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2017 Vol.32(6)

Chikungunya (chik-un-GUN-yuh) virus (CHIKV) is a mosquito-borne virus that belongs to the genus Alphavirus in family Togaviridae, which is named from the symptom it causes (meaning “disease that bends up the joints” in Tanzanian). Typically, the disease begins with a sudden high fever, maculopapular rash, and severe joint pain. Other symptoms will subside within 1~2 weeks, but the continued arthritis would be lasted from a few months to several years, exerting substantial impact on the quality of life of patients. CHIKV has two transmission cycles: one is normally maintained in a sylvatic cycle involving Aedes mosquitos and wild primates living in forests of western Africa; the other is direct human-mosquito-human transmission which occurs in Asia and Americas, especially in India and Indian Ocean regions. This issue presents three articles on the CHIKV topic, reporting the CHIKVs from the recent outbreak in Pakistan, analyzing the evolution and transmission, and reviewing the recent progress on CHIKV’s epidemiology, clinical presentation, molecular virology, diagnostic approaches, host immune response, vaccine development, and available animal models. It is hoped this special collection could shed lights on future studies of CHIKV. The cover image is courtesy of Jin Sun and Xia Jin (Institut Pasteur of Shanghai).



Recent progress on chikungunya virus research

Wenxi An, Ningning Ge, Yilin Cao, Jin Sun, Xia Jin

2017, 32(6): 441 doi: 10.1007/s12250-017-4072-x

Received: 23 August 2017 Accepted: 11 November 2017 Published: 28 November 2017
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Chikungunya virus (CHIKV) is an arbovirus transmitted by Aedes mosquitos in tropical and subtropical regions across the world. After decades of sporadic outbreaks, it re-emerged in Africa, Asia, India Ocean and America suddenly, causing major regional epidemics recently and becoming a notable global health problem. Infection by CHIKV results in a spectrum of clinical diseases including an acute self-limiting febrile illness in most individuals, a chronic phase of recurrent join pain in a proportion of patients, and long-term arthralgia for months to years for the unfortunate few. No specific anti-viral drugs or licensed vaccines for CHIKV are available so far. A better understanding of virus-host interactions is essential for the development of therapeutics and vaccines. To this end, we reviewed the existing knowledge on CHIKV’s epidemiology, clinical presentation, molecular virology, diagnostic approaches, host immune response, vaccine development, and available animal models. Such a comprehensive overview, we believe, will shed lights on the promises and challenges in CHIKV vaccine development.

Recent advances in the study of hepatitis B virus covalently closed circular DNA

Mengying Ji, , Kanghong Hu

2017, 32(6): 454 doi: 10.1007/s12250-017-4009-4

Received: 28 April 2017 Accepted: 27 November 2017 Published: 21 December 2017
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Chronic hepatitis B infection is caused by hepatitis B virus (HBV) and a total cure is yet to be achieved. The viral covalently closed circular DNA (cccDNA) is the key to establish a persistent infection within hepatocytes. Current antiviral strategies have no effect on the pre-existing cccDNA reservoir. Therefore, the study of the molecular mechanism of cccDNA formation is becoming a major focus of HBV research. This review summarizes the current advances in cccDNA molecular biology and the latest studies on the elimination or inactivation of cccDNA, including three major areas: (1) epigenetic regulation of cccDNA by HBV X protein, (2) immune-mediated degradation, and (3) genome-editing nucleases. All these aspects provide clues on how to finally attain a cure for chronic hepatitis B infection.
Research Article

Productive HBV infection of well-differentiated, hNTCP-expressing human hepatoma-derived (Huh7) cells

Ming Zhou, Kaitao Zhao, Yongxuan Yao, Yifei Yuan, Rongjuan Pei, Yun Wang, Jizheng Chen, Xue Hu, Yuan Zhou, Xinwen Chen, Chunchen Wu

2017, 32(6): 465 doi: 10.1007/s12250-017-3983-x

Received: 24 March 2017 Accepted: 17 July 2017 Published: 29 September 2017
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Feasible and effective cell models for hepatitis B virus (HBV) infection are required for investigating the complete lifecycle of this virus, including the early steps of viral entry. Resistance to dimethyl sulfoxide/polyethylene glycol (DMSO/PEG), hNTCP expression, and a differentiated state are the limiting factors for successful HBV infection models. In the present study, we used a hepatoma cell line (Huh7DhNTCP) to overcome these limiting factors so that it exhibits excellent susceptibility to HBV infection. To achieve this goal, different hepatoma cell lines were tested with 2.5% DMSO / 4% PEG8000, and one resistant cell line (Huh7D) was used to construct a stable hNTCP-expressing cell line (Huh7DhNTCP) using a recombinant lentivirus system. Then, the morphological characteristics and differentiation molecular markers of Huh7DhNTCP cells with or without DMSO treatment were characterized. Finally, the susceptibility of Huh7DhNTCP cells to HBV infection was assessed. Our results showed that Huh7D cells were resistant to 2.5% DMSO / 4% PEG8000, whereas the others were not. Huh7DhNTCP cells were established to express a high level of hNTCP compared to liver extracts, and Huh7DhNTCP cells rapidly transformed into a non-dividing, well-differentiated polarized phenotype under DMSO treatment. Huh7DhNTCP cells fully supported the entire lifecycle of HBV infection. This cell culture system will be useful for the analysis of host-virus interactions, which should facilitate the discovery of antiviral drugs and vaccines.

Developing a bacteriophage cocktail for biocontrol of potato bacterial wilt

Cuihua Wei, Junli Liu, Alice Nyambura Maina, Francis B Mwaura, Junping Yu, Chenghui Yan, Ruofang Zhang, Hongping Wei

2017, 32(6): 476 doi: 10.1007/s12250-017-3987-6

Received: 28 March 2017 Accepted: 18 October 2017 Published: 16 November 2017
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Bacterial wilt is a devastating disease of potato and can cause an 80% production loss. To control wilt using bacteriophage therapy, we isolated and characterized twelve lytic bacteriophages from different water sources in Kenya and China. Based on the lytic curves of the phages with the pathogen Ralstonia solanacearum, one optimal bacteriophage cocktail, P1, containing six phage isolations was formulated and used for studying wilt prevention and treatment efficiency in potato plants growing in pots. The preliminary tests showed that the phage cocktail was very effective in preventing potato bacterial wilt by injection of the phages into the plants or decontamination of sterilized soil spiked with R. solanacearum. Eighty percent of potato plants could be protected from the bacterial wilt (caused by R. solanacearum reference strain GIM1.74 and field isolates), and the P1 cocktail could kill 98% of live bacteria spiked in the sterilized soil at one week after spraying. However, the treatment efficiencies of P1 depended on the timing of application of the phages, the susceptibility of the plants to the bacterial wilt, as well as the virulence of the bacteria infected, suggesting that it is important to apply the phage therapy as soon as possible once there are early signs of the bacterial wilt. These results provide the basis for the development of bacteriophage-based biocontrol of potato bacterial wilt as an alternative to the use of antibiotics.

In vitro inhibition of HIV-1 replication in autologous CD4+ T cells indicates viral containment by multifactorial mechanisms

Ting Tu, Jianbo Zhan, Danlei Mou, Wei Li, Bin Su, Tong Zhang, Tao Li, Ning Li, Hao Wu, Cong Jin, Huabiao Chen

2017, 32(6): 485 doi: 10.1007/s12250-017-3992-9

Received: 05 April 2017 Accepted: 27 July 2017 Published: 15 September 2017
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HIV-1-specific cytotoxic T lymphocytes (CTLs) and neutralizing antibodies (NAbs) are present during chronic infection, but the relative contributions of these effector mechanisms to viral containment remain unclear. Here, using an in vitro model involving autologous CD4+ T cells, primary HIV-1 isolates, HIV-1-specific CTLs, and neutralizing monoclonal antibodies, we show that b12, a potent and broadly neutralizing monoclonal antibody to HIV-1, was able to block viral infection when preincubated with virus prior to infection, but was much less effective than CTLs at limiting virus replication when added to infected cell cultures. However, the same neutralizing antibody was able to contain viruses by antibody-dependent cell-mediated virus inhibition in vitro, which was mediated by natural killer cells (NKs) and dependent on an Fc-Fc receptor interaction. Meanwhile, bulk CTLs from HIV-1 controllers were more effective in suppression of virus replication than those from progressors. These findings indicate that control of HIV-1 replication in activated CD4+ T cells is ineffectively mediated by neutralizing antibodies alone, but that both CTLs and antibody-dependent NK-mediated immune mechanisms contribute to viral containment. Our study systemically compared three major players in controlling HIV-1 infection, CTLs, NAbs, and NKs, in an autologous system and highlighted the multifactorial mechanisms for viral containment and vaccine success.

DC-SIGN promotes Japanese encephalitis virus transmission from dendritic cells to T cells via virological synapses

Ping Wang, Mei Li, Wei Lu, Di Zhang, Qinxue Hu, Yalan Liu

2017, 32(6): 495 doi: 10.1007/s12250-017-4034-3

Received: 14 June 2017 Accepted: 18 August 2017 Published: 31 August 2017
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Skin-resident dendritic cells (DCs) likely encounter incoming viruses in the first place, and their migration to lymph nodes following virus capture may promote viral replication. However, the molecular mechanisms underlying these processes remain unclear. In the present study, we found that compared to cell-free viruses, DC-bound viruses showed enhanced capture of JEV by T cells. Additionally, JEV infection was increased by co-culturing DCs and T cells. Blocking the C-type lectin receptor DC-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) with neutralizing antibodies or antagonists blocked JEV transmission to T cells. Live-cell imaging revealed that DCs captured and transferred JEV viral particles to T cells via virological synapses formed at DC-T cell junctions. These findings indicate that DC-SIGN plays an important role in JEV transmission from DCs to T cells and provide insight into how JEV exploits the migratory and antigen-presenting capabilities of DCs to gain access to lymph nodes for dissemination and persistence in the host.

Hepatitis C virus-induced prion protein expression facilitates hepatitis C virus replication

Huixia Zhang, Shanshan Gao, Rongjuan Pei, Xinwen Chen, Chaoyang Li

2017, 32(6): 503 doi: 10.1007/s12250-017-4039-y

Received: 22 June 2017 Accepted: 18 September 2017 Published: 25 October 2017
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Hepatitis C virus (HCV) infects approximately 180 million people worldwide. Significant progress has been made since the establishment of in vitro HCV infection models in cells. However, the replication of HCV is complex and not completely understood. Here, we found that the expression of host prion protein (PrP) was induced in an HCV replication cell model. We then showed that increased PrP expression facilitated HCV genomic replication. Finally, we demonstrated that the KKRPK motif on the N-terminus of PrP bound nucleic acids and facilitated HCV genomic replication. Our results provided important insights into how viruses may harness cellular protein to achieve propagation.

Detection, isolation, and characterization of chikungunya viruses associated with the Pakistan outbreak of 2016–2017

Si-Qing Liu, Xiao Li, Ya-Nan Zhang, Ai-Li Gao, Cheng-Lin Deng, Jun-Hua Li, Shoukat Jehan, Nadia Jamil, Fei Deng, Hongping Wei, Bo Zhang

2017, 32(6): 511 doi: 10.1007/s12250-017-4059-7

Received: 04 September 2017 Accepted: 12 December 2017 Published: 21 December 2017
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The chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus, which has infected millions of people in Africa, Asia, Americas, and Europe since it reemerged in India and Indian Ocean regions in 2005–2006. Starting in the middle of November 2016, CHIKV has been widely spread, and more than 4,000 cases of infections in humans were confirmed in Pakistan. Here, we report the first isolation and characterization of CHIKV from the Pakistan outbreak. Eight CHIKV strains were newly isolated from human serum samples using a cell culture procedure. A full-length genome sequence and eight complete envelope (E1) sequences of CHIKV from Pakistan were obtained in this study. Alignment of the CHIKV E1 sequences revealed that the eight new CHIKV isolates were highly homogeneous, with only two nonsynonymous substitutions found at generally conserved sites (E99 and Q235). Based on the comparison of 342 E1 sequences, the two nonsynonymous mutations were located in well-recognized domains associated with viral functions such as the cell fusion and vector specificity, suggesting their potential functional importance. Phylogenetic analysis indicated that the CHIKV strains from Pakistan originated from CHIKV circulating in the Indian region. This study helps elucidate the epidemics of CHIKV in Pakistan and also provides a foundation for studies of evolution and expansion of CHIKV in South Asia.

Extensive evolution analysis of the global chikungunya virus strains revealed the origination of CHIKV epidemics in Pakistan in 2016

Junming Shi, Zhengyuan Su, Zhaojun Fan, Jun Wang, Siqing Liu, Bo Zhang, Hongping Wei, Shoukat Jehan, Nadia Jamil, Shu Shen, Fei Deng

2017, 32(6): 520 doi: 10.1007/s12250-017-4077-5

Received: 04 September 2017 Accepted: 28 November 2017 Published: 11 December 2017
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Chikungunya virus (CHIKV) is a mosquito-borne virus that causes epidemics widely in the world especially in the tropical and subtropical regions. Phylogenetic analysis has found that the CHIKV lineages were associated with the spatial and temporal distributions, which were related to the virus adaption to the major mosquito species and their distributions. In this study, we reported the complete genome sequences of eight CHIKV isolates from the outbreak in Pakistan last year. Then we reviewed the evolutionary history using extensive phylogenetic analysis, analyzed lineagespecific substitutions in viral proteins, and characterized the spreading pathway of CHIKV strains including the Pakistani strains. The results showed that the Pakistani stains belonged to the ECSA.IOL sub-lineage and derived from India. The genetic properties of the Pakistani strains including the adaptive substitution to vectors were further characterized, and the potential risks from the occurrence of CHIKV infection in Pakistan were discussed. These results provided better understanding of CHIKV evolution and transmission in the world and revealed the possible origination of the CHIKV outbreak and epidemic in Pakistan, which would promote the disease prevention and control in the identified countries and territories with the history of CHIKV infections as well as new regions with potential risk of CHIKV outbreaks.

Interplay between hepatitis C virus and ARF4

Na Zhang, Youyang Ke, Leiliang Zhang

2017, 32(6): 533 doi: 10.1007/s12250-017-4000-0

Received: 14 April 2017 Accepted: 17 July 2017 Published: 18 August 2017
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In summary, we show here that HCV infection is associated with an upregulation of ARF4, which promotes HCV replication. Upon HCV infection, CREB3 was redistributed to nucleus and activated ARF4 transcription. Our studies demonstrate a host factor ARF4 upregulated in HCV replication, which may provide new therapeutic targets for antiviral therapy.

An improved method for identifying SUMOylation sites of viral proteins

Yan Liu, Zhongyuan Tan, Bo Shu, Yuan Zhang, Caishang Zheng, Xianliang Ke, Xi Chen, Hanzhong Wang, Zhenhua Zheng

2017, 32(6): 537 doi: 10.1007/s12250-017-4018-3

Received: 16 May 2017 Published: 31 August 2017
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In this study, we improved the most commonly used methods for MS detection of SUMOylated sites and used an E. coli recombination SUMOylation system with SUMO-1 (T95R). This system provides fast enrichment of SUMOylated viral protein in less than 2 days, and shows advantage over the method of collecting modified protein from cells in convenience and sensitivity. Furthermore, this method provides an option for rapid and accurate identification of the potential viral protein SUMOylation sites.

Identification of a novel torque teno mini virus in cerebrospinal fluid from a child with encephalitis

Yan-Jun Kang, Mei-Fang Zhou, Wei Huang, Chao Deng, Gen Yan, Zhong-Hua Lu

2017, 32(6): 541 doi: 10.1007/s12250-017-4042-3

Received: 16 August 2017 Accepted: 20 September 2017 Published: 17 October 2017
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Anelloviruses are single-strand circular DNA viruses and ubiquitous within the human population. Although there is no direct evidence, many studies have suggested the anelloviruses may be associated with a variety of diseases. In this study, a novel torque teno mini virus (TTMV) was detected in a child with unexplained encephalitis. The detected virus had a circular genome of 2943 nt in length and 3 open reading frames. It shared 45.4% – 35.9% nucleotide identities with known TTMV species and anellovirus, which suggested it might belong to a new species within the genus Betatorquevirus. Phylogenetic analysis based on the amino acid sequences of ORF1 showed that this virus represented a distinct branch within the diversity of anellovirus. Whether this novel anellovirus strain is associated with encephalitis requires further study.

Antiviral activity of quercetin-3-β-O-D-glucoside against Zika virus infection

Gary Wong, Shihua He, Vinayakumar Siragam, Yuhai Bi, Majambu Mbikay, Michel Chretien, Xiangguo Qiu

2017, 32(6): 545 doi: 10.1007/s12250-017-4057-9

Received: 04 August 2017 Accepted: 23 August 2017 Published: 05 September 2017
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Q3G is a natural derivative of quercetin and is already widely used in various foods and drinks. Our results clearly demonstrated that Q3G exerts antiviral activity against ZIKV in both tissue culture and knockout mice, and that post-exposure in vivo treatment with Q3G could have a beneficial effect. In the future,Q3G should be tested in human cell lines (such as Huh-7,HeLa,or K048, a fetal brain neural stem cell line) to provide further data supporting its potential efficacy in humans; in addition, live viral loads or viremia should be tested in treated animals to supplement the survival results observed in this study. Although the treatment regimens will need to be further optimized (i.e.,dosage, frequency of treatment, and administration routes), our results support the results of Q3G efficacy studies in nonhuman primates against ZIKV infection. Further studies will also be needed to investigate the mechanism of Q3G antiviral action, in order to obtain valuable insights into the design of novel targets for antiviral therapeutics in the future.

Crystal structure of the coxsackievirus A16 RNA-dependent RNA polymerase elongation complex reveals novel features in motif A dynamics

Peng Bi, Bo Shu, Peng Gong

2017, 32(6): 548 doi: 10.1007/s12250-017-4066-8

Published: 20 November 2017
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The RNA-dependent RNA polymerases (RdRPs) encoded by RNA viruses represent a unique class of nucleic acid polymerases. Unlike other classes of single-subunit polymerases, viral RdRPs have evolved a unique conformational change in their palm domain to close the active site during catalysis. The hallmark of this conformational change is the backbone shift of the polymerase motif A from an “open” state to a “closed” state, allowing two universally conserved aspartic acid residues to orient toward each other for divalent metal binding and catalysis. The “closed” motif A conformation was only observed upon the binding of correct NTP in RdRP catalytic complexes or under rare conditions such as induced by a bound lutetium ion or a bound glutamate molecule. By solving the crystal structure of the catalytic elongation complex of the coxsackievirus RdRP, we in this work observed for the first time the “closed” motif A conformation in the absence of an NTP substrate or other conformational-change-inducing factors. This observation emphasizes the intrinsic dynamic features of viral RdRP motif A, and solidifies the structural basis for how this important structural element participates in catalytic events of the RdRPs.