H9N2 avian influenza viruses pose a persistent zoonotic risk owing to their broad host adaptability. Between 2024 and 2025, two H9N2 isolates (ZHY1022B2 and ZHY0417A11) were recovered from quail flocks in Hebei, China. Phylogenetic analysis clustered their HA genes within the dominant B4.7.2 subclade, but diverged into two subgroups (B4.7.2.2 and B4.7.2.1). Of particular interest was ZHY0417A11, which displayed a multigenic reassortment pattern—its PB1 originated from an H3N8 virus, PA and PB2 from H3N3, NS from H10N3, and the M gene was nearly identical (99.32%) to a human H3N8 isolate, whereas the remaining HA, NA and NP segments maintained the H9N2 backbone. Despite the presence of the HA mammalian-adaptive markers (H191N, A198V and Q234L), both strains showed marked antigenic drift from the vaccine strain SS (BJ/94-like; R < 0.5), while retaining reactivity with currently circulating field strains. These findings argue for heightened vigilance in quail populations, given their role as mixing vessels, and highlight the limitations of current vaccine matching in light of ongoing H9N2 evolution.

Avian influenza viruses (AIVs) of the H6 subtype demonstrate broad host tropism and represent a persistent concern for both poultry and public health due to their documented potential for cross-species transmission. This study aimed to isolate and characterize two novel H6 subtype AIVs from duck farms in China, focusing on their genetic evolution, molecular characterization, and antigenic properties. Virus isolation was performed from AIV-positive cloacal swabs. Whole genomes were sequenced and phylogenetically analyzed. Bioinformatic tools were employed to identify critical amino acid motifs, glycosylation patterns, and reassortment events. Antigenic cross-reactivity was evaluated through hemagglutination inhibition (HI) assays. Two strains, designated as H6N6 (SHT) and H6N2 (GXG), were successfully isolated. Phylogenetic analysis classified their hemagglutinin (HA) genes into the ST/2853-like and ST/339-like lineages, respectively. The GXG strain was identified as a triple-reassortant virus, with its matrix (M) gene derived from an H9N2 AIV, and contained a unique arginine insertion at residue 169 of the HA. In contrast, the SHT strain possessed a deletion in the neuraminidase (NA) stalk region (residues 58-68). Asymmetric antigenic cross-reactivity was observed: antiserum against GXG partially inhibited SHT, while SHT antiserum showed no inhibition of GXG. Epidemiological data confirmed the dominance of the ST/2853-like lineage in China, with N6 as the predominant NA subtype from 2015 to 2022. In conclusion, the two H6 AIV isolates exhibit distinct genetic and antigenic characteristics, carry mammalian adaptation markers, and highlight a potential cross-species transmission risk. These findings underscore the necessity for continued surveillance and the development of appropriately matched vaccine candidates.

The H3 subtype avian influenza virus (AIV) poses a substantial global public health threat due to its high host adaptability and ongoing evolution. The recent emergence of novel H3N8 and H3N3 AIVs associated with cross-species transmission underscores the urgent need for enhanced epidemiological surveillance. In this study, we conducted surveillance and characterization of H3 AIVs based on a total of 737 poultry samples collected across 21 Chinese provinces from November 2022 to December 2023. Of these, 69 (9.4%) tested positive for H3 AIV by RT-qPCR, and one H3N8 isolate and ten H3N3 isolates were obtained for whole-genome characterization. We performed whole-genome sequencing, phylogenetic analysis, reassortment inference, and evaluation of key amino acid substitutions, alongside antigenic characterization using hemagglutination inhibition (HI) assays and an in vivo mouse challenge experiment. The H3N8 isolate was identified as a triple-reassortant virus possessing the Eurasian avian H3 gene, the North American avian N8 gene, and H9N2-derived internal genes. The H3N3 isolates represented reassortant viruses that had acquired the HA gene from the novel H3N8 AIV lineage, the NA gene from H10N3 AIV, and internal genes from H9N2 AIV. All isolates exhibited HA cleavage sites characteristic of low pathogenic avian influenza viruses. Additionally, several amino acid substitutions previously associated with enhanced mammalian adaptation were identified, including L89V and I292V in PB2 and H436Y in PB1. In a BALB/c mouse challenge experiment, the representative H3N8 virus established infection without prior adaptation and replicated predominantly in the upper respiratory tract, with detectable viral RNA in respiratory tissues and limited extrapulmonary dissemination. Antigenic analysis revealed no cross-reactivity between the novel H3 AIVs and H5, H7, or H9 AIVs as measured by HI. Based on molecular and phylogenetic characterization, antigenic assessment, and preliminary mammalian infection data, our findings provide evidence suggesting a potential public health risk. We recommend intensified surveillance of H3 AIVs in poultry and accelerated vaccine development to curb viral spread and improve public health preparedness.

Duck circovirus (DuCV) infection is an immunosuppressive disease that affects ducks and causes severe damage to their immune system. To elucidate the epidemiological characteristics of DuCV infection in China, a total of 2944 waterfowl samples were collected from 17 provinces from January to October 2022, and 612 DuCV-positive samples were identified. A descriptive statistical analysis was subsequently conducted. Furthermore, 51 near-full-length DuCV genome sequences were obtained, and molecular genetic evolution and recombinant analyses were performed. Geographically, Fujian Province had the highest rate of DuCV positivity (54.8%), followed by the Guangxi Zhuang Autonomous Region (30.4%). The rate of DuCV positivity was highest in samples from 21–40-day-old ducklings, accounting for 66.5% of the total positive samples. The most common pathogen involved in mixed infections with DuCV was parvovirus or Riemerella anatipestifer . Genetic and evolutionary analyses of the full genome sequences of 51 DuCV strains revealed that DuCV-1b and DuCV-2c were the most prevalent strains in China. Genetic recombination analysis suggested that the major parental sequences involved in the recombination of DuCV strains in ducks are present in Anhui, Sichuan, Shandong, and Guangxi. In addition, DuCV recombination events have occurred between strains with different genotypes or strains isolated from different countries. In summary, the DuCV epidemic in China is complex. There are two main co-circulating genotypes, those of the DuCV-1b and DuCV-2c strains, and coinfection of DuCV with other pathogens is a very common phenomenon in clinical practice. There is an urgent demand for vaccines against DuCV, and the protective efficacy of these vaccines against different DuCV genotypes needs to be carefully evaluated.

ABSTRACT African swine fever (ASF) is a highly contagious disease of domestic and wild pigs caused by the African swine fever virus (ASFV). The current research on ASF vaccines focuses on the development of naturally attenuated, isolated, or genetically engineered live viruses that have been demonstrated to produce reliable immunity. As a result, a genetically engineered virus containing five genes deletion was synthesized based on ASFV Chinese strain GZ201801, named ASFV-GZΔI177LΔCD2vΔMGF. The five-gene-deleted ASFV was safe and fully attenuated in pigs and provides reliable protection against the parental ASFV strain challenge. This indicates that the five-gene-deleted ASFV is a potential candidate for a live attenuated vaccine that could control the spread of ASFV.

为构建塞内卡病毒(Senecavirus A)CH/ZZ/2016株的感染性克隆,采用RT-PCR技术分3个cDNA片段对SVA/CH/ZZ/2016株全基因组进行扩增,并克隆至pcDNA3.1(+)真核表达质粒中.获得重组质粒pSVA-CH/ZZ经NheⅠ、KpnⅠ酶切及测序鉴定后,转染BHK-21细胞并转至PK-15细胞上进行盲传,直至出现细胞病变(CPE).对出现CPE的细胞上清液进行RT-PCR扩增、酶切、测序、间接免疫荧光试验鉴定.结果显示,成功构建了含SVA全长cDNA克隆的重组质粒,转染BHK-21细胞经PK-15细胞盲传至F5代出现SVA的典型CPE.细胞上清液经RT-PCR扩增、酶切、测序结果表明拯救病毒含有不同于亲本病毒的分子标记.间接免疫荧光试验进一步表明拯救了SVA病毒.病毒蚀斑形成和生长曲线表明,拯救毒株和亲本毒株的复制能力及增殖特性相似.本研究构建的CH/ZZ/2016株感染性克隆为进一步研究SVA的致病机理、基因功能及疫苗的研发奠定基础.

African swine fever virus (ASFV) is a large, double-stranded DNA virus that causes a fatal disease in pigs, posing a threat to the global pig industry. Whereas some ASFV proteins have been found to play important roles in ASFV-host interaction, the functional roles of many proteins are still largely unknown. In this study, we identified I73R, an early viral gene in the replication cycle of ASFV, as a key virulence factor. Our findings demonstrate that pI73R suppresses the host innate immune response by broadly inhibiting the synthesis of host proteins, including antiviral proteins. Crystallization and structural characterization results suggest that pI73R is a nucleic-acid-binding protein containing a Z & alpha; domain. It localizes in the nucleus and inhibits host protein synthesis by suppressing the nuclear export of cellular messenger RNA (mRNAs). While pI73R promotes viral replication, the deletion of the gene showed that it is a nonessential gene for virus replication. In vivo safety and immuno-genicity evaluation results demonstrate that the deletion mutant ASFV-GZ & UDelta;I73R is completely nonpathogenic and provides effective protection to pigs against wild-type ASFV. These results reveal I73R as a virulence-related gene critical for ASFV patho-genesis and suggest that it is a potential target for virus attenuation. Accordingly, the deletion mutant ASFV-GZ & UDelta;I73R can be a potent live-attenuated vaccine candidate.

为了建立一种能够方便、高效检测溶血性曼氏杆菌(Mannheimia haemolytica)的方法,试验将溶血性曼氏杆菌重组质粒pET-32a-PlpE转化至BL21(DE3)感受态细胞,然后经IPTG诱导表达并纯化.以重组蛋白PlpE为包被抗原,通过优化抗原最佳包被浓度、一抗及二抗最佳稀释度、最佳反应时间和最佳显色时间等试验条件,建立了检测PlpE抗体的间接ELISA方法.结果 表明:重组质粒pET-32a-PlpE转化至感受态细胞BL21 (DE3)后利用IPTG诱导,重组蛋白主要在沉淀中大量表达,经包涵体纯化试剂盒纯化后获得纯度较高的目的 蛋白.重组蛋白PlpE具有良好的反应性,且蛋白大小符合预期.确定的检测条件为抗原包被浓度2μg/mL,一抗稀释度1∶300,酶标二抗稀释度1∶80000,反应时间45 min,一抗反应时间30 min,显色时间15 min.用该方法检出溶血性曼氏杆菌阳性血清的最低效价可达1∶1280,检测牛病毒性腹泻病毒阳性血清、牛传染性鼻气管炎病毒阳性血清、口蹄疫病毒阳性血清、副结核分枝杆菌阳性血清、布鲁氏杆菌阳性血清,结果均为阴性;批内重复性试验和批间重复性试验变异系数分别为0.70％～4.70％和0.50％～5.60％,均小于6.00％,该方法重复性较好.说明本试验建立的检测PlpE抗体的间接ELISA方法具有较高的敏感性和特异性,可用于临床牛血清抗体的检测.

为了给规模化生产牛病毒性腹泻病毒(BVDV)和牛传染性鼻气管炎病毒(IBRV)提供悬浮细胞培养方法,本试验采用4种降血清方法,使贴壁MDBK细胞逐步适应了悬浮培养环境,最终获得了 1株MDBK悬浮细胞株,同时摸索出MDBK悬浮细胞株培养BVDV和IBRV抗原的最佳参数;将MDBK悬浮细胞株扩大到60 L和500 L后连同MDBK贴壁细胞一同接种BVDV和IBRV,比较3种细胞培养后的病毒滴度;通过使用悬浮培养方法制备BVDV-IBRV二联灭活疫苗,按照2.00、1.00、0.50 mL/头和0.25 mL/头4个剂量免疫健康牛,确定其攻毒保护效果,继而免疫不同年龄黑白花奶牛群体(包括犊牛、后备母牛和成年母牛),检测免疫后牛群的BVDV和IBRV中和抗体效价.结果显示:在60 L和500 L规模化放大培养过程中,IBRV 病毒滴度各为 8.25 lgTCID50/mL 和 8.00 lgTCID50/mL,BVDV 病毒滴度各为 8.00 lgTCID50/mL 和 7.75 lgTCID50/mL,与MDBK贴壁细胞株产毒毒价几乎相同;免疫攻毒保护试验中,最低免疫剂量为1.0 mL/头,免疫保护率能在80％以上;二联灭活苗免疫的牛群试验中,IBRV和BVDV中和抗体几何平均值均显著高于未免疫组,达到了免疫保护效果.结果表明,本试验成功筛选到1株MDBK悬浮培养细胞株,并且通过悬浮培养工艺制备的BVDV-IBRV二联灭活疫苗免疫效果良好,该MDBK细胞全悬浮培养株的成功驯化为疫苗规模化制备提供了科学依据.

猪圆环病毒(PCV)为圆环病毒科圆环病毒属成员是目前已知最小的哺乳动物病毒.目前已确认PCV有4种基因型PCV1、PCV2、PCV3、PCV4.PCV1不能引起猪发病,PCV2不仅可引起断奶仔猪多系统衰竭综合征,且还与猪皮炎与肾病综合征(PDNS)、猪先天性震颤、母猪繁殖障碍、猪增生性和坏死性肺炎等疾病密切相关.通过流行病学调查发现PCV3感染多见于具有PDNS、繁殖障碍症状猪群,导致猪只心脏和多器官炎症.我国湖北、广东等多个省市猪群中也检测出PCV3.近年来,波兰和韩国相继报道存在PCV3感染猪群.作为一种被新发现的病原体,PCV3对猪致病性,现有商品化PCV2疫苗对其免疫效力等有待进一步研究, PCV3现已成为猪圆环病毒家族的又一重要新成员而引起大家重视.

为研究比较5种塞内卡病毒疫苗佐剂,分别用4种自主研发佐剂和进口ISA206佐剂配制塞内卡病毒灭活疫苗,检测疫苗的理化性质、安全性及免疫效力,并对检测结果进行对比分析.结果显示,进口ISA206佐剂疫苗黏度为43.02 cP,自主研发佐剂疫苗黏度均在27.88 cP以下,其他理化性质无差异;5批疫苗安全性试验均未见明显异常;5批疫苗免疫动物后均可诱导机体产生中和抗体,二免后14 d抗体滴度在211以上,4批自主研发佐剂疫苗免疫组抗体滴度水平高于进口ISA206佐剂疫苗约1个滴度,攻毒保护结果均在4/5以上,2批自主研发佐剂疫苗免疫组保护率可达5/5.结果表明,用4种自主研发佐剂制备的塞内卡病毒灭活疫苗质量不低于用进口ISA206佐剂制备的塞内卡病毒灭活疫苗.

为了明确猪场常用的油佐剂灭活疫苗与活疫苗混合后的免疫效果,本研究采用油佐剂猪口蹄疫0型、A型二价灭活疫苗与猪瘟活疫苗、猪伪狂犬病活疫苗混合后免疫60日龄商品猪,比较上述疫苗混合免疫与单独免疫之间的差异;通过检测各组免疫前后的抗体水平变化、细胞因子水平变化,评估混合免疫效果.结果显示,无论是混合免疫组还是单独免疫组,在二次免疫后4周,口蹄疫病毒抗体水平100％达到＞1 ∶ 128,混合免疫不影响口蹄疫疫苗的免疫效果;与单独免疫组相比,混合免疫组猪瘟病毒抗体水平无显著差异(P＞0.05),而猪伪狂犬病病毒gB抗体水平极显著提升(P＜0.01),表明油佐剂猪口蹄疫0型、A型二价灭活疫苗混合猪伪狂犬病活疫苗免疫能有效提升猪伪狂犬病活疫苗的免疫效果.结果表明,采用油佐剂口蹄疫疫苗稀释猪瘟、猪伪狂犬病活疫苗进行混合免疫不影响口蹄疫疫苗、猪瘟活疫苗的免疫效果,但可以极显著(P＜0.01)的提升猪伪狂犬病活疫苗的免疫效果.

为建立一种鉴别非洲猪瘟野毒株与基因缺失疫苗株的TaqMan探针荧光定量PCR检测方法,根据非洲猪瘟病毒(ASFV)的B646L、EP402R、MGF360-13L基因序列,分别设计PCR引物和TaqMan探针,绘制标准曲线,并进行重复性试验、特异性试验、敏感性试验与临床样品检测,建立三重TaqMan探针荧光定量PCR检测方法.结果显示,以B646L、EP402R和MGF360-13L重组质粒为标准品绘制的标准曲线具有良好的线性关系,线性相关系数(R2)分别为0.995、0.997和0.997;建立的方法与多种猪常见病原不存在交叉反应,特异性良好;对B646L、MGF360-13L与EP402R基因的检测下限均为10 copies/μL,变异系数均＜2％,该方法灵敏度高;当临床样品稀释至10-5时,即滴度为102.5TCID50/mL时仍能检测到病毒粒子,具有较高的临床使用价值.本研究建立了一种高效、灵敏、特异的ASFV分子检测方法,对ASF风险预警具有重要意义.

Newcastle disease virus (NDV), the causative agent that generally causes severe disease in poultry, continues to mutate and has thus evolved into 21 genotypes. We previously isolated a velogenic genotype III NDV JS/7/05/Ch that evolved from the vaccine strain Mukteswar, accompanying by amino acid mutations in Hemagglutinin-Neuraminidase (HN). Here, we sought to investigate the role of the mutant HN protein in NDV virulence. The HN genes of Mukteswar and JS/7/05/Ch were replaced reciprocally via reverse genetics, yielding two recombinant viruses rJS/MHN and rMu/JHN, respectively. rMu/JHN, in which the endogenous HN protein was replaced with the HN protein of JS/7/05/Ch, had a higher intravenous pathogenicity index (IVPI) value in chickens. Moreover, dual aa mutations (A494D and E495K from JS/7/05/Ch-type HN) were introduced into the HN protein of Mukteswar to generate the recombinant virus rMukHN494+495 JS . This virus showed an equivalent IVPI value to that of rJS/7/05/Ch (generated from parental JS/7/05/Ch via reverse genetics). In vitro and in vivo assays further showed that A494D and E495K in HN induced antigenic changes, a higher replication level and a more intense inflammatory response. Taken together, these findings indicate that aa mutations in HN are crucial for the virulence of the genotype III Newcastle disease (ND) vaccine strain after intravenous inoculation. Our study further highlights that close surveillance is needed to monitor the genetic variation of ND vaccine strains.

African swine fever virus (ASFV) represents a serious threat to the global swine industry, and there are no safe or commercially available vaccines. Previous studies have demonstrated that inactivated vaccines do not provide sufficient protection against ASFV and that attenuated vaccines are effective, but raise safety concerns. Here, we first constructed a deletion mutant in which EP153R and EP402R gene clusters were knocked out. Based on the deletion mutant, a further deletion from the MGF_360-12L, MGF_360-13L to MGF_360-14L genes was obtained. The five-genes knockout virus was designated as ASFV-ΔECM3. To investigate the efficacy and safety of the ASFV-ΔECM3 virus as a vaccine candidate, the evaluation of the virus was subsequently carried out in pigs. The results showed that the ASFV-ΔECM3 virus could induce homologous protection against the parental isolate, and no significant clinical signs or viremia were observed. These results show that the contiguous deletion mutant, ASFV-ΔECM3 encompassing the EP153R/EP402R and MGF_360-12L/13L/14L genes, could be a potential live-attenuated vaccine candidate for the prevention of ASFV infection.

猪圆环病毒是圆环病毒科圆环病毒属的DNA病毒,目前国内主要流行的为PCV-2.PCV-2感染后会出现断奶仔猪多系统衰竭综合征(PMWS)、猪皮炎肾病综合征(PDNS)、猪呼吸系统混合疾病、繁殖障碍综合征等.本实验在同一家猪场同一批次仔猪中,采用随机分组随机采样的方式,进行国产亚单位圆环病毒Ⅱ型疫苗和进口亚单位圆环病毒Ⅱ型疫苗免疫效果评估的实验,并对生产性能数据进行统计和分析,结果表明:免疫国产亚单位圆环病毒Ⅱ型圆环疫苗仔猪的生产性能略优于免疫进口亚单位圆环病毒Ⅱ型疫苗的仔猪.本实验为广大养殖户选择圆环疫苗提供了一定的实践依据.

为研究免疫塞内卡病毒(SVA)灭活疫苗后豚鼠、家兔与猪血清中和抗体的相关性,分别用0.25、0.5、1.0、2.0 mL的SVA灭活疫苗接种豚鼠和家兔,同时以2.0 mL的SVA灭活疫苗接种猪,分别于免疫前以及免疫后第7天、第14天、第21天、第28天采血液,测定各动物血清中SVA中和抗体,利用Microsoft Excel软件对所得结果进行整理并分析豚鼠与猪血清SVA中和抗体、家兔与猪血清SVA中和抗体之间的线性关系,进一步采用IBM SPSS Statistics 19软件对线性关系的显著性进行分析.结果显示,豚鼠和家兔均可产生SVA中和抗体,且免疫剂量越大,免疫动物产生的SVA中和抗体滴度越高.猪免疫后产生了SVA中和抗体,监测期内随着时间的延长,SVA中和抗体水平逐渐升高.当家兔和豚鼠免疫剂量为1.0 mL时,二者产生的SVA中和抗体与猪SVA中和抗体呈正相关,相关性分别为0.990和0.998,相关性极显著.研究表明,采用1.0 mL的剂量免疫豚鼠或家兔,能间接反应SVA灭活疫苗在猪体的SVA中和抗体水平.

为检测从内蒙古发病牛场分离到的3株牛支原体(HS2019、HSZ2019、HSS2019)的致病性,对其进行本动物回归试验,通过观察攻毒后的临床症状、病理变化,以及应用实时荧光定量PCR确定组织器官中支原体载量,分析3株支原体的毒力.结果显示,3株牛支原体回归牛体后均使试验牛出现体温升高、咳嗽、呼吸困难等临床症状,解剖可观察到试验牛肺部损伤以及肺脏与胸腔粘连的病理变化,其中HS2019株较其他两株引起的症状与病变更为明显,组织脏器中的载菌量最高.结果表明,这3株支原体均有致病性,其中HS2019株致病性最强,可作为今后疫苗研制的预备菌株.本研究既为国内疫苗的研制提供了菌株资源,也为今后牛支原体的免疫攻毒试验提供了评价标准.

African swine fever virus (ASFV) is a large nucleoplasmic DNA virus, in which the genome is around 170-198 kilobases (kb). More than 50 % genes have unknown functions. Here, MGF100-1R gene is chosen to study the primary function and sublocalization. The gene was located at the left variable region of the ASFV genome that belongs to MGF100 families. It located at the cytoplasm without cytotoxic activities. However, it related to induce the transcriptional levels of pro-inflammatory cytokines. A deletion mutant of MGF100-1R gene was constructed based on ASFV Chinese strain GZ201801. The recombinant deletion mutant (ASFV△MGF100-1R) was demonstrated in vitro that the gene is non-essential for virus replication with a similar replication kinetics in bone marrow-derived macrophages (BMDMs) cell cultures when compared to parental virus. In vivo evaluation, ASFV△MGF100-1R was inoculated intramuscularly and led to a similar pathogenesis that caused by the parental ASFV GZ201801, confirming that deletion of MGF100-1R gene from the ASFV genome does not impact virulence.