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For Selected: Download Citations EndNote Ris BibTeX Toggle Thumbnails Select Development and evaluation of a RT-RAA-combined CRISPR/Cas12a assay for the detection of African horse sickness virus Yingzhi Zhang, Lei Na, Kui Guo, Jinhui Wang, Zhe Hu, Cheng Du, Xuefeng Wang, Xiaojun Wang 2024, 23 (12): 4267-4271.   DOI: 10.1016/j.jia.2024.08.012 Abstract （116）      PDF in ScienceDirect       African horse sickness (AHS) is an acute and fatal vector-borne infectious disease of equids, caused by the African horse sickness virus (AHSV).  The World Organization for Animal Health (WOAH) has classified AHS as a notifiable animal disease, and AHS has also been classified as a Class I animal infectious disease in China.  AHS is mainly found in Africa, the Middle East and the Arabian Peninsula.  China is currently recognized by the WOAH as an AHS-free zone.  However, in 2020, there were outbreaks of AHS in 2 countries neighboring China, Thailand and Malaysia (Bunpapong et al. 2021), which increases the risk of the introduction of AHS into China.  Therefore, in order to prevent the occurrence of AHS in China and to further monitor the spread of the disease, the development of rapid, accurate and cost-effective diagnostic methods for the detection of AHSV is essential.   AHSV is a segmented double-stranded RNA virus belonging to the genus Orbivirus in the family Reoviridae.  It is mainly transmitted by midges (Maurer et al. 2022), and is able to infect all members of the Equidae, including horses, mules, donkeys, and zebras.  AHSV infection causes severe morbidity and mortality (up to 90%) in horses, while mules, donkeys and zebras are less susceptible than horses to the disease (Barnard 1998).   The AHSV genome contains 10 double-stranded RNA segments, encoding 7 structural proteins (VP1–7) and 4 non-structural proteins (NS1–4).  AHSV is a complex non-enveloped virus with an icosahedral capsid comprising 3 distinct concentric protein layers.  VP2 and VP5 are the components of the outer capsid of the virion.  VP2 is the major determinant of AHSV serotype, and 9 serotypes (AHSV-1 to AHSV-9) have been identified according to the VP2 antigenicity; VP3 and VP7 are the components of the major inner capsid of the virion; VP1, VP4 and VP6 constitute the minor inner capsid of AHSV.   AHSV RNA segment 7 (vp7) is highly conserved among all AHSV serotypes and is the primary molecular diagnostic target of AHSV.  The VP7 protein encoded by this segment is the major antigen of AHSV and is commonly used as a serological diagnostic for AHSV.  Real-time RT-PCR targeting vp7 is capable of detecting all known types of AHSV and is recommended by the WOAH for the detection of this virus (Aguero et al. 2008; Guthrie et al. 2013; WOAH 2019).   Recently, molecular diagnostics for infectious diseases have been developed based on clustered regularly interspaced short palindromic repeats-associated Cas endonucleases (CRISPR/Cas) systems combined with isothermal amplification techniques (Chen J S et al. 2018; Gootenberg et al. 2018; Myhrvold et al. 2018).  Some Cas proteins with non-specific endonuclease activity, such as Cas12a, activate auxiliary (non-specific) cleavage of nearby single-stranded non-target nucleic acids upon recognition of the target.  By modifying a single-stranded nucleic acid with a fluorophore quencher, which fluoresces upon cleavage of the Cas12a and crRNA complex, this activity can be used to detect the presence of specific cleavage.  The CRISPR/Cas12-based detection system has certain advantages over traditional nucleic acid diagnostic methods (qPCR), including rapidity, simplicity, low cost, and low equipment requirements.  In this study, we developed a sensitive detection method for AHSV using the CRISPR/Cas12a system combined with reverse transcription-recombinase-assisted amplification (RT-RAA) (CRISPR/Cas12a-RT-RAA), which specifically targets the vp7 RNA of AHSV.   To generate a CRISPR/Cas12-based AHSV detection system, a Cas12a from the Lachnospiraceae bacterium, LbCas12a protein, was first expressed in an Escherichia coli system and purified with Strep-Tactin Sepharose resin (Appendix A).  A single-stranded DNA (ssDNA) reporter labeled with a fluorophore and a quencher at the 2 termini (5´-6-FAM-TTATT-BHQ-3´) was synthesized by Sangon Biotech (Shanghai, China).  Ten crRNAs were designed to target the conserved region of the vp7 sequence of all AHSV strains (Appendices B and C).  These crRNAs with a repeat sequence were prepared using in vitro transcription following a previously described method (Wang et al. 2023).  In order to screen for an optimal crRNA for the sensitive detection of AHSV, 10 crRNAs were individually tested using a 25 μL CRISPR/Cas12-based reaction volume containing 0.4 μmol L–1 LbCas12a, 0.4 μmol L–1 ssDNA reporter, 1.2 μmol L–1 crRNA, 109 copies μL–1 vp7 plasmid DNA (pMD18-T-vp7, containing the entire vp7 sequence) and 2.5 μL NEBuffer 2.1 (10×).  The reaction was performed at 37°C for 50 min on a qPCR thermal cycler (Applied Biosystems QuantStusio 5 Real-Time PCR System, USA) with fluorescence measurements taken every 30 s.  Fluorescence detection suggested that crRNA10 showed the highest efficiency in this reaction system (Fig. 1-A).  Therefore, crRNA10 was identified as the best option for the AHSV CRISPR/Cas12a detection platform and was used in the subsequent experiments. Recombinase-assisted amplification (RAA) is an isothermal amplification technique that has been widely used to detect microbial pathogens (Chen C et al. 2018; Wang et al. 2020; Xue et al. 2020).  Recent studies have increasingly integrated the RAA assay with the CRISPR-Cas system, which provides a second detection step for amplification products, increasing detection sensitivity and specificity, and enabling more convenient and intuitive determination of detection results (Li et al. 2023).  Six specific RAA primers specifically targeting vp7 were designed based on the flanking sequence of the crRNA10 region (Appendix D).  To screen for the optimal RAA primer pair for the sensitive detection of AHSV, a standard RAA reaction was performed with pMD18-T-vp7 (at a concentration of 109 copies μL–1) as a template, using the RAA Nucleic Acid Amplification Kit (Qitian, China) and following the manufacturer’s instructions.  Following RAA amplification at 37°C for 30 min, the products of the RAA amplification were used as substrates for the CRISPR/Cas12a system detection.  As shown in Fig. 1-B, the strongest fluorescence signals for the CRISPR/Cas12a-RAA assay were detected when the F3/R3 primer pair was used.  The results showed that the F3/R3 primer pair had the best amplification efficiency and was therefore selected for the establishment of the CRISPR/Cas12a-RAA detection platform and used for subsequent experiments. To evaluate the sensitivity of the CRISPR/Cas12a-RAA detection platform, we prepared vp7 RNA in vitro using the HiScribe T7 Quick High Yield RNA Synthesis Kit (New England Biolabs, USA), using vp7 PCR products and with T7 promoter sequences as templates.  A total of 1 μL of vp7 RNA at different concentrations was used as a template for the RT-RAA reaction using RT-RAA Nucleic Acid Amplification Kit (Qitian, Wuxi, China) for 30 min, and then 1 μL of RAA amplification product was extracted and used as a substrate for the CRISPR/Cas12a detection system and reacted for 30 min.  The CRISPR/Cas12a-RT-RAA assay was developed in this way.  As shown in Fig. 1-C, the detection limit of the CRISPR/Cas12a-RT-RAA assay was 10 copies of the vp7 mRNA molecule per reaction.  However, the detection limit of the real-time RT-PCR assay established by Aguero in 2008 was 100 copies of the vp7 RNA molecule per reaction (Aguero et al. 2008) (Fig. 1-D).  In 2015, WOAH organized the AHS reference laboratory to conduct a comparison of trials to evaluate different conventional detection methods, and confirmed that the real-time RT-PCR established by Aguero in 2008 was one of the best detection methods for diagnosing AHSV (WOAH 2019).  Our results suggest that the CRISPR/Cas12a-RT-RAA assay has higher sensitivity compared to the real-time RT-PCR assay. To test the specificity of the CRISPR/Cas12a-RT-RAA assay to AHSV, other equine viral and bacterial pathogens were tested, including equine infectious anemia virus (EIAV), equine influenza virus (EIV), equine arthritis virus (EAV), equine herpesvirus-1 (EHV-1), equine herpesvirus-4 (EHV-4), Streptococcus equi subspecies equi (S. equi), and Salmonella enterica subsp. enterica serovar Abortusequi (S. Abortusequi).  All of these pathogens were stored in our laboratory and RNA/DNA from these pathogens was prepared as previously described (Chen et al. 2022).  As shown in Fig. 1-E, no fluorescence was observed when these equine pathogens were tested using the CRISPR/Cas12a-RT-RAA assay, whereas significant fluorescence was observed when the vp7 mRNA was tested, indicating that this CRISPR/Cas12a-RT-RAA method is highly specific for the detection of AHSV. Due to the lack of AHSV-positive samples, we evaluated the performance of the CRISPR/Cas12a-RT-RAA assay in clinical practice by adding vp7 mRNAs to mRNAs extracted from equine blood or tissue samples as positive controls, and equine blood or equine tissue mRNAs without vp7 mRNAs as negative controls.  A total of 20 equine mRNA samples, including 5 equine blood cell mRNA samples (S1–5), 5 equine blood cell mRNA samples with vp7 mRNA (S6–10), 5 equine tissue (heart, liver, spleen, lung and kidney) mRNA (S11–15), and 5 equine tissue mRNA with vp7 mRNA (S16–20), were prepared and assessed using the CRISPR/Cas12a-RT-RAA assay.  As shown in Fig. 1-F, strong fluorescence signals were detected in all equine mRNA samples with vp7 mRNA, but not in any equine mRNA samples without vp7 mRNA.  This result demonstrates that the CRISPR/Cas12a-RT-RAA assay is able to detect vp7 mRNA efficiently in samples collected under complex conditions and can be used as a back-up technology for the early field detection of AHSV. In conclusion, we reported the development and validation of a CRISPR/Cas12a-combined RT-RAA-based detection assay for AHSV with high specificity, sensitivity and convenience.  This assay targets the vp7 mRNA, a highly conserved segment of the AHSV genome that has not been observed to cross-react with the nucleic acids of 7 common equine pathogens, including EIAV, EIV, EAV, EHV-1, EHV-4, S. equi, and S. Abortusequi.  Notably, this assay was 10 times more sensitive than real-time RT-PCR.  In addition, the signal generated by the assay would be directly visible to the naked eye under UV light without the need for special instrumentation.  Therefore, the CRISPR/Cas12a combination RT-RAA assay developed here has the potential to be used as an alternative to traditional real-time RT-PCR assays for the rapid diagnosis of AHSV infection.  We will continue to optimize and improve the assay and expect that it will allow the detection of AHSV in the field and improve the early warning and diagnosis of AHS. Reference | Related Articles | Metrics Select Genetic and biological properties of H10Nx influenza viruses in China Yina Xu, Hailing Li, Haoyu Leng, Chaofan Su, Siqi Tang, Yongtao Wang, Shiwei Zhang, Yali Feng, Yanan Wu, Daxin Wang, Ying Zhang 2024, 23 (11): 3860-3869.   DOI: 10.1016/j.jia.2023.10.028 Abstract （107）      PDF in ScienceDirect       H10 subtype avian influenza viruses (AIV) have been circulating in China for 40 years.  H10 AIVs in China have expanded their host range from wild birds to domestic poultry and mammals, even human.  Most of the H10 subtype AIVs reported in China were isolate from the southeast part.  We isolated an H10N3 AIV, A/Chicken/Liaoning/SY1080/2021 (SY1080), from live poultry market (LPM) in Liaoning Province of the Northeast China.  SY1080 replicated efficiently in mice lungs and nasal turbinates without prior adaptation.  We systematically compared SY1080 with other H10 subtype isolates in China.  Phylogenetic analysis showed that SY1080 and most of the H10 strains belonged to the Eurasian lineage.  H10 AIVs in China have formed 63 genotypes.  SY1080 as well as the H10N3 strains from human infections belonged to G60 genotype.  H10Nx AIV acquired multiple mammalian adaptive and virulence related mutations during circulation and the recent reassortants derived internal genes from chicken H9N2 AIVs.  The H10Nx subtypes AIVs posed potential threat to public health.  These results suggested we should strengthen the surveillance and evaluation of H10 subtype strains. Reference | Related Articles | Metrics Select Role of feline ANP32 proteins in regulating polymerase activity of influenza A virus Gang Lu, Feiyan Zheng, Yuqing Xiao, Ran Shao, Jiajun Ou, Xin Yin, Shoujun Li, Guihong Zhang 2024, 23 (9): 3145-3158.   DOI: 10.1016/j.jia.2023.11.008 Abstract （100）      PDF in ScienceDirect       Recently, increasing natural infection cases and experimental animal challenge studies demonstrated domestic cats are susceptible to multiple subtypes influenza A virus (IAV) infections.  Notably, some subtype IAV strains could circulate in domestic cats after cross-species transmission and even infected humans, posing a threat to public health.  Host factors related to viral polymerase activity could determine host range of IAV and acidic nuclear phosphoprotein 32 (ANP32) is the most important one among them.  However, role of cat-derived ANP32 on viral polymerase activity and host range of IAV is still unknown.  In the present study, a total of 10 feline ANP32 (feANP32) splice variants (including 5 feANP32A, 3 feANP32B, and 2 feANP32E) were obtained from domestic cats by RT-PCR.  Sequence alignment results demonstrated amino acid deletions and/or insertions occurred among feANP32 variants, but all feANP32 proteins were primarily localized to cell nucleus.  Minigenome replication systems for several representative IAV strains were established and the support ability of feANP32 on IAV polymerase activity was estimated.  The results indicated that most feANP32A and feANP32B splice variants were able to support all the tested IAV strains, though the support activity of a single feANP32 protein on polymerase activity varied among different IAV strains.  In addition, the role of feANP32 in supporting H3N2 canine influenza virus was determined by investigating viral replication in vitro.  Collectively, our study systematically investigated the support activity of feANP32 on IAV, providing a clue for further exploring the mechanism of susceptibility of cats to IAV. Reference | Related Articles | Metrics Select Genetic and biological properties of H9N2 avian influenza viruses isolated in central China from 2020 to 2022 Libin Liang, Yaning Bai, Wenyan Huang, Pengfei Ren, Xing Li, Dou Wang, Yuhan Yang, Zhen Gao, Jiao Tang, Xingchen Wu, Shimin Gao, Yanna Guo, Mingming Hu, Zhiwei Wang, Zhongbing Wang, Haili Ma, Junping Li 2024, 23 (8): 2778-2791.   DOI: 10.1016/j.jia.2024.03.055 Abstract （133）      PDF in ScienceDirect       The H9N2 subtype of avian influenza virus (AIV) is widely prevalent in poultry and wild birds globally, and has become the predominant subtype circulating in poultry in China.  The H9N2 AIV can directly or indirectly (by serving as a “donor virus”) infect humans, posing a significant threat to public health.  Currently, there is a lack of in-depth research on the prevalence of H9N2 viruses in Shanxi Province, central China.  In this study, we isolated 14 H9N2 AIVs from October 2020 to April 2022 in Shanxi Province, and genetic analysis revealed that these viruses belonged to 7 different genotypes.  Our study on animals revealed that the H9N2 strains we identified displayed high transmission efficiency among chicken populations, and exhibited diverse replication abilities within these birds.  These viruses could replicate efficiently in the lungs of mice, with one strain also demonstrating the capacity to reproduce in organs like the brain and kidneys.  At the cellular level, the replication ability of different H9N2 strains was evaluated using plaque formation assays and multi-step growth curve assays, revealing significant differences in the replication and proliferation efficiency of the various H9N2 viruses at the cellular level.  The antigenicity analysis suggested that these isolates could be classified into 2 separate antigenic clusters.  Our research provides crucial data to help understand the prevalence and biological characteristics of H9N2 AIVs in central China.  It also highlights the necessity of enhancing the surveillance of H9N2 AIVs. Reference | Related Articles | Metrics Select A CRISPR/Cas12a-based platform for rapid on-site bovine viral diarrhea virus diagnostics Meixi Wang, Jitao Chang, Yuxin Han, Chaonan Wang, Songkang Qin, Jun Wang, Lulu Zhang, Yuanmao Zhu, Fei Xue, Fang Wang, Hongliang Chai, Yulong Wang, Xinjie Wang, Xin Yin 2024, 23 (8): 2872-2876.   DOI: 10.1016/j.jia.2024.03.074 Abstract （106）      PDF in ScienceDirect       Reference | Related Articles | Metrics Select Genetic and pathogenic characterization of new infectious bronchitis virus strains in the GVI-1 and GI-19 lineages isolated in central China Yuhan Yang, Dou Wang, Yaning Bai, Wenyan Huang, Shimin Gao, Xingchen Wu, Ying Wang, Jianle Ren, Jinxin He, Lin Jin, Mingming Hu, Zhiwei Wang, Zhongbing Wang, Haili Ma, Junping Li, Libin Liang 2024, 23 (7): 2407-2420.   DOI: 10.1016/j.jia.2023.10.029 Abstract （157）      PDF in ScienceDirect       Avian infectious bronchitis (IB) is a highly contagious infectious disease caused by infectious bronchitis virus (IBV), which is prevalent in many countries worldwide and causes serious harm to the poultry industry.  At present, many commercial IBV vaccines have been used for the prevention and control of IB; however, IB outbreaks occur frequently.  In this study, two new strains of IBV, SX/2106 and SX/2204, were isolated from two flocks which were immunized with IBV H120 vaccine in central China.  Phylogenetic and recombination analysis indicated that SX/2106, which was clustered into the GI-19 lineage, may be derived from recombination events of the GI-19 and GI-7 strains and the LDT3-A vaccine.  Genetic analysis showed that SX/2204 belongs to the GVI-1 lineage, which may have originated from the recombination of the GI-13 and GVI-1 strains and the H120 vaccine.  The virus cross-neutralization test showed that the antigenicity of SX/2106 and SX/2204 was different from H120.  Animal experiments found that both SX/2106 and SX/2204 could replicate effectively in the lungs and kidneys of chickens and cause disease and death, and H120 immunization could not provide effective protection against the two IBV isolates.  It is noteworthy that the pathogenicity of SX/2204 has significantly increased compared to the GVI-1 strains isolated previously, with a mortality rate up to 60%.  Considering the continuous mutation and recombination of the IBV genome to produce new variant strains, it is important to continuously monitor epidemic strains and develop new vaccines for the prevention and control of IBV epidemics. Reference | Related Articles | Metrics Select Substitutions of stem-loop subdomains in internal ribosome entry site of Senecavirus A: Impacts on rescue of sequence-modifying viruses Qianqian Wang, Jie Wang, Lei Zhang, Xiaoxiao Duan, Lijie Zhu, Youming Zhang, Yan Li, Fuxiao Liu 2024, 23 (7): 2391-2406.   DOI: 10.1016/j.jia.2024.04.019 Abstract （97）      PDF in ScienceDirect       Senecavirus A (SVA) has a positive-sense, single-stranded RNA genome. Its 5´ untranslated region harbors an internal ribosome entry site (IRES), comprising 10 larger or smaller stem-loop structures (including a pseudoknot) that have been demonstrated to be well conserved. However, it is still unclear whether each stem-loop subdomain, such as a single stem or loop, is also highly conserved. To clarify this issue in the present study, a set of 29 SVA cDNA clones were constructed by site-directed mutagenesis (SDM) on the IRES. The SDM-modified scenarios included: (1) stem-formed complementary sequences exchanging with each other; (2) loop transversion; (3) loop transition; and (4) point mutations. All cDNA clones were separately transfected into cells for rescuing viable viruses, whereas only four SVAs of interest could be recovered, and were genetically stable during 20 passages. One progeny grew significantly slower than the other three did. The dual-luciferase reporter assay showed that none of the SDM-modified IRESes significantly inhibited the IRES activity. Our previous study indicated that a single motif from any of the ten stem structures, if completely mutated, would cause the failure of virus recovery. Interestingly, our present study revealed three stem structures, whose individual complementary sequences could exchange with each other to rescue sequence-modifying SVAs. Moreover, one apical loop was demonstrated to have the ability to tolerate its own full-length transition, also having no impact on the recovery of sequence-modifying SVA. The present study suggested that not every stem-loop structure was strictly conserved in its conformation, while the full-length IRES itself was well conserved. This provides a new research direction on interaction between the IRES and many factors. Reference | Related Articles | Metrics Select Antibodies elicited by Newcastle disease virus-vectored H7N9 avian influenza vaccine are functional in activating the complement system Zenglei Hu, Ya Huang, Jiao Hu, Xiaoquan Wang, Shunlin Hu, Xiufan Liu 2024, 23 (6): 2052-2064.   DOI: 10.1016/j.jia.2023.11.007 Abstract （85）      PDF in ScienceDirect       H7N9 subtype avian influenza virus poses a great challenge for poultry industry.  Newcastle disease virus (NDV)-vectored H7N9 avian influenza vaccines (NDVvecH7N9) are effective in disease control because they are protective and allow mass administration.  Of note, these vaccines elicit undetectable H7N9-specific hemagglutination-inhibition (HI) but high IgG antibodies in chickens.  However, the molecular basis and protective mechanism underlying this particular antibody immunity remain unclear.  Herein, immunization with an NDVvecH7N9 induced low anti-H7N9 HI and virus neutralization titers but high levels of hemagglutinin (HA)-binding IgG antibodies in chickens.  Three residues (S150, G151 and S152) in HA of H7N9 virus were identified as the dominant epitopes recognized by the NDVvecH7N9 immune serum.  Passively transferred NDVvecH7N9 immune serum conferred complete protection against H7N9 virus infection in chickens.  The NDVvecH7N9 immune serum can mediate a potent lysis of HA-expressing and H7N9 virus-infected cells and significantly suppress H7N9 virus infectivity.  These activities of the serum were significantly impaired after heat-inactivation or treatment with complement inhibitor, suggesting the engagement of the complement system.  Moreover, mutations in the 150-SGS-152 sites in HA resulted in significant reductions in cell lysis and virus neutralization mediated by the NDVvecH7N9 immune serum, indicating the requirement of antibody-antigen binding for complement activity.  Therefore, antibodies induced by the NDVvecH7N9 can activate antibody-dependent complement-mediated lysis of H7N9 virus-infected cells and complement-mediated neutralization of H7N9 virus.  Our findings unveiled a novel role of the complement in protection conferred by the NDVvecH7N9, highlighting a potential benefit of engaging the complement system in H7N9 vaccine design. Reference | Related Articles | Metrics Select A rapid and visual detection method for Crimean-Congo hemorrhagic fever virus by targeting S gene Xingqi Liu, Zengguo Cao, Boyi Li, Pei Huang, Yujie Bai, Jingbo Huang, Zanheng Huang, Yuanyuan Zhang, Yuanyuan Li, Haili Zhang, Hualei Wang 2024, 23 (6): 2149-2153.   DOI: 10.1016/j.jia.2024.03.050 Abstract （107）      PDF in ScienceDirect       Reference | Related Articles | Metrics Select Inactivated H9N2 vaccines developed with early strains do not protect against recent H9N2 viruses: Call for a change in H9N2 control policy Yanjing Liu, Qingqing Yu, Xiangyu Zhou, Wenxin Li, Xinwen He, Yan Wang, Guohua Deng, Jianzhong Shi, Guobin Tian, Xianying Zeng, Hualan Chen 2024, 23 (6): 2144-2148.   DOI: 10.1016/j.jia.2024.05.001 Abstract （84）      PDF in ScienceDirect       Reference | Related Articles | Metrics Select A novel live attenuated vaccine candidate protects chickens against subtype B avian metapneumovirus Lingzhai Meng, Mengmeng Yu, Suyan Wang, Yuntong Chen, Yuanling Bao, Peng Liu, Xiaoyan Feng, Tana He, Ru Guo, Tao Zhang, Mingxue Hu, Changjun Liu, Xiaole Qi, Kai Li, Li Gao, Yanping Zhang, Hongyu Cui, Yulong Gao 2024, 23 (5): 1658-1670.   DOI: 10.1016/j.jia.2023.10.025 Abstract （151）      PDF in ScienceDirect       Avian metapneumovirus (aMPV) is a highly contagious pathogen that causes acute upper respiratory tract diseases in chickens and turkeys, resulting in serious economic losses.  Subtype B aMPV has recently become the dominant epidemic strain in China.  We developed an attenuated aMPV subtype B strain by serial passaging in Vero cells and evaluated its safety and efficacy as a vaccine candidate.  The safety test showed that after the 30th passage, the LN16-A strain was fully attenuated, as clinical signs of infection and histological lesions were absent after inoculation.  The LN16-A strain did not revert to a virulent strain after five serial passages in chickens.  The genomic sequence of LN16-A differed from that of the parent wild-type LN16 (wtLN16) strain and had nine amino acid mutations.  In chickens, a single immunization with LN16-A induced robust humoral and cellular immune responses, including the abundant production of neutralizing antibodies, CD4+ T lymphocytes, and the Th1 (IFN-γ) and Th2 (IL-4 and IL-6) cytokines.  We also confirmed that LN16-A provided 100% protection against subtype B aMPV and significantly reduced viral shedding and turbinate inflammation.  Our findings suggest that the LN16-A strain is a promising live attenuated vaccine candidate that can prevent infection with subtype B aMPV. Reference | Related Articles | Metrics Select Control of highly pathogenic avian influenza through vaccination Xianying Zeng, Jianzhong Shi, Hualan Chen 2024, 23 (5): 1447-1453.   DOI: 10.1016/j.jia.2024.03.044 Abstract （201）      PDF in ScienceDirect       The stamping-out strategy has been used to control highly pathogenic avian influenza viruses in many countries, driven by the belief that vaccination would not be successful against such viruses and fears that avian influenza virus in vaccinated birds would evolve more rapidly and pose a greater risk to humans.  In this review, we summarize the successes in controlling highly pathogenic avian influenza in China and make suggestions regarding the requirements for vaccine selection and effectiveness.  In addition, we present evidence that vaccination of poultry not only eliminates human infection with avian influenza virus, but also significantly reduces and abolishes some harmful characteristics of avian influenza virus. Reference | Related Articles | Metrics Select A nanobody-based blocking enzyme-linked immunosorbent assay for detecting antibodies against pseudorabies virus glycoprotein E Huanhuan Lü, Pinpin Ji, Siyu Liu, Ziwei Zhang, Lei Wang, Yani Sun, Baoyuan Liu, Lizhen Wang, Qin Zhao 2024, 23 (4): 1354-1368.   DOI: 10.1016/j.jia.2023.09.033 Abstract （149）      PDF in ScienceDirect       Pseudorabies (PR) is an acute infectious disease of pigs caused by the PR virus (PRV) and results in great economic losses to the pig industry worldwide.  PRV glycoprotein E (gE)-based enzyme-linked immunosorbent assay (ELISA) has been used to distinguish gE-deleted vaccine-immunized pigs from wild-type virus-infected pigs to eradicate PR in some countries.  Nanobody has the advantages of small size and easy genetic engineering and has been a promising diagnostic reagent.  However, there were few reports about developing nanobody-based ELISA for detecting anti-PRV-gE antibodies.  In the present study, the recombinant PRV-gE was expressed with a bacterial system and used to immunize the Bactrian camel.  Then, two nanobodies against PRV-gE were screened from the immunized camel by phage display technique.  Subsequently, two nanobody-HRP fusion proteins were expressed with HEK293T cells.  The PRV-gE-Nb36-HRP fusion protein was selected as the probe for developing the blocking ELISA (bELISA) to detect anti-PRV-gE antibodies.  Through optimizing the conditions of bELISA, the amount of coated antigen was 200 ng per well, and dilutions of the fusion protein and tested pig sera were separately 1:320 and 1:5.  The cut-off value of bELISA was 24.20%, and the sensitivity and specificity were 96.43 and 92.63%, respectively.  By detecting 233 clinical pig sera with the developed bELISA and a commercial kit, the results showed that the coincidence rate of two assays was 93.99%.  Additionallly, epitope mapping showed that PRV-gE-Nb36 recognized a conserved conformational epitope in different reference PRV strains.  Simple, great stability and low-cost nanobody-based bELISA for detecting anti-PRV-gE antibodies were developed.  The bELISA could be used for monitoring and eradicating PR. Reference | Related Articles | Metrics Select First identification of a novel Aichivirus D in goats with diarrhea Chen Yang, Keha-mo Abi, Hua Yue, Falong Yang, Cheng Tang 2024, 23 (4): 1442-1446.   DOI: 10.1016/j.jia.2023.11.041 Abstract （125）      PDF in ScienceDirect       Reference | Related Articles | Metrics Select Porcine enteric alphacoronavirus infection increases lipid droplet accumulation to facilitate the virus replication Qi Gao, Yongzhi Feng, Ting Gong, Dongdong Wu, Xiaoyu Zheng, Yizhuo Luo, Yunlong Yang, Zebu Song, Lang Gong, Guihong Zhang 2024, 23 (3): 988-1005.   DOI: 10.1016/j.jia.2023.10.010 Abstract （129）      PDF in ScienceDirect       Coronaviruses are widely transmissible between humans and animals, causing diseases of varying severity.  Porcine enteric alphacoronavirus (PEAV) is a newly-discovered pathogenic porcine enteric coronavirus in recent years, which causes watery diarrhea in newborn piglets.  The host inflammatory responses to PEAV and its metabolic regulation mechanisms remain unclear, and no antiviral studies have been reported.  Therefore, we investigated the pathogenic mechanism and antiviral drugs of PEAV.  The transcriptomic analysis of PEAV-infected host cells revealed that PEAV could upregulate lipid metabolism pathways.  In lipid metabolism, steady-state energy processes, which can be mediated by lipid droplets (LDs), are the main functions of organelles.  LDs are also important in viral infection and inflammation.  In infected cells, PEAV increased LD accumulation, upregulated NF-κB signaling, promoted the production of the inflammatory cytokines IL-1β and IL-8, and induced cell death.  Inhibiting LD accumulation with a DGAT-1 inhibitor significantly inhibited PEAV replication, downregulated the NF-κB signaling pathway, reduced the production of IL-1β and IL-8, and inhibited cell death.  The NF-κB signaling pathway inhibitor BAY11-7082 significantly inhibited LD accumulation and PEAV replication.  Metformin hydrochloride also exerted anti-PEAV effects and significantly inhibited LD accumulation, downregulated the NF-κB signaling pathway, reduced the production of IL-1β and IL-8, and inhibited cell death.  LD accumulation in the lipid metabolism pathway therefore plays an important role in the replication and pathogenesis of PEAV, and metformin hydrochloride inhibits LD accumulation and the inflammatory response to exert anti-PEAV activity and reducing pathological injury.  These findings contribute new targets for developing treatments for PEAV infections. Reference | Related Articles | Metrics Select Establishment of an indirect immunofluorescence assay for the detection of African swine fever virus antibodies Wan Wang, Zhenjiang Zhang, Weldu Tesfagaber, Jiwen Zhang, Fang Li, Encheng Sun, Lijie Tang, Zhigao Bu, Yuanmao Zhu, Dongming Zhao 2024, 23 (1): 228-238.   DOI: 10.1016/j.jia.2023.05.021 Abstract （203）      PDF in ScienceDirect       African swine fever (ASF) continues to cause enormous economic loss to the global pig industry.  Since there is no safe and effective vaccine, accurate and timely diagnosis of ASF is essential to implement control measures.  Indirect immunofluorescence assay (IFA) is a gold standard serological method recommended by the World Organization for Animal Health (WOAH).  In this study, we used primary fetal kidney cells to establish a wild boar cell line (BK2258) that supported the efficient replication of ASF virus (ASFV) SD/DY-I/21 and showed visible cytopathic effect (CPE).  Moreover, using BK2258, we established a sensitive and specific IFA for ASFV antibody detection.  To standardize and evaluate the performance of this assay, we used serum samples from pigs infected with the low virulent genotype I SD/DY-I/21 and genotype II HLJ/HRB1/20, and immunized with the vaccine candidate HLJ/18-7GD, field samples, and negative serum samples.  The IFA reacted with the ASFV-positive sera and displayed bright fluorescence foci.  There was no non-specific green fluorescence due to cellular senescence or other cell damage-causing factors.  Compared to a commercial indirect enzyme-linked immunosorbent assay (iELISA), ASFV antibodies were detected 1–4 days earlier using our IFA.  The detection limits of the IFA and iELISA for the same ASFV-antibody positive serum samples were 1:25,600 and 1:6,400, respectively, indicating that the IFA is more sensitive than iELISA.  The newly established IFA was highly specific and did not cross-react with sera positive for six other important porcine pathogens (i.e., Classical swine fever virus (CSFV), Porcine reproductive and respiratory syndrome virus (PRRSV), Porcme circovirus type 2 (PCV2), Pseudorabies virus (PRV), Foot-and-Mouth disease virus type O (FMDV/O), and Porcine epidemic diarrhea virus (PEDV)).  This study thus provides a sensitive, specific, and reliable detection method that is suitable for the serological diagnosis of ASF. 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