Pseudorabies virus (PRV) is a large double-stranded DNA alphaherpesvirus with a genome of approximately 145kb containing nearly 70 genes. To systematically profile gene functions, we constructed a genome-wide mutant library by performing Tn5 transposon mutagenesis on the PRV infectious clone pBAC-JS2012. Through transposon sequencing, 277 BAC plasmids (pBAC-JS2012) containing a single-copy transposon insertion in the PRV genome were screened from 352 clones, achieving mutations in 52 viral genes. Functional characterization demonstrated that 20 genes were essential for viral replication, while 8 genes, though non-essential, significantly modulated replication efficiency in vitro. Mutations in other 22 genes showed no significant effect on viral replication. In a mouse infection model, eight genes were identified as critical determinants of viral pathogenicity. Collectively, this genome-wide analysis delineates the functional landscape of PRV, classifying genes essential for replication and those contributing to virulence. This study not only advances the understanding of PRV genome organization and function, but also provides a rational basis for developing targeted antiviral interventions and attenuated vaccines.

African swine fever, which is induced by the African swine fever virus (ASFV), poses a significant threat to the global pig industry due to its high lethality in domestic pigs and wild boars. Despite the severity of the disease, there is a lack of effective vaccines and drugs against the ASFV. The p72 protein, constituting 31 to 33% of the total virus particle mass, serves as the primary capsid protein of ASFV. It is a crucial antigen for the development of ASF subunit vaccines and serological diagnostic methods. In this investigation, 27 monoclonal antibodies (mAbs) were generated through mouse immunization with the truncated C-terminal p72 protein expressed by Escherichia coli. Among these, six mAbs exhibited binding to the p72 trimer, with their respective recognized epitopes identified as 542VTAHGINLIDKF553, 568GNAIKTP574, and 584FALKPREEY592. All three epitopes were situated within the interval sequences of functional units of the C-terminal jelly-roll barrel of p72. Notably, two epitopes, 568GNAIKTP574 and 584FALKPREEY592, were internal to the p72 trimer, while the epitope 542VTAHGINLIDKF553 was exposed on the surface of the trimer and consistently conserved across all ASFV genotypes. These findings enhance our comprehension of the antigenic function and structure of the p72 protein, facilitating the utilization of p72 in the development of diagnostic techniques for ASFV.

H9N2亚型禽流感病毒(AIV)抗原变异迅速,相继于2009年及2013年分别出现新的抗原亚群病毒,对该病毒的防控带来了极大的挑战.目前,关于H9N2禽流感病毒形成不同抗原亚群机制,尚不清楚.流感病毒血凝素蛋白(HA)的单克隆抗体是研究该机制的基础工具.为此,本研究通过血凝抑制(HI)实验筛选到1株新型的抗原亚群H9N2病毒H514.通过杂交瘤细胞融合技术以及间接免疫荧光的方法,筛选到5株抗H514病毒HA蛋白的单抗,均属于IgG亚型.Western blot结果表明5株单克隆抗体中只有5E9作用的是线性表位,其余4株皆是针对构象表位.进一步地研究发现,这5株单克隆抗体对H514株皆有HI活性,最高达到28.这些结果为H9N2亚型禽流感病毒的抗原性分子研究提供了重要材料.

近几年,禽流感病毒(AIV)跨越禽-犬屏障产生新型H3N2犬流感病毒,后者在亚洲多国的犬中广泛流行,造成潜在的公共卫生安全威胁.尽管如此,H3N2AIV如何跨宿主传播至犬的分子机制尚不清楚.为此,本研究利用早期筛选到的一株不感染犬和鼠,但感染禽的H3N2禽流感病毒A/Duck/Shanghai/01/2009(H3N2)(简称为SH01),通过RT-PCR技术扩增了病毒的8个全基因片段,并分别克隆至PLLB双向表达载体上.将构建成功的8个质粒纯化后共转染293T细胞,转染48 h后加入TPCK胰酶作用2h,将上清液和细胞一同接种9～11日龄SPF鸡胚,检测其血凝效价.经测序分析,确定获救病毒的8个基因片段序列与亲本毒株的序列完全一致,表明该病毒拯救成功.H3N2禽流感病毒反向遗传操作平台的成功建立为探索该病毒跨宿主传播犬的分子机制提供了工具.

The H9N2 subtype avian influenza virus (AIV) has become endemic in poultry globally; however due to its low pathogenicity, it is not under primary surveillance and control in many countries. Recent reports of human infection caused by H9N2 AIV has increased public concern. This study investigated the genetic and antigenic characteristics of H9N2 AIV isolated from local markets in nine provinces in Southern China from 2013 to 2018. We detected an increasing annual isolation rate of H9N2 AIV. Phylogenetic analyses of hemagglutinin (HA) genes suggests that isolated strains were rooted in B.194 lineage but have evolved into new subgroups (II and III), which derived from subgroup I. The estimated substitution rate of the subgroup III strains was 6.23 x 10(-3) substitutions/site/year, which was 1.5-fold faster than that of the average H9N2 HA rate (3.95 x 10(-3 )substitutions/site/year). Based on the antigenic distances, subgroup II and III strains resulted in two clear antigenic clusters 2 and 3, separated from the vaccine strain F98, cluster 1. New antigenic properties of subgroup III viruses were associated with 11 amino acid changes in the HA protein, suggesting antigenic drift in H9N2 viruses. Our phylogenetic and antigenic analyses of the H9N2 strains circulating in local markets in Southern China provide new insights on the antigenic diversification of H9N2 viruses. IMPORTANCE The H9N2 low pathogenicity avian influenza (LPAI) virus has become endemic in poultry globally. In several Asian countries, vaccination against H9N2 avian influenza virus (AIV) was approved to reduce economic losses in the poultry industry. However, surveillance programs initiated after the introduction of vaccination identified the persistence of H9N2 AIV in poultry (especially in chicken in South Korea and China). Recent reports of human infection caused by H9N2 AIV has increased public concern. Surveillance of H9N2 circulating in poultry in the fields or markets was essential to update the vaccination strategies. This study investigated the genetic and antigenic characteristics of H9N2 ATVs isolated from local markets in nine provinces in Southern China from 2013 to 2018. The discovery of mutations in the hemagglutinin (HA) gene that result in antigenic changes provides a baseline reference for evolutionary studies of H9N2 viruses and vaccination strategies in poultry.

细胞培养流感病毒生产疫苗过程中细胞系的选择至关重要,本研究建立了可控表达H9N2亚型禽流感病毒(AIV)HA蛋白的MDCK细胞系,能有效支持H9N2流感病毒的扩增.本研究应用插入四环素调控元件和H9N2亚型禽流感病毒HA基因的重组质粒V2-H9,与辅助质粒pMDSV、psPAX2共转染293T细胞包装慢病毒,将包装出的慢病毒感染MDCK细胞,通过添加嘌呤霉素筛选出阳性的单克隆细胞.再用PR8 SH441病毒感染筛选出来的MDCK单克隆细胞.结果显示,流感病毒在其中4株MDCK单克隆细胞的HA效价接近于8;生长曲线结果显示,流感病毒在第34株MDCK单克隆细胞中复制效果最好;间接免疫荧光(IFA)和Western blot检验结果显示,第34株MDCK单克隆细胞H9 HA的表达量最高;将第34株MDCK单克隆细胞驯化后进行悬浮培养,每隔24 h测定细胞密度,结果表明,第34株MDCK单克隆细胞可以悬浮培养,且流感病毒在第34株MDCK悬浮细胞中HA效价可达9log2.以上结果表明,本研究获得了一株可高效扩增H9亚型禽流感病毒的MDCK细胞株,为流感弱毒疫苗的研发提供了新的研究方法和实验基础.