2016 Vol.31(2)

Recent large outbreaks of Zika virus (ZIKV) in Oceania’s islands and the Americas have linked the ZIKV infection with occurrence of microcephaly in newborns. The virus can be passed from a pregnant woman to her fetus. Even worse, ZIKV has now spread to nearly 30 areas worldwide and become a global health threat. In this issue, the virological, epidemiological and clinical essentials of ZIKV infection is reviewed; the evolution and spread pattern of ZIKV is analyzed; an infectious ZIKV strain from in imported case in mainland China is reported. The cover shows the immunofluorescence image of C6/36 mosquito cells infected with the isolated ZIKV strain at 24 hour post-infection. See page 176–179 for details.

News & Views

Zika virus: a flavivirus caused pandemics in Latin America

Si-Qing Liu, Bo Zhang

2016, 31(2): 101 doi: 10.1007/s12250-016-3738-0

Published: 08 March 2016
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Zika virus and Zika fever

Zhaoyang Wang, Peigang Wang, Jing An

2016, 31(2): 103 doi: 10.1007/s12250-016-3780-y

Received: 30 March 2016 Accepted: 21 April 2016 Published: 26 April 2016
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An emerging mosquito-borne arbovirus named Zika virus (ZIKV), of the family Flaviviridae and genus Flavivirus, is becoming a global health threat. ZIKV infection was long neglected due to its sporadic nature and mild symptoms. However, recently, with its rapid spread from Asia to the Americas, affecting more than 30 countries, accumulating evidences have demonstrated a close association between infant microcephaly and Zika infection in pregnant women. Here, we reviewed the virological, epidemiological, and clinical essentials of ZIKV infection.

Recent advances in synthetic carbohydrate-based human immunodeficiency virus vaccines

Zhenyuan Wang, Chunjun Qin, Jing Hu, Xiaoqiang Guo, Jian Yin

2016, 31(2): 110 doi: 10.1007/s12250-015-3691-3

Received: 11 December 2015 Accepted: 02 March 2016 Published: 17 March 2016
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An effective vaccine for human immunodeficiency virus (HIV) is urgently needed to prevent HIV infection and progression to acquired immune deficiency syndrome (AIDS). As glycosylation of viral proteins becomes better understood, carbohydrate-based antiviral vaccines against special viruses have attracted much attention. Significant efforts in carbohydrate synthesis and immunogenicity research have resulted in the development of multiple carbohydrate-based HIV vaccines. This review summarizes recent advances in synthetic carbohydrate-based vaccines design strategies and the applications of these vaccines in the prevention of HIV.
Research Article

Phylogenetic analysis revealed the central roles of two African countries in the evolution and worldwide spread of Zika virus

Shu Shen, Junming Shi, Jun Wang, Shuang Tang, Hualin Wang, Zhihong Hu, Fei Deng

2016, 31(2): 118 doi: 10.1007/s12250-016-3774-9

Received: 18 March 2016 Accepted: 21 April 2016 Published: 26 April 2016
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Recent outbreaks of Zika virus (ZIKV) infections in Oceania's islands and the Americas were characterized by high numbers of cases and the spread of the virus to new areas. To better understand the origin of ZIKV, its epidemic history was reviewed. Although the available records and information are limited, two major genetic lineages of ZIKV were identified in previous studies. However, in this study, three lineages were identified based on a phylogenetic analysis of all virus sequences from GenBank, including those of the envelope protein (E) and non-structural protein 5 (NS5) coding regions. The spatial and temporal distributions of the three identified ZIKV lineages and the recombination events and mechanisms underlying their divergence and evolution were further elaborated. The potential migration pathway of ZIKV was also characterized. Our findings revealed the central roles of two African countries, Senegal and Cote d'Ivoire, in ZIKV evolution and genotypic divergence. Furthermore, our results suggested that the outbreaks in Asia and the Pacific islands originated from Africa. The results provide insights into the geographic origins of ZIKV outbreaks and the spread of the virus, and also contribute to a better understanding of ZIKV evolution, which is important for the prevention and control of ZIKV infections.

The long non-coding RNA expression profile of Coxsackievirus A16 infected RD cells identified by RNA-seq

Yingying Shi, Huilin Tu, Xiong Chen, Yingying Zhang, Liujun Chen, Zhongchun Liu, Jiqun Sheng, Song Han, Jun Yin, Biwen Peng, Xiaohua He, Wanhong Liu

2016, 31(2): 131 doi: 10.1007/s12250-015-3693-1

Received: 27 November 2015 Accepted: 02 March 2016 Published: 31 March 2016
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Coxsackievirus A16 (CVA16) is one of major pathogens of hand, foot and mouth disease (HFMD) in children. Long non-coding RNAs (IncRNAs) have been implicated in various biological processes, but they have not been associated with CVA16 infection. In this study, we comprehensively characterized the landscape of IncRNAs of normal and CVA16 infected rhabdomyosarcoma (RD) cells using RNA-Seq to investigate the functional relevance of IncRNAs. We showed that a total of 760 IncRNAs were upregulated and 1210 IncRNAs were downregulated. Out of these dysregulated IncRNAs, 43.64% were intergenic, 22.31% were sense, 15.89% were intronic, 8.67% were bidirectional, 5.59% were antisense, 3.85% were sRNA host IncRNAs and 0.05% were enhancer. Six dysregulated IncRNAs were validated by quantitative PCR assays and the secondary structures of these IncRNAs were projected. Moreover, we conducted a bioinformatics analysis of an IncRNAs (ENST00000602478) to elucidate the diversity of modification and functions of IncRNAs. In summary, the current study compared the dysregulated IncRNAs profile upon CVA16 challenge and illustrated the intricate relationship between coding and IncRNAs transcripts. These results may not only provide a complete picture of transcription in CVA16 infected cells but also provide novel molecular targets for treatments of HFMD.

Lysine residues K66, K109, and K110 in the bovine foamy virus transactivator protein are required for transactivation and viral replication

Suzhen Zhang, Xiaoxu Cui, Jing Li, Zhibin Liang, Wentao Qiao, Juan Tan

2016, 31(2): 142 doi: 10.1007/s12250-015-3652-x

Received: 18 September 2015 Accepted: 14 January 2016 Published: 11 March 2016
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Bovine foamy virus (BFV) is a complex retrovirus that infects cattle. Like all retroviruses, BFV encodes a transactivator Tas protein (BTas) that increases gene transcription from viral promoters. BFV encodes two promoters that can interact with BTas, a conserved promoter in the 5' long terminal repeat (LTR) and a unique internal promoter (IP). Our previous study showed that BTas is acetylated by p300 at residues K66, K109, and K110, which markedly enhanced the ability of BTas to bind to DNA. However, whether these residues are important for BFV replication was not determined. Therefore, in this study we provide direct evidence that BTas is required for BFV replication and demonstrate that residues K66, K109, and K110 are critical for BTas function and BFV replication. Full-length infectious clones were generated, which were BTas deficient or contained lysine to arginine (K→R) mutations at position 66, 109, and/or 110. In vivo data indicated that K→R mutations at positions 66, 109, and 110 in BTas impaired transactivation of both the LTR and IP promoters. In addition, the K→R mutations in full-length infectious clones reduced expression of viral proteins, and the triple mutant and BTas deletion completely abrogated viral replication. Taken together, these results indicate that lysine residues at positions 66, 109, and 110 in the BTas protein are crucial for BFV replication and suggest a potential role for BTas acetylation in regulating the viral life cycle.

Recombinant Newcastle disease virus expressing African swine fever virus protein 72 is safe and immunogenic in mice

Xinxin Chen, Jifei Yang, Yanhong Ji, Edward Okoth, Bin Liu, Xiaoyang Li, Hong Yin, Qiyun Zhu

2016, 31(2): 150 doi: 10.1007/s12250-015-3692-2

Received: 28 November 2015 Accepted: 25 January 2016 Published: 10 March 2016
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African swine fever (ASF) is a lethal hemorrhagic disease that affects wild and domestic swine. The etiological agent of ASF is African swine fever virus (ASFV). Since the first case was described in Kenya in 1921, the disease has spread to many other countries. No commercial vaccines are available to prevent ASF. In this study, we generated a recombinant Newcastle disease virus (rNDV) expressing ASFV protein 72 (p72) by reverse genetics and evaluated its humoral and cellular immunogenicity in a mouse model. The recombinant virus, rNDV/p72, replicated well in embryonated chicken eggs and was safe to use in chicks and mice. The p72 gene in rNDV/p72 was stably maintained through ten passages. Mice immunized with rNDV/p72 developed high titers of ASFV p72 specific IgG antibody, and had higher levels of IgG1 than IgG2a. Immunization also elicited T-cell proliferation and secretion of IFN-γ and IL-4. Taken together, these results indicate that rNDV expressing ASFV p72 might be a potential vaccine candidate for preventing ASF.

Assembly of long DNA sequences using a new synthetic Escherichia coli-yeast shuttle vector

Zheng Hou, Zheng Zhou, Zonglin Wang, Gengfu Xiao

2016, 31(2): 160 doi: 10.1007/s12250-016-3730-8

Received: 21 January 2016 Accepted: 17 March 2016 Published: 11 April 2016
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Synthetic biology is a newly developed field of research focused on designing and rebuilding novel biomolecular components, circuits, and networks. Synthetic biology can also help understand biological principles and engineer complex artificial metabolic systems. DNA manipulation on a large genome-wide scale is an inevitable challenge, but a necessary tool for synthetic biology. To improve the methods used for the synthesis of long DNA fragments, here we constructed a novel shuttle vector named pGF (plasmid Genome Fast) for DNA assembly in vivo. The BAC plasmid pCC1BAC, which can accommodate large DNA molecules, was chosen as the backbone. The sequence of the yeast artificial chromosome (YAC) regulatory element CEN6-ARS4 was synthesized and inserted into the plasmid to enable it to replicate in yeast. The selection sequence HIS3, obtained by polymerase chain reaction (PCR) from the plasmid pBS313, was inserted for screening. This new synthetic shuttle vector can mediate the transformation-associated recombination (TAR) assembly of large DNA fragments in yeast, and the assembled products can be transformed into Escherichia coli for further amplification. We also conducted in vivo DNA assembly using pGF and yeast homologous recombination and constructed a 31-kb long DNA sequence from the cyanophage PP genome. Our findings show that this novel shuttle vector would be a useful tool for efficient genome-scale DNA reconstruction.

Immunogenicity of multi-epitope-based vaccine candidates administered with the adjuvant Gp96 against rabies

Yange Niu, Ye Liu, Limin Yang, Hongren Qu, Jingyi Zhao, Rongliang Hu, Jing Li, Wenjun Liu

2016, 31(2): 168 doi: 10.1007/s12250-016-3734-4

Received: 29 January 2016 Accepted: 16 March 2016 Published: 06 April 2016
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Rabies, a zoonotic disease, causes > 55,000 human deaths globally and results in at least 500 million dollars in losses every year. The currently available rabies vaccines are mainly inactivated and attenuated vaccines, which have been linked with clinical diseases in animals. Thus, a rabies vaccine with high safety and efficacy is urgently needed. Peptide vaccines are known for their low cost, simple production procedures and high safety. Therefore, in this study, we examined the efficacy of multi-epitope-based vaccine candidates against rabies virus. The ability of various peptides to induce epitope-specific responses was examined, and the two peptides that possessed the highest antigenicity and conservation, i.e., AR16 and hPAB, were coated with adjuvant canineGp96 and used to prepare vaccines. The peptides were prepared as an emulsion of oil in water (O/W) to create three batches of bivalent vaccine products. The vaccine candidates possessed high safety. Virus neutralizing antibodies were detected on the day 14 after the first immunization in mice and beagles, reaching 5–6 IU/mL in mice and 7–9 IU/mL in beagles by day 28. The protective efficacy of the vaccine candidates was about 70%–80% in mice challenged by a virulent strain of rabies virus. Thus, a novel multi-epitope-based rabies vaccine with Gp96 as an adjuvant was developed and validated in mice and dogs. Our results suggest that synthetic peptides hold promise for the development of novel vaccines against rabies.

Isolation and characterization of Zika virus imported to China using C6/36 mosquito cells

Chenglin Deng, Siqing Liu, Qiuyan Zhang, Mingyue Xu, Honglei Zhang, Dayong Gu, Lei Shi, Jian'an He, Gengfu Xiao, Bo Zhang

2016, 31(2): 176 doi: 10.1007/s12250-016-3778-5

Published: 22 April 2016
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Here, we describe a cell culture-based procedure for isolating the infectious ZIKV (GenBank KU963796) from a human serum sample (ca. 50 μL) with an extremely low viral load (Ct value = 32).

Using the inverse Poisson distribution to calculate multiplicity of infection and viral replication by a high-throughput fluorescent imaging system

Robert William Figliozzi, Feng Chen, Albert Chi, Hsia Shao-Chung Victor

2016, 31(2): 180 doi: 10.1007/s12250-015-3662-8

Published: 26 January 2016
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Here, we introduce an additional method that we believe has merits with respect to reducing labor, decreasing wait times, improving objectivity, and decreasing long-term costs. This new method, called fluorescently labeled infected cell inoculum titration (FLICIT), utilizes high-throughput fluorescent microscopy instrumentation such as Cytation 3 from Biotek, and is therefore only applicable to transgenic viruses that cause host cells to express fluorescent proteins. For the purpose of developing this method, we used the recombinant virus HSV-1 strain 17-Syn+, which expresses green fluorescent protein (GFP) (Figure 1A) and exhibits the same replication pattern as the wild type counterpart (Foster et al., 1998). This new method relies on titrating the inoculum so that dilution ensures that the rate of infection is less than 50%, with cells infected by nearly one viral particle each; thus, each fluorescent signal can be attributed to a single viral particle.

Identification and characterization of two human immunodeficiency virus type 1 unique recombinant forms from Yunnan, China

Jianjian Li, Lin Li, Shaomin Yang, Jingyun Li, Mi Zhang, Cuixian Yang, Jiafa Liu, Huiqin Li

2016, 31(2): 184 doi: 10.1007/s12250-015-3671-7

Published: 31 January 2016
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Recombination contributes greatly to the diversity of human immunodeficiency virus type 1 (HIV-1). A large number of recombinant strains have been found in China, particularly in Yunnan, which is considered the HIV-1 epicenter of China. Surveillance of unique recombinant forms is helpful for prediction of new circulating recombinant forms. In this study, we identified two unique recombinant forms of HIV-1 in Yunnan Province. The near full-length genomes of HIV-1 strains were amplified in three parts, and the genomic structures of two strains (12YN10159 and 12YN10192) were analyzed. Strain 12YN10159 was comprised of subtypes C and CRF01_AE, while strain 12YN10192 was comprised of subtypes B and C. Genomic breakpoints were different from all previously reported strains. The data emphasized that more surveillance of unique strains of HIV is needed in Yunnan.

Narrow distribution of cyanophage psbA genes observed in two paddy waters of Northeast China by an incubation experiment

Xinzhen Wang, Ruiyong Jing, Junjie Liu, Zhenhua Yu, Jian Jin, Xiaobing Liu, Xiaojuan Wang, Guanghua Wang

2016, 31(2): 188 doi: 10.1007/s12250-015-3673-5

Published: 26 January 2016
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we successfully isolated 152 cyanophage psbA clones from two paddy waters of NE China. Although the majority of psbA sequences observed in this study had relative high similarity with those from Japanese paddy water, two and seven specific cyanophage psbA groups were constructed inJPWs and this study, respectively, which suggested that cyanophage psbA assemblages in paddy waters, to some extent were different between two countries. In addition, we also found that the majority of psbA clones from paddy waters of both countries fell into the subcluster α-2, suggesting that the distribution of cyanophage psbA genes in paddy waters is narrow, butfar away from those from environmental freshwater and seawater.