caveolae-associated Cav1 and Dyn2, as well as specific Rho GTPases and downstream effectors. IMPORTANCE: In general, it is known that the paramyxovirus gains access to host cells through direct penetration at the plasma membrane; however, emerging evidence suggests more complex entry mechanisms for paramyxoviruses. The endocytic entry of Newcastle disease virus (NDV), a representative member of the paramyxovirus family, into multiple types of cells has been recently reported. Herein, we demonstrate the binding of NDV to induce ganglioside-activated Src signaling, which is responsible for the endocytic entry of NDV through caveolae-mediated endocytosis. This process involved Src-dependent activation of the caveolae-associated Cav1 and Dyn2, as well as specific Rho GTPase and downstream effectors, thereby orchestrating the endocytic entry process of NDV. Our findings uncover a novel molecular mechanism of endocytic entry of NDV into host cells and provide novel insight into paramyxovirus mechanisms of entry.

Development of a Recombinant Enzyme-Linked Immunosorbent Assay for the Detection of Antibodies Against Infectious Bronchitis Virus.

Infectious bronchitis virus (IBV), a gammacoronavirus within the Coronaviridae family, is an economically important etiological disease agent in chickens. Both early diagnosis and determination of the immune status of chickens are important for controlling IBV outbreaks in chicken flocks. The N protein is the most abundantly expressed virus-derived protein during IBV infection and can induce a strong immune response by producing antibodies during early infection or immunization. In this study, we found that the amino acid sequences of the N protein between CK/CH/LJL/04I and the other 22 IBVs were conserved, especially in the 1-160 amino acid region. Based on the sequence similarities, the three recombinant proteins, rN160 (amino acid positions 1-160), rN266 (144-409), and rN409 (1-409), were expressed using the Escherichia coli system and subsequently purified. The results demonstrated that the antigenicity and reactivity of rN160 were better than those of rN266 and rN409. As a result, an indirect enzyme-linked immunosorbent assay (ELISA) (rN160 ELISA) was developed to detect the IBV antibody based on the rN160 protein. Using 1,500 clinical field serum samples, the relative sensitivity, specificity, and accuracy of the rN160 ELISA were 98.97%, 92.34%, and 97.93%, respectively, compared to those of a commercial ELISA kit (IDEXX), indicating a strong positive correlation between the two methods. Taken together, these results reveal that the rN160 ELISA is a rapid, simple, and sensitive method for detecting group-specific IBV antibodies for epidemiological investigation and antibody-level monitoring.

Characterization of the Effects of Host p53 and Fos on Gallid Alpha Herpesvirus 1 Replication.

Treatment options for herpesvirus infections that target the interactions between the virus and the host have been identified as promising. Our previous studies have shown that transcription factors p53 and Fos are essential host determinants of gallid alpha herpesvirus 1 (ILTV) infection. The impact of p53 and Fos on ILTV replication has 'not been fully understood yet. Using the sole ILTV-permissive chicken cell line LMH as a model, we examined the effects of hosts p53 and Fos on all phases of ILTV replication, including viral gene transcription, viral genome replication, and infectious virion generation. We achieved this by manipulating the expression of p53 and Fos in LMH cells. Our results demonstrate that the overexpression of either p53 or Fos can promote viral gene transcription at all stages of the temporal cascade of ILTV gene expression, viral genome replication, and infectious virion production, as assessed through absolute quantitative real-time PCR, ILTV-specific RT-qPCR assays, and TCID50 assays. These findings are consistent with our previous analyses of the effects of Fos and p53 knockdowns on virus production and also suggest that both p53 and Fos may be dispensable for ILTV replication. Based on the synergistic effect of regulating ILTV, we further found that there is an interaction between p53 and Fos. Interestingly, we found that p53 also has targeted sites upstream of ICP4, and these sites are very close to the Fos sites. In conclusion, our research offers an in-depth understanding of how hosts p53 and Fos affect ILTV replication. Understanding the processes by which p53 and Fos regulate ILTV infection will be improved by this knowledge, potentially paving the way for the development of novel therapeutics targeting virus-host interactions as a means of treating herpesvirus infections.

Image Quality Assessment for Realistic Zoom Photos.

New CMOS imaging sensor (CIS) techniques in smartphones have helped user-generated content dominate our lives over traditional DSLRs. However, tiny sensor sizes and fixed focal lengths also lead to more grainy details, especially for zoom photos. Moreover, multi-frame stacking and post-sharpening algorithms would produce zigzag textures and over-sharpened appearances, for which traditional image-quality metrics may over-estimate. To solve this problem, a real-world zoom photo database is first constructed in this paper, which includes 900 tele-photos from 20 different mobile sensors and ISPs. Then we propose a novel no-reference zoom quality metric which incorporates the traditional estimation of sharpness and the concept of image naturalness. More specifically, for the measurement of image sharpness, we are the first to combine the total energy of the predicted gradient image with the entropy of the residual term under the framework of free-energy theory. To further compensate for the influence of over-sharpening effect and other artifacts, a set of model parameters of mean subtracted contrast normalized (MSCN) coefficients are utilized as the natural statistics representatives. Finally, these two measures are combined linearly. Experimental results on the zoom photo database demonstrate that our quality metric can achieve SROCC and PLCC over 0.91, while the performance of single sharpness or naturalness index is around 0.85. Moreover, compared with the best tested general-purpose and sharpness models, our zoom metric outperforms them by 0.072 and 0.064 in SROCC, respectively.

Pathogenicity and innate immune responses induced by fowl adenovirus serotype 8b in specific pathogen-free chicken.

Fowl adenovirus serotype 8b (FAdV-8b), as causative agent of inclusion body hepatitis (IBH), poses a great threat to the poultry industry. Considering the importance of innate immune response in host against viral infections, we investigated pathogenicity of a FAdV-8b strain HLJ/151129 in 1-mo-old specific pathogen-free (SPF) chickens and immune responses of host to FAdV-8b infection in this study. The results demonstrated that no obvious clinical signs were observed in infected birds. Neither mobility nor mortality was observed in both FAdV-8b infected and control chickens, as well. However, hepatic necrosis and a small amount of inflammatory cell infiltration were observed by pathological analysis. Viral load was detected in bursa of Fabricius, cecal tonsils, liver, heart, spleen, Harderian glands, and thymus. Virus shedding and viremia generated as early as 3 days postinfection (dpi) (9/10) and reached the peak at 7 dpi (10/10). In addition, the infected birds had developed FAdV-specific antibodies at 7 dpi, and the antibody titers reached the peak at 14 dpi. Furthermore, the results demonstrated that the mRNA expression levels of most of toll-like receptors (TLRs), most of avian ß-defensins (AvBDs), and cytokines [interleukin (IL)-2, IL-6, and interferon (IFN)-γ], were significantly upregulated in most tissues at early phases of FAdV-8b infection, especially in liver and spleen. In contrast, FAdV-8b infection results in downregulation of TLR4, TLR5, and TLR21 expressions in some tissues of infected chickens. In addition, FAdV-8b infection upregulated myeloid differentiation factor 88 (MyD88), nuclear factor-kappa B (NF-κB) p65, and TIR-domain-containing adapter inducing interferon-ß (TRIF) expression in some tissues, while decreased NF-κBp65 and TRIF in spleen at both 72 hpi and 21 dpi. Taken together, these results confirmed that FAdV-8b could replicate in all investigated tissues of infected birds, and then, result in production of FAdV-specific antibody titers. Meanwhile, the FAdV-8b infection induces strong innate immune responses at early stage in chickens, which may associate with the viral pathogenesis.

Effect of oral vitamin A supplementation on host immune response to infectious bronchitis virus infection in specific pathogen-free chicken.

Vitamin A is a fat-soluble vitamin that is a crucial mediator of the immune system. In this study, we evaluated the effect of oral vitamin A supplementation on host immune responses to infectious bronchitis virus (IBV) infection in chickens. Forty 1-day-old specific pathogen-free (SPF) chickens were fed a basal diet and randomly divided into 2 groups (n = 20 birds per group). Chickens in the experimental group were treated orally with vitamin A (dissolved in 0.1 mL soybean oil, at a dose of 8,000 IU per kg diet) daily. Birds in the control group were orally administered 0.1 mL soybean oil without vitamin A until 21 d of age. On d 21 after birth, all chickens were infected with 0.1 mL of 106.5 50% median embryo infectious dose of a pathogenic IBV strain (CK/CH/LDL/091022) by intraocular and intranasal routes. The results demonstrated that oral vitamin A supplementation did not affect the clinical course of disease and growth performance of SPF chickens. However, vitamin A supplementation increased the IBV-specific IgG serum levels and decreased the viral load in some tissues of IBV-infected chickens. In addition, the results demonstrated that vitamin A supplementation decreased the expression levels of most immune-related molecules in some tissues of IBV-infected chickens. Vitamin A supplementation decreased the mRNA expression levels of some avian ß-defensins (AvBD2, 3, 6, 7, 11, and 13) and increased the expression levels of AvBD9 and AvBD12 in some tissues of IBV-infected chickens. Similarly, vitamin A supplementation decreased the mRNA expression levels of some cytokines (interferon-γ, interleukin-1ß [IL-1ß], and IL-6) and increased the mRNA expression levels of IL-2 in some tissues of IBV-infected chickens. Furthermore, vitamin A supplementation decreased the mRNA expression levels of myeloid differentiation primary response protein 88, nuclear factor-κB p65, toll-like receptor 3, toll-like receptor 7, and CD4. In summary, the present study suggests that vitamin A supplementation enhances the immune function of SPF chickens against IBV infection by inhibiting viral replication, increasing the IBV-specific antibody titer, and suppressing the excessive inflammatory responses to IBV infection.

Surveillance of Class I Newcastle Disease Virus at Live Bird Markets in China and Identification of Variants with Increased Virulence and Replication Capacity.

In this study, 232 class I Newcastle disease viruses (NDVs) were identified from multiple bird species at nationwide live bird markets (LBMs) from 2017 to 2019 in China. Phylogenetic analysis indicated that all 232 isolates were clustered into genotype 1.1.2 of class I on the basis of the fusion (F) gene sequences, which were distinct from the genotypes identified in other countries. Most of the isolates (212/232) were shown to have the typical F gene molecular characteristics of class I NDVs, while a few (20/232) contained mutations at the site of the conventional start codon of the F gene, which resulted in open reading frames (ORFs) altered in length. The isolates with ACG, CTA, and ATA mutations showed different levels of increased virulence and replication capacity, suggesting that these viruses may be transitional types during the evolution of class I NDVs from avirulent to virulent. Further evaluation of biological characteristics with recombinant viruses obtained by reverse genetics demonstrated that the ATG located at genomic positions 4523 to 4525 was the authentic start codon in the F gene of class I NDV, and the specific ATA mutations which contributed to the expression of F protein on the surface of infected cells were the key determinants of increased replication capacity and virulence. Interestingly, the mutation at the corresponding site of genotype II LaSota of class II had no effects on the virulence and replication capacity in chickens. Our results suggest that the alteration of virulence and replication capacity caused by specific mutations in the F gene could be a specific characteristic of class I NDVs and indicate the possibility of the emergence of virulent NDVs due to the persistent circulation of class I NDVs. IMPORTANCE The available information on the distribution, genetic diversity, evolution, and biological characteristics of class I Newcastle disease viruses (NDVs) in domestic poultry is currently very limited. Here, identification of class I NDVs at nationwide live bird markets (LBMs) in China was performed and representative isolates were characterized. A widespread distribution of genotype 1.1.2 of class I NDVs was found in multiple bird species at LBMs in China. Though most isolates demonstrated typical molecular characteristics of class I NDVs, a few that contained specific mutations at the site of the conventional start codon of the fusion gene with increased virulence and replication capacity were identified for the first time. Our findings indicate that the virulence of class I NDVs could have evolved, and the widespread transmission and circulation of class I NDVs may represent a potential threat for disease outbreaks in poultry.

Replication and vaccine protection of multiple infectious bronchitis virus strains in pheasants (Phasianus colchicus).

This study demonstrates that infectious bronchitis virus (IBV) strain M41, which is pathogenic for chickens, is nonpathogenic for pheasants. However, M41 replicated in the respiratory tracts of most inoculated pheasants and the virus was shed from their respiratory tracts in the early stages of infection (4 and 8 dpc). Similarly, the attenuated IBV H120 vaccine strain also replicated and the virus was shed from their respiratory tracts of most inoculated pheasants, whereas the pheasant coronavirus (PhCoV) I0623/17 replicated in the respiratory tracts of all challenged pheasants, which then shed virus for a long period of time. Strain M41 also replicated in selected tissues of the inoculated pheasants, including the lung, kidney, proventriculus, and cecal tonsil, although the viral titers were very low. Therefore, it was important to establish whether the H120 vaccine, which has a limited replication capacity in pheasants, induces a protective immune response to both "homologous" M41 and "heterologous" I0623/17 challenge. Vaccination with H120 induced humoral responses, and the replication of M41 was reduced or restricted in the tissues of the H120-vaccinated pheasants compared with its replication in unvaccinated birds. This implies that partial protection was conferred on pheasants by vaccination with the H120 vaccine. Prolonged viral replication and a large number of birds shedding virus into the respiratory tract were also observed in the unvaccinated pheasants after inoculation with M41. However, only limited protection against challenge with PhCoV I0623/17 was conferred on pheasants vaccinated with H120, largely because the replication of H120 in pheasants was limited, thus, limiting the immune responses induced by it. The low amino acid identity of the S1 subunit of the S proteins of H120 and I0623/17 might also account, at least in part, for the poor cross-protective immunity induced by H120. These results suggest that further work is required to rationally design vaccines that confer effective protection against PhCoV infection in commercial pheasant stocks.

Newcastle Disease Virus Entry into Chicken Macrophages via a pH-Dependent, Dynamin and Caveola-Mediated Endocytic Pathway That Requires Rab5.

The cellular entry pathways and the mechanisms of Newcastle disease virus (NDV) entry into cells are poorly characterized. In this study, we demonstrated that chicken interferon-induced transmembrane protein 1 (chIFITM1), which is located in the early endosomes, could limit the replication of NDV in chicken macrophage cell line HD11, suggesting the endocytic entry of NDV into chicken macrophages. Then, we presented a systematic study about the entry mechanism of NDV into chicken macrophages. First, we demonstrated that a low-pH condition and dynamin were required during NDV entry. However, NDV entry into chicken macrophages was independent of clathrin-mediated endocytosis. We also found that NDV entry was dependent on membrane cholesterol. The NDV entry and replication were significantly reduced by nystatin and phorbol 12-myristate 13-acetate treatment, overexpression of dominant-negative (DN) caveolin-1, or knockdown of caveolin-1, suggesting that NDV entry depends on caveola-mediated endocytosis. However, macropinocytosis did not play a role in NDV entry into chicken macrophages. In addition, we found that Rab5, rather than Rab7, was involved in the entry and traffic of NDV. The colocalization of NDV with Rab5 and early endosome suggested that NDV virion was transported to early endosomes in a Rab5-dependent manner after internalization. Of particular note, the caveola-mediated endocytosis was also utilized by NDV to enter primary chicken macrophages. Moreover, NDV entered different cell types using different pathways. Collectively, our findings demonstrate for the first time that NDV virion enters chicken macrophages via a pH-dependent, dynamin and caveola-mediated endocytosis pathway and that Rab5 is involved in the traffic and location of NDV. IMPORTANCE Although the pathogenesis of Newcastle disease virus (NDV) has been extensively studied, the detailed mechanism of NDV entry into host cells is largely unknown. Macrophages are the first-line defenders of host defense against infection of pathogens. Chicken macrophages are considered one of the main types of target cells during NDV infection. Here, we comprehensively investigated the entry mechanism of NDV in chicken macrophages. This is the first report to demonstrate that NDV enters chicken macrophages via a pH-dependent, dynamin and caveola-mediated endocytosis pathway that requires Rab5. The result is important for our understanding of the entry of NDV in chicken macrophages, which will further advance the knowledge of NDV pathogenesis and provide useful clues for the development of novel preventive or therapeutic strategies against NDV infection. In addition, this information will contribute to our further understanding of pathogenesis with regard to other members of the Avulavirus genus in the Paramyxoviridae family.

Innate immune responses of domestic pigeons to the infection of pigeon paramyxovirus type 1 virus.

Pigeon paramyxovirus type 1 (PPMV-1) is a globally distributed, virulent member of the avian paramyxovirus type-1. The PPMV-1-associated disease poses a great threat to the pigeon industry. The innate immune response is crucial for antiviral infections and revealing the pathogenic mechanisms of PPMV-1. In this study, we evaluated the pathogenicity of a PPMV-1 strain LHLJ/110822 in one-month-old domestic pigeons, as well as the host immune responses in PPMV-1-infected pigeons. We observed typically clinical sign in infected pigeons by 3 dpi. The morbidity rate and the mortality in pigeons inoculated with the PPMV-1 strain were up to 100% and 30%, respectively. The virus could replicate in all of the examined tissues, namely trachea, lung, liver, spleen, and bursa of Fabricius. In addition, the infected pigeons had developed anti-PPMV-1 antibodies as early as 8 dpi; and the antibody level increased over the time in this study. The expression level of toll-like receptor (TLR) 2, TLR3 TLR15, IFN-γ, and IL-6 were significantly upregulated by the PPMV-1 infection in some tissues of pigeons. By contrast, PPMV-1 infection results in downregulation of IL-18 expression in most of investigated tissues except for bursa of Fabricius in this study. The current results confirmed that this virus could replicate in pigeons and induce host immune responses, then leading to produce serum antibody titers. Meanwhile, the PPMV-1 infection induces strong innate immune responses and intense inflammatory responses at early stage in pigeon which may associate with the viral pathogenesis.

Genetic and antigenic heterogeneity of GI-1/Massachusetts lineage infectious bronchitis virus variants recently isolated in China.

Four GI-1/Massachusetts-type (GI-1/Mass-type) infectious bronchitis virus (IBV) strains were isolated and the complete genomes of these isolates, coupled with the Mass-type live-attenuated vaccine H120 and the Mass-type pathogenic M41 strains, were sequenced in the present study. Our results show that isolates LJL/140820 and I0306/17 may be derived from the Ma5 (another Mass-type live-attenuated vaccine strain) and H120 vaccine strains, respectively. The I1124/16 strain was found to be a M41 variant that likely resulted from nucleotide accumulated mutations in the genome. Consistently, the results of the virus neutralization test showed that isolate I1124/16 was antigenically related but slight different from the M41. Our results from the protection experiments pointed out that chickens immunized with H120 failed to eliminate viral shedding after infection with the isolate I1124/16, which was different from that of M41; this result was consistent to the field observation and further implicated that the variant IBV isolate I1124/16 was antigenic different from the M41 strain. Furthermore, the I1124/16 was found to have comparable but slightly lower pathogenicity with the M41 strain. More studies based on the reverse genetic techniques are needed to elucidate the amino acids in the S1 subunit of spike protein contributing to the altered antigenicity of the isolate I1124/16. In addition, an IBV isolate, LJL/130609, was found to be originated from recombination events between the I1124/16- and Connecticut-like strains. Our results from the virus neutralization test also showed that isolates LJL/130609 and I1124/16 were antigenic closely related. Hence, there are at least 3 different genetic evolution patterns for the circulation of the GI-1/Mass-type IBV field strains in China. The differences of vaccines used, the field conditions and genetic pressures between different flocks, likely account for the emergence, evolution patterns, and characteristics of the Mass-type IBV strains.

Construction and immune protection evaluation of recombinant virus expressing Newcastle disease virus F protein by the largest intergenic region of fowlpox virus NX10.

Fowlpox virus (FPV) is used as a vaccine vector to prevent diseases in poultry and mammals. The insertion site is considered as one of the main factors influencing foreign gene expression. Therefore, the identification of insertion sites that can stably and efficiently express foreign genes is crucial for the construction of recombinant vaccines. In this study, we found that the insertion of foreign genes into ORF054 and the ORF161/ORF162 intergenic region of the FPV genome did not affect replication, and that the foreign genes inserted into the intergenic region were more efficiently expressed than when they were inserted into a gene. Based on these results, the recombinant virus rFPVNX10-NDV F-E was constructed and immune protection against virulent FPV and Newcastle disease virus (NDV) was evaluated. Tests for anti-FPV antibodies in the vaccinated chickens were positive within 14 days post-vaccination. After challenge with FPV102, no clinical signs of FP were observed in vaccinated chickens, as compared to that in the control group (unvaccinated), which showed 100% morbidity. Low levels of NDV-specific neutralizing antibodies were detected in vaccinated chickens before challenge. After challenge with NDV ck/CH/LHLJ/01/06, all control chickens died within 4 days post-challenge, whereas 5/15 vaccinated chickens died between 4 and 12 days post-challenge. Vaccination provided an immune protection rate of 66.7%, whereas the control group showed 100% mortality. These results indicate that the ORF161/ORF162 intergenic region of FPVNX10 can be used as a recombination site for foreign gene expression in vivo and in vitro.

Glycoprotein-C-gene-deleted recombinant infectious laryngotracheitis virus expressing a genotype VII Newcastle disease virus fusion protein protects against virulent infectious laryngotracheitis virus and Newcastle disease virus.

To develop an alternative vectored vaccine against both Newcastle disease virus (NDV) and infectious laryngotracheitis virus (ILTV), the glycoprotein C (gC) gene was first deleted from an avirulent ILTV. Based on this gC-deleted ILTV mutant, a recombinant ILTV expressing the fusion protein (F) of a genotype VII NDV (designated ILTV-ΔgC-F) was then constructed. Expression of the NDV F protein in ILTV-ΔgC-F-infected LMH cells was examined with an immunofluorescence assay and western blotting. The F gene was stably maintained in the genome of ILTV-ΔgC-F and the F protein was stably expressed. Compared with the parental virus, ILTV-ΔgC-F demonstrated an increased penetration capacity in vitro, and an increased replication rate in vitro and in vivo. Both the parental virus and ILTV-ΔgC-F were avirulent in chickens. Vaccination of specific-pathogen-free chickens with ILTV-ΔgC-F induced ILTV-specific antibodies, detected with an enzyme-linked immunosorbent assay (ELISA), and provided complete clinical protection against virulent ILTV, although viral shedding and replication were detected in the respiratory tract in the early stage of infection in a very small number of birds. Vaccination with ILTV-ΔgC-F also provided significant protection against challenge with a virulent genotype VII NDV, although the level of NDV-specific antibodies detected with an ELISA was low. Notably, the numbers of birds that were positive for the virulent genotype VII NDV and the replication of the challenge virus NDV in selected target tissues were significantly lower in the ILTV-ΔgC-F-vaccinated chickens than in the control birds. Our results indicate that ILTV-ΔgC-F has potential utility as a bivalent candidate vaccine against both infectious laryngotracheitis and Newcastle disease.

Protection of chicks from Newcastle disease by combined vaccination with a plasmid DNA and the pre-fusion protein of the virulent genotype VII of Newcastle disease virus.

In this study, four codon optimized plasmids (designated as pCAG-optiF-1, 2, -3, and -4) containing modified F genes from the epidemic and virulent NDV genotype VII strain isolated in China that is expected to express the pre-fusion conformation of the F protein were constructed. The expression of these F variants in chicken-derived cells was detected by an indirect immunofluorescence assay and western blot analysis. Two soluble F variants (roptiF-1 and 2) potentially with the pre-fusion conformation were expressed and purified from suspended cells. Vaccination with each of the plasmids as a DNA vaccine conferred partial clinical protection to chicks against NDV. Comparatively, the plasmid pCAG-optiF-2 encoded a soluble protein with a mutant cleavage site and the potential pre-fusion conformation provided better protection than the other plasmids. Further investigation of the combined vaccinations with the plasmid DNA pCAG-optiF-2 prime + protein roptiF-2 boost vaccination strategy elicited more robust immunity, as confirmed by the detection of antibodies against NDV using enzyme-linked immunosorbent assay and virus neutralization assay, as compared to those vaccinated with only the plasmid pCAG-optiF-2 or protein roptiF-2. More importantly, the DNA prime + protein boost vaccination provided more efficacious protection against virulent NDV challenge, as evidenced by the complete clinical protection, reduced viral shedding, and limited virus replication in tissues of the challenge chicks. These results indicated that the pre-fusion conformation of the F protein could be considered as the target immunogen for the development of novel NDV vaccines.

Global exploration of the metabolic requirements of gallid alphaherpesvirus 1.

Although therapeutics targeting viral metabolic processes have been considered as promising strategies to treat herpesvirus infection, the metabolic requirements of gallid alphaherpesvirus 1 (ILTV), which is economically important to the poultry industry worldwide, remain largely unknown. Using the ILTV-susceptible but nonpermissive chicken cell line DF-1 and the ILTV-permissive chicken cell line LMH as models, the present study explored the metabolic requirements of ILTV by global transcriptome analysis and metabolome assays of ILTV infected cell lines in combination with a set of functional validations. The extensive metabolic exploration demonstrated that ILTV infection tended to promote a metabolic shift from glycolysis to fatty acid (FA) and nucleotide biosynthesis and utilizes glutamine independently of glutaminolysis, without significant general effect on the TCA cycle. In addition, different metabolic pathways were found to be required for distinct stages of ILTV replication. Glucose and glutamine were required for the transcription of viral immediate early gene ICP4 and subsequent steps of viral replication. However, FA synthesis was essential for assembly but not required for other upstream steps of ILTV replication. Moreover, the metabolic requirements of ILTV infection revealed in chicken cell lines were further validated in chicken primary cells isolated from chicken embryo kidneys and chicken embryo livers. The present study, to the best of our knowledge, provides the first global metabolic profile of animal herpesviruses and illustrates the main characteristics of the metabolic program of ILTV.

Genetic, Antigenic, and Pathogenic Characteristics of Infectious Bronchitis Virus GI-7/TW-II in China.

Nine infectious bronchitis virus (IBV) strains belonging to the GI-7 lineage were isolated between 2009 and 2017 in China. Phylogenetic analysis and comparisons of full-length sequences of the S1 gene suggested that the GI-7 lineage should be further classified as Taiwan (TW)-I and TW-II sublineages, which correspond to the previous TW-I and TW-II genotypes. The nine IBV strains were clustered in the TW-II sublineage. Further investigation revealed that viruses in the TW-I and TW-II were not only genetically but also antigenically different. Moreover, the TW-II sublineage contained various clades and recombinants. A recombinant was found to originate from recombination events between field strains (TW-II ck/CH/LJL/090608- and GI-19 ck/ CH/LDL/091022-like viruses) in which the recombination in the S1 subunit coding sequences had led to changes in antigenicity of the viruses. A more in-depth investigation demonstrated that TW-II viruses appear to have undergone a significant evolution following introduction in mainland China, which resulted in the viruses diverging into different clades. The viruses between the different clades in TW-II sublineage exhibited a significant change in genetic and antigenic characteristics. In addition, the five TW-II viruses selected on the basis of the results of S1 nucleotide sequence phylogenetic trees showed different pathogenicity to specific-pathogen-free chickens, although they could induce nephritis in the infected chickens and thus were identified as nephropathogenic strains.

Características genéticas, antigénicas y patógenas del virus de la bronquitis infecciosa GI-7/TW-II en China. Nueve cepas del virus de la bronquitis infecciosa (IBV) que pertenecen al linaje GI-7 se aislaron entre 2009 y 2017 en China. El análisis filogenético y las comparaciones de las secuencias completas del gene S1 sugirieron que el linaje GI-7 debería ser clasificado además como sublinajes TW-I y TW-II, que corresponden a los anteriores genotipos TW-T y TW-II. Las nueve cepas del virus de la bronquitis infecciosa se agruparon en el sublinaje TW-II. La investigación adicional reveló que los virus en TW-I y TW-II no solo eran tanto genéticamente como antigénicamente diferentes. Además, el sublinaje TW-II contenía varios clados y recombinantes. Se descubrió que un recombinante se originaba a partir de eventos de recombinación entre cepas de campo (virus similares a las cepas TW-II ck/CH/LJL/090608 y GI-19 ck/CH/LDL/091022) en los que la recombinación en las secuencias de codificación de la subunidad de S1 condujo a cambios en la antigenicidad de los virus. Una investigación más profunda demostró que los virus TW-II parecen haber experimentado una evolución significativa después de su introducción en China continental, lo que resultó en la divergencia de los virus en diferentes clados. Los virus entre los diferentes clados en el sublinaje TW-II exhibieron un cambio significativo en las características genéticas y antigénicas. Además, los cinco virus TW-II seleccionados con base en los resultados de los árboles filogenéticos de las secuencias de nucleótidos de S1 mostraron patogenicidad diferente en los pollos libres de patógenos específicos, aunque pudieron inducir nefritis en los pollos infectados y, por lo tanto, se identificaron como cepas nefropatógenas.

A highly pathogenic GI-19 lineage infectious bronchitis virus originated from multiple recombination events with broad tissue tropism.

In the present study, an IBV strain I0305/19 was isolated from a diseased commercial broiler flock in 2019 in China with high morbidity and mortality. The isolate I0305/19 was clustered together with viruses in sublineage D of GI-19 lineage on the basis of the complete S1 sequence analysis. Isolate I0305/19 and other GI-19 viruses isolated in China have the amino acid sequence MIA at positions 110-112 in the S protein. Further analysis based on the complete genomic sequence showed that the isolate emerged through at least four recombination events between GI-19 ck/CH/LJS/120848- and GI-13 4/91-like strains, in which the S gene was found to be similar to that of the GI-19 ck/CH/LJS/120848-like strain. Pathological assessment showed the isolate was a nephropathogenic IBV strain that caused high morbidity of 100 % and mortality of 80 % in 1-day-old specific-pathogen-free (SPF) chicks. The isolate I0305/19 exhibited broader tropisms in different tissues, including tracheas, lungs, bursa of Fabricius, spleen, liver, kidneys, proventriculus, small intestines, large intestines, cecum, and cecal tonsils. Furthermore, subpopulations of the virus were found in tissues of infected chickens; this finding is important in understanding how the virulent IBV strains can potentially replicate and evolve to cause disease. This information is also valuable for understanding the mechanisms of replication and evolution of other coronaviruses such as the newly emerged SARS-CoV-2.

Multiple recombination events between field and vaccine strains resulted in the emergence of a novel infectious bronchitis virus with decreased pathogenicity and altered replication capacity.

In this study, we isolated and identified 2 infectious bronchitis virus (IBV) strains from layer chickens soon after vaccination with the Massachusetts-Connecticut bivalent vaccine (Conn) and H120 and 4/91 booster vaccines in China in 2011. The results of cross-virus-neutralization tests and phylogenetic analysis of the S1 subunit of spike gene of these vaccine strains and other reference strains showed that strain LJL/110302 was of GI-19 lineage, whereas LLN/111169 was of the GI-1 lineage of the Conn serotype. Further comparative genomic analysis revealed that LLN/111169, an IBV strain with novel traits, originated from multiple recombination events (at least 3 recombination sites) between GI-19 and the Conn and 4/91 vaccine strains. LLN/111169 was pathogenic to specific pathogen-free (SPF) chickens. This is of prime importance because while IBV prevention measures worldwide are mainly dependent on modified live vaccine strains, our results showed that recombination between field and vaccine strains has produced a novel pathogenic IBV strain. In addition, LLN/111169 showed relatively broad tissue tropism (trachea, lungs, kidneys, and cecal tonsils) in infected SPF chickens. These results emphasize the importance of IBV surveillance in chicken flocks.

Genetic, antigenic and pathogenic characterization of avian coronaviruses isolated from pheasants (Phasianus colchicus) in China.

Two viruses were isolated in 2017 from commercial pheasants with severe clinical signs and mortality in Shandong and Anhui provinces, China, respectively. We examined the pathogenic effects of the viruses in chicken embryos and the size and morphology of the virus particles, performed phylogenetic analysis based on the S1 gene and complete genomic sequences, and examined the antibody responses against infectious bronchitis virus (IBV). The results suggested that the viruses I0623/17 and I0710/17 were avian coronaviruses and were identified as pheasant coronaviruses (PhCoV), with greatest similarity to IBV. Further investigations of the antigenicity, complete genome organization, substitutions in multiple genes, and viral pathogenicity, replication, and shedding in chickens and pheasants showed obvious differences between PhCoV and IBV in terms of antigenicity, and viral pathogenicity, replication, and shedding in chickens and pheasants. The close genetic relationship, but obvious differences between PhCoVs and IBVs suggested the IBVs could be the ancestors of PhCoVs, and that PhCoVs isolated from different outbreaks may have evolved independently from IBVs circulating in the specific region by adaption in pheasants. This hypothesis was supported by analysis of the S1 gene fragments of the two PhCoVs isolated in the current study, as well as PhCoVs isolated in the UK and selected IBV strains. Such analyses indicated different evolution patterns and different tissue tropisms between PhCoVs isolated in different outbreaks. Further studies are needed to confirm this hypothesis by studying the complete genomic sequences of PhCoVs from different outbreaks and the pathogenicity of IBVs in pheasants to compare and clarify the relationships between PhCoVs and IBVs.

Fowl adenoviruse-4 infection induces strong innate immune responses in chicken.

Fowl adenovirus (FAdV), as the causative agent of hepatitis-hydropericardium syndrome (HHS), poses a significant threat to the poultry industry in China in recent years. In this study, we investigated the immunopathogenesis of a FAdV-4 strain HN/151025 in 60-day-old chickens. The virus was highly virulent in chickens, with a broader tissue tropism in chickens, causing 60 % mortality. Postmortem findings of dead chickens showed mild HHS and liver degeneration and necrosis. Importantly, FAdV-4 infection induced significant upregulation of genes encoding most toll-like receptors, some cytokines (interleukin-1ß, 2, 6, 8, and 18, and interferon-γ), most of avian ß-defensins, myeloid differentiation primary response protein 88, p38 mitogen-activated protein kinases, and inducible nitric oxide synthase, in tissues of infected chicken, especially in spleen and bursa of Fabricius. There was also a significant positive correlation between FAdV-4 genome load and the mRNA expression levels of most of these factors in specific infected tissues. The results indicated the potential role of these proteins in host immune response against FAdV-4 infection. However, overexpression of these proteins might contribute to tissue damage of FAdV-4 infected chickens, and eventually lead to chicken death.