Bacterial Artificial Chromosome System Research Articles

Regulation viral RNA transcription and replication by higher-order RNA structures within the nsp1 coding region of MERS coronavirus

Coronavirus (CoV) possesses numerous functional cis-acting elements in its positive-strand genomic RNA. Although most of these RNA structures participate in viral replication, the functions of RNA structures in the genomic RNA of CoV in viral replication remain unclear. In this study, we investigated the functions of the higher-order RNA stem-loop (SL) structures SL5B, SL5C, and SL5D in the ORF1a coding region of Middle East respiratory syndrome coronavirus (MERS-CoV) in viral replication. Our approach, using reverse genetics of a bacterial artificial chromosome system, revealed that SL5B and SL5C play essential roles in the discontinuous transcription of MERS-CoV. In silico analyses predicted that SL5C interacts with a bulged stem-loop (BSL) in the 3′ untranslated region, suggesting that the RNA structure of SL5C is important for viral RNA transcription. Conversely, SL5D did not affect transcription, but mediated the synthesis of positive-strand genomic RNA. Additionally, the RNA secondary structure of SL5 in the revertant virus of the SL5D mutant was similar to that of the wild-type, indicating that the RNA structure of SL5D can finely tune RNA replication in MERS-CoV. Our data indicate novel regulatory mechanisms of viral RNA transcription and replication by higher-order RNA structures in the MERS-CoV genomic RNA.

Reverse genetic systems of SARS-CoV-2 for antiviral research

Reverse genetic systems are widely used to engineer recombinant viruses with desired mutations. In response to the COVID-19 pandemic, four types of reverse genetic systems have been developed for SARS-CoV-2: (i) a full-length infectious clone that can be used to prepare recombinant SARS-CoV-2 at biosafety level 3 (BSL3), (ii) a trans-complementation system that can be used to produce single-round infectious SARS-CoV-2 at BSL2, (iii) an attenuated SARS-CoV-2 vaccine candidate (with deletions of viral accessory genes) that may be developed for veterinary use as well as for antiviral screening at BSL2, and (iv) replicon systems with deletions of viral structural genes that can be used at BSL2. Each of these genetic systems has its advantages and disadvantages that can be used to address different questions for basic and translational research. Due to the long genomic size and bacteria-toxic sequences of SARS-CoV-2, several experimental approaches have been established to rescue recombinant viruses and replicons, including (i) in vitro DNA ligation, (ii) bacterial artificial chromosome (BAC) system, (iii) yeast artificial chromosome (YAC) system, and (iv) circular polymerase extension reaction (CPER). This review summarizes the current status of SARS-CoV-2 genetic systems and their applications for studying viral replication, pathogenesis, vaccines, and therapeutics.

Construction of an IL12 and CXCL11 armed oncolytic herpes simplex virus using the CRISPR/Cas9 system for colon cancer treatment

Oncolytic viruses are an emerging cancer treatment modality with promising results in clinical trials. The new generation of oncolytic viruses are genetically modified to enhance virus selectivity for tumor cells and allow local expression of therapeutic genes in tumors. The traditional technique for viral genome engineering based on homologous recombination using a bacterial artificial chromosome (BAC) system is laborious and time-consuming. With the advent of the CRISPR/Cas9 system, the efficiency of gene editing in human cells and other organisms has dramatically increased. In this report, we successfully applied the CRISPR/Cas9 technique to construct an HSV-based oncolytic virus, where the ICP34.5 coding region was replaced with the therapeutic genes murine interleukin 12 (IL12, p40-p35) and C-X-C motif chemokine ligand 11 (CXCL11), and ICP47 gene was deleted. The combination of IL12 and CXCL11 in oncolytic viruses showed considerable promise in colorectal cancer (CRC) treatment. Overall, our study describes genetic modification of the HSV-1 genome using the CRISPR/Cas9 system and provides evidence from principle studies for engineering of the HSV genome to express foreign genes.

A Recombinant Porcine Reproductive and Respiratory Syndrome Virus Stably Expressing DsRed Protein Based on Bacterial Artificial Chromosome System.

Recombinant viruses possessing reporter proteins as tools are widely applied in investigating viral biology because of the convenience for observation. Previously, we generated a recombinant pathogenic porcine reproductive and respiratory syndrome virus (PRRSV) with enhanced green fluorescent protein (EGFP) reporter for monitoring virus spread and screening of neutralizing antibodies. PRRSV with different kinds of reporters can support more application scenarios. Here, we described a new genetically stable infectious clones of a highly pathogenic PRRSV (HP-PRRSV) harboring the DsRed (a red fluorescent protein isolated from the coral Discosoma) gene. In the recombinant infectious clone, the transcription regulatory sequence 2 (TRS2) of PRRSV was inserted between the open reading frame 7 (ORF7) and 3′UTR to drive the transcription of DsRed gene, which makes it a separate transcription unit in the viral genome. Using the bacterial artificial chromosome (BAC) system and cytomegalovirus (CMV) promoter, the recombinant HP-PRRSV with the DsRed insertion was successfully rescued and showed similar growth and replication patterns compared with the wild-type virus in the MARC-145 cells. In addition, the DsRed protein was stably expressed in the recombinant virus for at least 10 passages with consistent fluorescence intensity and density. Using the recombinant HP-PRRSV with DsRed protein, the virus tracking in MARC-145 was observed by live-cell imaging. Meanwhile, quantification of the DsRed fluorescence positive cells by flow cytometry provides an alternative to standard methods for testing the level of PRRSV infection. This recombinant PRRSV with DsRed fluorescence protein expression could be a useful tool for fundamental research on the viral biology and shows the new design for stable expression of foreign genes in PRRSV.

A novel and accurate full-length HTT mouse model for Huntington's disease.

Here, we report the generation and characterization of a novel Huntington's disease (HD) mouse model BAC226Q by using a bacterial artificial chromosome (BAC) system, expressing full-length human HTT with ~226 CAG-CAA repeats and containing endogenous human HTT promoter and regulatory elements. BAC226Q recapitulated a full-spectrum of age-dependent and progressive HD-like phenotypes without unwanted and erroneous phenotypes. BAC226Q mice developed normally, and gradually exhibited HD-like psychiatric and cognitive phenotypes at 2 months. From 3 to 4 months, BAC226Q mice showed robust progressive motor deficits. At 11 months, BAC226Q mice showed significant reduced life span, gradual weight loss and exhibited neuropathology including significant brain atrophy specific to striatum and cortex, striatal neuronal death, widespread huntingtin inclusions, and reactive pathology. Therefore, the novel BAC226Q mouse accurately recapitulating robust, age-dependent, progressive HD-like phenotypes will be a valuable tool for studying disease mechanisms, identifying biomarkers, and testing gene-targeting therapeutic approaches for HD.

Triple‐mutated oncolytic herpes virus for treating both fast‐ and slow‐growing tumors

Oncolytic virus therapy has emerged as a promising treatment option against cancer. To date, oncolytic viruses have been developed for malignant tumors, but the need for this new therapeutic modality also exists for benign and slow‐growing tumors. G47∆ is an oncolytic herpes simplex virus type 1 (HSV‐1) with an enhanced replication capability highly selective to tumor cells due to genetically engineered, triple mutations in the γ34.5, ICP6 and α47 genes. To create a powerful, but safe oncolytic HSV‐1 that replicates efficiently in tumors regardless of growth speed, we used a bacterial artificial chromosome system that allows a desired promoter to regulate the expression of the ICP6 gene in the G47∆ backbone. Restoration of the ICP6 function in a tumor‐specific manner using the hTERT promoter led to a highly capable oncolytic HSV‐1. T‐hTERT was more efficacious in the slow‐growing OS‐RC‐2 and DU145 tumors than the control viruses, while retaining a high efficacy in the fast‐growing U87MG tumors. The safety features are also retained, as T‐hTERT proved safe when inoculated into the brain of HSV‐1 sensitive A/J mice. This new technology should facilitate the use of oncolytic HSV‐1 for all tumors irrespective of growth speed.

Oncolytic virotherapy with SOCS3 enhances viral replicative potency and oncolysis for gastric cancer.

Oncolytic virotherapy is an encouraging treatment using herpes simplex virus (HSV) for gastric cancer patients. To treat gastric cancer, we generated and evaluated the efficacy of an attractive type of oncolytic HSV expressing the suppressor of cytokine signaling 3 (SOCS3). We constructed a third-generation type of oncolytic HSV (T-SOCS3) arming with SOCS3 by a bacterial artificial chromosome (BAC) system. We examined the viral replicative intensification and oncolysis of T-SOCS3 for human gastric cancer cell lines ex vivo. T-SOCS3 enhanced its replication and potentiated its cell-killing effect for MKN1 human gastric cancer cell lines, which are resistant to a non-armed third-generation type of oncolytic HSV (T-01) ex vivo. T-SOCS3 also induced the destruction within human gastric cancer specimens. Armed oncolytic HSVs expressing SOCS3 may be an efficacious therapeutic agent for gastric cancer treatment.

A bacterial artificial chromosome (BAC)-vectored noninfectious replicon of SARS-CoV-2

Vaccines and antiviral agents are in urgent need to stop the COVID-19 pandemic. To facilitate antiviral screening against SARS-CoV-2 without requirement for high biosafety level facility, we developed a bacterial artificial chromosome (BAC)-vectored replicon of SARS-CoV-2, nCoV-SH01 strain, in which secreted Gaussia luciferase (sGluc) was encoded in viral subgenomic mRNA as a reporter gene. The replicon was devoid of structural genes spike (S), membrane (M), and envelope (E). Upon transfection, the replicon RNA replicated in various cell lines, and was sensitive to interferon alpha (IFN-α), remdesivir, but was resistant to hepatitis C virus inhibitors daclatasvir and sofosbuvir. Replication of the replicon was also sensitive overexpression to zinc-finger antiviral protein (ZAP). We also constructed a four-plasmid in-vitro ligation system that is compatible with the BAC system, which makes it easy to introduce desired mutations into the assembly plasmids for in-vitro ligation. This replicon system would be helpful for performing antiviral screening and dissecting virus-host interactions.

A strain of porcine deltacoronavirus: Genomic characterization, pathogenicity and its full-length cDNA infectious clone.

As a novel enteropathogenic coronavirus, porcine deltacoronavirus (PDCoV) warrants further investigation. In this study, a Chinese PDCoV strain, designated CHN-HN-1601, was isolated from the faeces of a diarrhoeic piglet. After plaque purification, the genome was determined which shared 97.5%-99.5% nucleotide identities with 71 representative PDCoV strains available in the GenBank. The pathogenic properties of CHN-HN-1601 were evaluated using 5-day-old piglets. All inoculated piglets developed severe diarrhoea from 2days post-infection (dpi) onwards. To our surprise, two periods of diarrhoea starting from 2 to 7 dpi and from 13 to 19dpi were observed in affected piglets during the experiment. Faecal viral shedding of the inoculated piglets was detected by real-time RT-PCR, with viral shedding peaked at 4 and 16dpi, respectively. At necropsy at 5dpi, the main gross lesions included transparent, thin-walled and gas-distended intestines containing yellow watery contents. Further histopathological examinations, including haematoxylin and eosin staining, immunohistochemistry and RNAscope in situ hybridization, revealed that the virus infection caused severe villous atrophy of the small intestines, with PDCoV antigen and RNA mainly distributed in the cytoplasm of the villous epithelial cells of jejunum and ileum in piglets. The dynamic production of PDCoV-specific IgG and neutralizing antibodies in serum of the affected piglets was also assessed using a whole virus-based ELISA and an immunofluorescence assay-based neutralization test, respectively. Furthermore, a full-length cDNA infectious clone of CHN-HN-1601 was constructed using a bacterial artificial chromosome system. The rescued virus exhibited in vitro growth and pathogenic properties similar to the parental virus. Taken together, our study not only enriches the information of PDCoV, but also provides a useful reverse genetics platform for further pathogenesis exploration of the virus.

Human herpesvirus 6A U27 plays an essential role for the virus propagation.

Human herpesvirus 6A (HHV-6A) is a member of the genus Roseolovirus and the subfamily Betaherpesvirinae. It is similar to and human cytomegalovirus (HCMV). HHV-6A encodes a 41 kDa nuclear phosphoprotein, U27, which acts as a processivity factor in the replication of the viral DNA. HHV-6A U27 has 43% amino acid sequence homology with HCMV UL44, which is important for DNA replication. A previous study on HHV-6A U27 revealed that it greatly increases the in vitro DNA synthesis activity of HHV-6A DNA polymerase. However, the role of U27 during the HHV-6A virus replication process remains unclear. In this study, we constructed a U27-deficient HHV-6A mutant (HHV-6ABACU27mut) with a frameshift insertion at the U27 gene using an HHV-6A bacterial artificial chromosome (BAC) system. Viral reconstitution from the mutant BAC DNA was not detected, in contrast to the wild type and the revertant from the U27 mutant. This suggests that U27 plays a critical role in the life cycle of HHV-6A.

Molecular analysis of lymphoid tissue from rhesus macaque rhadinovirus-infected monkeys identifies alterations in host genes associated with oncogenesis.

Rhesus macaque (RM) rhadinovirus (RRV) is a simian gamma-2 herpesvirus closely related to human Kaposi’s sarcoma-associated herpesvirus (KSHV). RRV is associated with the development of diseases in simian immunodeficiency virus (SIV) co-infected RM that resemble KSHV-associated pathologies observed in HIV-infected humans, including B cell lymphoproliferative disorders (LPD) and lymphoma. Importantly, how de novo KSHV infection affects the expression of host genes in humans, and how these alterations in gene expression affect viral replication, latency, and disease is unknown. The utility of the RRV/RM infection model provides a novel approach to address these questions in vivo, and utilizing the RRV bacterial artificial chromosome (BAC) system, the effects of specific viral genes on host gene expression patterns can also be explored. To gain insight into the effects of RRV infection on global host gene expression patterns in vivo, and to simultaneously assess the contributions of the immune inhibitory viral CD200 (vCD200) molecule to host gene regulation, RNA-seq was performed on pre- and post-infection lymph node (LN) biopsy samples from RM infected with either BAC-derived WT (n = 4) or vCD200 mutant RRV (n = 4). A variety of genes were identified as being altered in LN tissue samples due to RRV infection, including cancer-associated genes activation-induced cytidine deaminase (AICDA), glypican-1 (GPC1), CX3C chemokine receptor 1 (CX3CR1), and Ras dexamethasone-induced 1 (RasD1). Further analyses also indicate that GPC1 may be associated with lymphomagenesis. Finally, comparison of infection groups identified the differential expression of host gene thioredoxin interacting protein (TXNIP), suggesting a possible mechanism by which vCD200 negatively affects RRV viral loads in vivo.

Initial Characterization of the Epstein⁻Barr Virus BSRF1 Gene Product.

Epstein–Barr virus (EBV) is a ubiquitous virus that causes infectious mononucleosis and several types of cancer, such as Burkitt lymphoma, T/NK-cell lymphoma, and nasopharyngeal carcinoma. As a herpesvirus, it encodes more than 80 genes, many of which have not been characterized. EBV BamHI S rightward reading frame 1 (BSRF1) encodes a tegument protein that, unlike its homologs herpes simplex virus unique long 51 (UL51) and human cytomegalovirus UL71, has not been extensively investigated. To examine the role of BSRF1, we prepared knockout and revertant strains using the bacterial artificial chromosome system. Unexpectedly, the disruption of the gene had little or no effect on EBV lytic replication and the transformation of primary B cells. However, the knockdown of BSRF1 in B95-8 cells decreased progeny production. An immunofluorescence assay revealed that BSRF1 localized to the Golgi apparatus in the cytoplasm, as did its homologs. BSRF1 also associated with BamHI G leftward reading frame 3.5 (BGLF3.5), BamHI B rightward reading frame 2 (BBRF2), and BamHI A leftward reading frame 1 (BALF1), and BALF1 was incorporated into the tegument fraction with BSRF1. Taken together, our results indicate that BSRF1 plays a role in secondary envelopment or virion egress in the cytoplasm, as do its homolog genes.

Acyclovir Sensitivity and Neurovirulence of Herpes Simplex Virus Type 1 with Amino Acid Substitutions in the Viral Thymidine Kinase Gene, Which Were Detected in the Patients with Intractable Herpes Simplex Encephalitis Previously Reported.

Several cases of herpes simplex encephalitis (HSE) caused by acyclovir (ACV)-resistant herpes simplex virus type 1 (HSV-1) have been reported. Amino acid substitutions of R41H, Q125H, and A156V in the viral thymidine kinase (vTK) gene have been reported to confer ACV resistance. Recombinant HSV-1 clones, containing each amino acid substitution in the vTK gene, were generated using the bacterial artificial chromosome system. A recombinant HSV-1 with the Q125H substitution showed ACV resistance while the R41H or A156V substitutions were ACV-sensitive. Furthermore, the Q125H recombinant HSV-1 was less virulent than the repaired virus, but it maintained neurovirulence in mice at relatively high levels. Substitution of Q125H, which was detected in the neonatal HSE patient, conferred ACV resistance, but the substitutions of R41H and A156V, which were detected in immunocompetent adult HSE patients, did not. This suggests that HSE caused by ACV-resistant HSV-1 might be a very rare event to occur during the course of ACV treatment in immunocompetent patients. Showing resistance to ACV treatment does not always indicate emergence of ACV-resistant HSV-1 in HSE patients.

Effects of Shield1 on the viral replication of varicella‑zoster virus containing FKBP‑tagged ORF4 and 48.

The present study aimed to explore the effects of a stabilizing ligand, Shield-1, on the replication of recombinant varicella-zoster virus (VZV) containing FK506 binding protein (FKPB) tags in essential open reading frames (ORF) 4 and 48. A specific galactokinase (galK) selection method was conducted, following the addition of galK labels to VZV ORF4 and 48, using a SW102 VZV bacterial artificial chromosome (BAC) system. Subsequently, recombinant VZV containing FKPB tags in ORF4 and 48 was constructed by counterselection and homologous recombination. Recombinant viral plasmids containing FKPB-tagged VZV ORF4 and 48 were extracted and transfected into human acute retinal pigment epithelial ARPE-19 cells. The results demonstrated that the FKPB-tagged viral protein was rapidly degraded by proteases in recombinant virus-infected ARPE-19 cells. In addition, the recombinant VZVORF4-FKBP-ORF48-FKBP virus could not grow if a synthetic ligand of FKBP, Shield1, was not added to the ARPE-19 cell culture medium; however, the degradation of FKPB-tagged viral protein was prevented if Shield1 was added to the ARPE-19 cell culture medium, thereby allowing viral replication in ARPE-19 cells. These results indicated that Shield1 may regulate replication of recombinant VZVORF4-FKBP-ORF48-FKBP following transfection into human epithelial cells.

Bovine herpesvirus 1 tegument protein UL21 plays critical roles in viral secondary envelopment and cell-to-cell spreading.

Bovine herpesvirus 1 (BoHV-1) UL21 is a tegument protein thought to be indispensable for efficient viral growth but its precise function in BoHV-1 is currently unknown. To determine the function of UL21 in BoHV-1 replication, we constructed a mutant virus bearing a UL21 deletion (vBoHV-1-∆UL21) and its revertant virus, vBoHV-1-∆UL21R, in which the UL21 gene was restored using a bacterial artificial chromosome system. The replication of vBoHV-1-∆UL21 was 1,000-fold lower and its plaque size was 85% smaller than those of the wild-type virus (BoHV-1). An ultrastructural analysis showed that deletion of UL21 led to an un-enveloped capsid accumulation in the cytoplasm, whereas nucleocapsid egress was not impaired, suggesting that UL21 is critical for secondary envelopment in BoHV-1. Co-immunoprecipitation assays revealed that HA-tagged UL21 pulled down UL16, suggesting that these two proteins form a complex, and this was further confirmed by a co-immunofluorescence assay. Taken together, these data provide evidence that UL21 plays critical roles in BoHV-1 secondary envelopment, and UL16 is likely to be involved in these activities.

A Domain of Herpes Simplex Virus pUL33 Required To Release Monomeric Viral Genomes from Cleaved Concatemeric DNA.

ABSTRACTMonomeric herpesvirus DNA is cleaved from concatemers and inserted into preformed capsids through the actions of the viral terminase. The terminase of herpes simplex virus (HSV) is composed of three subunits encoded by UL15, UL28, and UL33. The UL33-encoded protein (pUL33) interacts with pUL28, but its precise role in the DNA cleavage and packaging reaction is unclear. To investigate the function of pUL33, we generated a panel of recombinant viruses with either deletions or substitutions in the most conserved regions of UL33 using a bacterial artificial chromosome system. Deletion of 11 amino acids (residues 50 to 60 or residues 110 to 120) precluded viral replication, whereas the truncation of the last 10 amino acids from the pUL33 C terminus did not affect viral replication or the interaction of pUL33 with pUL28. Mutations that replaced the lysine at codon 110 and the arginine at codon 111 with alanine codons failed to replicate, and the pUL33 mutant interacted with pUL28 less efficiently. Interestingly, genomic termini of the large (L) and small (S) components were detected readily in cells infected with these mutants, indicating that concatemeric DNA was cleaved efficiently. However, the release of monomeric genomes as assessed by pulsed-field gel electrophoresis was greatly diminished, and DNA-containing capsids were not observed. These results suggest that pUL33 is necessary for one of the two viral DNA cleavage events required to release individual genomes from concatemeric viral DNA.IMPORTANCE This paper shows a role for pUL33 in one of the two DNA cleavage events required to release monomeric genomes from concatemeric viral DNA. This is the first time that such a phenotype has been observed and is the first identification of a function of this protein relevant to DNA packaging other than its interaction with other terminase components.

The Epstein-Barr Virus BRRF1 Gene Is Dispensable for Viral Replication in HEK293 cells and Transformation

The Epstein-Barr virus (EBV) is a gamma-herpesvirus associated with several malignancies. It establishes a latent infection in B lymphocytes and is occasionally reactivated to enter the lytic cycle. Here we examined the role of the EBV gene BRRF1, which is expressed in the lytic state. We first confirmed, using a DNA polymerase inhibitor, that the BRRF1 gene is expressed with early kinetics. A BRRF1-deficient recombinant virus was constructed using a bacterial artificial chromosome system. No obvious differences were observed between the wild-type, BRRF1-deficient mutant and the revertant virus in HEK293 cells in terms of viral lytic protein expression, viral DNA synthesis, progeny production, pre-latent abortive lytic gene expression and transformation of primary B cells. However, reporter assays indicated that BRRF1 may activate transcription in promoter- and cell type-dependent manners. Taken together, BRRF1 is dispensable for viral replication in HEK293 cells and transformation of B cells, but it may have effects on transcription.

Kaposi\u2019s sarcoma-associated herpesvirus ORF34 is essential for late gene expression and virus production

Kaposi’s sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi’s sarcoma, primary effusion lymphoma, and multicentric Castleman’s disease. KSHV establishes a life-long infection in its host and alternates between a latent and lytic infection state. During lytic infection, lytic-related genes are expressed in a temporal manner and categorized as immediate early, early, and late gene transcripts. ORF34 is an early-late gene that interacts with several viral transcription-associated factors, however its physiological importance remains poorly understood. Here, we investigated the role of ORF34 during KSHV infection by generating ORF34-deficient KSHV, using a bacterial artificial chromosome system. Our results reveal that ORF34-deficient KSHV exhibited significantly attenuated late gene expression and viral production but did not affect viral DNA replication. ORF34 interacted with transcription factors ORF18, ORF24, ORF31, and ORF66, and a novel ORF34-interaction partner, ORF23. The C-terminal region of ORF34 was important for interaction with ORF24 and viral production. Our data support a model, in which ORF34 serves as a hub for recruiting a viral transcription complex to ORF24 to promote late viral gene expression.

The Kaposi's-sarcoma-associated herpesvirus orf35 gene product is required for efficient lytic virus reactivation

Kaposi's sarcoma-associated herpesvirus (KSHV) is implicated in the etiology of several human malignancies. KSHV open reading frame (orf) 35 encodes a conserved gammaherpesvirus protein with an, as yet, unknown function. Employing the bacterial artificial chromosome (BAC) system, we generated a recombinant viral clone that fails to express ORF35 (BAC16-ORF35-stop) but preserves intact adjacent and overlapping reading frames. Using this construct, we studied the role of this previously uncharacterized gene product during lytic reactivation of KSHV. Upon lytic reactivation, the ORF35-stop recombinant virus displayed significantly reduced lytic viral gene expression, viral DNA replication, and progeny virus production as compared to control wild-type virus. Exogenous expression of ORF35-Flag reversed the effects of ORF35 deficiency. These results demonstrate that ORF35 is important for efficient lytic virus reactivation.

Application of genome editing technologies in researches on herpes simplex virus 1

The viral genome editing technologies have been shown to largely contribute to the studies of viral gene functions and to the treatment of infectious diseases. With the development of genetic engineering technologies, a variety of viral genome mutation technologies have been established. The homologous recombination is traditionally used to edit the viral genome, but the low frequency of homologous recombination limits its application. Large genome DNA viruses including herpes simplex virus 1 can be edited by bacterial artificial chromosome system. However, the cloning of viral genome to bacterial artificial chromosome plasmid is both laborious and time-consuming, and parts of the plasmid genes or drug selection markers may remain in the genome of virus and then affect the function of virus. Fortunately, the CRISPR-cas9 system which was discovered in 2013 can be used to edit the viral genome efficiently and accurately. Furthermore, the experiment is simple and will not leave any trail of the viral genome when the genetic mutation is performed. Here, we briefly review the application of homologous recombination, bacterial artificial chromosome system and CRISPR-cas9 system in researches on herpes simplex virus 1. Key words: Genome editing technologies; Herpes simplex virus 1