2021 Vol.36(4)

Hepatitis B virus (HBV) is a hepatotropic, small, enveloped double-stranded DNA virus that belongs to the Hepadnaviridae family and causes anacute or chronic infection in humans. Chronic hepatitis B (CHB) infection ismore common in clinic, and worldwide, over 450 million people have CHB and areat risk for the development of liver cirrhosis, liver failure, and hepatocellularcarcinoma. Although CHB can be controlled by nucleos(t)ide analogs andinterferon, CHB still cannot be completely cured currently because of theexistence of covalently closed circular DNA in the cell nucleus. In this issue,Jun Zhang et al. found that ATP1B3, aβ3-subunit of the Na+/KATPasein the cell, could restrict HBVreplication via inducing the degradation of the HBV S proteins throughproteasomal pathway. Identification of ATP1B3 anti-HBV mechanisms would help usto develop more precise targeted therapeutic strategies for HBV infectedpatients. The cover artwork ismodified from the realistic molecular mechanism of ATP1B3-mediated proteasomaldegradation of the HBV S proteins (kindly provided by Baisong Zheng and WenyanZhang). See page 678–691 for details.


Monocyte/Macrophage-Mediated Innate Immunity in HIV-1 Infection: From Early Response to Late Dysregulation and Links to Cardiovascular Diseases Onset

Eman Teer, Danzil E. Joseph, Richard H. Glashoff, M.Faadiel Essop

2021, 36(4): 565 doi: 10.1007/s12250-020-00332-0

Received: 29 July 2020 Accepted: 26 October 2020 Published: 05 January 2021
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Although monocytes and macrophages are key mediators of the innate immune system, the focus has largely been on the role of the adaptive immune system in the context of human immunodeficiency virus (HIV) infection. Thus more attention and research work regarding the innate immune system—especially the role of monocytes and macrophages during early HIV-1 infection—is required. Blood monocytes and tissue macrophages are both susceptible targets of HIV-1 infection, and the early host response can determine whether the nature of the infection becomes pathogenic or not. For example, monocytes and macrophages can contribute to the HIV reservoir and viral persistence, and influence the initiation/extension of immune activation and chronic inflammation. Here the expansion of monocyte subsets (classical, intermediate and non-classical) provide an increased understanding of the crucial role they play in terms of chronic inflammation and also by increasing the risk of coagulation during HIV-1 infection. This review discusses the role of monocytes and macrophages during HIV-1 pathogenesis, starting from the early response to late dysregulation that occurs as a result of persistent immune activation and chronic inflammation. Such changes are also linked to downstream targets such as increased coagulation and the onset of cardiovascular diseases.

Herpesviruses and the Type Ⅲ Interferon System

Yue Yin, Herman W. Favoreel

2021, 36(4): 577 doi: 10.1007/s12250-020-00330-2

Received: 09 September 2020 Accepted: 27 October 2020 Published: 05 January 2021
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Type Ⅲ interferons (IFNs) represent the most recently discovered group of IFNs. Together with type Ⅰ IFNs (e.g. IFN-α/β), type Ⅲ IFNs (IFN-λ) are produced as part of the innate immune response to virus infection, and elicit an anti-viral state by inducing expression of interferon stimulated genes (ISGs). It was initially thought that type Ⅰ IFNs and type Ⅲ IFNs perform largely redundant functions. However, it has become evident that type Ⅲ IFNs particularly play a major role in antiviral protection of mucosal epithelial barriers, thereby serving an important role in the first-line defense against virus infection and invasion at contact areas with the outside world, versus the generally more broad, potent and systemic antiviral effects of type Ⅰ IFNs. Herpesviruseses are large DNA viruses, which enter their host via mucosal surfaces and establish lifelong, latent infections. Despite the importance of mucosal epithelial cells in the pathogenesis of herpesviruses, our current knowledge on the interaction of herpesviruses with type Ⅲ IFN is limited and largely restricted to studies on the alphaherpesvirus herpes simplex virus (HSV). This review summarizes the current understanding about the role of IFN-λ in the immune response against herpesvirus infections.

Current Status and Challenge of Pseudorabies Virus Infection in China

Lei Tan, Jun Yao, Yadi Yang, Wei Luo, Xiaomin Yuan, Lingchen Yang, Aibing Wang

2021, 36(4): 588 doi: 10.1007/s12250-020-00340-0

Received: 20 July 2020 Accepted: 17 September 2020 Published: 22 February 2021
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Pseudorabies (PR), also called Aujeszky's disease, is a highly infectious disease caused by pseudorabies virus (PRV). Without specific host tropism, PRV can infect a wide variety of mammals, including pig, sheep, cattle, etc., thereby causing severe clinical symptoms and acute death. PRV was firstly reported in China in 1950s, while outbreaks of emerging PRV variants have been documented in partial regions since 2011, leading to significant economic losses in swine industry. Although scientists have been devoting to the design of diagnostic approaches and the development of vaccines during the past years, PR remains a vital infectious disease widely prevalent in Chinese pig industry. Especially, its potential threat to human health has also attracted the worldwide attention. In this review, we will provide a summary of current understanding of PRV in China, mainly focusing on PRV history, the existing diagnosis methods, PRV prevalence in pig population and other susceptible mammals, molecular characteristics, and the available vaccines against its infection. Additionally, promising agents including traditional Chinese herbal medicines and novel inhibitors that may be employed to treat this viral infection, are also discussed.
Research Article

Foot-and-Mouth Disease Virus Inhibits RIP2 Protein Expression to Promote Viral Replication

Huisheng Liu, Qiao Xue, Zixiang Zhu, Fan Yang, Weijun Cao, Xiangtao Liu, Haixue Zheng

2021, 36(4): 608 doi: 10.1007/s12250-020-00322-2

Received: 10 July 2020 Accepted: 17 September 2020 Published: 05 January 2021
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Receptors interaction protein 2 (RIP2) is a specific adaptor molecule in the downstream of NOD2. The role of RIP2 during foot-and-mouth disease virus (FMDV) infection remains unknown. Here, our results showed that RIP2 inhibited FMDV replication and played an important role in the activation of IFN-β and NF-κB signal pathways during FMDV infection. FMDV infection triggered RIP2 transcription, while it reduced the expression of RIP2 protein. Detailed analysis showed that FMDV 2B, 2C, 3Cpro, and Lpro proteins were responsible for inducing the reduction of RIP2 protein. 3Cpro and Lpro are viral proteinases that can induce the cleavage or reduction of many host proteins and block host protein synthesis. The carboxyl terminal 105-C114 and 135-C144 regions of 2B were essential for reduction of RIP2. Our results also showed that the N terminal 1-61 region of 2C were essential for the reduction of RIP2. The 2C-induced reduction of RIP2 was dependent on inducing the reduction of poly(A)-binding protein 1 (PABPC1). The interaction between RIP2 and 2C was observed in the context of viral infection, and the residues 1-61 were required for the interaction. These data clarify novel mechanisms of reduction of RIP2 mediated by FMDV.

Metagenomic Profiling of Viruses Associated with Rhipicephalus microplus Ticks in Yunnan Province, China

Junming Shi, Shu Shen, Hui Wu, Yunzhi Zhang, Fei Deng

2021, 36(4): 623 doi: 10.1007/s12250-020-00319-x

Received: 22 August 2020 Accepted: 10 October 2020 Published: 05 January 2021
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Ticks are well known as vectors of many viruses which usually do great harm to human and animal health. Yunnan Province, widely covered by flourishing vegetation and mainly relying on farming husbandry, is abundant with Rhipicephalus microplus ticks. Therefore, it is of great significance to characterize the viral profile present in R. microplus parasitizing on cattle in Yunnan Province. In this study, a total of 7387 R. microplus ticks were collected from cattle and buffalo in the northwest and southeast areas of Yunnan Province from 2015 to 2017. We investigated the virome of R. microplus using next-generation sequencing (NGS) and the prevalence of important identified viruses among tick groups by RT-PCR. It revealed the presence of diverse virus concerning chu-, rhabdo-, phlebo-, flavi- and parvo- viruses in Yunnan. These viruses consist of single-stranded, circular and segmented sense RNAs, showing a greatly diversity in genomic organization. Furthermore, continuous epidemiological survey among ticks reveals broad prevalence of three viruses (Yunnan mivirus 1, Wuhan tick vrius 1 and YN tick-associated phlebovirus 1) and two possible prevalent viruses including a flavivirus-like segmented virus (Jingmen tick virus) and a bovine hokovirus 2 in Yunnan. Serological investigation among cattle indicates that these identified viruses may be infectious to cattle and can elicit corresponding antibody. Our findings on R. microplus-associated viral community will contribute to the prevention of viral disease and tracking the viral evolution. Further analysis is needed to better elucidate the pathogenicity and natural circulation of these viruses.

Phylogenetic Analysis of the Dengue Virus Strains Causing the 2019 Dengue Fever Outbreak in Hainan, China

Jiang Du, Liyuan Zhang, Xiaoyuan Hu, Ruoyan Peng, Gaoyu Wang, Yi Huang, Wenqi Wang, Kunliang Wu, Qiang Wang, Haoxiang Su, Fan Yang, Yun Zhang, Chuanning Tang, Xiuji Cui, Lina Niu, Gang Lu, Meifang Xiao, Yongguo Du, Feifei Yin

2021, 36(4): 636 doi: 10.1007/s12250-020-00335-x

Received: 16 August 2020 Accepted: 03 December 2020 Published: 05 January 2021
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Dengue virus is an arthropod-borne pathogen that is transmitted to humans primarily by Aedes spp. mosquitos, causing the acute infectious disease, dengue fever (DF). Until 2019, no dengue outbreak had been reported in Hainan Province for over 20 years. However, in early September of 2019, an increasing number of infected cases appeared and the DF outbreak lasted for over one month in Haikou City, Hainan Province. In our study, we collected 97 plasma samples from DF patients at three hospitals, as well as 1585 mosquito larvae samples from puddles in different areas of Haikou. There were 49 (50.5%) plasma samples found to be strongly positive and 9 (9.3%) plasma samples were weakly positive against the NS1 antigen. We discovered DENV both in the patient's plasma samples and mosquito larvae samples, and isolated the virus from C6/36 cells inoculated with the acute phase serum of patients. Phylogenetic analysis revealed that the new strains were the most closely related to the epidemic strain in the southern regions of China, belonging to lineage Ⅳ, genotype Ⅰ, DENV-1. Compared to the seven closest strains from neighboring countries and provinces, a total of 18 amino acid mutations occurred in the coding sequences (CDS) of the new isolated strain, DENV1 HMU-HKU-2. Our data shows that dengue virus is re-emerged in Hainan, and pose new threats for public health. Thus regular molecular epidemiological surveillance is necessary for control and prevention of DENV transmission.

Laboratory-based Surveillance and Clinical Profile of Sporadic HEV Infection in Shanghai, China

Jie Lu, Qing Li, Jiayuan Jiang, Ziqiang Li, Peiyun Wang, Zike Sheng, Rongtao Lai, Huijuan Zhou, Wei Cai, Hui Wang, Qing Guo, Honglian Gui, Qing Xie

2021, 36(4): 644 doi: 10.1007/s12250-020-00336-w

Received: 17 September 2020 Accepted: 30 October 2020 Published: 12 January 2021
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The study aimed to describe the epidemiological, virological and clinical features of sporadic HEV infection in eastern China. A total of 6112 patient sera were tested for anti-HEV IgG or anti-HEV IgM during one consecutive year (between August 2018 and July 2019). HEV RNA presence was evaluated by RT-PCR and HEV sequences were phylogenetically analyzed. Clinical features of confirmed HEV-infected patients were delineated. The sero-positivity rate of anti-HEV IgG maintained stable around 40%, while an obvious winter spike of anti-HEV IgM prevalence was observed. A total of 111 patients were confirmed of HEV viremia by molecular diagnosis. Subtype 4d was predominant. Phylogenetic analyses suggest that certain strains circulate across species and around the country. Subjects with confirmed current HEV infection had a high median age (58 years) and males were predominant (62.2%). Most patients presented with jaundice (75.7%) and anorexia (68.0%). Significantly elevated levels of liver enzymes and bilirubin were observed. Remarkably, the baseline bilirubin level was positively correlated with illness severity. Pre-existing HBV carriage may deteriorate illness. The clinical burden caused by locally acquired HEV infection is increasing. Surveillance should be enforced especially during the transition period from winter to spring. Patients with higher level of bilirubin at disease onset had slower recovery from HEV infection.

Visualization of the Oncolytic Alphavirus M1 Life Cycle in Cancer Cells

Jia Dan, Lin Nie, Xudong Jia, Cuiying Xu, Jing Cai, Yuan Lin, Jun Hu, Wenbo Zhu, Yinyin Li, Dong Chen, Ying Liu, Cheng Hu, Guangmei Yan, Jiankai Liang, Qinfen Zhang

2021, 36(4): 655 doi: 10.1007/s12250-020-00339-7

Received: 11 July 2020 Accepted: 26 October 2020 Published: 22 January 2021
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Oncolytic alphavirus M1 has been shown to selectively target and kill cancer cells, but cytopathic morphologies induced by M1 virus and the life cycle of the M1 strain in cancer cells remain unclear. Here, we study the key stages of M1 virus infection and replication in the M1 virus-sensitive HepG2 liver cancer cell line by transmission electron microscopy, specifically examining viral entry, assembly, maturation and release. We found that M1 virus induces vacuolization of cancer cells during infection and ultimately nuclear marginalization, a typical indicator of apoptosis. Specifically, our results suggest that the endoplasmic reticulum participates in the assembly of nucleocapsids. In the early and late stage of infection, three kinds of special cytopathic vacuoles are formed and appear to be involved in the replication, maturation and release of the virus. Taken together, our data displayed the process of M1 virus infection of tumor cells and provide the structural basis for the study of M1 virus-host interactions.

Axl Alleviates Neuroinflammation and Delays Japanese Encephalitis Progression in Mice

Zhao-Yang Wang, Zi-Da Zhen, Dong-Ying Fan, Pei-Gang Wang, Jing An

2021, 36(4): 667 doi: 10.1007/s12250-020-00342-y

Received: 06 July 2020 Accepted: 07 December 2020 Published: 03 February 2021
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Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus, which causes the most commonly diagnosed viral encephalitis named Japanese encephalitis (JE) in the world with an unclear pathogenesis. Axl, a receptor tyrosine kinase from TAM family, plays crucial role in many inflammatory diseases. We have previously discovered that Axl deficiency resulted in more severe body weight loss in mice during JEV infection, which we speculate is due to the anti-inflammatory effect of Axl during JE. Currently, the role of Axl in regulating the neuroinflammation and brain damage during JE has not been investigated yet. In this study, by using Axl deficient and heterozygous control mice, we discovered that Axl deficient mice displayed accelerated JE progression and exacerbated brain damage characterized by increased neural cell death, extended infiltration of inflammatory cells, and enhanced production of pro-inflammatory cytokines, in comparison to control mice. Additionally, consistent with our previous report, Axl deficiency had no impact on the infection and target cell tropism of JEV in brain. Taken together, our results suggest that Axl plays an anti-inflammatory and neuroprotective role during the pathogenesis of JE.

ATP1B3 Restricts Hepatitis B Virus Replication Via Reducing the Expression of the Envelope Proteins

Jun Zhang, Tianhang Zheng, Xiaolei Zhou, Hong Wang, Zhaolong Li, Chen Huan, Baisong Zheng, Wenyan Zhang

2021, 36(4): 678 doi: 10.1007/s12250-021-00346-2

Received: 06 August 2020 Accepted: 23 November 2020 Published: 03 February 2021
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Our recent study reported that ATP1B3 inhibits hepatitis B virus (HBV) replication via inducing NF-κB activation. However, ATP1B3 mutants which were defective in NF-κB activation still maintained the moderate degree of suppression on HBV replication, suggesting that another uncharacterized mechanism is also responsible for ATP1B3-mediated HBV suppression. Here, we demonstrated that ATP1B3 reduced the expression of HBV envelope proteins LHBs, MHBs and SHBs, but had no effect on intracellular HBV DNA, RNA levels as well as HBV promoter activities. Further investigation showed that proteasome inhibitor MG132 rescued ATP1B3-mediated envelope proteins degradation, demonstrating that proteasome-dependent pathway is involved in ATP1B3-induced degradation of envelope proteins. Co-IP showed that ATP1B3 interacts with LHBs and MHBs and induces LHBs and MHBs polyubiquitination. Immunofluorescence colocalization analysis confirmed LHBs and MHBs colocalized with ATP1B3 together. Our work provides important information for targeting ATP1B3 as a potential therapeutic molecule for HBV infection.

Rearrangement of Actin Cytoskeleton by Zika Virus Infection Facilitates Blood–Testis Barrier Hyperpermeability

Yiwen Nie, Lixia Hui, Moujian Guo, Wei Yang, Rui Huang, Junsen Chen, Xinyue Wen, Meng Zhao, Ying Wu

2021, 36(4): 692 doi: 10.1007/s12250-020-00343-x

Received: 27 August 2020 Accepted: 24 November 2020 Published: 03 February 2021
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In recent years, various serious diseases caused by Zika virus (ZIKV) have made it impossible to be ignored. Confirmed existence of ZIKV in semen and sexually transmission of ZIKV suggested that it can break the blood–testis barrier (BTB), or Sertoli cell barrier (SCB). However, little is known about the underlying mechanism. In this study, interaction between actin, an important component of the SCB, and ZIKV envelope (E) protein domain Ⅲ (EDⅢ) was inferred from coimmunoprecipitation (Co-IP) liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis. Confocal microscopy confirmed the role of actin filaments (F-actin) in ZIKV infection, during which part of the stress fibers, the bundles that constituted by paralleled actin filaments, were disrupted and presented in the cell periphery. Colocalization of E and reorganized actin filaments in the cell periphery of transfected Sertoli cells suggests a participation of ZIKV E protein in ZIKV-induced F-actin rearrangement. Perturbation of F-actin by cytochalasin D (CytoD) or Jasplakinolide (Jas) enhanced the infection of ZIKV. More importantly, the transepithelial electrical resistance (TEER) of an in vitro mouse SCB (mSCB) model declined with the progression of ZIKV infection or overexpression of E protein. Co-IP and confocal microscopy analyses revealed that the interaction between F-actin and tight junction protein ZO-1 was reduced after ZIKV infection or E protein overexpression, highlighting the role of E protein in ZIKV-induced disruption of the BTB. We conclude that the interaction between ZIKV E and F-actin leads to the reorganization of F-actin network, thereby compromising BTB integrity.

Evaluation of the Safety and Immune Efficacy of Recombinant Human Respiratory Syncytial Virus Strain Long Live Attenuated Vaccine Candidates

Li-Nan Wang, Xiang-Lei Peng, Min Xu, Yuan-Bo Zheng, Yue-Ying Jiao, Jie-Mei Yu, Yuan-Hui Fu, Yan-Peng Zheng, Wu-Yang Zhu, Zhong-Jun Dong, Jin-Sheng He

2021, 36(4): 706 doi: 10.1007/s12250-021-00345-3

Received: 28 February 2020 Accepted: 18 September 2020 Published: 09 February 2021
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Human respiratory syncytial virus (RSV) infection is the leading cause of lower respiratory tract illness (LRTI), and no vaccine against LRTI has proven to be safe and effective in infants. Our study assessed attenuated recombinant RSVs as vaccine candidates to prevent RSV infection in mice. The constructed recombinant plasmids harbored (5′ to 3′) a T7 promoter, hammerhead ribozyme, RSV Long strain antigenomic cDNA with cold-passaged (cp) mutations or cp combined with temperature-sensitive attenuated mutations from the A2 strain (A2cpts) or further combined with SH gene deletion (A2cptsΔSH), HDV ribozyme (δ), and a T7 terminator. These vectors were subsequently co-transfected with four helper plasmids encoding N, P, L, and M2-1 viral proteins into BHK/T7-9 cells, and the recovered viruses were then passaged in Vero cells. The rescued recombinant RSVs (rRSVs) were named rRSV-Long/A2cp, rRSV-Long/A2cpts, and rRSV-Long/A2cptsΔSH, respectively, and stably passaged in vitro, without reversion to wild type (wt) at sites containing introduced mutations or deletion. Although rRSV-Long/A2cpts and rRSV-Long/A2cptsΔSH displayed temperature-sensitive (ts) phenotype in vitro and in vivo, all rRSVs were significantly attenuated in vivo. Furthermore, BALB/c mice immunized with rRSVs produced Th1-biased immune response, resisted wtRSV infection, and were free from enhanced respiratory disease. We showed that the combination of ΔSH with attenuation (att) mutations of cpts contributed to improving att phenotype, efficacy, and gene stability of rRSV. By successfully introducing att mutations and SH gene deletion into the RSV Long parent and producing three rRSV strains, we have laid an important foundation for the development of RSV live attenuated vaccines.

Artesunate and Dihydroartemisinin Inhibit Rabies Virus Replication

Jun Luo, Yue Zhang, Yang Wang, Qing Liu, Jiesen Li, Hongling He, Yongwen Luo, Shile Huang, Xiaofeng Guo

2021, 36(4): 721 doi: 10.1007/s12250-021-00349-z

Received: 05 August 2020 Accepted: 16 November 2020 Published: 04 March 2021
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Rabies is caused by infection of rabies virus (RABV) and remains a serious threat to the global public health. Except for the requirement for cold chain and high cost of human rabies immune globulin, no small molecule drugs are currently available for clinical treatment of rabies. So, it is of great importance to identify novel compounds that can effectively inhibit RABV infection. Artesunate (ART) and dihydroartemisinin (DHA), two derivatives of artemisinin, are widely used for treatment of malaria in adults and children, showing high safety. In this study, we found that both ART and DHA were able to inhibit RABV replication in host cells at a low concentration (0.1 μmol/L). The antiviral effects of ART and DHA were independent of viral strains and cell lines. Pre-treatment with ART or DHA for 2 h in vitro did not affect the viral replication in host cells, implying that ART and DHA neither reduced the viability of RABV directly nor inhibited the binding and entrance of the virus to host cells. Further studies revealed that ART and DHA inhibited RABV genomic RNA synthesis and viral gene transcription. Treatment with ART or DHA (5 mg/kg) by intramuscular injection improved, to some extent, the survival rate of RABV-challenged mice. Combination treatment with derivatives of artemisinin and mannitol significantly improved the survival rate of RABV-challenged mice. The results suggest that ART and DHA have a great potential to be explored as new anti-rabies agents for treatment of rabies.

Development of A MERS-CoV Replicon Cell Line for Antiviral Screening

Jing Chen, Bing-Jie Hu, Kai Zhao, Yun Luo, Hao-Feng Lin, Zheng-Li Shi

2021, 36(4): 730 doi: 10.1007/s12250-020-00341-z

Received: 17 October 2020 Accepted: 23 November 2020 Published: 22 February 2021
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Middle East respiratory syndrome coronavirus (MERS-CoV) is the causative agent of a severe respiratory disease with a high mortality of ~ 35%. The lack of approved treatments for MERS-CoV infection underscores the need for a user-friendly system for rapid drug screening. In this study, we constructed a MERS-CoV replicon containing the Renilla luciferase (Rluc) reporter gene and a stable luciferase replicon-carrying cell line. Using this cell line, we showed that MERS-CoV replication was inhibited by combined application of lopinavir and ritonavir, indicating that this cell line can be used to screen inhibitors of MERS-CoV replication. Importantly, the MERS-replicon cell line can be used for high-throughput screening of antiviral drugs without the need for live virus handling, providing an effective and safe tool for the discovery of antiviral drugs against MERS-CoV.

HRV16 Infection Induces Changes in the Expression of Multiple piRNAs

Jie Li, Xinling Wang, Yanhai Wang, Juan Song, Qinqin Song, Yanbin Wang, Jun Han

2021, 36(4): 736 doi: 10.1007/s12250-021-00344-4

Received: 20 May 2020 Accepted: 30 October 2020 Published: 22 February 2021
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Human rhinovirus (HRV) is one of the most important cold-causing pathogens in humans. Piwi-interacting RNAs (piRNAs) are a recently discovered class of small non-coding RNAs whose best-understood function is to repress mobile element (ME) activity in animal germline. However, the profile of human/host piRNA during HRV infection is largely unknown. Here we performed high-throughput sequencing of piRNAs from H1-HeLa cells infected with HRV16 at 12 h, 24 h, and 36 h. The results showed that 22, 151, 664, 24, 362, 486 and 22, 726, 546 piRNAs displayed differential expression after HRV16 infection for three time points. A significant differential expression of 21 piRNAs was found in all time points and further verified by RT-qPCR, including 7 known piRNAs and 14 newly found piRNAs. In addition, piRNA prediction was performed on Piano using the SVM algorithm and transposon information. It found that novel_pir78110, novel_pir78107, novel_pir78097, novel_pir78094 and novel_pir76584 are associated with the DNA/hobo of Drosophila, Ac of maize and Tam3 of snapdragon (hAT)-Charlie transposon. The novel_pir97924, novel_pir105705 and novel_pir105700 recognize long interspersed nuclear elements 1 (LINE-1). The novel_pir33182 and novel_pir46604 are related to the long terminal repeat (LTR)/(Endogenous Retrovirus1) ERV1 repetitive element. The novel_pir73855 is related to the LTR/ERVK repetitive element. Both novel_pir70108 and novel_pir70106 are associated with the LTR/ERVL-MaLR repetitive element. The novel_pir15900 is associated with the DNA/hAT-Tip100 repetitive element. Overall, our results indicated that rhinovirus infection could reduce the expression of some piRNAs to facilitate upregulation of LINE-1 transcription or retrotransposons' expression, which is helpful to further explore the mechanism of rhinovirus infection.

A Novel 2-dimensional Multiplex qPCR Assay for Single-Tube Detection of Nine Human Herpesviruses

Yingxue Li, Zhenzhou Wan, Lulu Zuo, Shenwei Li, Honglian Liu, Yingying Ma, Lianqun Zhou, Xia Jin, Yuye Li, Chiyu Zhang

2021, 36(4): 746 doi: 10.1007/s12250-021-00354-2

Received: 26 September 2020 Accepted: 28 December 2020 Published: 26 February 2021
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Human herpesviruses are double-stranded DNA viruses that are classified into nine species. More than 90% of adults are ever infected with one or more herpesviruses. The symptoms of infection with different herpesviruses are diverse ranging from mild or asymptomatic infections to deadly diseases such as aggressive lymphomas and sarcomas. Timely and accurate detection of herpesvirus infection is critical for clinical management and treatment. In this study, we established a single-tube nonuple qPCR assay for detection of all nine herpesviruses using a 2-D multiplex qPCR method with a house-keeping gene as the internal control. The novel assay can detect and distinguish different herpesviruses with 30 to 300 copies per 25 µL single-tube reaction, and does not cross-react with 20 other human viruses, including DNA and RNA viruses. The robustness of the novel assay was evaluated using 170 clinical samples. The novel assay showed a high consistency (100%) with the single qPCR assay for HHVs detection. The features of simple, rapid, high sensitivity, specificity, and low cost make this assay a high potential to be widely used in clinical diagnosis and patient treatment.

Effects of Overwintering on the Survival and Vector Competence of Aedes albopictus in the Urban Life Cycle of Dengue Virus in Guangzhou, China

Yue Chen, Ronghua Chen, Jianrong Gao, Chunyuan Li, Jun Liu, Zhijian Zhou, Ruiwen Ren

2021, 36(4): 755 doi: 10.1007/s12250-021-00356-0

Received: 27 September 2020 Accepted: 21 December 2020 Published: 05 March 2021
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The Pearl River Delta, where Aedes albopictus (Ae. albopictus) is the only vector for dengue transmission, has exhibited one of the highest dengue burdens in southern China in recent decades. However, whether dengue virus (DENV) can overwinter in Ae. albopictus in the Pearl River Delta has not been determined to date. In this study, 300 field-derived Ae. albopictus mosquitoes from Guangzhou that were infected with the predominant endemic DENV-1 strain were investigated under simulated urban balcony environment from October 16, 2016, to June 16, 2017. The vertical transmission of DENV in the infected overwintering Ae. albopictus was analyzed. The DENV infected overwintering mosquitoes were evaluated for viral load at nine-time points using reverse transcription-quantitative PCR. The vector competence of the infected overwintering Ae. albopictus was also investigated by using suckling mice. Adult mosquitoes and larvae were found during the observation period. The vertical transmission of DENV-1 was documented. The DENV-1-positive rates between overwintering males and females had no difference. The proportion of DENV-1-positive overwintering mosquitoes decreased over time and had no difference beyond three months after the experiment. Overwintering mosquitoes can spread DENV-1 to hosts. No engorged mosquitoes at an ambient temperature below 15 ℃ were observed. The ratio of engorged mosquitoes was positively correlated with the ambient temperature ranging from 15 to 30 ℃. Our results demonstrated that DENV can overwinter in Ae. albopictus in the Pearl River Delta, Ae. albopictus is the competent vector for DENV, and maintain autochthonous dengue outbreaks in the Pearl River Delta through vertical transmission.

Systematic Analysis of 42 Autographa Californica Multiple Nucleopolyhedrovirus Genes Identifies An Additional Six Genes Involved in the Production of Infectious Budded Virus

Tong Chen, Xiaoyan Duan, Hengrui Hu, Yu Shang, Yangbo Hu, Fei Deng, Hualin Wang, Manli Wang, Zhihong Hu

2021, 36(4): 762 doi: 10.1007/s12250-021-00355-1

Received: 30 July 2020 Accepted: 29 December 2020 Published: 08 March 2021
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Baculoviruses have been widely used as a vector for expressing foreign genes. Among numerous baculoviruses, Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is the most frequently used and it encodes 155 open reading frames (ORFs). Here, we systematically investigated the impact of 42 genes of AcMNPV on the production of infectious budded viruses (BVs) by constructing gene-knockout bacmids and subsequently conducting transfection and infection assays. The results showed that among the 39 functionally unverified genes and 3 recently reported genes, 36 are dispensable for infectious BV production, as the one-step growth curves of the gene-knockout viruses were not significantly different from those of the parental virus. Three genes (ac62, ac82 and ac106/107) are essential for infectious BV production, as deletions thereof resulted in complete loss of infectivity while the repaired viruses showed no significant difference in comparison to the parental virus. In addition, three genes (ac13, ac51 and ac120) are important but not essential for infectious BV production, as gene-knockout viruses produced significantly lower BV levels than that of the parental virus or repaired viruses. We then grouped the 155 AcMNPV genes into three categories (Dispensable, Essential, or Important for infectious BV production). Based on our results and previous publications, we constructed a schematic diagram of a potential mini-genome of AcMNPV, which contains only essential and important genes. The results shed light on our understanding of functional genomics of baculoviruses and provide fundamental information for future engineering of baculovirus expression system.

Effects of N-Linked Glycan on Lassa Virus Envelope Glycoprotein Cleavage, Infectivity, and Immune Response

Xueqin Zhu, Yang Liu, Jiao Guo, Junyuan Cao, Zonglin Wang, Gengfu Xiao, Wei Wang

2021, 36(4): 774 doi: 10.1007/s12250-021-00358-y

Received: 02 November 2020 Accepted: 13 January 2021 Published: 10 March 2021
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Lassa virus (LASV) belongs to the Mammarenavirus genus (family Arenaviridae) and causes severe hemorrhagic fever in humans. The glycoprotein complex (GPC) contains eleven N-linked glycans that play essential roles in GPC functionalities such as cleavage, transport, receptor recognition, epitope shielding, and immune response. We used three mutagenesis strategies (asparagine to glutamine, asparagine to alanine, and serine/tyrosine to alanine mutants) to abolish individual glycan chain on GPC and found that all the three strategies led to cleavage inefficiency on the 2nd (N89), 5th (N119), or 8th (N365) glycosylation motif. To evaluate N to Q mutagenesis for further research, it was found that deletion of the 2nd (N89Q) or 8th (N365Q) glycan completely inhibited the transduction efficiency of pseudotyped particles. We further investigated the role of individual glycan on GPC-mediated immune response by DNA immunization of mice. Deletion of the individual 1st (N79Q), 3rd (N99Q), 5th (N119Q), or 6th (N167Q) glycan significantly enhanced the proportion of effector CD4+ cells, whereas deletion of the 1st (N79Q), 2nd (N89Q), 3rd (N99Q), 4th (N109Q), 5th (N119Q), 6th (N167Q), or 9th (N373Q) glycan enhanced the proportion of CD8+ effector T cells. Deletion of specific glycan improves the Th1-type immune response, and abolishment of glycan on GPC generally increases the antibody titer to the glycan-deficient GPC. However, the antibodies from either the mutant or WT GPC-immunized mice show little neutralization effect on wild-type LASV. The glycan residues on GPC provide an immune shield for the virus, and thus represent a target for the design and development of a vaccine.

Exploration of a Sequential Gp140-Gp145 Immunization Regimen with Heterologous Envs to Induce a Protective Cross-Reactive HIV Neutralizing Antibody Response In Non-human Primates

Xiangqing Ding, Kangli Cao, Jing Wang, Yanmin Wan, Qinyun Chen, Yanqin Ren, Yongtang Zheng, Mingzhao Zhu, Renrong Tian, Wenjun Wang, Chen Zhao, Xiaoyan Zhang, Jianqing Xu

2021, 36(4): 784 doi: 10.1007/s12250-021-00361-3

Received: 10 November 2020 Accepted: 13 January 2021 Published: 15 March 2021
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Raising a heterologous tier 2 neutralizing antibody (nAb) response remains a daunting task for HIV vaccine development. In this study, we explored the utility of diverse HIV-1 envelope (Env) immunogens in a sequential immunization scheme as a solution to this task. This exploration stemmed from the rationale that gp145, a membrane-bound truncation form of HIV Env, may facilitate the focusing of induced antibody response on neutralizing epitopes when sequentially combined with the soluble gp140 form as immunogens in a prime-boost mode. We first showed that gp140 DNA prime-gp145 Tiantan vaccinia (TV) boost likely represents a general format for inducing potent nAb response in mice. However, when examined in rhesus macaque, this modality showed little effectiveness. To improve the efficacy, we extended the original modality by adding a strong protein boost, namely native-like SOSIP.664 trimer displayed on ferritin-based nanoparticle (NP), which was generated by a newly developed click approach. The resulting three-immunization regimen succeeded in eliciting tier-2 nAb response with substantial breadth when implemented in rhesus macaque over a short 8-week schedule. Importantly, the elicited nAb response was able to effectively contain viremia upon a heterologous SHIV challenge. Collectively, our studies highlighted that diversification of Env immunogens, in both types and formulations, under the framework of a sequential immunization scheme might open new opportunity toward HIV vaccine development.

The CREB Regulated Transcription Coactivator 2 Suppresses HIV-1 Transcription by Preventing RNA Pol Ⅱ from Binding to HIV-1 LTR

Ling Ma, Shumin Chen, Zhen Wang, Saisai Guo, Jianyuan Zhao, Dongrong Yi, Quanjie Li, Zhenlong Liu, Fei Guo, Xiaoyu Li, Pingping Jia, Jiwei Ding, Chen Liang, Shan Cen

2021, 36(4): 796 doi: 10.1007/s12250-021-00363-1

Received: 25 September 2020 Accepted: 09 December 2020 Published: 15 March 2021
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The CREB-regulated transcriptional co-activators (CRTCs), including CRTC1, CRTC2 and CRTC3, enhance transcription of CREB-targeted genes. In addition to regulating host gene expression in response to cAMP, CRTCs also increase the infection of several viruses. While human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) promoter harbors a cAMP response element and activation of the cAMP pathway promotes HIV-1 transcription, it remains unknown whether CRTCs have any effect on HIV-1 transcription and HIV-1 infection. Here, we reported that CRTC2 expression was induced by HIV-1 infection, but CRTC2 suppressed HIV-1 infection and diminished viral RNA expression. Mechanistic studies revealed that CRTC2 inhibited transcription from HIV-1 LTR and diminished RNA Pol Ⅱ occupancy at the LTR independent of its association with CREB. Importantly, CRTC2 inhibits the activation of latent HIV-1. Together, these data suggest that in response to HIV-1 infection, cells increase the expression of CRTC2 which inhibits HIV-1 gene expression and may play a role in driving HIV-1 into latency.

Cell Cycle Arrest Protein CDKN2C Is Not an HBV Host Factor

Guiwen Guan, Liwei Zheng, Jingyuan Xi, Xingwen Yang, Xiangmei Chen, Fengmin Lu

2021, 36(4): 810 doi: 10.1007/s12250-020-00337-9

Received: 27 July 2020 Accepted: 16 November 2020 Published: 05 January 2021
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In summary, we demonstrated here that elevated CDKN2C expression was positively correlated with the proliferation state of the parenchymal liver cells, suggesting that CDKN2C expression in necro-inflammatory liver tissues, even though markedly upregulated, was not enough to facilitate the expression of the HBV host factor genes since it failed to repress cell cycle progression. Likewise, no correlation between CDKN2C expression and HBV replication in HBV-infected patients owing to the absence of CDKN2C expression in hepatocytes dominated in quiescent status and animal experiments also confirmed that CDKN2C cannot promote HBV replication in vivo. In conclusion, it is neither CDKN2C nor other single cell cycle specific gene itself, but instead the cell cycle arrest induced by them promotes HBV replication in patients with HBV infection. Hence, it is improper to term these genes as HBV host factors.

Conferring Resistance to Plant RNA Viruses with the CRISPR/CasRx System

Yongsen Cao, Huanbin Zhou, Xueping Zhou, Fangfang Li

2021, 36(4): 814 doi: 10.1007/s12250-020-00338-8

Received: 19 August 2020 Accepted: 21 November 2020 Published: 18 January 2021
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In summary, we have established a CasRx-mediated RNA interference system which is capable of attenuating plant RNA virus infection and suppressing the expression of the target RNA. Our results greatly expand the applicability of CasRx and provide a new tool for the design of anti-viral strategies as well as for RNA functional studies in plants (Fig. 1M).

Surveillance of Class Ⅰ Newcastle Disease Virus at Live Bird Markets and Commercial Poultry Farms in Eastern China Reveals the Epidemic Characteristics

Xiaolong Lu, Xiaoquan Wang, Tiansong Zhan, Yifan Sun, Xin Wang, Naiqing Xu, Tianxing Liao, Yu Chen, Min Gu, Shunlin Hu, Xiaowen Liu, Xiufan Liu

2021, 36(4): 818 doi: 10.1007/s12250-021-00357-z

Received: 25 October 2020 Accepted: 21 January 2021 Published: 15 March 2021
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Human PRV Infection in China: An Alarm to Accelerate Eradication of PRV in Domestic Pigs

Zhenhua Guo, Xin-Xin Chen, Gaiping Zhang

2021, 36(4): 823 doi: 10.1007/s12250-021-00347-1

Received: 12 October 2020 Accepted: 21 December 2020 Published: 04 February 2021
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Correction to: Hantavirus Infection during Pregnancy

Deng-Hui Lu, Hong Jiang, Jian-Qi Lian

2021, 36(4): 829 doi: 10.1007/s12250-021-00421-8

Published: 08 July 2021
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Correction to: Visualization of the Oncolytic Alphavirus M1 Life Cycle in Cancer Cells

Jia Dan, Lin Nie, Xudong Jia, Cuiying Xu, Jing Cai, Yuan Lin, Jun Hu, Wenbo Zhu, Yinyin Li, Dong Chen, Ying Liu, Cheng Hu, Guangmei Yan, Jiankai Liang, Qinfen Zhang

2021, 36(4): 830 doi: 10.1007/s12250-021-00429-0

Published: 15 July 2021
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