2019 Vol.34(6)

Coxsackievirus B3 (CVB3) is a pathogenic enterovirus of Picornaviridae family. LncRNAs are an important subgroup of non-coding RNAs and actively transcribed in large numbers in human genome. They are widely involved in various physiological and pathological processes, including viral infection. In this issue, Tong et al. profiled lncRNAs and mRNA expression in CVB3-infected HeLa cells by lncRNA-mRNA integrated microarrays, established the correlation network of lncRNAs and mRNAs, revealed a negative correlation between the lncRNA XLOC_001188 and the antiviral gene NFAT5, and identified four critical lncRNAs, SNHG11, RP11-145F16.2, RP11-1023L17.1 and RP11-1021N1.2, which potentially affect CVB3 replication. See page 618–630 for details.

Research Article

Genome Characteristics and Evolution of Pseudorabies Virus Strains in Eastern China from 2017 to 2019

Xiaofeng Zhai, Wen Zhao, Kemang Li, Cheng Zhang, Congcong Wang, Shuo Su, Jiyong Zhou, Jing Lei, Gang Xing, Haifeng Sun, Zhiyu Shi, Jinyan Gu

2019, 34(6): 601 doi: 10.1007/s12250-019-00140-1

Received: 06 March 2019 Accepted: 24 April 2019 Published: 05 July 2019
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Since late 2011, outbreaks of pseudorabies virus (PRV) have occurred in southern China causing major economic losses to the pig industry. We previously reported that variant PRV forms and recombination in China could be the source of continued epidemics. Here, we analyzed samples from intensive pig farms in eastern China between 2017 and 2019, and sequenced the main glycoproteins (gB, gC, gD, and gE) to study the evolution characteristics of PRV. Based on the gC gene, we found that PRV variants belong to clade 2 and detected a founder effect during by the PRV epidemic. In addition, we detected inter- and intra-clade recombination; in particular, inter-clade recombination in the gB genes of strains FJ-ZXF and FJ-W2, which were recombinant with clade 1 strains. We also found specific amino-acid changes and positively selected sites, possibly associated with functional changes. This analysis of the emergence of PRV in China illustrates the need for continuous monitoring and the development of vaccines against specific variants of PRV.

The DEAD-Box RNA Helicase DDX1 Interacts with the Viral Protein 3D and Inhibits Foot-and-Mouth Disease Virus Replication

Qiao Xue, Huisheng Liu, Qiaoying Zeng, Haixue Zheng, Qinghong Xue, Xuepeng Cai

2019, 34(6): 610 doi: 10.1007/s12250-019-00148-7

Received: 20 January 2019 Accepted: 17 May 2019 Published: 29 July 2019
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Foot-and-mouth disease virus (FMDV) can infect domestic and wild cloven-hoofed animals. The non-structural protein 3D plays an important role in FMDV replication and pathogenesis. However, the interaction partners of 3D, and the effects of those interactions on FMDV replication, remain incompletely elucidated. In the present study, using the yeast two-hybrid system, we identified a porcine cell protein, DEAD-box RNA helicase 1 (DDX1), which interacted with FMDV 3D. The DDX1-3D interaction was further confirmed by co-immunoprecipitation experiments and an indirect immunofluorescence assay (IFA) in porcine kidney 15 (PK-15) cells. DDX1 was reported to either inhibit or facilitate viral replication and regulate host innate immune responses. However, the roles of DDX1 during FMDV infection remain unclear. Our results revealed that DDX1 inhibited FMDV replication in an ATPase/helicase activity-dependent manner. In addition, DDX1 stimulated IFN-β activation in FMDV-infected cells. Together, our results expand the body of knowledge regarding the role of DDX1 in FMDV infection.

Expression Profile and Function Analysis of Long Non-coding RNAs in the Infection of Coxsackievirus B3

Lei Tong, Ye Qiu, Hui Wang, Yunyue Qu, Yuanbo Zhao, Lexun Lin, Yan Wang, Weizhen Xu, Wenran Zhao, Hongyan He, Guangze Zhao, Mary H. Zhang, Decheng Yang, Xingyi Ge, Zhaohua Zhong

2019, 34(6): 618 doi: 10.1007/s12250-019-00152-x

Received: 13 February 2019 Accepted: 17 June 2019 Published: 06 August 2019
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The roles of lncRNAs in the infection of enteroviruses have been barely demonstrated. In this study, we used coxsackievirus B3 (CVB3), a typical enterovirus, as a model to investigate the expression profiles and functional roles of lncRNAs in enterovirus infection. We profiled lncRNAs and mRNA expression in CVB3-infected HeLa cells by lncRNA-mRNA integrated microarrays. As a result, 700 differentially expressed lncRNAs (431 up-regulated and 269 down-regulated) and 665 differentially expressed mRNAs (299 up-regulated and 366 down-regulated) were identified in CVB3 infection. Then we performed lncRNA-mRNA integrated pathway analysis to identify potential functional impacts of the differentially expressed mRNAs, in which lncRNA-mRNA correlation network was built. According to lncRNA-mRNA correlation, we found that XLOC-001188, an lncRNA down-regulated in CVB3 infection, was negatively correlated with NFAT5 mRNA, an anti-CVB3 gene reported previously. This interaction was supported by qPCR detection following siRNA-mediated knockdown of XLOC-001188, which showed an increase of NFAT5 mRNA and a reduction of CVB3 genomic RNA. In addition, we observed that four most significantly altered lncRNAs, SNHG11, RP11-145F16.2, RP11-1023L17.1 and RP11-1021N1.2 share several common correlated genes critical for CVB3 infection, such as BRE and IRF2BP1. In all, our studies reveal the alteration of lncRNA expression in CVB3 infection and its potential influence on CVB3 replication, providing useful information for future studies of enterovirus infection.

Nsp2 and GP5-M of Porcine Reproductive and Respiratory Syndrome Virus Contribute to Targets for Neutralizing Antibodies

Jia Su, Lei Zhou, Bicheng He, Xinhui Zhang, Xinna Ge, Jun Han, Xin Guo, Hanchun Yang

2019, 34(6): 631 doi: 10.1007/s12250-019-00149-6

Received: 03 March 2019 Accepted: 23 May 2019 Published: 25 July 2019
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Porcine reproductive and respiratory syndrome virus (PRRSV) is characterized by its genetic variation and limited cross protection among heterologous strains. Even though several viral structural proteins have been regarded as inducers of neutralizing antibodies (NAs) against PRRSV, the mechanism underlying limited cross-neutralization among heterologous strains is still controversial. In the present study, examinations of NA cross reaction between a highly pathogenic PRRSV (HP-PRRSV) strain, JXwn06, and a low pathogenic PRRSV (LP-PRRSV) strain, HB-1/3.9, were conducted with viral neutralization assays in MARC-145 cells. None of the JXwn06-hyperimmuned pigs' sera could neutralize HB-1/3.9 in vitro and vice versa. To address the genetic variation between these two viruses that are associated with limited crossneutralization, chimeric viruses with coding regions swapped between these two strains were constructed. Viral neutralization assays indicated that variations in nonstructural protein 2 (nsp2) and structural proteins together contribute to weak cross-neutralization activity between JXwn06 and HB-1/3.9. Furthermore, we substituted the nsp2-, glycoprotein2 (GP2)-, GP3-, and GP4-coding regions together, or nsp2-, GP5-, and membrane (M) protein-coding regions simultaneously between these two viruses to construct chimeric viruses to test cross-neutralization reactivity with hyperimmunized sera induced by their parental viruses. The results indicated that the swapped nsp2 and GP5-M viruses increased the neutralization reactivity with the donor strain antisera in MARC-145 cells. Taken together, these results show that variations in nsp2 and GP5-M correlate with the limited neutralization reactivity between the heterologous strains HP-PRRSV JXwn06 and LP-PRRSV HB-1/3.9.

A Novel DT40 Antibody Library for the Generation of Monoclonal Antibodies

Bei Wang, Fei Wang, He Huang, Zhendong Zhao

2019, 34(6): 641 doi: 10.1007/s12250-019-00142-z

Received: 01 April 2019 Accepted: 30 April 2019 Published: 08 July 2019
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Early etiological diagnosis is very important for the control of sudden viral infections, and requires antibodies with both high sensitivity and high specificity. Traditional antibody preparation methods have limitations, such as a long and arduous cycle, complicated operation, and high expenses. A chicken lymphoma cell line, DT40, is known to produce IgM-type antibodies and undergo gene conversion and somatic mutation in the variable region of the immunoglobulin gene during culture. Here, the DT40 cell line was developed to produce antibody libraries and prepare antibody rapidly in vitro. Since hypermutation in DT40 cells was regulated by the activation-induced cytidine deaminase (AID) gene, AID expression needs to be controlled to either fix the Ig sequence by stopping mutation or improve affinity by resuming mutation after the antibodies have been selected. In this study, we generated a novel AID-inducible DT40 cell line (DT40-H7), in which the endogenous AID gene was knocked out using the CRISPR/Cas9 genome editing system, and an inducible AID gene, based on the Tet-Off expression system, was stably transfected. AID expression was controlled in DT40-H7 cells in a simple and efficient manner; gene conversion and point mutations were observed only when AID was expressed. Using the antibody library generated from this cell line, we successfully obtained monoclonal antibodies against the NS1 protein of Zika virus. The DT40-H7 cell line represents a useful tool for the selection and evolution of antibodies and may also be a powerful tool for the rapid selection and generation of diagnostic antibodies for emerging infectious diseases.

The Establishment and Validation of the Human U937 Cell Line as a Cellular Model to Screen Immunomodulatory Agents Regulating Cytokine Release Induced by Influenza Virus Infection

Ge Liu, Si Chen, Ao Hu, Li Zhang, Wenyu Sun, Jungang Chen, Wei Tang, Haiwei Zhang, Chunlan Liu, Chang Ke, Xulin Chen

2019, 34(6): 648 doi: 10.1007/s12250-019-00145-w

Received: 11 March 2019 Accepted: 17 May 2019 Published: 08 July 2019
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Severe influenza infections are often associated with the excessive induction of pro-inflammatory cytokines, which is also referred to as "cytokine storms". Several studies have shown that cytokine storms are directly associated with influenza-induced fatal acute lung injury and acute respiratory distress syndrome. Due to the narrow administration window, current antiviral therapies are often inadequate. The efforts to use immunomodulatory agents alone or in combination with antiviral agents in the treatment of influenza in animal models have resulted in the achievement of protective effects accompanied with reduced cytokine production. Currently, there are no immunomodulatory drugs for influenza available for clinical use. Animal models, despite being ideal to study the anti-inflammatory responses to influenza virus infection, are very costly and time-consuming. Therefore, there is an urgent need to establish fast and economical screening methods using cell-based models to screen and develop novel immunomodulatory agents. In this study, we screened seven human cell lines and found that the human monocytic cell U937 supports the replication of different subtypes of influenza viruses as well as the production of the important pro-inflammatory cytokines and was selected to develop the cell-based model. The U937 cell model was validated by testing a panel of known antiviral and immunomodulatory agents and screening a drug library consisting of 1280 compounds comprised mostly of FDA-approved drugs. We demonstrated that the U937 cell model is robust and suitable for the high-throughput screening of immunomodulators and antivirals against influenza infection.

A Recombinant Rabies Virus Expressing Fms-like Tyrosine Kinase 3 Ligand (Flt3L) Induces Enhanced Immunogenicity in Mice

Yachun Zhang, Jie Yang, Mingming Li, Min Cui, Zhen F. Fu, Ling Zhao, Ming Zhou

2019, 34(6): 662 doi: 10.1007/s12250-019-00144-x

Received: 08 January 2019 Accepted: 29 May 2019 Published: 28 June 2019
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Rabies is a zoonotic disease that still causes 59,000 human deaths each year, and rabies vaccine is the most effective way to control the disease. Our previous studies suggested that the maturation of DC plays an important role in enhancing the immunogenicity of rabies vaccine. Flt3L has been reported to own the ability to accelerate the DC maturation, therefore, in this study, a recombinant rabies virus expressing mouse Flt3L, designated as LBNSE-Flt3L, was constructed, and its immunogenicity was characterized. It was found that LBNSE-Flt3L could enhance the maturation of DC both in vitro and in vivo, and significantly more TFH cells and Germinal Center B (GC B) cells were generated in mice immunized with LBNSE-Flt3L than those immunized with the parent virus LBNSE. Consequently, expressing of Flt3L could elevate the level of virus-neutralizing antibodies (VNA) in immunized mice which provides a better protection from a lethal rabies virus challenge. Taken together, our study extends the potential of Flt3L as a good adjuvant to develop novel rabies vaccine by enhancing the VNA production through activating the DC-TFH-GC B axis in immunized mice.

Reducing Viral Inhibition of Host Cellular Apoptosis Strengthens the Immunogenicity and Protective Efficacy of an Attenuated HSV-1 Strain

Xingli Xu, Yufeng He, Shengtao Fan, Min Feng, Guorun Jiang, Lichun Wang, Ying Zhang, Yun Liao, Qihan Li

2019, 34(6): 673 doi: 10.1007/s12250-019-00156-7

Received: 28 January 2019 Accepted: 17 June 2019 Published: 10 September 2019
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Herpes simplex virus 1 (HSV-1), a member of α herpesviruses, shows a high infectivity rate of 30%-60% in populations of various ages. Some herpes simplex (HSV) vaccine candidates evaluated during the past 20 years have not shown protective efficacy against viral infection. An improved understanding of the immune profile of infected individuals and the associated mechanism is needed. HSV uses an immune evasion strategy during viral replication, and various virus-encoded proteins, such as ICP47 and Vhs, participate in this process through limiting the ability of CD8+ cytotoxic T lymphocytes to recognize target cells. Other proteins, e.g., Us3 and Us5, also play a role in viral immune evasion via interfering with cellular apoptosis. In this work, to study the mechanism by which HSV-1 strain attenuation interferes with the viral immune evasion strategy, we constructed a mutant strain, M5, with deletions in the Us3 and Us5 genes. M5 was shown to induce higher neutralizing antibody titers and a stronger cellular immune response than our previously reported M3 strain, and to prevent virus infection more effectively than the M3 strain in an in vivo mouse challenge test.

Blue-White Colony Selection of Virus-Infected Isogenic Recipients Based on a Chrysovirus Isolated from Penicillium italicum

Tingfu Zhang, Na Li, Yongze Yuan, Qianwen Cao, Yanfen Chen, Binglan Tan, Guoqi Li, Deli Liu

2019, 34(6): 688 doi: 10.1007/s12250-019-00150-z

Received: 16 February 2019 Accepted: 14 May 2019 Published: 02 August 2019
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Mycoviruses have been found to infect more than 12 species of Penicillium, but have not been isolated from Penicillium italicum (P. italicum). In this study, we isolated and characterized a new double-stranded RNA (dsRNA) virus, designated Penicillium italicum chrysovirus 1 (PiCV1), from the citrus pathogen P. italicum HSPi-YN1. Viral genome sequencing and molecular characterization indicated that PiCV1 was highly homologous to the previously described Penicillium chrysogenum virus. We further constructed the mutant HSPi-YN1ΔpksP defective in the polyketide synthase gene (pksP), which is involved in pigment biosynthesis, and these mutants formed albino (white) colonies. Then we applied hyphal anastomosis method to horizontally transmit PiCV1 from the white virus-donors (i.e., HSPi-YN1 mutants) to wild-type recipients (i.e., P. italicum strains HSPi-CQ54, HSPi-HB4, and HSPi-HN1), and the desirable PiCV1-infected isogenic recipients, a certain part of blue wild-type strains, can be eventually selected and confirmed by viral genomic dsRNA profile analysis. This blue-white colony screening would be an easier method to select virus-infected P. italicum recipients, according to distinguishable color phenotypes between blue virus-recipients and white virus-donors. In summary, the current work newly isolated and characterized PiCV1, verified its horizontal transmission among dually cultured P. italicum isolates, and based on these, established an effective and simplified approach to screen PiCV1-infected isogenic recipients.

Functional Characterization of the Group Ⅰ Alphabaculovirus Specific Gene ac73

Wei Shao, Lihong He, Qingxiu Chen, Jiang Li, Fei Deng, Hualin Wang, Zhihong Hu, Manli Wang

2019, 34(6): 701 doi: 10.1007/s12250-019-00146-9

Received: 13 March 2019 Accepted: 22 April 2019 Published: 17 July 2019
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Baculoviridae is a family of large DNA viruses that specifically infect insects. It contains four genera, Alpha-, Beta-, Gamma-, and Deltabaculovirus. Alphabaculovirus is further divided into Group Ⅰ and Ⅱ, and Group Ⅰ appears to be emerged most recently among all baculoviruses. Interestingly, there are 12 Group Ⅰ specific genes that are only found in this lineage. Studying these genes is helpful to understand how baculoviruses evolved. Here, we reported the functional analyzing results of ac73, a function unknown Group Ⅰ specific gene of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) which is the type species of baculovirus. The AC73 protein encoded by ac73 was found to be expressed during the late stage of infection and incorporated into the nucleocapsids of budded virus (BV) and occlusion-derived virus (ODV). In infected cells, AC73 resided mainly in the ring zone region of the nucleus, and appeared to be assembled into occlusion bodies (OBs). The ac73 knockout and repaired viruses were constructed and studied by in vitro and in vivo infection. Although ac73 was not essential for BV and ODV or OB formation, the BV titer and viral infectivity in insect larvae of ac73 knockout AcMNPV decreased by about 5-8 and 3-4 fold compared to those of wild type virus, respectively, suggesting ac73 contributed to infectious BV production and viral infectivity in vivo. This research provides new insight into the function of this Group Ⅰ specific gene.

Autographa Californica Multiple Nucleopolyhedrovirus P48 (Ac103) Is Required for the Efficient Formation of Virus-Induced Intranuclear Microvesicles

Yan Wang, Qingyun Cai, Jiannan Chen, Zhihong Huang, Wenbi Wu, Meijin Yuan, Kai Yang

2019, 34(6): 712 doi: 10.1007/s12250-019-00147-8

Received: 19 April 2019 Accepted: 14 May 2019 Published: 10 July 2019
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Our previous study has shown that the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) P48 (ac103) gene is essential for the nuclear egress of nucleocapsids and the formation of occlusion-derived virions (ODVs). However, the exact role of P48 in the morphogenesis of ODVs remains unknown. In this study, we demonstrated that P48 was required for the efficient formation of intranuclear microvesicles. To further understand its functional role in intranuclear microvesicle formation, we characterized the distribution of the P48 protein, which was found to be associated with the nucleocapsid and envelope fractions of both budded virions and ODVs. In AcMNPV-infected cells, P48 was predominantly localized to nucleocapsids in the virogenic stroma and the nucleocapsids enveloped in ODVs, with a limited but discernible distribution in the plasma membrane, nuclear envelope, intranuclear microvesicles, and ODV envelope. Furthermore, coimmunoprecipitation assays showed that among the viral proteins required for intranuclear microvesicle formation, P48 associated with Ac93 in the absence of viral infection.

Novel Recombinant Seneca Valley Virus Isolated from Slaughtered Pigs in Guangdong Province

Jianxin Liu, Yunfeng Zha, Huizi Li, Yanwei Sun, Fuguang Wang, Rong Lu, Zhangyong Ning

2019, 34(6): 722 doi: 10.1007/s12250-019-00139-8

Received: 14 March 2019 Accepted: 05 May 2019 Published: 25 June 2019
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Seneca valley virus (SVV) associated porcine idiopathic vesicular disease showed increasing geographic distribution in major pig-raising countries and it is important to monitor the epidemic strains. Here, two newly identified recombinant SVV strains, CH-GDFS-2018 and CH-GDJY-2018, which were isolated from slaughtered pigs in Guangdong Province, were performed genetic, phylogenetic and recombination analyses to determine their characterization. Genetic analysis revealed that CH-GDFS-2018 was closely related to KS15-01 and US-15-39812IA, while CH-GDJY-2018 was closely related to SVA/CHN/07/2017. Phylogenetic analysis indicated that these two strains were clustered with previous strains circulating in China in 2017 and belonged to the US SVV branch. Recombination analysis showed that the genome of CH-GDFS-2018 consisted of two fragments from USA-IA46008/2015-Passage 1 and one fragment from CH-GDYD-2017. Similarly, the recombinant genome of CH-GDJY-2018 consisted of two fragments from SVA-CHN-07-2017 and one fragment from GD01-2017. Although the significance of recombination in different regions of SVV genome needs further research, the findings of recombinant strains may help in developing better prevention strategies against outbreaks.

High-Efficiency Rescue of Equine Infectious Anemia Virus from a CMV-Driven Infectious Clone

Xue-Feng Wang, Bowen Bai, Yuezhi Lin, Ting Qi, Cheng Du, Mingxin Song, Xiaojun Wang

2019, 34(6): 725 doi: 10.1007/s12250-019-00153-w

Received: 14 May 2019 Accepted: 01 July 2019 Published: 02 August 2019
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In this study, the strong promoter of CMV was introduced in place of the 5' LTR U3 of EIAV to improve the expression level of the molecular clone and to promote the rescue efficiency of the virus. We demonstrated that the EIAV infectious clone pCMV3-8 have a strong capacity to rescue EIAV by viral RT activity assay, Western blotting and TEM. Taken together, these results indicate that pCMV3-8 could be used for genetic manipulation of EIAV and can be applied to the researches on the mechanism of viral replication and attenuated EIAV vaccine.

Correction to: Oxymatrine Inhibits Bocavirus MVC Replication, Reduces Viral Gene Expression and Decreases Apoptosis Induced by Viral Infection

Yanqin Ding, Na Li, Jinhan Sun, Linran Zhang, Jianhui Guo, Xueqi Hao, Yuning Sun

2019, 34(6): 729 doi: 10.1007/s12250-019-00132-1

Published: 03 July 2019
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