Recombinant Hemagglutinin Protein from H9N2 Avian Influenza Virus Exerts Good Immune Effects in Mice.

The H9N2 subtype of avian influenza virus (AIV) causes enormous economic losses and poses a significant threat to public health; the development of vaccines against avian influenza is ongoing. To study the immunogenicity of hemagglutinin (HA) protein, we constructed a recombinant pET-32a-HA plasmid, induced HA protein expression with isopropyl ß-D-1-thiogalactopyranoside (IPTG), verified it by SDS-PAGE and Western blotting, and determined the sensitivity of the recombinant protein to acid and heat. Subsequently, mice were immunized with the purified HA protein, and the immunization effect was evaluated according to the hemagglutination inhibition (HI) titer, serum IgG antibody titer, and cytokine secretion level of the mice. The results showed that the molecular weight of the HA protein was approximately 84 kDa, and the protein existed in both soluble and insoluble forms; in addition, the HA protein exhibited good acid and thermal stability, the HI antibody titer reached 6 log2-8 log2, and the IgG-binding antibody titer was 1:1,000,000. Moreover, the levels of IL-2, IL-4, and IL-5 in the immunized mouse spleen cells were significantly increased compared with those in the control group. However, the levels of IL-1ß, IL-6, IL-13, IFN-γ, IL-18, TNF-α, and GM-CSF were decreased in the immunized group. The recombinant HA protein utilized in this study exhibited good stability and exerted beneficial immune effects, providing a theoretical basis for further research on influenza vaccines.

Simultaneous differential detection of H5, H7 and H9 subtypes of avian influenza viruses by a triplex fluorescence loop-mediated isothermal amplification assay.

H5, H7, and H9 are pivotal avian influenza virus (AIV) subtypes that cause substantial economic losses and pose potential threats to public health worldwide. In this study, a novel triplex fluorescence reverse transcription-loop-mediated isothermal amplification (TLAMP) assay was developed in which traditional LAMP techniques were combined with probes for detection. Through this innovative approach, H5, H7, and H9 subtypes of AIV can be simultaneously identified and differentiated, thereby offering crucial technical support for prevention and control efforts. Three primer sets and composite probes were designed based on conserved regions of the haemagglutinin gene for each subtype. The probes were labelled with distinct fluorophores at their 3' ends, which were detached to release the fluorescence signal during the amplification process. The detection results were interpreted based on the colour of the TLAMP products. Then, the reaction conditions were optimized, and three primer sets and probes were combined in the same reaction system, resulting in a TLAMP detection assay for the differential diagnosis of AIV subtypes. Sensitivity testing with in vitro-transcribed RNA revealed that the detection limit of the TLAMP assay was 205 copies per reaction for H5, 360 copies for H7, and 545 copies for H9. The TLAMP assay demonstrated excellent specificity, no cross-reactivity with related avian viruses, and 100% consistency with a previously published quantitative polymerase chain reaction (qPCR) assay. Therefore, due to its simplicity, rapidity, sensitivity, and specificity, this TLAMP assay is suitable for epidemiological investigations and is a valuable tool for detecting and distinguishing H5, H7, and H9 subtypes of AIV in clinical samples.

Avian Orthoreoviruses: A Systematic Review of Their Distribution, Dissemination Patterns, and Genotypic Clustering.

Avian orthoreviruses have become a global challenge to the poultry industry, causing significant economic impacts on commercial poultry. Avian reoviruses (ARVs) are resistant to heat, proteolytic enzymes, a wide range of pH values, and disinfectants, so keeping chicken farms free of ARV infections is difficult. This review focuses on the global prevalence of ARVs and associated clinical signs and symptoms. The most common signs and symptoms include tenosynovitis/arthritis, malabsorption syndrome, runting-stunting syndrome, and respiratory diseases. Moreover, this review also focused on the characterization of ARVs in genotypic clusters (I-VI) and their relation to tissue tropism or viral distribution. The prevailing strains of ARV in Africa belong to all genotypic clusters (GCs) except for GC VI, whereas all GCs are present in Asia and the Americas. In addition, all ARV strains are associated with or belong to GC I-VI in Europe. Moreover, in Oceania, only GC V and VI are prevalent. This review also showed that, regardless of the genotypic cluster, tenosynovitis/arthritis was the predominant clinical manifestation, indicating its universal occurrence across all clusters. Globally, most avian reovirus infections can be prevented by vaccination against four major strains: S1133, 1733, 2408, and 2177. Nevertheless, these vaccines may not a provide sufficient defense against field isolates. Due to the increase in the number of ARV variants, classical vaccine approaches are being developed depending on the degree of antigenic similarity between the vaccine and field strains, which determines how successful the vaccination will be. Moreover, there is a need to look more closely at the antigenic and pathogenic properties of reported ARV strains. The information acquired will aid in the selection of more effective vaccine strains in combination with biosecurity and farm management methods to prevent ARV infections.

Differences in the pathogenicity and molecular characteristics of fowl adenovirus serotype 4 epidemic strains in Guangxi Province, southern China.

Starting in 2015, the widespread prevalence of hydropericardium-hepatitis syndrome (HHS) has led to considerable financial losses within China's poultry farming industry. In this study, pathogenicity assessments, whole-genome sequencing, and analyses were conducted on 10 new isolates of the novel genotype FAdV-4 during a HHS outbreak in Guangxi Province, China, from 2019 to 2020. The results indicated that strains GX2019-010 to GX2019-013 and GX2019-015 to GX2019-018 were highly virulent, while strain GX2020-019 exhibited moderate virulence. Strain GX2019-014 was characterized as a wild-type strain with low virulence, displaying no pathogenic effects when 0.5 mL containing 106 TCID50 virus was inoculated into the muscle of specific pathogen-free (SPF) chickens at 4 weeks of age, while 107 TCID50 and 108 TCID50 resulted in mortality rates of 80 and 100%, respectively. The whole genomes of strains GX2019-010 to GX2019-013, GX2019-015 to GX2019-018, and GX2020-019 showed high homology with other Chinese newly emerging highly pathogenic FAdV-4 strains, whereas GX2019-014 was closer to nonmutant strains and shared the same residues with known nonpathogenic strains (B1-7, KR5, and ON1) at positions 219AA and 380AA of the Fiber-2 protein. Our work enriches the research on prevalent strains of FAdV-4 in China, expands the knowledge on the virulence diversity of the novel genotype FAdV-4, and provides valuable reference material for further investigations into the key virulence-associated genetic loci of FAdV-4.

Biological features of fowl adenovirus serotype-4.

Fowl adenovirus serotype 4 (FAdV-4) is highly pathogenic to broilers aged 3 to 5 weeks and has caused considerable economic loss in the poultry industry worldwide. FAdV-4 is the causative agent of hydropericardium-hepatitis syndrome (HHS) or hydropericardium syndrome (HPS). The virus targets mainly the liver, and HPS symptoms are observed in infected chickens. This disease was first reported in Pakistan but has now spread worldwide, and over time, various deletions in the FAdV genome and mutations in its major structural proteins have been detected. This review provides detailed information about FAdV-4 genome organization, physiological features, epidemiology, coinfection with other viruses, and host immune suppression. Moreover, we investigated the role and functions of important structural proteins in FAdV-4 pathogenesis. Finally, the potential regulatory effects of FAdV-4 infection on ncRNAs are also discussed.

Analysis of Chicken IFITM3 Gene Expression and Its Effect on Avian Reovirus Replication.

Interferon-inducible transmembrane protein 3 (IFITM3) is an antiviral factor that plays an important role in the host innate immune response against viruses. Previous studies have shown that IFITM3 is upregulated in various tissues and organs after avian reovirus (ARV) infection, which suggests that IFITM3 may be involved in the antiviral response after ARV infection. In this study, the chicken IFITM3 gene was cloned and analyzed bioinformatically. Then, the role of chicken IFITM3 in ARV infection was further explored. The results showed that the molecular weight of the chicken IFITM3 protein was approximately 13 kDa. This protein was found to be localized mainly in the cytoplasm, and its protein structure contained the CD225 domain. The homology analysis and phylogenetic tree analysis showed that the IFITM3 genes of different species exhibited great variation during genetic evolution, and chicken IFITM3 shared the highest homology with that of Anas platyrhynchos and displayed relatively low homology with those of birds such as Anser cygnoides and Serinus canaria. An analysis of the distribution of chicken IFITM3 in tissues and organs revealed that the IFITM3 gene was expressed at its highest level in the intestine and in large quantities in immune organs, such as the bursa of Fabricius, thymus and spleen. Further studies showed that the overexpression of IFITM3 in chicken embryo fibroblasts (DF-1) could inhibit the replication of ARV, whereas the inhibition of IFITM3 expression in DF-1 cells promoted ARV replication. In addition, chicken IFITM3 may exert negative feedback regulatory effects on the expression of TBK1, IFN-γ and IRF1 during ARV infection, and it is speculated that IFITM3 may participate in the innate immune response after ARV infection by negatively regulating the expression of TBK1, IFN-γ and IRF1. The results of this study further enrich the understanding of the role and function of chicken IFITM3 in ARV infection and provide a theoretical basis for an in-depth understanding of the antiviral mechanism of host resistance to ARV infection.

Development of a Double-Antibody Sandwich ELISA Based on a Monoclonal Antibody against the Viral NS1 Protein for the Detection of Chicken Parvovirus.

Chicken parvovirus (ChPV) infection can cause runting-stunting syndrome (RSS) in chickens. There is currently no commercially available vaccine for controlling ChPV, and ChPV infection in chickens is widespread globally. The rapid detection of ChPV is crucial for promptly capturing epidemiological data on ChPV. Two monoclonal antibodies (mAbs), 1B12 and 2B2, against the ChPV NS1 protein were generated. A double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) was developed for detecting ChPV based on the mAb 1B12 and an anti-chicken polyclonal antibody against the ChPV NS1 protein. The detection limit for the ChPV recombinant pET32a-NS1 protein was approximately 31.2 ng/mL. A total of 192 throat and cloaca swab samples were analyzed for ChPV by the established DAS-ELISA and nested PCR methods. The concordance rate between the DAS-ELISA and the nested PCR method was 89.1%. The DAS-ELISA can detect the ChPV antigen without any cross-reaction with FAdV-4, FAdV-1, NDV, AIV, MS, CIAV, aMPV, EDSV, IBV, or AGV2. The method also has high repeatability, with a coefficient of variation (CV) of less than 5%. These findings indicate that the DAS-ELISA exhibits high accuracy, good sensitivity, and specificity, making it suitable for viral detection, field surveillance, and epidemiological studies.

Avian infectious bronchitis virus (AIBV) review by continent.

Infectious bronchitis virus (IBV) is a positive-sense, single-stranded, enveloped RNA virus responsible for substantial economic losses to the poultry industry worldwide by causing a highly contagious respiratory disease. The virus can spread quickly through contact, contaminated equipment, aerosols, and personal-to-person contact. We highlight the prevalence and geographic distribution of all nine genotypes, as well as the relevant symptoms and economic impact, by extensively analyzing the current literature. Moreover, phylogenetic analysis was performed using Molecular Evolutionary Genetics Analysis (MEGA-6), which provided insights into the global molecular diversity and evolution of IBV strains. This review highlights that IBV genotype I (GI) is prevalent worldwide because sporadic cases have been found on many continents. Conversely, GII was identified as a European strain that subsequently dispersed throughout Europe and South America. GIII and GV are predominant in Australia, with very few reports from Asia. GIV, GVIII, and GIX originate from North America. GIV was found to circulate in Asia, and GVII was identified in Europe and China. Geographically, the GVI-1 lineage is thought to be restricted to Asia. This review highlights that IBV still often arises in commercial chicken flocks despite immunization and biosecurity measures because of the ongoing introduction of novel IBV variants and inadequate cross-protection provided by the presently available vaccines. Consequently, IB consistently jeopardizes the ability of the poultry industry to grow and prosper. Identifying these domains will aid in discerning the pathogenicity and prevalence of IBV genotypes, potentially enhancing disease prevention and management tactics.

Simultaneous Differential Detection of H5, H7, H9 and Nine NA Subtypes of Avian Influenza Viruses via a GeXP Assay.

H5, H7 and H9 are the most important subtypes of avian influenza viruses (AIVs), and nine neuraminidase (NA) subtypes (N1-N9) of AIVs have been identified in poultry. A method that can simultaneously detect H5, H7, H9 and the nine NA subtypes of AIVs would save time and effort. In this study, 13 pairs of primers, including 12 pairs of subtype-specific primers for detecting particular subtypes (H5, H7, H9 and N1-N9) and one pair of universal primers for detecting all subtypes of AIVs, were designed and screened. The 13 pairs of primers were mixed in the same reaction, and the 13 target genes were simultaneously detected. A GeXP assay using all 13 pairs of primers to simultaneously detect H5, H7, H9 and the nine NA subtypes of AIVs was developed. The GeXP assay showed specific binding to the corresponding target genes for singlet and multiplex templates, and no cross-reactivity was observed between AIV subtypes and other related avian pathogens. Detection was observed even when only 102 copies of the 13 target genes were present. This study provides a high-throughput, rapid and labor-saving GeXP assay for the simultaneous rapid identification of three HA subtypes (H5, H7 and N9) and nine NA subtypes (N1-N9) of AIVs.

A sandwich amperometric immunosensor for the detection of fowl adenovirus group I based on bimetallic Pt/Ag nanoparticle-functionalized multiwalled carbon nanotubes.

An enzyme-free sandwich amperometric immunosensor based on bimetallic Pt/Ag nanoparticle (Pt/AgNPs)-functionalized chitosan (Chi)-modified multiwalled carbon nanotubes (MWCNTs) as dual signal amplifiers and Chi-modified MWCNTs (MWCNTs-Chi) as substrate materials was developed for ultrasensitive detection of fowl adenovirus group I (FAdV-I). MWCNTs have a large specific surface area, and many accessible active sites were formed after modification with Chi. Hence, MWCNTs-Chi, as a substrate material for modifying glassy carbon electrodes (GCEs), could immobilize more antibodies (fowl adenovirus group I-monoclonal antibody, FAdV-I/MAb). Multiple Pt/AgNPs were attached to the surface of MWCNTs-Chi to generate MWCNTs-Chi-Pt/AgNPs with high catalytic ability for the reaction of H2O2 and modified active sites for fowl adenovirus group I-polyclonal antibody (FAdV-I/PAb) binding. Amperometric i-t measurements were employed to characterize the recognizability of FAdV-I. Under optimal conditions, and the developed immunosensor exhibited a wide linear range (100.93 EID50 mL-1 to 103.43 EID50 mL-1), a low detection limit (100.67 EID50 mL-1) and good selectivity, reproducibility and stability. This immunosensor can be used in clinical sample detection.

Roles and functions of IAV proteins in host immune evasion.

Influenza A viruses (IAVs) evade the immune system of the host by several regulatory mechanisms. Their genomes consist of eight single-stranded segments, including nonstructural proteins (NS), basic polymerase 1 (PB1), basic polymerase 2 (PB2), hemagglutinin (HA), acidic polymerase (PA), matrix (M), neuraminidase (NA), and nucleoprotein (NP). Some of these proteins are known to suppress host immune responses. In this review, we discuss the roles, functions and underlying strategies adopted by IAV proteins to escape the host immune system by targeting different proteins in the interferon (IFN) signaling pathway, such as tripartite motif containing 25 (TRIM25), inhibitor of nuclear factor κB kinase (IKK), mitochondrial antiviral signaling protein (MAVS), Janus kinase 1 (JAK1), type I interferon receptor (IFNAR1), interferon regulatory factor 3 (IRF3), IRF7, and nuclear factor-κB (NF-κB). To date, the IAV proteins NS1, NS2, PB1, PB1-F2, PB2, HA, and PA have been well studied in terms of their roles in evading the host immune system. However, the detailed mechanisms of NS3, PB1-N40, PA-N155, PA-N182, PA-X, M42, NA, and NP have not been well studied with respect to their roles in immune evasion. Moreover, we also highlight the future perspectives of research on IAV proteins.

Transcriptomic and Translatomic Analyses Reveal Insights into the Signaling Pathways of the Innate Immune Response in the Spleens of SPF Chickens Infected with Avian Reovirus.

Avian reovirus (ARV) infection is prevalent in farmed poultry and causes viral arthritis and severe immunosuppression. The spleen plays a very important part in protecting hosts against infectious pathogens. In this research, transcriptome and translatome sequencing technology were combined to investigate the mechanisms of transcriptional and translational regulation in the spleen after ARV infection. On a genome-wide scale, ARV infection can significantly reduce the translation efficiency (TE) of splenic genes. Differentially expressed translational efficiency genes (DTEGs) were identified, including 15 upregulated DTEGs and 396 downregulated DTEGs. These DTEGs were mainly enriched in immune regulation signaling pathways, which indicates that ARV infection reduces the innate immune response in the spleen. In addition, combined analyses revealed that the innate immune response involves the effects of transcriptional and translational regulation. Moreover, we discovered the key gene IL4I1, the most significantly upregulated gene at both the transcriptional and translational levels. Further studies in DF1 cells showed that overexpression of IL4I1 could inhibit the replication of ARV, while inhibiting the expression of endogenous IL4I1 with siRNA promoted the replication of ARV. Overexpression of IL4I1 significantly downregulated the mRNA expression of IFN-ß, LGP2, TBK1 and NF-κB; however, the expression of these genes was significantly upregulated after inhibition of IL4I1, suggesting that IL4I1 may be a negative feedback effect of innate immune signaling pathways. In addition, there may be an interaction between IL4I1 and ARV σA protein, and we speculate that the IL4I1 protein plays a regulatory role by interacting with the σA protein. This study not only provides a new perspective on the regulatory mechanisms of the innate immune response after ARV infection but also enriches the knowledge of the host defense mechanisms against ARV invasion and the outcome of ARV evasion of the host's innate immune response.

Chicken IFI6 inhibits avian reovirus replication and affects related innate immune signaling pathways.

Interferon-alpha inducible protein 6 (IFI6) is an important interferon-stimulated gene. To date, research on IFI6 has mainly focused on human malignant tumors, virus-related diseases and autoimmune diseases. Previous studies have shown that IFI6 plays an important role in antiviral, antiapoptotic and tumor-promoting cellular functions, but few studies have focused on the structure or function of avian IFI6. Avian reovirus (ARV) is an important virus that can exert immunosuppressive effects on poultry. Preliminary studies have shown that IFI6 expression is upregulated in various tissues and organs of specific-pathogen-free chickens infected with ARV, suggesting that IFI6 plays an important role in ARV infection. To analyze the function of avian IFI6, particularly in ARV infection, the chicken IFI6 gene was cloned, a bioinformatics analysis was conducted, and the roles of IFI6 in ARV replication and the innate immune response were investigated after the overexpression or knockdown of IFI6 in vitro. The results indicated that the molecular weight of the chicken IFI6 protein was approximately 11 kDa and that its structure was similar to that of the human IFI27L1 protein. A phylogenetic tree analysis of the IFI6 amino acid sequence revealed that the evolution of mammals and birds was clearly divided into two branches. The evolutionary history and homology of chickens are similar to those of other birds. Avian IFI6 localized to the cytoplasm and was abundantly expressed in the chicken lung, intestine, pancreas, liver, spleen, glandular stomach, thymus, bursa of Fabricius and trachea. Further studies demonstrated that IFI6 overexpression in DF-1 cells inhibited ARV replication and that the inhibition of IFI6 expression promoted ARV replication. After ARV infection, IFI6 modulated the expression of various innate immunity-related factors. Notably, the expression patterns of MAVS and IFI6 were similar, and the expression patterns of IRF1 and IFN-ß were opposite to those of IFI6. The results of this study further advance the research on avian IFI6 and provide a theoretical basis for further research on the role of IFI6 in avian virus infection and innate immunity.

Molecular characterization of emerging chicken and turkey parvovirus variants and novel strains in Guangxi, China.

Avian parvoviruses cause several enteric poultry diseases that have been increasingly diagnosed in Guangxi, China, since 2014. In this study, the whole-genome sequences of 32 strains of chicken parvovirus (ChPV) and 3 strains of turkey parvovirus (TuPV) were obtained by traditional PCR techniques. Phylogenetic analyses of 3 genes and full genome sequences were carried out, and 35 of the Guangxi ChPV/TuPV field strains were genetically different from 17 classic ChPV/TuPV reference strains. The nucleotide sequence alignment between ChPVs/TuPVs from Guangxi and other countries revealed 85.2-99.9% similarity, and the amino acid sequences showed 87.8-100% identity. The phylogenetic tree of these sequences could be divided into 6 distinct ChPV/TuPV groups. More importantly, 3 novel ChPV/TuPV groups were identified for the first time. Recombination analysis with RDP 5.0 revealed 15 recombinants in 35 ChPV/TuPV isolates. These recombination events were further confirmed by Simplot 3.5.1 analysis. Phylogenetic analysis based on full genomes showed that Guangxi ChPV/TuPV strains did not cluster according to their geographic origin, and the identified Guangxi ChPV/TuPV strains differed from the reference strains. Overall, whole-genome characterizations of emerging Guangxi ChPV and TuPV field strains will provide more detailed insights into ChPV/TuPV mutations and recombination and their relationships with molecular epidemiological features.

Global review of the H5N8 avian influenza virus subtype.

Orthomyxoviruses are negative-sense, RNA viruses with segmented genomes that are highly unstable due to reassortment. The highly pathogenic avian influenza (HPAI) subtype H5N8 emerged in wild birds in China. Since its emergence, it has posed a significant threat to poultry and human health. Poultry meat is considered an inexpensive source of protein, but due to outbreaks of HPAI H5N8 from migratory birds in commercial flocks, the poultry meat industry has been facing severe financial crises. This review focuses on occasional epidemics that have damaged food security and poultry production across Europe, Eurasia, the Middle East, Africa, and America. HPAI H5N8 viral sequences have been retrieved from GISAID and analyzed. Virulent HPAI H5N8 belongs to clade 2.3.4.4b, Gs/GD lineage, and has been a threat to the poultry industry and the public in several countries since its first introduction. Continent-wide outbreaks have revealed that this virus is spreading globally. Thus, continuous sero- and viro-surveillance both in commercial and wild birds, and strict biosecurity reduces the risk of the HPAI virus appearing. Furthermore, homologous vaccination practices in commercial poultry need to be introduced to overcome the introduction of emergent strains. This review clearly indicates that HPAI H5N8 is a continuous threat to poultry and people and that further regional epidemiological studies are needed.

Pathogenicity and molecular characteristics of fowl adenovirus serotype 4 with moderate virulence in Guangxi Province, China.

The GX2020-019 strain of fowl adenovirus serotype 4 (FAdV-4) was isolated from the liver of chickens with hydropericardium hepatitis syndrome in Guangxi Province, China, and was purified by plaque assay three times. Pathogenicity studies showed that GX2020-019 can cause typical FAdV-4 pathology, such as hydropericardium syndrome and liver yellowing and swelling. Four-week-old specific pathogen-free (SPF) chickens inoculated with the virus at doses of 103 median tissue culture infectious dose (TCID50), 104 TCID50, 105 TCID50, 106 TCID50, and 107 TCID50 had mortality rates of 0, 20, 60, 100, and 100%, respectively, which were lower than those of chickens inoculated with other highly pathogenic Chinese isolates, indicating that GX2020-019 is a moderately virulent strain. Persistent shedding occurred through the oral and cloacal routes for up to 35 days postinfection. The viral infection caused severe pathological damage to the liver, kidney, lung, bursa of Fabricius, thymus, and spleen. The damage to the liver and immune organs could not be fully restored 21 days after infection, which continued to affect the immune function of chickens. Whole genome analysis indicated that the strain belonged to the FAdV-C group, serotype 4, and had 99.7-100% homology with recent FAdV-4 strains isolated from China. However, the amino acid sequences encoded by ORF30 and ORF49 are identical to the sequences found in nonpathogenic strains, and none of the 32 amino acid mutation sites that appeared in other Chinese isolates were found. Our research expands understanding of the pathogenicity of FAdV-4 and provides a reference for further studies.

Comparative mutational analysis of the Zika virus genome from different geographical locations and its effect on the efficacy of Zika virus-specific neutralizing antibodies.

The Zika virus (ZIKV), which originated in Africa, has become a significant global health threat. It is an RNA virus that continues to mutate and accumulate multiple mutations in its genome. These genetic changes can impact the virus's ability to infect, cause disease, spread, evade the immune system, and drug resistance. In this study genome-wide analysis of 175 ZIKV isolates deposited at the National Center for Biotechnology Information (NCBI), was carried out. The comprehensive mutational analysis of these isolates was carried out by DNASTAR and Clustal W software, which revealed 257 different substitutions at the proteome level in different proteins when compared to the reference sequence (KX369547.1). The substitutions were capsid (17/257), preM (17/257), envelope (44/257), NS1 (34/257), NS2A (30/257), NS2B (11/257), NS3 (37/257), NS4A (6/257), 2K (1/257), NS4B (15/257), and NS5 (56/257). Based on the coexisting mutational analysis, the MN025403.1 isolate from Guinea was identified as having 111 substitutions in proteins and 6 deletions. The effect of coexisting/reoccurring mutations on the structural stability of each protein was also determined by I-mutant and MUpro online servers. Furthermore, molecular docking and simulation results showed that the coexisting mutations (I317V and E393D) in Domain III (DIII) of the envelope protein enhanced the bonding network with ZIKV-specific neutralizing antibodies. This study, therefore, highlighted the rapid accumulation of different substitutions in various ZIKV proteins circulating in different geographical regions of the world. Surveillance of such mutations in the respective proteins will be helpful in the development of effective ZIKV vaccines and neutralizing antibody engineering.

A multiplex fluorescence-based loop-mediated isothermal amplification assay for identifying chicken parvovirus, chicken infectious anaemia virus, and fowl aviadenovirus serotype 4.

Chicken parvovirus (ChPV), chicken infectious anaemia virus (CIAV) and fowl adenovirus serotype 4 (FAdV-4) are avian viruses that have emerged in recent years and have endangered the global poultry industry, causing great economic loss. In this study, a multiplex fluorescence-based loop-mediated isothermal amplification (mLAMP) assay for detecting ChPV, CIAV and FAdV-4 was developed to simultaneously diagnose single and mixed infections in chickens. Three primer sets and composite probes were designed according to the conserved regions of the NS gene of ChPV, VP1 gene of CIAV and hexon gene of FAdV-4. Each composite probe was labelled with a different fluorophore, which was detached to release the fluorescence signal after amplification. The target viruses were distinguished based on the colour of the mLAMP products. The mLAMP assay was shown to be sensitive, with detection limits of 307 copies of recombinant plasmids containing the ChPV target genes, 749 copies of CIAV and 648 copies of FAdV-4. The assay exhibited good specificity and no cross-reactivity with other symptomatically related avian viruses. When used on field materials, the results of the mLAMP assay were in 100% agreement with those of the previously published PCR assay. The mLAMP assay is rapid, economical, sensitive and specific, and the results of amplification are directly observable by eye. Therefore, the mLAMP assay is a useful tool for the clinical detection of ChPV, CIAV and FAdV-4 and can be applied in rural areas.

Explore how immobilization strategies affected immunosensor performance by comparing four methods for antibody immobilization on electrode surfaces.

Among the common methods used for antibody immobilization on electrode surfaces, which is the best available option for immunosensor fabrication? To answer this question, we first used graphene-chitosan-Au/Pt nanoparticle (G-Chi-Au/PtNP) nanocomposites to modify a gold electrode (GE). Second, avian reovirus monoclonal antibody (ARV/MAb) was immobilized on the GE surface by using four common methods, which included glutaraldehyde (Glu), 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide/N-hydroxysuccinimide (EDC/NHS), direct incubation or cysteamine hydrochloride (CH). Third, the electrodes were incubated with bovine serum albumin, four different avian reovirus (ARV) immunosensors were obtained. Last, the four ARV immunosensors were used to detect ARV. The results showed that the ARV immunosensors immobilized via Glu, EDC/NHS, direct incubation or CH showed detection limits of 100.63 EID50 mL-1, 100.48 EID50 mL-1, 100.37 EID50 mL-1 and 100.46 EID50 mL-1 ARV (S/N = 3) and quantification limits of 101.15 EID50 mL-1, and 101.00 EID50 mL-1, 100.89 EID50 mL-1 and 100.98 EID50 mL-1 ARV (S/N = 10), respectively, while the linear range of the immunosensor immobilized via CH (0-105.82 EID50 mL-1 ARV) was 10 times broader than that of the immunosensor immobilized via direct incubation (0-104.82 EID50 mL-1 ARV) and 100 times broader than those of the immunosensors immobilized via Glu (0-103.82 EID50 mL-1 ARV) or EDC/NHS (0-103.82 EID50 mL-1 ARV). And the four immunosensors showed excellent selectivity, reproducibility and stability.

Development of a visual multiplex fluorescent LAMP assay for the detection of foot-and-mouth disease, vesicular stomatitis and bluetongue viruses.

Loop-mediated isothermal amplification (LAMP) is a nucleic acid amplification technique that can be used to amplify target genes at a constant temperature, and it has several advantages, including convenience, specificity and sensitivity. However, due to the special interpretation methods of this technology for reaction results, all the previously reported LAMP detection methods have been restricted to identifying a single target, which limits the application of this technology. In this study, we modified conventional LAMP to include a quencher-fluorophore composite probe complementary to the F1c segment of the inner primer FIP; upon strand separation, a gain in the visible fluorescent signal was observed. The probes could be labeled with different fluorophores, showing different colors at the corresponding wavelengths. Therefore, this multiplex LAMP (mLAMP) assay can simultaneously detect 1-3 target sequences in a single LAMP reaction tube, and the results are more accurate and intuitive. In this study, we comprehensively demonstrated a single-reaction mLAMP assay for the robust detection of three cattle viruses without nonspecific amplification of other related pathogenic cattle viruses. The detection limit of this mLAMP assay was as low as 526-2477 copies/reaction for the recombinant plasmids. It is expected that this mLAMP assay can be widely used in clinical diagnosis.