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1.
Transbound Emerg Dis ; 2020 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-32037673

RESUMO

Porcine respiratory and reproductive syndrome virus (PRRSV) causes an economically important disease affecting commercial pork production worldwide. NADC34-like PRRSV has had a strong impact on the U.S. and Peruvian pig industries in recent years and also emerged in northeastern China in 2017. However, the endemic status of NADC34-like PRRSV in China is unclear. In this study, we examined 650 tissue samples collected from 16 Provinces in China from 2018 to 2019. Six NADC34-like PRRSV strains were detected in samples from 3 Provinces, and the complete genomes of four of these strains were sequenced. Phylogenetic analysis showed that these novel PRRSV strains belong to sublineage 1.5 (or NADC34-like PRRSV), forming two groups in China. Sequence alignment suggested that these novel strains share the same 100-aa deletion in the Nsp2 protein that was identified in IA/2014/NADC34 isolated from the U.S in 2014. Recombination analysis revealed that five of eight complete genome sequences are derived from recombination between IA/2014/NADC34 and ISU30 or NADC30. The number and distribution of NADC34-like PRRSVs is increasing in China. Importantly, compared with the currently endemic strain NADC30-like PRRSV, NADC34-like PRRSV has the potential to be an endemic strain in China. This study will help us understand the epidemic status of NADC34-like PRRSV in China and provide data for further monitoring this type of PRRSV in China.

2.
Nano Lett ; 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31930919

RESUMO

Labeling viruses with high-photoluminescence quantum dots (QDs) for single virus tracking provides a visual tool to aid our understanding of viral infection mechanisms. However, efficiently labeling internal viral components without modifying the viral envelope and capsid remains a challenge, and existing strategies are not applicable to most viruses. Here, we have devised a strategy using the clustered regularly interspaced short palindromic repeats (CRISPR) imaging system to label the nucleic acids of Pseudorabies virus (PRV) with QDs. In this strategy, QDs were conjugated to viral nucleic acids with the help of nuclease-deactivated Cas9/gRNA complexes in the nuclei of living cells and then packaged into PRV during virion assembly. The processes of PRV-QD adsorption, cytoplasmic transport along microtubules, and nuclear entry were monitored in real time in both Vero and HeLa cells, demonstrating the utility and efficiency of the strategy in the study of viral infection.

3.
Viruses ; 12(1)2020 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-31906441

RESUMO

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.

4.
J Virol ; 2020 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-31896589

RESUMO

Porcine reproductive and respiratory syndrome virus (PRRSV), an important pathogen that affects the pig industry, is a highly genetically-diverse RNA virus. However, the phylogenetic and genomic recombination properties of this virus have not been completely elucidated. In this study, comparative analyses of all available genomic sequences of North American (NA) type PRRSVs (n = 355, including 138 PRRSV genomes sequenced in this study) in China and the United States during 2014-2018 revealed a high frequency of inter-lineage recombination hotspots in non-structural protein 9 (NSP9) and the GP2-GP3 regions. Lineage 1 (L1) PRRSV was found to be susceptible to recombination among PRRSVs both in China and the US. The recombinant major parent between the 1991-2013 data and the 2014-2018 data showed a trend from complex to simple. The major recombination pattern changed from an L8 to L1 backbone during 2014-2018 for Chinese PRRSVs, whereas L1 was always the major parent for US PRRSVs. Intra-lineage recombination hotspots were not as concentrated as inter-lineage recombination hotspots. In the two main clades with differential diversity in L1, NADC30-like PRRSVs is undergoing a decrease in population genetic diversity, NADC34-like PRRSVs has been relatively stable in population genetic diversity for years. Systematic analyses of insertion and deletion (indel) polymorphisms of NSP2 divided PRRSVs into 25 patterns, which could generate novel references for the classification of PRRSVs. The results of this study contribute to a deeper understanding of the recombination of PRRSVs and indicate the need for coordinated epidemiological investigations among countries.IMPORTANCE Porcine reproductive and respiratory syndrome (PRRS) is one of the most significant swine diseases. However, the phylogenetic and genomic recombination properties of the PRRS virus (PRRSV) have not been completely elucidated. In this study, we systematically compared differences in the lineage distribution, recombination, NSP2 polymorphisms, and evolutionary dynamics between NA-type PRRSVs in China and in the US. Strikingly, we found high frequency of inter-lineage recombination hotspots in non-structural protein 9 (NSP9) and in the GP2-GP3 region. Also, intra-lineage recombination hotspots were scattered across the genome between Chinese and US strains. Furthermore, we proposed novel methods based on NSP2 indel patterns for the classification of PRRSVs. Evolutionary dynamics analysis revealed that NADC30-like PRRSV is undergoing a decrease in population genetic diversity, suggesting that a dominant population may occur and cause an outbreak. Our findings offer important insights into the recombination of PRRSVs, and suggest the need for coordinated international epidemiological investigations.

5.
Emerg Microbes Infect ; 8(1): 1501-1510, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31631782

RESUMO

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.


Assuntos
Regulação Viral da Expressão Gênica , Vírus da Síndrome Respiratória e Reprodutiva Suína/metabolismo , RNA Mensageiro/genética , RNA Viral/genética , Proteínas não Estruturais Virais/metabolismo , Animais , Fases de Leitura Aberta , Síndrome Respiratória e Reprodutiva Suína/virologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/genética , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , Suínos , Transcrição Genética , Proteínas não Estruturais Virais/genética , Replicação Viral
6.
Transbound Emerg Dis ; 66(6): 2592-2600, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31379138

RESUMO

Porcine reproductive and respiratory syndrome virus (PRRSV) is an agent of porcine reproductive and respiratory syndrome (PRRS), which causes substantial economic losses to the swine industry. PRRSV displays rapid variation, and five lineages coexist in mainland China. Lineage 3 PRRSVs emerged in mainland China in 2005 and prevailed in southern China after 2010. In the present study, two lineage 3 PRRSV strains, which are named SD110-1608 and SDWH27-1710, were isolated from northern China in 2017. To explore the characteristics and origins of the two strains, we divided lineage 3 into five sublineages (3.1-3.5) based on 146 open reading frame (ORF) 5 sequences. Both strains and the strains isolated from mainland China were classified into sublineage 3.5. Lineage 3 PRRSVs isolated from Taiwan and Hong Kong were classified into sublineages 3.1-3.3 and sublineage 3.4, respectively. Recombination analysis revealed that SD110-1608 and SDWH27-1710 were derived from recombination of QYYZ (major parent strain) and JXA1 (minor parent strain). Sequence alignment showed that SD110-1608 and SDWH27-1710 shared a 36-aa insertion in Nsp2 with QYYZ isolated from Guangdong Province in 2010. Based on the evolutionary relationship among GP2a, GP3, GP4, GP5 and N proteins between sublineages 3.2 (FJ-1) and 3.5 (FJFS), we speculated that sublineage 3.5 (mainland China) originated from sublineage 3.2 (Taiwan, China). This study provides important information regarding the classification and transmission of lineage 3 PRRSVs.

7.
Vet Res ; 49(1): 103, 2018 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-30290850

RESUMO

In the original publication of this article [1], the author found the brand of vimentin antibody was wrong in Fig. 3. The legend of Fig. 3, 'mouse anti-vimentin mAb (Cell Signaling Technology) at 4 °C overnight' should be 'mouse anti-vimentin mAb (Sigma-Aldrich) at 4 °C overnight'.

8.
Vet Microbiol ; 222: 105-108, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30080663

RESUMO

Porcine reproductive and respiratory syndrome viruses (PRRSVs) pose a serious threat to the porcine industry of China, and the importation of novel strain(s) makes it challenging to control these viruses. Several NADC30-like PRRSV outbreaks have occurred in mainland China since 2013. In the current study, we report two novel PRRSVs, designated LNWK96 and LNWK130, which belong to lineage 1 and are closely related to US strains with ORF5 restriction fragment length polymorphism (RFLP) 1-7-4. The two viruses had a 100-aa deletion in the nsp2 gene corresponding to positions 328-427 in the VR-2332 strain, which was consistent with most of the ORF5 RFLP 1-7-4 viruses. Recombination analyses indicated that both viruses derived from the recombination of 1-7-4 isolates and ISU30 or NADC30, which were isolated in the United States. Taken together, these results demonstrate the emergence of ORF5 RFLP 1-7-4-like (NADC34-like) PRRSVs in China for the first time.


Assuntos
Doenças Transmissíveis Importadas/veterinária , Surtos de Doenças/veterinária , Síndrome Respiratória e Reprodutiva Suína/epidemiologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/genética , Vírus da Síndrome Respiratória e Reprodutiva Suína/isolamento & purificação , Proteínas do Envelope Viral , Animais , China/epidemiologia , Mapeamento Cromossômico , Doenças Transmissíveis Importadas/epidemiologia , Doenças Transmissíveis Importadas/transmissão , Doenças Transmissíveis Importadas/virologia , Fazendas , Variação Genética , Genoma Viral , Filogenia , Polimorfismo de Fragmento de Restrição/genética , Síndrome Respiratória e Reprodutiva Suína/virologia , Análise de Sequência de DNA , Deleção de Sequência , Suínos/virologia , Doenças dos Suínos/epidemiologia , Doenças dos Suínos/transmissão , Doenças dos Suínos/virologia , Estados Unidos/epidemiologia , Proteínas do Envelope Viral/genética
9.
Vet Microbiol ; 222: 46-54, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30080672

RESUMO

The recent rapid evolution of PRRSVs has resulted in certain biological characteristic changes, such as the fact that an increasing number of field PRRSVs can be isolated from PAMs but not from Marc-145 cells. In this study, we first isolated Marc-145-unadaptive field PRRSV strains from PAMs; sequence analysis showed that these PRRSVs belong to the HP-PRRSV (lineage 8) branch or NADC30-Like (lineage 1) branch. We further found major variations in ORF2-4 regions. To explore the viral adaptation mechanisms in detail, we constructed a full-length cDNA clone of MY-376, a Marc-145-unadaptive PRRSV. Construction of serially chimeric viruses of HuN4-F112 (a Marc-145-adaptive strain) and MY-376 demonstrated that variation in the minor envelope protein (GP2a and GP3) complex is a main determinant of PRRSV tropism for Marc-145 cells.


Assuntos
Células Epiteliais/virologia , Variação Genética , Vírus da Síndrome Respiratória e Reprodutiva Suína/genética , Vírus da Síndrome Respiratória e Reprodutiva Suína/isolamento & purificação , Proteínas do Envelope Viral , Animais , Técnicas de Cultura de Células , Linhagem Celular , DNA Complementar , Fases de Leitura Aberta/genética , Síndrome Respiratória e Reprodutiva Suína/virologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/química , Suínos , Proteínas do Envelope Viral/genética , Tropismo Viral
10.
Vet Res ; 49(1): 75, 2018 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-30053894

RESUMO

Porcine reproductive and respiratory syndrome virus (PRRSV) is an important globally distributed and highly contagious pathogen that has restricted cell tropism in vivo and in vitro. In the present study, we found that annexin A2 (ANXA2) is upregulated expressed in porcine alveolar macrophages infected with PRRSV. Additionally, PRRSV replication was significantly suppressed after reducing ANXA2 expression in Marc-145 cells using siRNA. Bioinformatics analysis indicated that ANXA2 may be relevant to vimentin, a cellular cytoskeleton component that is thought to be involved in the infectivity and replication of PRRSV. Co-immunoprecipitation assays and confocal analysis confirmed that ANXA2 interacts with vimentin, with further experiments indicating that the B domain (109-174 aa) of ANXA2 contributes to this interaction. Importantly, neither ANXA2 nor vimentin alone could bind to PRRSV and only in the presence of ANXA2 could vimentin interact with the N protein of PRRSV. No binding to the GP2, GP3, GP5, nor M proteins of PRRSV was observed. In conclusion, ANXA2 can interact with vimentin and enhance PRRSV growth. This contributes to the regulation of PRRSV replication in infected cells and may have implications for the future antiviral strategies.


Assuntos
Anexina A2/metabolismo , Síndrome Respiratória e Reprodutiva Suína/virologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/fisiologia , Vimentina/metabolismo , Replicação Viral , Animais , Ligação Proteica , Suínos
11.
FASEB J ; 32(8): 4293-4301, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29509513

RESUMO

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.


Assuntos
Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Vírus de DNA/genética , RNA Guia/genética , Animais , Linhagem Celular , Edição de Genes/métodos , Técnicas de Inativação de Genes/métodos , Genoma Viral/genética , Herpesvirus Suídeo 1/genética , Transfecção/métodos , Células Vero
12.
Oncotarget ; 9(15): 12174-12185, 2018 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-29552301

RESUMO

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.

13.
J Virol ; 92(7)2018 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-29321316

RESUMO

Highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) possesses greater replicative capacity and pathogenicity than classical PRRSV. However, the factors that lead to enhanced replication and pathogenicity remain unclear. In our study, an alignment of all available full-length sequences of North American-type PRRSVs (n = 204) revealed two consistent amino acid mutations that differed between HP-PRRSV and classical PRRSV and were located at positions 519 and 544 in nonstructural protein 9. Next, a series of mutant viruses with either single or double amino acid replacements were generated from HP-PRRSV HuN4 and classical PRRSV CH-1a infectious cDNA clones. Deletion of either of the amino acids led to a complete loss of virus viability. In both Marc-145 and porcine alveolar macrophages, the replicative efficiencies of mutant viruses based on HuN4 were reduced compared to the parent, whereas the replication level of CH-1a-derived mutant viruses was increased. Plaque growth assays showed clear differences between mutant and parental viruses. In infected piglets, the pathogenicity of HuN4-derived mutant viruses, assessed through clinical symptoms, viral load in sera, histopathology examination, and thymus atrophy, was reduced. Our results indicate that the amino acids at positions 519 and 544 in NSP9 are involved in the replication efficiency of HP-PRRSV and contribute to enhanced pathogenicity. This study is the first to identify specific amino acids involved in PRRSV replication or pathogenicity. These findings will contribute to understanding the molecular mechanisms of PRRSV replication and pathogenicity, leading to better therapeutic and prognostic options to combat the virus.IMPORTANCE Porcine reproductive and respiratory syndrome (PRRS), caused by porcine reproductive and respiratory syndrome virus (PRRSV), is a significant threat to the global pig industry. Highly pathogenic PRRSV (HP-PRRSV) first emerged in China in 2006 and has subsequently spread across Asia, causing considerable damage to local economies. HP-PRRSV strains possess a greater replication capacity and higher pathogenicity than classical PRRSV strains, although the mechanisms that underlie these characteristics are unclear. In the present study, we identified two mutations in HP-PRRSV strains that distinguish them from classical PRRSV strains. Further experiments that swapped the two mutations in an HP-PRRSV strain and a classical PRRSV strain demonstrated that they are involved in the replication efficiency of the virus and its virulence. Our findings have important implications for understanding the molecular mechanisms of PRRSV replication and pathogenicity and also provide new avenues of research for the study of other viruses.


Assuntos
Mutação de Sentido Incorreto , Síndrome Respiratória e Reprodutiva Suína , Vírus da Síndrome Respiratória e Reprodutiva Suína , Proteínas não Estruturais Virais , Replicação Viral/genética , Substituição de Aminoácidos , Animais , Linhagem Celular , Síndrome Respiratória e Reprodutiva Suína/genética , Síndrome Respiratória e Reprodutiva Suína/metabolismo , Síndrome Respiratória e Reprodutiva Suína/patologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/patogenicidade , Vírus da Síndrome Respiratória e Reprodutiva Suína/fisiologia , Suínos , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo
14.
Vet Microbiol ; 208: 164-172, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28888632

RESUMO

Porcine reproductive and respiratory syndrome (PRRS) has caused huge economic losses to Chinese swine industry and remains a major threat since it was first reported in 1996. However, investigations of molecular epidemiological and genetic diversity of PRRS viruses (PRRSVs) in China were limited to a small number of representative strains collected in several areas. Moreover, lineage classifications reported by individual researchers were quite different. In the present study, we sequenced ORF5 sequences of 217 PRRSVs from clinical samples, retrieved all the available ORF5 sequences of PRRSVs isolated in China in 1996-2016 (n=2213) from GenBank, and systematically analyzed corresponding epidemiological data. NA-type PRRSVs in China were classified into five lineages: lineage 1, lineage 3, lineage 5, lineage 8, and lineage 9. Most strains in China belonged to lineage 8 (85.6%), with dominant strains being classified as sublineage 8.3 (78.3%). Importantly, the emerging lineage 1 and lineage 3 strains spread rapidly, and their proportions among circulating PRRSVs have significantly increased in recent years. The geographical distribution of different PRRSV lineages in each province was analyzed and possible inter-province transmission routes were outlined for main lineages and sublineages. To our knowledge, this study is the most comprehensive and extensive phylogeographical analysis of PRRSVs in China since PRRS outbreak in 1996. Our dataset can serve as a canonical standard for PRRSV classification and will help to study genetic evolution of PRRSV. The results of the present study may also improve prevention of PRRS in China.


Assuntos
Síndrome Respiratória e Reprodutiva Suína/virologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/genética , Animais , China/epidemiologia , Variação Genética , Genótipo , Filogenia , Síndrome Respiratória e Reprodutiva Suína/epidemiologia , RNA Viral/genética , Suínos
15.
Arch Virol ; 162(12): 3881-3886, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28900740

RESUMO

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.


Assuntos
Antivirais/farmacologia , Produtos Biológicos/farmacologia , Herpesvirus Suídeo 1/efeitos dos fármacos , Herpesvirus Suídeo 1/fisiologia , RNA Guia/farmacologia , Replicação Viral/efeitos dos fármacos , Animais , Antivirais/isolamento & purificação , Produtos Biológicos/isolamento & purificação , Sistemas CRISPR-Cas , Linhagem Celular , Marcação de Genes , RNA Guia/isolamento & purificação , Suínos
16.
Sci Rep ; 7(1): 7783, 2017 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-28798304

RESUMO

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.


Assuntos
Herpesvirus Suídeo 1/genética , Pseudorraiva/virologia , Proteínas do Envelope Viral/genética , Animais , China , Surtos de Doenças , Feminino , Herpesvirus Suídeo 1/isolamento & purificação , Herpesvirus Suídeo 1/patogenicidade , Camundongos , Camundongos Endogâmicos BALB C , Pseudorraiva/epidemiologia , Timidina Quinase/genética , Células Vero , Virulência/genética
17.
Virus Res ; 225: 33-39, 2016 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-27619840

RESUMO

Currently, pseudorabies virus (PRV) variant strains are outbreaking in China; these variants belong to genotype II PRV. The traditional Bartha-K61 vaccine has failed to provide complete protection against the emergent variant strains. Therefore, rapid attenuation of current epidemic strains is needed for effective PRV control. In this study, we report a rapid method for editing the PRV genome using the CRISPR-Cas9 system. We developed a triple gE/gI/TK gene-inactivated HeN1 PRV strain, because mice were more susceptible to PRV infection, we then evaluated the attenuation of PRV in the mice and demonstrated that modified PRV was fully attenuated. Furthermore, the attenuated strain also induced immune protection in response to a parental PRV challenge. Overall, we showed that PRVs can be rapidly attenuated using CRISPR-Cas9 technology, which will be critical for PRV control, especially when new variant PRV strains emerge.


Assuntos
Sistemas CRISPR-Cas , Vetores Genéticos/genética , Herpesvirus Suídeo 1/genética , Vacinas contra Pseudorraiva/genética , Vacinas Atenuadas/genética , Animais , Feminino , Edição de Genes , Marcação de Genes , Herpesvirus Suídeo 1/imunologia , Camundongos , Vacinas contra Pseudorraiva/imunologia , RNA Guia , Deleção de Sequência , Vacinas Atenuadas/imunologia , Células Vero , Replicação Viral
18.
Viruses ; 8(4): 90, 2016 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-27043610

RESUMO

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.


Assuntos
Antivirais/farmacologia , Sistemas CRISPR-Cas , Avaliação Pré-Clínica de Medicamentos/métodos , Expressão Gênica , Vetores Genéticos , Herpesvirus Suídeo 1/efeitos dos fármacos , Luciferases de Vaga-Lume/genética , RNA Guia , Animais , Linhagem Celular , Ordem dos Genes , Genes Reporter , Vetores Genéticos/genética , Herpesvirus Suídeo 1/genética , Replicação Viral/efeitos dos fármacos
19.
Virology ; 491: 56-63, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26874017

RESUMO

Pseudorabies virus (PRV), the causative agent of Aujeszky's disease, has gained increased attention in China in recent years as a result of the outbreak of emergent pseudorabies. Several genomic and partial sequences are available for Chinese emergent and European-American strains of PRV, but limited sequence data exist for the earlier Chinese strains. In this study, we determined the complete genomic sequence of one earlier Chinese strain SC and one emergent strain HLJ8. Compared with other known sequences, we demonstrated that PRV strains from distinct geographical regions displayed divergent evolution. Additionally, we report for the first time, a recombination event between PRV strains, and show that strain SC is a recombinant of an endemic Chinese strain and a Bartha-vaccine-like strain. These results contribute to our understanding of PRV evolution.


Assuntos
Genoma Viral , Herpesvirus Suídeo 1/genética , Pseudorraiva/virologia , Doenças dos Suínos/virologia , Vacinas Virais/genética , Animais , China , Genômica , Herpesvirus Suídeo 1/classificação , Herpesvirus Suídeo 1/imunologia , Herpesvirus Suídeo 1/isolamento & purificação , Dados de Sequência Molecular , Filogenia , Recombinação Genética , Análise de Sequência de DNA , Suínos , Vacinas Virais/imunologia
20.
Front Microbiol ; 7: 2110, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-28066407

RESUMO

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.

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