Hepatitis caused by fowl adenovirus serotype 4 (FAdV-4) is a serious avian disease associated with huge losses to the poultry industry worldwide. Herein, we document the first outbreak of FAdV-4 infection in blue-breasted quail (Excalfactoria chinensis). The causative agent, designated as HNSQ2023, was isolated and characterized as an FAdV-4 epidemic strain using PCR amplification, DNA sequencing, and IFA staining. Comparative genomic analysis of the viral genomes of 79 virulent or non-pathogenic FAdV-4 strains showed that the gene order and shared open reading frames of HNSQ2023 were similar to those of previously reported virulent FAdV-4 strains. It also contains 100% conserved amino acid mutations in major structural proteins such as hexon and fiber-2. This is similar to the virulent FAdV-4 strains, except for an irregular mutation at aa46 (A to T) in fiber-1. Notably, the newly identified HNSQ2023 isolated from Excalfactoria chinensis and the other five epidemic FAdV-4 strains previously isolated from peacocks, chickens, ducks, and pigeons during 2018-2023 formed a close evolutionary sub-branch, distinct from the main branch, mostly composed of Chinese FAdV-4 strains originating from chickens. Furthermore, animal experiments demonstrated that HNSQ2023 retained high pathogenicity to SPF chickens and Excalfactoria chinensis, with 100% morbidity and over 90% mortality in both species. Our data shed new light on the high risk of cross-species transmission of FAdV-4 in domestic poultry and ornamental pet birds, stressing the importance of stricter biosecurity measures and necessary vaccinations for future control of this disease.

Outbreaks of joint and visceral gout in goslings caused by goose astrovirus (GAstV) continue to affect the goose industry in China. Currently, there is a lack of vaccines and drugs to treat GAstV-associated diseases. Due to the wide spread of GAstV through fecal-oral, vertical, and cross-host transmissions, the need to develop effective antiviral agents is urgent. In this study, the spike domain of the capsid protein of GAstV XX strain was targeted for the development of potential peptide ligands that possessed antiviral effect on GAstV infection. Molecular docking was performed to select the peptides with potential binding to the capsid spike of GAstV. The binding was then validated using an enzyme-linked immunosorbent assay (ELISA). After screening based on immuoperoxidase monolayer assay (IPMA), immunofluorescence assay (IFA), and Western blot, peptide AP21 was demonstrated to inhibit GAstV infection in LMH cells in a dose-dependent manner. The expression of capsid spike reduced significantly in GAstV-infected cells upon pre-incubation of the virus with AP21. In addition, AP21 reduced viral infectivity by more than 10-fold as determined by the 50% tissue culture infectious dose (TCID50) assay. Structural analysis showed that the binding sites of AP21 on capsid spike were located on the surface of the protein and these sites were not linear but discontinued. In summary, the identification of antiviral peptides based on virtual screening and experimental validation greatly facilitated the development of antiviral peptides, which is important for the prevention and control of GAstV-associated gout diseases.

BackgroundFowl adenovirus serotype 4 (FAdV-4) is the main pathogen of hepatitis-hydropericardium syndrome (HHS), which brings huge economic losses to the poultry industry worldwide. Fiber-1 protein plays an important role in viral infection and pathogenesis by binding directly to cellular receptors of FAdV-4. In particular, the knob domain of fiber-1 protein has been reported to induce the production of neutralizing antibodies and arouse protection against the lethal challenge of chickens with FAdV-4.MethodsThe fiber-1 knob (F1K) protein was expressed in a prokaryotic expression system and purified using Ni-NTA affinity chromatography. Monoclonal antibodies (mAbs) against FAdV-4 were generated by immunizing BALB/c mice with the purified F1K protein and screened using a series of immunoassays. Potential B cell epitopes on the knob domain of fiber-1 protein were mapped using enzyme-linked immunosorbent assay (ELISA) and dot-blot. Precious location and crucial amino acids of the identified epitopes were determined using peptide array scanning, truncations and alanine-scanning mutagenesis. The epitopes were analyzed and visualized on the knob trimer of FAdV-4 fiber-1 protein using the PyMOL software.ResultsWater-soluble recombinant fiber-1 knob (F1K) protein was obtained with the assistance of chaperone. Four monoclonal antibodies (5C10, 6F8, 8D8, and 8E8) against FAdV-4 were generated and characterized using indirect ELISA, Western blot, dot-blot, and immunological fluorescence assay (IFA). The mAbs were demonstrated to be from different hybridoma cell lines based on the sequences of the variable regions. Meanwhile, three distinct novel linear B-cell epitopes (319SDVGYLGLPPH329, 328PHTRDNWYV336, and 407VTTGPIPFSYQ417) on the knob domain of fiber-1 protein were identified and the key amino acid residues in the epitopes were determined. Structural analysis showed that the two adjacent epitopes 319SDVGYLGLPPH329 and 328PHTRDNWYV336 were exposed on the surface of the fiber-1 knob trimer, whereas the epitope 407VTTGPIPFSYQ417 was located inside of the spatial structure.ConclusionThis was the first identification of B-cell epitopes on the knob domain of fiber-1 protein and these findings provided a sound basis for the development of subunit vaccines, therapeutics, and diagnostic methods to control FAdV infections.

Classical swine fever (CSF), caused by CSF virus (CSFV), is a highly contagious disease affecting pigs and causing massive pig production losses with severe global economic recession. The immunization of live-attenuated vaccines is still one of the key measures to CSFV management in endemic countries. However, there are also strong controversies about the usage of live-attenuated vaccines, particularly in pregnant sows and young pigs, such as in Europe, where domestic pigs are routinely not vaccinated until severe outbreaks occur. Here, we report a CSF outbreak in a pig farm in China, which affected more than 90% of the delivery sows and led to ∼45% birth loss. Surprisingly, phylogenetic analysis showed that the CSFV isolate (named CSFV/HeNLY2022, GenBank No. OR195698) was clustered into subgenotype 1.1a, closely together with the live-attenuated vaccine strains. Further genomic analysis also revealed that the isolate CSFV/HeNLY2022 shared the highest nucleotide identity of 99.7% with the C/HVRI vaccine strain (C-strain, GenBank No. AY805221). Moreover, compared to the C/HVRI strain, a total of eight amino acid mutations, distributed in Erns (H436thY and S476thR), E1 (T502thI and P581thT), E2 (M979thK and A1061thS), NS5A (A2980thT), and NS5B (I3818thM), were characterized in the CSFV/HeNLY2022 isolate. Our results suggested that the CSF outbreak was most likely caused by the live-attenuated CSFV vaccine or its derivative. It raises concern that the unscientific application of CSFV vaccines could potentially lead to CSFV spread in pigs. It is needed to perform a more rigorous evaluation of the safety of the C-strain-derived vaccines in combination with other different live-attenuated vaccines.

(1) Goose astrovirus (GAstV) is a novel emerging pathogen that causes significant economic losses in waterfowl farming. A convenient, sensitive, and specific detection method for GAstV in field samples is important in order to effectively control GAstV. Droplet digital polymerase chain reaction (ddPCR) is a novel, sensitive, good-precision, and absolute quantitation PCR technology which does not require calibration curves. (2) In this study, we developed a ddPCR system for the sensitive and accurate quantification of GAstV using the conserved region of the ORF2 gene. (3) The detection limit of ddPCR was 10 copies/µL, ~28 times greater sensitivity than quantitative real-time PCR (qPCR). The specificity of the test was determined by the failure of amplification of other avian viruses. Both ddPCR and qPCR tests showed good repeatability and linearity, and the established ddPCR method had high sensitivity and good specificity to GAstV. Clinical sample test results showed that the positive rate of ddPCR (88.89%) was higher than that of qPCR (58.33%). (4) As a result, our results suggest that the newly developed ddPCR method might offer improved analytical sensitivity and specificity in its GAstV measurements. The ddPCR could be widely applied in clinical tests for GAstV infections.

RESEARCH HIGHLIGHTS:A sandwich ELISA was developed to detect EDSV using the mAbs 5G4 and HRP-6G6.The sandwich ELISA maintained high specificity and sensitivity.The sandwich ELISA had equivalent consistency with real-time PCR assay.

Background Porcine deltacoronavirus (PDCoV) is one of the emerging swine enteric coronaviruses (SECoVs), which has been widely prevalent in the North America and Asia. In addition to causing severe diarrhea in piglets, PDCoV also shows the potential to infect diverse host species, including calves, chickens, turkey poults, and humans. However, the clinical pathogenicity and genetic evolution of PDCoV is still not fully understood. Results Here, we recorded an outbreak of a novel recombinant PDCoV strain (CHN-HeN06-2022) in a large nursery fattening pig farm. Genomic analysis showed that the CHN-HeN06-2022 strain shared 98.3-98.7% sequence identities with the Chinese and American reference strains. To clarify the evolutionary relationships, phylogenetic analysis was performed using the PDCoV genome sequences available in the GenBank database. Based on genetic distance and geographical distribution, the phylogenetic tree clearly showed that all the PDCoV sequences could be divided into lineage 1 and lineage 2, which were further classified into sublineage 1.1 (Chinese strains), 1.2 (the North American strains), 2.1 (the Southeast Asian strains), and 2.2 (Chinese strains). Corresponding to the evolutionary tree, we found that, compared to lineage 1, lineage 2 strains usually contain a continuous 6-nt deletion in Nsp2 and a 9-nt deletion in Nsp 3, respectively. Furthermore, recombination analysis suggested that the CHN-HeN06-2022 occurred segments exchange crossed Nsp2 and Nsp3 region between sublineage 1.1 and sublineage 2.1. Combined with previously reported recombinant strains, the highest recombination frequency occurred in Nsp2 , Nsp3 , and S gene. Additionally, we identified a total of 14 amino acid sites under positive selection in spike protein, most of which are located in the regions related with the viral attachment, receptor binding, and membrane fusion. Conclusions Taken together, our studies provide novel insights into the genetic diversity and adaptive evolution of PDCoV. It would be helpful to the development of vaccine and potential antiviral agent.

Goose astrovirus (GAstV) is a newly discovered astrovirus. GAstV causes gout and death in 4- to -16-day-old goslings. For the past few years, fatal gout, the cardinal clinical symptom of gosling infected with GAstV, has been spreading rapidly in some goose Chinese farms, which caused continuous economic losses to the goose breeding industry in China. Currently, several underlying mechanisms involved in viral replication, inflammatory reaction, virions release, and viral pathogenesis of GAstV remain to be elucidated. In this study, we explored the mechanisms of GAstV-host interactions, the transcriptome and proteome profiles of GAstV-infected LMH cells were sequenced by RNA-seq and data-independent acquisition (DIA) techniques, respectively, and followed using an integrative analysis. Compared with uninfected LMH cells, a total of 322 differentially expressed genes (DEG) (195 up-regulated, 127 down-regulated) and 36 differentially expressed proteins (DEP) (31 up-regulated, 5 down-regulated) were detected. Nine DEGs were randomly selected for further validation by quantitative real-time polymerase chain reaction (qPCR). Through GO and KEGG enrichment analysis, DEG and DEP were significantly enriched in several important cellular signaling pathways, including MAPK, PI3K-Akt, cAMP, chemokine, calcium, phospholipase D, Ras, TNF, IL-17, Rap1, NF-kappa B signaling pathways, indicating that GAstV affects cell growth and immune signaling. This study provided an overview of changes in transcriptome and proteome profiles of GAstV-infected LMH cells, therefore, providing a crucial basis to further explore the mechanisms of GAstV-host interactions.

猪瘟(classical swine fever,CSF)是危害猪健康的重要传染病之一,具有急性、热性和高度接触性等特性,给世界养猪业造成了巨大的经济损失.PCR-ELISA 检测方法是在PCR和 ELISA 基础上创新的一种新技术,其灵敏性和准确性都高于单纯的 PCR 检测和ELISA检测.根据GenBank中猪瘟石门株序列,设计特异性引物,分别在 5'端标记生物素和地高辛,经过PCR扩增后,加入到包被有链霉亲和素的ELISA反应板上,然后通过DIG-HRP抗体进行显色反应,建立PCR-ELISA检测方法.结果表明:PCR-ELISA 最低检测到 1.81×104 拷贝/μL,普通 PCR 最低检测到 1.81×106 拷贝/μL,因此 PCR-ELISA 灵敏性比普通PCR高约 100 倍;对 27 份未知样品进行检测,普通 PCR 检测出阳性样品 15 份,阳性率为55.5%,PCR-ELISA检出 20 份阳性样品,阴性 7 份,阳性率为 74%,阳性检出率增加 18.5%;用建立的Real-Time PCR进行验证,检测结果与 PCR-ELISA 一致,两者的符合度为 100%;然后用PK15 细胞对普通PCR 未检测出的阳性样品进行病毒分离培养,通过间接免疫荧光(IFA)及普通PCR检测,结果显示该 5 份样品均为阳性.说明本研究所建立的 PCR-ELISA方法具有灵敏、特异、准确、快速、安全等优势,可应用于 CSFV 的快速诊断,为 CSFV 的流行病学监测提供有力保障.

Coronaviruses (CoVs) are a wide group of animal pathogens, which can cause a variety of diseases, and sometimes pose a great challenge on public health.1 CoVs belong to the Nidovirales order, Coronaviridae family, and Coronavirinae subfamily, which contains four genera-Alphacoronavirus (α-CoV), Betacoronavirus (β-CoV), Gammacoronavirus (γ-CoV) and Deltacoronavirus (δ-CoV).2 To date, seven human CoVs (HCoVs) have been identified: HCoV-NL63 and HCoV-229E belong to α-CoV, HCoV-OC43, HKU1, severe acute respiratory syndrome coronavirus (SARS-CoV), MERS-CoV, and the latest SARS-CoV-2 belong to β-CoV.1, 3 Importantly, all of them are descended from animal reservoirs. HCoV-OC43 and HKU1 are considered likely to be originated from rodents, HCoV-NL63 HCoV-229E, SARS-CoV, MERS-CoV, and SARS-CoV-2 are very likely passed from bats to humans in a zoonotic event, although the supposed origins of SARS-CoV and SARS-CoV-2 are still in debate.2, 3 Wild animals (e.g., palm civets for SARS-CoV, camels for MERS-CoV) usually play important roles as intermediate hosts that enable virus spill over from natural hosts to humans.2 Although the exact intermediate host for SARS-CoV-2 spread to humans are controversial, wild animals, including raccoon dogs, bamboo rats, and pangolin, are thought to have the highest likehood.1, 3 The ongoing SARS-CoV-2 pandemic has brought the zoonotic CoVs, especially those with a potential for cross-species transmission, into unprecedented attention worldwide. Swine CoVs (SCoVs) are the major enteric and respiratory pathogens in pigs. These include transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV), porcine respiratory coronavirus (PRCV), porcine hemagglutinating encephalomyelitis virus (PHEV), and the latest emerging SCoVs, porcine delta coronavirus (PDCoV), and swine acute diarrhea syndrome coronavirus (SADS-CoV).3 Among them, TGEV, PEDV, PRCV, SADS-CoV belong to α-CoV, PHEV belongs to β-CoV while PDCoV belongs to δ-CoV.2 All these SCoVs usually infect pigs. However, the newly emerging PDCoV and SADS-CoV have also shown broad host tropism,4, 5 suggesting a potential challenge to public health. Here, we discussed the recent progress of cross-species transmission and zoonotic potential of PDCoV and SADS-CoV. We also highlight the urgency of implementing preventive interventions.3 PDCoV was first reported in Hong Kong in 2012 and associated with severe diarrhea in suckling piglets in 2014 in the United States,6 while SADS-CoV was first detected from piglets with severe diarrhea in China in 2016.2 Retrospective studies showed that PDCoV was detectable in clinical porcine samples as early as 2004 and SADS-related CoVs (SADSr-CoVs) with 96−98% sequence identity to SADS-CoV were detected in 9.8% of anal swabs collected from different Rhinolophus species during 2013-2016.2, 6 Currently, PDCoV is widely prevalent in pigs in the North America, East Asia and Southeast Asia.3 Studies suggest that aquatic birds may serve as natural reservoirs for δ-CoVs and terrestrial birds may represent spill over hosts (Figure 1).6 Phylogenetic analyses suggest that PDCoV originated relatively recently from a host-switching event between birds and mammals, and sparrows may play a role as intermediate host for the adaptive evolution of PDCoV.6, 7 However, SADS-CoV was initially identified as a HKU2-related bat coronavirus at the time of its first finding. The transmission directions of SADS-CoV from bats to pigs are unclear and there are still no clues of the potential intermediate host.2 To date, SADS-CoV was limited in China and only four outbreaks have been independently reported in Guangdong, Jiangxi, Fujian and Guangxi provinces.8 The prevalent status of SADS-CoV suggest that the HKU2-related bat coronavirus may have jumped directly from bats to pigs through accidental events (Figure 1), so its adaptive evolution is still incomplete, which limits the transmission of SADS-CoV in pigs. Recent studies show that the newly emerging SCoVs have some biological properties that raised researchers concerns about their potential as zoonotic agents (Table 1, Figure 1). First, both PDCoV and SADS-CoV can infect and multiply in various cell lines derived from diverse host species, including swine, chicken, bovine, feline, monkey, and human.4, 5, 9, 11, 12, 15, 16, 18 Second, PDCoV and SADS-CoV have been demonstrated to be able to infect other species under experimental conditions, for example, the chicks, turkey poults, calves, and mice. Further, the infected animals also showed some clinical symptoms and pathological lesions, including diarrhea, mild respiratory symptoms, lesions in intestinal and lung tissues, and neuroinflammatory.3, 6, 10, 11, 15-18 Third, although SADS-CoV has not been detected in humans, independent natural infections of PDCoV to human has been identified in three Haitian children with fever, cough, and abdominal pain between 2014 and 2015.14 All these results imply that these emerging SCoVs possess broad species tropism and have a great potential to pose harm to human health and public safety. Thus, it is warranted to implement One Health approach coronavirus surveillance to identify the etiology of patients with fever, respiratory and/or enteric infections caused by unknown pathogens. This will become clinically feasible when economic and convenient new diagnostic methods that can detect many pathogens simultaneously from a small amount of biological sample (e.g., nasopharyngeal/oropharyngeal swab sample, and blood) become available. Meanwhile, we can monitor persons who closely contact animals, such as farm workers, to detect both asymptomatic and symptomatic zoonotic infections. Another important concern is the biosafety and biosecurity issue of the proposed experiments. To evaluate their potential human compatibility, a series of experiments were conducted in vitro and vivo. On one hand, we did learn a great deal about the biological characteristics of these viruses and prepare us for future vaccines and therapeutics needs. On the other hand, the experiments make viruses easily accessible to different host species, inadvertently accelerating the adaptive evolution of viruses and possibly facilitating viruses to cross species barriers. For example, after going through passage in chickens and mice, the PDCoV HNZK-02 strain habored 2 amino acids mutation (D138H and Q641K) in S protein.13 SADS-CoV cell adapted strains also showed sequence differences. GDS04-P12 strain showed nucleotides insertion and deletion at multiple sites of the genome compared with CH/GD-01/2017 or CN/GDWT/2017 strains, although they were obtained from the same source in the first outbreaks.19 Moreover, most of the current research have been performed in biosafety level II (BSL-2) laboratories, which obviously increase the risk of virus spill over. Thus, researcher should be more cautious in conducting studies on these potentially high-risk viruses with the possibility of cross-species transmission, such as using pseudovirus system or in higher biosafety laboratories (BSL-3 or BSL-4), to avoid facilitating interspecies transmission and adaptation of the virus to a new species. Additionally, any research study involving potentially hazardous zoonotic viruses must be subject to the ethical principles of medical practice. In summary, current studies suggest that PDCoV and SADS-CoV represent two possible zoonotic animal viruses for human. It is necessary to take appropriate interventions to reduce the risk of cross species transmission, since poultry and livestock usually become suitable intermediate hosts or mixers for viruses (e.g., influenza viruses) to infect human beings. Considering the wide prevalence of PDCoV in pigs, it is a economically rational strategy to block the potential transmission routes by massive immunization with highly effective vaccine, similar to the successful experience in the H7N9 intervention.20 SADS-CoV is only limited and rarely outbroke in southern China, so it is more reasonable and efficient to adopt the strategy of eradication. Moreover, given that animal CoVs are important viruses seriously endangering human health in the past, present and future, it is also urgent to understand their pathogenic mechanisms and develop broad-spectrum anti-CoV drugs and pan-CoV vaccines. Zhenhua Guo: conceptualization, data collection and analysis, writing-original draft, and grant application. Qianyue Jin, Peng Li, and Guangxu Xing: proof reading and editing. Qingxia Lu and Gaiping Zhang: supervision, grant application and editing. All authors have read and approved the final manuscript. This work was supported by Natural Science Foundation of Henan (232300421170), Henan Major Science and Technology Projects (221100110600), Outstanding Youth Science Fund of Henan Natural Science Foundation (222300420051), and China Agriculture Research System of MOF and MARA (CARS-35). The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. The authors declare no conflict of interest. The data that support the findings of this study are available from the corresponding author upon reasonable request.

PRV belongs to the alphaherpesvirus and has recently re-emerged in China, causing severe economic losses. Recent studies also indicate that PRV may pose a potential public health challenge.

Outbreaks of hydropericardium hepatitis syndrome caused by fowl adenovirus serotype 4 (FAdV-4) with a novel genotype have been reported in China since 2015, with significant economic losses to the poultry industry. Fiber2 is one of the important structural proteins on FAdV-4 virions. In this study, the C-terminal knob domain of the FAdV-4 Fiber2 protein was expressed and purified, and its trimer structure (PDB ID: 7W83) was determined for the first time. A series of affinity peptides targeting the knob domain of the Fiber2 protein were designed and synthesized on the basis of the crystal structure using computer virtual screening technology. A total of eight peptides were screened using an immunoperoxidase monolayer assay and RT-qPCR, and they exhibited strong binding affinities to the knob domain of the FAdV-4 Fiber2 protein in a surface plasmon resonance assay. Treatment with peptide number 15 (P15; WWHEKE) at different concentrations (10, 25, and 50 μM) significantly reduced the expression level of the Fiber2 protein and the viral titer during FAdV-4 infection. P15 was found to be an optimal peptide with antiviral activity against FAdV-4 in vitro with no cytotoxic effect on LMH cells up to 200 μM. This study led to the identification of a class of affinity peptides designed using computer virtual screening technology that targeted the knob domain of the FAdV-4 Fiber2 protein and may be developed as a novel potential and effective antiviral strategy in the prevention and control of FAdV-4.

Pseudorabies virus (PRV) is an economically important viral agent affecting the swine industry in China. Accurate, rapid and simple detection is critical to PRV control and eradication. In the present study, a visible and low equipment-dependent recombinase-aid amplification assay integrated with lateral flow assay (RAA-LFA) was successfully developed to detect the PRV against the gE gene. The RAA-LFA did not react with the other swine pathogens, indicating the method has a good specificity. The limit of detection (LOD) for this RAA-LFA method was 21 copies per reaction against standard plasmids containing gE gene. Notably, the RAA-LFA can detect as low as 6.0 × 100 50 % tissue culture infective dose (TCID50) viral titer per reaction under nucleic-acid-extraction free condition. Clinical detection showed that the results detected by RAA-LFA were completely consistent with that of the qPCR assay. Taken together, the developed PRV RAA-LFA method provides approachability, comparable accuracy and sensitivity tool for PRV point-of-care testing (POCT), which is valuable to PRV control in areas where equipment and personnel resources are scarce.

US3蛋白激酶是伪狂犬病病毒(PRV)的一个重要毒力因子,研究显示US3参与了细胞骨架重排、病毒复制和传播、细胞凋亡和干扰素信号通路等多个生物学过程,鉴定新的和US3互作的宿主蛋白对于认识其生物学功能具有重要意义.本研究首先利用免疫共沉淀和pull-down技术验证了 US3和宿主膜联蛋白A2(ANXA2)的相互作用;然后利用RNAi技术,通过荧光定量PCR、病毒滴度测定和免疫印迹分析,发现敲低ANXA2的表达显著抑制了 PRV在PK-15细胞上的增殖;进一步发现过表达ANXA2可以抑制PRV或者凋亡刺激剂诱导的细胞凋亡,提示ANXA2具有负调控细胞凋亡的作用.本研究进一步完善了可以和US3蛋白互作的宿主蛋白成员,加深了人们对ANXA2生物学功能的理解.

Porcine circoviruses (PCVs) are distributed in swine herds worldwide and represent a threat to the health of domestic pigs and the profits of the swine industry. Currently, four PCV species, including PCV-1, PCV-2, PCV-3 and PCV-4, have been identified in China. Considering the ubiquitous characteristic of PCVs, the new emerged PCV-4 and the large scale of swine breeding in China, an overall analysis on codon usage bias for Chinese PCV sequences was performed by using the major proteins coding sequences (ORF1 and ORF2) to better understand the relationship of these viruses with their host. The data from genome nucleotide frequency composition and relative synonymous codon usage (RSCU) analysis revealed an overrepresentation of AT pair and the existence of a certain codon usage bias in all PCVs. However, the values of an effective number of codons (ENC) revealed that the bias was of low magnitude. Principal component analysis, ENC-plot, parity rule two analysis and correlation analysis suggested that natural selection and mutation pressure were both involved in the shaping of the codon usage patterns of PCVs. However, a neutrality plot revealed a stronger effect of natural selection than mutation pressure on codon usage patterns. Good host adaptation was also shown by the codon adaptation index analysis for all these viruses. Interestingly, obtained data suggest that PCV-4 might be more adapted to its host compared to other PCVs. The present study obtained insights into the codon usage pattern of PCVs based on ORF1 and ORF2, which further helps the understanding the molecular evolution of these swine viruses.

量子点具有独特的光学性质,近年已在材料科学、分析化学、生命科学、医学检验等学科领域得到广泛研究和应用.作为一种新型的荧光标记材料,量子点在兽医领域越来越受到重视.作者就量子点的特性、种类及其在兽医领域的研究做一综述,并提出了未来研究方向,为拓展量子点在动物疫病诊断、兽药研发、致病机制等方面的研究提供参考.

为进一步了解猪圆环病毒2型(PCV2)分子进化特点,以PCV2主要蛋白编码序列ORF1和ORF2为研究对象,对74株不同亚型(2a-2e)PCV2进行核酸组成、相对同义密码子使用频率(RSCU)、有效密码子数目(ENC)、中性绘图和密码自适应指数等生物信息学分析,以了解PCV2密码子使用模式及其影响因素.结果显示,相比AU含量,PCV2 GC含量较低(低于50％),但更倾向于使用以C和U结尾的密码子;RSCU值和对应分析显示在密码子使用上,各亚型PCV2间差异较小,但PCV2各亚型与寄主Sus.s间则存在较大的差异;ENC分析和中性分析显示,相比突变压力,各亚型PCV2密码子使用模式受到较大程度的自然选择的影响;CAI值分析结果进一步确定,相比PCV2 c和PCV2 e,PCV2 a、2 b和2 d在密码子使用上更适应其寄主.论文揭示了自然选择是影响PCV2密码子使用模式的重要因素,并分析了影响各PCV2亚型流行的因素,该结果有助于PCV2分子进化的研究和理解该病毒在其寄主内翻译的机制.

过氧化氢又称双氧水(H2O2),为常用的消毒灭菌剂,且含氧高、氧化能力强、具有良好的消毒杀菌功能、不产生二次污染等优点,但其因稳定性差、储存条件要求高、8％以上的双氧水被列入危化品管理的特点,极大地限制了其应用.因此对其进行改造并解决稳定性,将能极大地提高其临床应用范围.本试验研究了一种稳定性过氧化氢的制备方法,并对其杀菌效果及稳定性进行考察.

Goose astrovirus (GAstV), the major causative agent of visceral and joint gout in goslings, is a novel pathogen greatly threatening waterfowl industry. Importantly, the horizontal and vertical transmissibility of GAstV posed a great challenge for disease prevention and control. Given the absence of commercial vaccine, restricting vertical transmission and protecting susceptible goslings must be a priority. Although many detection methods have been established, there is no serological method to detect GAstV-specific antibody, greatly limiting inspection and elimination of infected breeding bird. In this study, the B-cell epitopes of GAstV capsid protein were predicted, and its core antigenic advantage domain (shCAP) was expressed and purified. After authenticating the antigenicity, the recombinant shCAP protein was taken as the coating antigen, and an easily accessible indirect enzyme-linked immunosorbent assay (ELISA) was established to detect GAstV-specific antibody. The working conditions, including antigen concentration, serum dilution and incubation time, blocking buffer concentration, and color developing time, were gradually optimized by checkerboard titration. The cut-off OD450 value of the indirect ELISA for positive sample was 0.379, and the analytical sensitivity was 1:800. There was no cross-reaction with sera against goose parvovirus (GPV), Tembusu virus (TUMV), H5 and H7 subtype avian influenza virus (AIV H5 + H7), and Newcastle disease virus (NDV). The assay was further applied to examine 73 breeding goose serum samples and shared excellent agreement of 93.5% (68/73) with western blot, which also suggested that GAstV is circulating in the goose population in China. In conclusion, the developed indirect ELISA is simple, specific, and sensitive, which will be greatly useful to screen GAstV infection and block vertical transmission. • B-cell epitopes of GAstV capsid protein were predicted and expressed as immunogen • A core antigenic advantage domain-based ELISA was established to detect GAstV-specific antibody • The established ELISA will contribute to inspection and elimination of infected breeding geese and provide a useful tool for large scale serological testing of GAstV in geese

针对非洲猪瘟病毒B646L、MGF505-2R和CD2v基因分别设计了引物和探针,经过条件优化,建立了基于Taq-Man探针技术的三重荧光定量PCR检测方法.结果显示,本研究建立的三重荧光定量PCR检测方法与猪场常见病毒的核酸不发生交叉反应,具有良好的特异性;敏感性分析显示,针对B646L、MGF505-2R和CD2v基因的最低检测下限分别为6.5,8.0和14.0 copies/μL;并且该方法在101～107 copies/μL模板范围内具有良好的线性关系,组间变异系数为0.05％～2.68％,组内变异系数为0.10％～1.17％,稳定性良好.进一步针对临床核酸样品的检测显示,本方法和OIE推荐的检测方法具有良好的一致性.本研究成功建立了非洲猪瘟病毒野毒株和基因缺失株的荧光定量PCR鉴别检测方法,为临床非洲猪瘟病毒的监测诊断提供了良好的技术支撑.