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2016 Vol.31(4)

Among six human coronaviruses, severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle-East respiratory syndrome coronavirus (MERSCoV) are connected with severe respiratory-tract infection, which lead to high case-fatality rates of ~10 and ~35%, respectively. The papain-like protease, a domain located in the middle part of the largest non-structural protein Nsp3, has proteolytic, deubiquitinating, and deISGylating activities. The latter two functions are involved in the suppression of the antiviral innate immune response of the host cell. In this issue, Lei and Hilgenfeld present the X-ray crystal structure of a complex between MERS-CoV PLpro and human ubiquitin (Ub) that is devoid of any covalent linkage between the two proteins. The cover shows five regions of the PLpro bind to two areas of the Ub. See page 288-299 for details.

Review

Virus like particle-based vaccines against emerging infectious disease viruses

Jinliang Liu, Shiyu Dai, Manli Wang, Zhihong Hu, Hualin Wang, Fei Deng

2016, 31(4): 279 doi: 10.1007/s12250-016-3756-y

Received: 29 February 2016 Accepted: 25 June 2016 Published: 11 July 2016
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Emerging infectious diseases are major threats to human health. Most severe viral disease outbreaks occur in developing regions where health conditions are poor. With increased international travel and business, the possibility of eventually transmitting infectious viruses between different countries is increasing. The most effective approach in preventing viral diseases is vaccination. However, vaccines are not currently available for numerous viral diseases. Viruslike particles (VLPs) are engineered vaccine candidates that have been studied for decades. VLPs are constructed by viral protein expression in various expression systems that promote the selfassembly of proteins into structures resembling virus particles. VLPs have antigenicity similar to that of the native virus, but are non-infectious as they lack key viral genetic material. VLP vaccines have attracted considerable research interest because they offer several advantages over traditional vaccines. Studies have shown that VLP vaccines can stimulate both humoral and cellular immune responses, which may offer effective antiviral protection. Here we review recent developments with VLP-based vaccines for several highly virulent emerging or re-emerging infectious diseases. The infectious agents discussed include RNA viruses from different virus families, such as the Arenaviridae, Bunyaviridae, Caliciviridae, Coronaviridae, Filoviridae, Flaviviridae, Orthomyxoviridae, Paramyxoviridae, and Togaviridae families.
Research Article

Structural and mutational analysis of the interaction between the Middle-East respiratory syndrome coronavirus (MERS-CoV) papain-like protease and human ubiquitin

Jian Lei, Rolf Hilgenfeld

2016, 31(4): 288 doi: 10.1007/s12250-016-3742-4

Received: 08 February 2016 Accepted: 10 March 2016 Published: 30 March 2016
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The papain-like protease (PLpro) of Middle-East respiratory syndrome coronavirus (MERS-CoV) has proteolytic, deubiquitinating, and deISGylating activities. The latter two are involved in the suppression of the antiviral innate immune response of the host cell. To contribute to an understanding of this process, we present here the X-ray crystal structure of a complex between MERS-CoV PLpro and human ubiquitin (Ub) that is devoid of any covalent linkage between the two proteins. Five regions of the PLpro bind to two areas of the Ub. The C-terminal five residues of Ub, RLRGG, are similar to the P5–P1 residues of the polyprotein substrates of the PLpro and are responsible for the major part of the interaction between the two macromolecules. Through sitedirected mutagenesis, we demonstrate that conserved Asp165 and non-conserved Asp164 are important for the catalytic activities of MERS-CoV PLpro. The enzyme appears not to be optimized for catalytic efficiency; thus, replacement of Phe269 by Tyr leads to increased peptidolytic and deubiquitinating activities. Ubiquitin binding by MERS-CoV PLpro involves remarkable differences compared to the corresponding complex with SARS-CoV PLpro. The structure and the mutational study help understand common and unique features of the deubiquitinating activity of MERS-CoV PLpro.

Highly pathogenic avian influenza H5N1 Clade 2.3.2.1c virus in migratory birds, 2014-2015

Yuhai Bi, Jianjun Chen, Zhenjie Zhang, Mingxin Li, Tianlong Cai, Kirill Sharshov, Ivan Susloparov, Alexander Shestopalov, Gary Wong, Yubang He, Zhi Xing, Jianqing Sun, Di Liu, Yingxia Liu, Lei Liu, Wenjun Liu, Fumin Lei, Shi Weifeng, Gao George F.

2016, 31(4): 300 doi: 10.1007/s12250-016-3750-4

Received: 22 February 2016 Accepted: 08 June 2016 Published: 30 June 2016
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A novel Clade 2.3.2.1c H5N1 reassortant virus caused several outbreaks in wild birds in some regions of China from late 2014 to 2015. Based on the genetic and phylogenetic analyses, the viruses possess a stable gene constellation with a Clade 2.3.2.1c HA, a H9N2-derived PB2 gene and the other six genes of Asian H5N1-origin. The Clade 2.3.2.1c H5N1 reassortants displayed a high genetic relationship to a human H5N1 strain (A/Alberta/01/2014). Further analysis showed that similar viruses have been circulating in wild birds in China, Russia, Dubai (Western Asia), Bulgaria and Romania (Europe), as well as domestic poultry in some regions of Africa. The affected areas include the Central Asian, East Asian-Australasian, West Asian-East African, and Black Sea/Mediterranean flyways. These results show that the novel Clade 2.3.2.1c reassortant viruses are circulating worldwide and may have gained a selective advantage in migratory birds, thus posing a serious threat to wild birds and potentially humans.

Association of single nucleotide polymorphism rs2065955 of the filaggrin gene with susceptibility to Epstein-Barr virus-associated gastric carcinoma and EBV-negative gastric carcinoma

Xiaojing Kuang, Lingling Sun, Shuzhen Liu, Zhenzhen Zhao, Danrui Zhao, Song Liu, Bing Luo

2016, 31(4): 306 doi: 10.1007/s12250-016-3721-9

Received: 08 January 2016 Accepted: 28 June 2016 Published: 10 August 2016
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The relationship between the Filaggrin gene (FLG) rs2065955 polymorphism and susceptibility to Epstein-Barr virus (EBV)-associated gastric carcinoma (EBVaGC) and EBV-negative gastric carcinoma (EBVnGC) was investigated in Shandong Province, China. We detected theFLG rs2065955 genotype and allele distribution by using PCR and restriction fragment length polymorphism (RFLP) in 64 EBVaGC, 82 EBVnGC, and 111 normal control samples. Immunohistochemistry was used to detect the level ofFLG protein in 35 EBVaGC and 51 EBVnGC tumor tissues. Compared with normal controls, the genotype CC and allele C ofFLG rs2065955 showed higher frequency in EBVaGC and EBVnGC. There was no significant difference between EBVaGC and EBVnGC in allele distribution ofFLG rs2065955, but the genotype CC was found more frequently in EBVaGC than in EBVnGC. The risk of developing either EBVaGC or EBVnGC in genotype CC was higher than in other genotypes. Furthermore, genotype CC ofFLG rs2065955 may contribute more to the risk of developing EBVaGC than EBVnGC. There was no significant difference in the expression level ofFLG protein between EBVaGC and EBVnGC. In conclusion, theFLG rs2065955 polymorphism was significantly related to gastric carcinoma. Allele C ofFLG rs2065955 could be a risk factor for EBVaGC or EBVnGC, while genotype CC ofFLG rs2065955 was especially associated with EBVaGC.

Monoclonal antibody-based serological methods for detecting Citrus tristeza virus in citrus groves

Zhen Liu, Zhe Chen, Jian Hong, Xuefeng Wang, Changyong Zhou, Xueping Zhou, Jianxiang Wu

2016, 31(4): 314 doi: 10.1007/s12250-016-3718-4

Received: 06 January 2016 Accepted: 12 June 2016 Published: 11 July 2016
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Aquareovirus species vary with respect to pathogenicity, and the nonstructural protein NS80 of aquareoviruses has been implicated in the regulation of viral replication and assembly, which can form viral inclusion bodies (VIBs) and recruit viral proteins to its VIBs in infected cells. NS80 consists of 742 amino acids with a molecular weight of approximately 80 kDa. Interestingly, a short specific fragment of NS80 has also been detected in infected cells. In this study, an approximately 58-kDa product of NS80 was confirmed in various infected and transfected cells by immunoblotting analyses using α-NS80C. Mutational analysis and time course expression assays indicated that the accumulation of the 58-kDa fragment was related to time and infection dose, suggesting that the fragment is not a transient intermediate of protein degradation. Moreover, another smaller fragment with a molecular mass of approximately 22 kDa was observed in transfected and infected cells by immunoblotting with a specific anti-FLAG monoclonal antibody or α-NS80N, indicating that the 58- kDa polypeptide is derived from a specific cleavage site near the amino terminus of NS80. Additionally, different subcellular localization patterns were observed for the 22-kDa and 58-kDa fragments in an immunofluorescence analysis, implying that the two cleavage fragments of NS80 function differently in the viral life cycle. These results provide a basis for additional studies of the role of NS80 played in replication and particle assembly of the Aquareovirus.

Identification and characterization of two cleavage fragments from the Aquareovirus nonstructural protein NS80

Qingxiu Chen, Jie Zhang, Fuxian Zhang, Hong Guo, Qin Fang

2016, 31(4): 321 doi: 10.1007/s12250-016-3723-7

Received: 11 January 2016 Accepted: 05 March 2016 Published: 06 June 2016
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Aquareovirus species vary with respect to pathogenicity, and the nonstructural protein NS80 of aquareoviruses has been implicated in the regulation of viral replication and assembly, which can form viral inclusion bodies (VIBs) and recruit viral proteins to its VIBs in infected cells. NS80 consists of 742 amino acids with a molecular weight of approximately 80 kDa. Interestingly, a short specific fragment of NS80 has also been detected in infected cells. In this study, an approximately 58-kDa product of NS80 was confirmed in various infected and transfected cells by immunoblotting analyses using α-NS80C. Mutational analysis and time course expression assays indicated that the accumulation of the 58-kDa fragment was related to time and infection dose, suggesting that the fragment is not a transient intermediate of protein degradation. Moreover, another smaller fragment with a molecular mass of approximately 22 kDa was observed in transfected and infected cells by immunoblotting with a specific anti-FLAG monoclonal antibody or α-NS80N, indicating that the 58-kDa polypeptide is derived from a specific cleavage site near the amino terminus of NS80. Additionally, different subcellular localization patterns were observed for the 22-kDa and 58-kDa fragments in an immunofluorescence analysis, implying that the two cleavage fragments of NS80 function differently in the viral life cycle. These results provide a basis for additional studies of the role of NS80 played in replication and particle assembly of the Aquareovirus.

Comparison of concentration methods for detection of hepatitis A virus in water samples

Yuting Qiao, Zhiwei Sui, Guoliang Hu, Huabin Cao, Guoxiang Yang, Yong Li, Yongsong Lei, Lihua Zhao, Quanjiao Chen

2016, 31(4): 331 doi: 10.1007/s12250-016-3786-5

Received: 12 April 2016 Accepted: 01 August 2016 Published: 10 August 2016
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Hepatitis A virus is a pathogen associated with water pollution. Contaminated drinking water can cause hepatitis A outbreaks, lead to economic losses, and even threaten human lives. It is difficult to detect low levels of hepatitis A virus in water, so the virus must be concentrated in order to quantify it accurately. Here, we present a simple, rapid, efficient technique for the concentration and detection of hepatitis A virus in water. Our data showed that adding phosphate-buffered saline to the water, pre-filtering the water, and adding Trizol reagent directly to the filtration membrane can significantly improve concentration efficiency. Of three types of filtration membranes studied (mixed cellulose ester membrane, polyvinylidene fluoride membrane, and nylon membrane), the concentration efficiency using mixed cellulose ester membrane with a 0.1-μm pore size was the highest, reaching 92.62 ± 5.17%. This method was used to concentrate hepatitis A virus in water samples from Donghu Lake. Using SYBR Green real-time reverse transcription polymerase chain reaction analysis, the detection sensitivity of this method reached 101 copies/μL and its concentration efficiency reached 79.45 ± 9.88%.
Letter

Improved plasmid-based recovery of coxsackievirus A16 infectious clone driven by human RNA polymerase Ⅰ promoter

Xiaoli Wang, Chaoyun Shen, Tan Chen, Ke Lan, Zhong Huang, Yunfang Zhang, Qingwei Liu

2016, 31(4): 339 doi: 10.1007/s12250-016-3716-6

Published: 19 April 2016
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Coxsackievirus A16 (CA16) is one of the major viral pathogens associated with hand, foot, and mouth disease. CA16 belongs to the Enterovirus genus of the Picornaviridae family and possesses a single-stranded positivesense RNA genome (Mao et al., 2014). Reverse genetics is an important tool for CA16 research. Previously, a reverse genetics T7 polymerase-based system was developed for poliovirus (Moss et al., 1989), foot-andmouth disease virus (FMDV) (Zibert et al., 1990), coxsackievirus B3 (Klump et al., 1990), enterovirus 71 (EV71) (Han et al., 2010; Shang et al., 2013), and even CA16 (Liu et al., 2011). However, in that system, cDNA must be transcribed to RNA by an exogenous T7 polymerase in vitro or in vivo. To develop a more rapid and simple method, a reverse genetics system based on human polymerase I (Pol I ) was developed for FMDV (Chang et al., 2009) and EV71 (Meng et al., 2012). In the current study, we developed a high-efficiency Pol I-driven plasmid-based reverse genetics system for CA16 (Gen- Bank: EU262658), and systemically characterized recovered CA16 particles.

Detection of infectious dengue virus by selective real-time quantitative polymerase chain reaction

Xin Huang, Xuan Zhou, Xiaoyan He, Pei Wang, Shuai Yue, Lixin Wu, Yu Zhang, Qian Xie, Bao Zhang, Wei Zhao

2016, 31(4): 342 doi: 10.1007/s12250-016-3757-x

Published: 30 March 2016
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The dengue virus (DENV) is a single-stranded positivesense RNA virus that belongs to the family Flaviviridae (Gubler, 2002), and has four serotypes, DENV1-DENV4, which are transmitted via Aedes aegypti and Aedes albopictus (Rodriguez-Roche and Gould, 2013). It has been reported that more than 50 million dengue infections occur each year (Guzman et al., 2010), and a serious outbreak occurred in the Southern Provinces of China in the summer of 2014. The clinical presentations of dengue infection range from mild febrile illness to severe dengue characterized by dengue hemorrhagic fever and shock syndrome, which make the accurate laboratory confirmation of the diagnosis challenging but crucial. Currently, serological assays and real-time quantitative polymerase chain reaction (qPCR) techniques are most commonly applied in the diagnosis of dengue infection (Guzman et al., 2010; B?ck and Lundkvist, 2013; Guzman and Harris, 2015). However, the current methods have some major drawbacks, such as falsepositive results owing to cross-reactivity with other arthropod- borne flaviviruses, and inability to determine the infectiousness in individual patients (Schwartz et al., 2000). Therefore, a simple and accurate method is needed to detect the virus, especially its associated infectious particles.

P33 of Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus is a functional homolog of AcP33

Wenhua Kuang, Huanyu Zhang, Dianhai Hou, Manli Wang, Fei Deng, Hualin Wang, Zhihong Hu

2016, 31(4): 346 doi: 10.1007/s12250-016-3771-z

Published: 09 March 2016
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Baculoviruses are insect-specific viruses with a circular double-stranded DNA genome ranging in size from 80–180 kb (Lu et al., 2012). Two distinct types of virions have been identified during the infectious cycle of baculoviruses, namely budded virions (BVs) and occlusion- derived virions (ODVs). BVs mediate infection from cell to cell, while ODVs initiate oral infection in the insect midgut (Braunagel and Summers, 2007).

PHYPred: a tool for identifying bacteriophage enzymes and hydrolases

Hui Ding, Wuritu Yang, Hua Tang, Peng-Mian Feng, Jian Huang, Wei Chen, Hao Lin

2016, 31(4): 350 doi: 10.1007/s12250-016-3740-6

Published: 04 March 2016
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Bacteriophages are viruses that attack bacteria and kill them through the lytic replication cycle. Many studies have reported that phages are more specific to bacteria than antibiotics are; thus, phage therapy has many potential applications in human medicine, with the advantage of having few side effects (Keen, 2012). Investigating the mechanisms of bacteria-killing phages will therefore aid in the development of antibacterial drugs.
LETTERS

Characterization and complete genome sequence of vB_EcoPBp4, a novel polyvalent N4-like bacteriophage that infects chicken pathogenic Escherichia coli

Can Zhang, Yanxiang Ma, Ting Wang, Huzhi Sun, Guomin Lu, Huiying Ren

2016, 31(4): 353 doi: 10.1007/s12250-016-3787-4

Published: 10 August 2016
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Pathogenic Escherichia coli cause chicken colibacillosis, which is economically devastating to the poultry industry worldwide (Bagheri et al., 2014). Owing to increasing antibiotic resistance, phage therapy reagents have been developed to treat bacterial infections (Xu et al., 2015).
INSIGHT

Accidental discovery and isolation of Zika virus in Uganda and the relentless epidemiologist behind the investigations

Hedi Zhou, Bryan Eaton, Zhihong Hu, Basil Arif

2016, 31(4): 357 doi: 10.1007/s12250-016-3821-6

Published: 18 July 2016
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Zika virus (ZIKV) has captured the attention of the world because of its potential to infect neural cells and its teratogenic effects on foetuses and the new born. The virus seems to have various modes of transmission and has been the subject of many reviews in the literature (example, Musso and Gubler, 2016, Wang et al., 2016). ZIKV was first isolated in 1947 but remained almost innocuous causing few and sporadic mild infections until 60 years later when an outbreak occurred in YAP State in the Federal State of Micronesia in 2007 infecting nearly 75% of the population (Duffy et al., 2009; Ai et al., 2016). A few years later (2013– 2014), there was an epidemic in the islands of French Polynesia located about half way between Mexico and Australia. Almost concomitantly, minor outbreaks occurred in other isolated Pacific islands such as Cook Islands, Samoa, Fiji and New Caledonia. It is truly remarkable how the virus has spread within a short time to these seemingly isolated islands. However, nothing brought ZIKV to the attention of the world more than the horrific images of newborn babies with microcephaly in north eastern Brazil (Adibi et al., 2016, Rubin et al., 2016; Schuler-Faccini et al., 2016). These images were the impetus for concerted efforts to study viral tropism and to put into motion efforts to combat its spread. The World Health Organization has deemed ZIKV a “public health emergency of international concern” (Cohn J, 2016). The purpose of this manuscript is to review the very early research that led to the discovery and partial characterization of the virus along with some current thoughts and add few personal anecdotes about the scientist who discovered it.