SARS-CoV-2 hijacks macropinocytosis to facilitate its entry and promote viral spike-mediated cell-to-cell fusion.

Revealing the mechanisms of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) entry and cell-to-cell spread might provide insights for understanding the underlying mechanisms of viral pathogenesis, tropism, and virulence. The signaling pathways involved in SARS-CoV-2 entry and viral spike-mediated cell-to-cell fusion remain elusive. In the current study, we found that macropinocytosis inhibitors significantly suppressed SARS-CoV-2 infection at both the entry and viral spike-mediated cell-to-cell fusion steps. We demonstrated that SARS-CoV-2 entry required the small GTPase Rac1 and its effector kinase p21-activated kinase 1 by dominant-negative and RNAi assays in human embryonic kidney 293T-angiotensin-converting enzyme 2 cells and that the serine protease transmembrane serine protease 2 reversed the decrease in SARS-CoV-2 entry caused by the macropinocytosis inhibitors. Moreover, in the cell-to-cell fusion assay, we confirmed that macropinocytosis inhibitors significantly decreased viral spike-mediated cell-to-cell fusion. Overall, we provided evidence that SARS-CoV-2 utilizes a macropinocytosis pathway to enter target cells and to efficiently promote viral spike-mediated cell-to-cell fusion.

Neuropilin-1 Facilitates Pseudorabies Virus Replication and Viral Glycoprotein B Promotes Its Degradation in a Furin-Dependent Manner.

Pseudorabies virus (PRV), which is extremely infectious and can infect numerous mammals, has a risk of spillover into humans. Virus-host interactions determine viral entry and spreading. Here, we showed that neuropilin-1 (NRP1) significantly potentiates PRV infection. Mechanistically, NRP1 promoted PRV attachment and entry, and enhanced cell-to-cell fusion mediated by viral glycoprotein B (gB), gD, gH, and gL. Furthermore, through in vitro coimmunoprecipitation (Co-IP) and bimolecular fluorescence complementation (BiFC) assays, NRP1 was found to physically interact with gB, gD, and gH, and these interactions were C-end Rule (CendR) motif independent, in contrast to currently known viruses. Remarkably, we illustrated that the viral protein gB promotes NRP1 degradation via a lysosome-dependent pathway. We further demonstrate that gB promotes NRP1 degradation in a furin-cleavage-dependent manner. Interestingly, in this study, we generated gB furin cleavage site (FCS)-knockout PRV (Δfurin PRV) and evaluated its pathogenesis; in vivo, we found that Δfurin PRV virulence was significantly attenuated in mice. Together, our findings demonstrated that NRP1 is an important host factor for PRV and that NRP1 may be a potential target for antiviral intervention. IMPORTANCE Recent studies have shown accelerated PRV cross-species spillover and that PRV poses a potential threat to humans. PRV infection in humans always manifests as a high fever, tonic-clonic seizures, and encephalitis. Therefore, understanding the interaction between PRV and host factors may contribute to the development of new antiviral strategies against PRV. NRP1 has been demonstrated to be a receptor for several viruses that harbor CendR, including SARS-CoV-2. However, the relationships between NRP1 and PRV are poorly understood. Here, we found that NRP1 significantly potentiated PRV infection by promoting PRV attachment and enhanced cell-to-cell fusion. For the first time, we demonstrated that gB promotes NRP1 degradation via a lysosome-dependent pathway. Last, in vivo, Δfurin PRV virulence was significantly attenuated in mice. Therefore, NRP1 is an important host factor for PRV, and NRP1 may be a potential target for antiviral drug development.

Comparative analysis of SARS-CoV-2 Omicron BA.2.12.1 and BA.5.2 variants.

The initial severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron subvariants, BA.1 and BA.2, are being progressively displaced by BA.5 in many countries. To provide insight on the replacement of BA.2 by BA.5 as the dominant SARS-CoV-2 variant, we performed a comparative analysis of Omicron BA.2.12.1 and BA.5.2 variants in cell culture and hamster models. We found that BA.5.2 exhibited enhanced replicative kinetics over BA.2.12.1 in vitro and in vivo, which is evidenced by the dominant BA.5.2 viral genome detected at different time points, regardless of immune selection pressure with vaccine-induced serum antibodies. Utilizing reverse genetics, we constructed a mutant SARS-CoV-2 carrying spike F486V substitution, which is an uncharacterized mutation that concurrently discriminates Omicron BA.5.2 from BA.2.12.1 variant. We noticed that the 486th residue does not confer viral replication advantage to the virus. We also found that 486V displayed generally reduced immune evasion capacity when compared with its predecessor, 486F. However, the surge of fitness in BA.5.2 over BA.2.12.1 was not due to stand-alone F486V substitution but as a result of the combination of multiple mutations. Our study upholds the urgency for continuous monitoring of SARS-CoV-2 Omicron variants with enhanced replication fitness.

Evaluating angiotensin-converting enzyme 2-mediated SARS-CoV-2 entry across species.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic represents a global threat, and the interaction between the virus and angiotensin-converting enzyme 2 (ACE2), the primary entry receptor for SARS-CoV-2, is a key determinant of the range of hosts that can be infected by the virus. However, the mechanisms underpinning ACE2-mediated viral entry across species remains unclear. Using infection assay, we evaluated SARS-CoV-2 entry mediated by ACE2 of 11 different animal species. We discovered that ACE2 of Rhinolophus sinicus (Chinese rufous horseshoe bat), Felis catus (domestic cat), Canis lupus familiaris (dog), Sus scrofa (wild pig), Capra hircus (goat), and Manis javanica (Malayan pangolin) facilitated SARS-CoV-2 entry into nonsusceptible cells. Moreover, ACE2 of the pangolin also mediated SARS-CoV-2 entry, adding credence to the hypothesis that SARS-CoV-2 may have originated from pangolins. However, the ACE2 proteins of Rhinolophus ferrumequinum (greater horseshoe bat), Gallus gallus (red junglefowl), Notechis scutatus (mainland tiger snake), or Mus musculus (house mouse) did not facilitate SARS-CoV-2 entry. In addition, a natural isoform of the ACE2 protein of Macaca mulatta (rhesus monkey) with the Y217N mutation was resistant to SARS-CoV-2 infection, highlighting the possible impact of this ACE2 mutation on SARS-CoV-2 studies in rhesus monkeys. We further demonstrated that the Y217 residue of ACE2 is a critical determinant for the ability of ACE2 to mediate SARS-CoV-2 entry. Overall, these results clarify that SARS-CoV-2 can use the ACE2 receptors of multiple animal species and show that tracking the natural reservoirs and intermediate hosts of SARS-CoV-2 is complex.

Aminopeptidase N Is an Entry Co-factor Triggering Porcine Deltacoronavirus Entry via an Endocytotic Pathway.

Porcine deltacoronavirus (PDCoV) is a recently discovered coronavirus that poses a potential threat to the global swine industry. Although we know that aminopeptidase N (APN) is important for PDCoV replication, it is unclear whether it is the primary functional receptor, and the mechanism by which it promotes viral replication is not fully understood. Here, we systematically investigated the roles of porcine APN (pAPN) during PDCoV infection of nonsusceptible cells, including in viral attachment and internalization. Using a viral entry assay, we found that PDCoV can enter nonsusceptible cells but then fails to initiate efficient replication. pAPN and PDCoV virions clearly colocalized with the endocytotic markers RAB5, RAB7, and LAMP1, suggesting that pAPN mediates PDCoV entry by an endocytotic pathway. Most importantly, our study shows that regardless of which receptor PDCoV engages, only entry by an endocytotic route ultimately leads to efficient viral replication. This knowledge should contribute to the development of efficient antiviral treatments, which are especially useful in preventing cross-species transmission. IMPORTANCE PDCoV is a pathogen with the potential for transmission across diverse species, although the mechanism of such host-switching events (from swine to other species) is poorly understood. Here, we show that PDCoV enters nonsusceptible cells but without efficient replication. We also investigated the key role played by aminopeptidase N in mediating PDCoV entry via an endocytotic pathway. Our results demonstrate that viral entry via endocytosis is a major determinant of efficient PDCoV replication. This knowledge provides a basis for future studies of the cross-species transmissibility of PDCoV and the development of appropriate antiviral drugs.

Isobavachalcone inhibits Pseudorabies virus by impairing virus-induced cell-to-cell fusion.

Pseudorabies virus (PRV) is an important pathogen that threatens the global swine industry. Currently, there is no effective drug that can clinically prevent or treat PRV infections. Isobavachalcone (IBC), a natural chalcone compound derived from Psoralea corylifolia, displays multiple biological activities, such as antibacterial, antifungal, and anticancer activities. Recently, it was found that IBC exhibited antiviral activity against an RNA virus, porcine reproductive and respiratory syndrome virus (PRRSV), in vitro. In the current study, we further demonstrated for the first time that IBC has a strong inhibitory effect on PRV. Through a viral luciferase expression assay, we showed that the inhibition step occurs mainly in the late stage of viral replication. Finally, via a cell-to-cell fusion assay, we demonstrated that IBC inhibits PRV by blocking virus-mediated cell fusion. Thus, IBC may be a candidate for further therapeutic evaluation against PRV infection in vivo.

CRISPR/Cas9-mediated 2-sgRNA cleavage facilitates pseudorabies virus editing.

Several groups have used CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) for DNA virus editing. In most cases, one single-guide RNA (sgRNA) is used, which produces inconsistencies in gene editing. In this study, we used a swine herpesvirus, pseudorabies virus, as a model to systematically explore the application of CRISPR/Cas9 in DNA virus editing. In our current report, we demonstrated that cotransfection of 2 sgRNAs and a viral genome resulted in significantly better knockout efficiency than the transfection-infection-based approach. This method could result in 100% knockout of ≤3500 bp of viral nonessential large fragments. Furthermore, knockin efficiency was significantly improved by using 2 sgRNAs and was also correlated with the number of background viruses. We also demonstrated that the background viruses were all 2-sgRNA-mediated knockout mutants. Finally, this study demonstrated that the efficacy of gene knockin is determined by the replicative kinetics of background viruses. We propose that CRISPR/Cas9 coupled with 2 sgRNAs creates a powerful tool for DNA virus editing and offers great potential for future applications.-Tang, Y.-D., Guo, J.-C., Wang, T.-Y., Zhao, K., Liu, J.-T., Gao, J.-C., Tian, Z.-J., An, T.-Q., Cai, X.-H. CRISPR/Cas9-mediated 2-sgRNA cleavage facilitates pseudorabies virus editing.

Isobavachalcone inhibits post-entry stages of the porcine reproductive and respiratory syndrome virus life cycle.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a pathogen of great economic significance that impacts the swine industry globally. Since the first report of a porcine reproductive and respiratory syndrome (PRRS) outbreak, tremendous efforts to control this disease, including various national policies and plans incorporating the use of multiple modified live-virus vaccines, have been made. However, PRRSV is still a significant threat to the swine industry, and new variants continually emerge as a result of PRRSV evolution. Several studies have shown that pandemic PRRSV strains have enormous genetic diversity and that commercial vaccines can only provide partial protection against these strains. Therefore, effective anti-PRRSV drugs may be more suitable and reliable for PRRSV control. In this study, we observed that isobavachalcone (IBC), which was first isolated from Psoralea corylifolia, had potent anti-PRRSV activity in vitro. Although many biological activities of IBC have been reported, this is the first report describing the antiviral activity of IBC. Furthermore, after a systematic investigation, we demonstrated that IBC inhibits PRRSV replication at the post-entry stage of PRRSV infection. Thus, IBC may be a candidate for further evaluation as a therapeutic agent against PRRSV infection of swine in vivo.

CRISPR/Cas9-mediated multiple single guide RNAs potently abrogate pseudorabies virus replication.

Pseudorabies virus (PRV) is a swine herpesvirus that causes significant morbidity and mortality in swine populations and has caused huge economic losses in the worldwide swine industry. Currently, there is no effective antiviral drug in clinical use for PRV infection; it is also difficult to eliminate PRV from infected swine. In our study, we set out to combat these swine herpesvirus infections by exploiting the CRISPR/Cas9 system. We designed 75 single guide RNAs (sgRNA) by targeting both essential and non-essential genes across the genome of PRV. We applied a firefly luciferase-tagged reporter PRV virus for high-throughput sgRNA screening and found that most of the sgRNAs significantly inhibited PRV replication. More importantly, using a transfection assay, we demonstrated that simultaneous targeting of PRV with multiple sgRNAs completely abolished the production of infectious viruses in cells. These data suggest that CRISPR/Cas9 could be a novel therapeutic agent against PRV in the future.

Open reading frames 1a and 1b of the porcine reproductive and respiratory syndrome virus (PRRSV) collaboratively initiate viral minus-strand RNA synthesis.

The porcine reproductive and respiratory syndrome virus (PRRSV) causes a persistent threat to the swine industry, especially when highly pathogenic PRRSV (HP-PRRSV) emerges. Previous studies have indicated that PRRSV RNA synthesis was correlated with HP-PRRSV virulence. PRRSV RNA synthesis includes genomic RNA and sub-genomic mRNA, and these processes require minus-strand RNA as a template. However, the mechanisms involved in PRRSV minus-strand RNA synthesis are not fully understood. A mini-genome system can be used to assess viral replication mechanisms and to evaluate the effects of potential antiviral drugs on viral replicase activities. In this study, we developed a mini-genome system that uses firefly luciferase as a reporter. Based on this system, we found that PRRSV RNA-dependent RNA polymerase nsp9 alone failed to activate virus minus-strand RNA synthesis. We also demonstrated that combinations of open reading frames 1a (ORF1a) and ORF1b are necessary for viral minus-strand RNA synthesis.

A novel viral vaccine platform based on engineered transfer RNA.

In recent years, an increasing number of emerging and remerging virus outbreaks have occurred and the rapid development of vaccines against these viruses has been crucial. Controlling the replication of premature termination codon (PTC)-containing viruses is a promising approach to generate live but replication-defective viruses that can be used for potent vaccines. Here, we used anticodon-engineered transfer RNAs (ACE-tRNAs) as powerful precision switches to control the replication of PTC-containing viruses. We showed that ACE-tRNAs display higher potency of reading through PTCs than genetic code expansion (GCE) technology. Interestingly, ACE-tRNA has a site preference that may influence its read-through efficacy. We further attempted to use ACE-tRNAs as a novel viral vaccine platform. Using a human immunodeficiency virus type 1 (HIV-1) pseudotyped virus as an RNA virus model, we found that ACE-tRNAs display high potency for read-through viral PTCs and precisely control their production. Pseudorabies virus (PRV), a herpesvirus, was used as a DNA virus model. We found that ACE-tRNAs display high potency for reading through viral PTCs and precisely controlling PTC-containing virus replication. In addition, PTC-engineered PRV completely attenuated and lost virulence in mice in vivo, and immunization with PRV containing a PTC elicited a robust immune response and provided complete protection against wild-type PRV challenge. Overall, replication-controllable PTC-containing viruses based on ACE-tRNAs provide a new strategy to rapidly attenuate virus infection and prime robust immune responses. This technology can be used as a platform for rapidly developing viral vaccines in the future.

CRISPR-Cas13d Exhibits Robust Antiviral Activity Against Seneca Valley Virus.

In recent years, Seneca Valley virus (SVV) as a newly identified pathogen of porcine vesicular disease spread quickly and has posed a potential threat to the swine industry in several countries resulting in economic losses. Considering the evolution of SVV, attention should be given to controlling SVV epidemics. So far there are no commercial vaccines or drugs available to combat SVV. Therefore, development of strategies for preventing and controlling SVV infection should be taken into account. In the current study, we evaluated whether the CRISPR-Cas13d system could be used as a powerful tool against SVV infection. Besides, selected crRNAs showed different capacity against SVV infection. Our study suggests the CRISPR-Cas13d system significantly inhibited SVV replication and exhibited potent anti-SVV activity. This knowledge may provide a novel alternative strategy to control epidemics of SVV in the future.

Generation of Premature Termination Codon (PTC)-Harboring Pseudorabies Virus (PRV) via Genetic Code Expansion Technology.

Despite many efforts and diverse approaches, developing an effective herpesvirus vaccine remains a great challenge. Traditional inactivated and live-attenuated vaccines always raise efficacy or safety concerns. This study used Pseudorabies virus (PRV), a swine herpes virus, as a model. We attempted to develop a live but replication-incompetent PRV by genetic code expansion (GCE) technology. Premature termination codon (PTC) harboring PRV was successfully rescued in the presence of orthogonal system MbpylRS/tRNAPyl pair and unnatural amino acids (UAA). However, UAA incorporating efficacy seemed extremely low in our engineered PRV PTC virus. Furthermore, we failed to establish a stable transgenic cell line containing orthogonal translation machinery for PTC virus replication, and we demonstrated that orthogonal tRNAPyl is a key limiting factor. This study is the first to demonstrate that orthogonal translation system-mediated amber codon suppression strategy could precisely control PRV-PTC engineered virus replication. To our knowledge, this is the first reported PTC herpesvirus generated by GCE technology. Our work provides a proof-of-concept for generating UAAs-controlled PRV-PTC virus, which can be used as a safe and effective vaccine.

Evasion of Antiviral Innate Immunity by Porcine Reproductive and Respiratory Syndrome Virus.

Innate immunity is the front line for antiviral immune responses and bridges adaptive immunity against viral infections. However, various viruses have evolved many strategies to evade host innate immunity. A typical virus is the porcine reproductive and respiratory syndrome virus (PRRSV), one of the most globally devastating viruses threatening the swine industry worldwide. PRRSV engages several strategies to evade the porcine innate immune responses. This review focus on the underlying mechanisms employed by PRRSV to evade pattern recognition receptors signaling pathways, type I interferon (IFN-α/ß) receptor (IFNAR)-JAK-STAT signaling pathway, and interferon-stimulated genes. Deciphering the antiviral immune evasion mechanisms by PRRSV will enhance our understanding of PRRSV's pathogenesis and help us to develop more effective methods to control and eliminate PRRSV.

Pseudorabies Virus UL24 Abrogates Tumor Necrosis Factor Alpha-Induced NF-κB Activation by Degrading P65.

The transcription factor NF-κB plays a critical role in diverse biological processes. The NF-κB pathway can be activated by incoming pathogens and then stimulates both innate and adaptive immunity. However, many viruses have evolved corresponding strategies to balance NF-κB activation to benefit their replication. Pseudorabies virus (PRV) is an economically important pathogen that belongs to the alphaherpesvirus group. There is little information about PRV infection and NF-κB regulation. This study demonstrates for the first time that the UL24 protein could abrogate tumor necrosis factor alpha (TNF-α)-mediated NF-κB activation. An overexpression assay indicated that UL24 inhibits this pathway at or downstream of P65. Furthermore, co-immunoprecipitation analysis demonstrated that UL24 selectively interacts with P65. We demonstrated that UL24 could significantly degrade P65 by the proteasome pathway. For the first time, PRV UL24 was shown to play an important role in NF-κB evasion during PRV infection. This study expands our understanding that PRV can utilize its encoded protein UL24 to evade NF-κB signaling.

The Nsp12-coding region of type 2 PRRSV is required for viral subgenomic mRNA synthesis.

As one of many nonstructural proteins of porcine reproductive and respiratory syndrome virus (PRRSV), nonstructural protein 12 (Nsp12) has received relatively little attention, and its role in virus replication, if any, is essentially unknown. By the application of reverse genetic manipulation of an infectious PRRSV clone, the current study is the first to demonstrate that Nsp12 is a key component of PRRSV replication. In addition, the biochemical properties of Nsp12 were evaluated, revealing that Nsp12 forms dimers when exposed to oxidative conditions. Furthermore, we systemically analyzed the function of Nsp12 in PRRSV RNA synthesis using a strand-specific PCR method. To our surprise, Nsp12 was not found to be involved in minus-strand genomic RNA (-gRNA) synthesis; importantly, our results indicate that Nsp12 is involved in the synthesis of both plus- and minus-strand subgenomic mRNAs (+sgmRNA and -sgmRNA). Finally, we found that the combination of cysteine 35 and cysteine 79 in Nsp12 is required for sgmRNA synthesis. To our knowledge, we are the first to report the biological role of Nsp12 in the PRRSV lifecycle, and we conclude that Nsp12 is involved in the synthesis of both + sgRNA and -sgRNA.

Porcine alveolar macrophage CD163 abundance is a pivotal switch for porcine reproductive and respiratory syndrome virus infection.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a problematic virus that is difficult to control. The principal target cells for PRRSV infection are porcine alveolar macrophages (PAMs). Increasing evidence has demonstrated that CD163 is the determinant receptor for PRRSV infection. However, the relationship between CD163 abundance and PRRSV infection is unclear. In this study, we first generated primary immortalized PAMs (iPAMs) using SV40 large T antigen and demonstrated that CD163 expression is suppressed by the alternative splicing of mRNA in iPAMs. Two forms of CD163 transcripts were discovered, and most iPAMs expressed a short-form CD163 transcript that lacked from scavenger receptor cysteine-rich tandem repeat 1 (SRCR1) to SRCR5 of the functional domain. More importantly, using flow cytometric cell sorting technology, we isolated CD163-positive single-cell-derived clones with varying CD163 abundances to investigate the relationship between CD163 abundance and PRRSV infection. For the first time, we showed that cells with low CD163 abundance (approximately 20%) do not initiate PRRSV infection, while cells with moderate CD163 abundance display limited infection. PRRSV initiated efficient infection only in cells with high CD163 abundances. Our results demonstrate that CD163 abundance is a pivotal switch for PRRSV replication.

Comparison of Pathogenicity-Related Genes in the Current Pseudorabies Virus Outbreak in China.

There is currently a pandemic of pseudorabies virus (PRV) variant strains in China. Despite extensive research on PRV variant strains in the past two years, few studies have investigated PRV pathogenicity-related genes. To determine which gene(s) is/are linked to PRV virulence, ten putative virulence genes were knocked out using clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 technology. The pathogenicity of these mutants was evaluated in a mouse model. Our results demonstrated that of the ten tested genes, the thymidine kinase (TK) and glycoprotein M (gM) knockout mutants displayed significantly reduced virulence. However, mutants of other putative virulence genes, such as glycoprotein E (gE), glycoprotein I (gI), Us2, Us9, Us3, glycoprotein G (gG), glycoprotein N (gN) and early protein 0 (EP0), did not exhibit significantly reduced virulence compared to that of the wild-type PRV. To our knowledge, this study is the first to compare virulence genes from the current pandemic PRV variant strain. This study will provide a valuable reference for scientists to design effective live attenuated vaccines in the future.

Recombinant Pseudorabies Virus (PRV) Expressing Firefly Luciferase Effectively Screened for CRISPR/Cas9 Single Guide RNAs and Antiviral Compounds.

A Pseudorabies virus (PRV) variant has emerged in China since 2011 that is not protected by commercial vaccines, and has not been well studied. The PRV genome is large and difficult to manipulate, but it is feasible to use clustered, regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology. However, identification of single guide RNA (sgRNA) through screening is critical to the CRISPR/Cas9 system, and is traditionally time and labor intensive, and not suitable for rapid and high throughput screening of effective PRV sgRNAs. In this study, we developed a recombinant PRV strain expressing firefly luciferase and enhanced green fluorescent protein (EGFP) as a reporter virus for PRV-specific sgRNA screens and rapid evaluation of antiviral compounds. Luciferase activity was apparent as soon as 4 h after infection and was stably expressed through 10 passages. In a proof of the principle screen, we were able to identify several PRV specific sgRNAs and confirmed that they inhibited PRV replication using traditional methods. Using the reporter virus, we also identified PRV variants lacking US3, US2, and US9 gene function, and showed anti-PRV activity for chloroquine. Our results suggest that the reporter PRV strain will be a useful tool for basic virology studies, and for developing PRV control and prevention measures.

Highly Efficient CRISPR/Cas9-Mediated Homologous Recombination Promotes the Rapid Generation of Bacterial Artificial Chromosomes of Pseudorabies Virus.

Bacterial artificial chromosomes (BACs) are powerful tools for the manipulation of the large genomes of DNA viruses, such as herpesviruses. However, the methods currently used to construct the recombinant viruses, an important intermediate link in the generation of BACs, involve the laborious process of multiple plaque purifications. Moreover, some fastidious viruses may be lost or damaged during these processes, making it impossible to generate BACs from these large-genome DNA viruses. Here, we introduce the CRISPR/Cas9 as a site-specific gene knock-in instrument that promotes the homologs recombination of a linearized transfer vector and the Pseudorabies virus genome through double incisions. The efficiency of recombination is as high as 86%. To our knowledge, this is the highest efficiency ever reported for Pseudorabies virus recombination. We also demonstrate that the positions and distances of the CRISPR/Cas9 single guide RNAs from the homology arms correlate with the efficiency of homologous recombination. Our work show a simple and fast cloning method of BACs with large genome inserted by greatly enhancing the HR efficiencies through CRISPR/Cas9-mediated homology-directed repair mechanism, and this method could be of helpful for manipulating large DNA viruses, and will provide a successful model for insertion of large DNA fragments into other viruses.