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2018 Vol.33(3)

The newly emerged mosquito-borne Zika virus (ZIKV) has posed a global threat to human health. Several ZIKV vaccine candidates have been developed, and demonstrated to be efficacious in preclinical studies in mice and nonhuman primates. In this issue, Shiyu Dai et al. report a new virus-like particle (VLP) ZIKV vaccine based on the baculovirus expression system. Those VLPs demonstrate good immunogenicity in immunized mice, as they stimulate high levels of virus neutralizing antibody titers, ZIKV-specific IgG titers and potent memory T cell responses. The cover is adopted from an electron micrograph of negative staining baculovirus-expressed ZIKV VLPs (kindly provided by Fei Deng) with artistic processing. See page 213–226 for details.

Research Article

Zika Virus Baculovirus-Expressed Virus-Like Particles Induce Neutralizing Antibodies in Mice

Shiyu Dai, Tao Zhang, Yanfang Zhang, Hualin Wang, Fei Deng

2018, 33(3): 213 doi: 10.1007/s12250-018-0030-5

Received: 13 December 2017 Accepted: 30 March 2018 Published: 17 May 2018
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The newly emerged mosquito-borne Zika virus (ZIKV) strains pose a global challenge owing to its ability to cause microcephaly and neurological disorders. Several ZIKV vaccine candidates have been proposed, including inactivated and live attenuated virus vaccines, vector-based vaccines, DNA and RNA vaccines. These have been shown to be efficacious in preclinical studies in mice and nonhuman primates, but their use will potentially be a threat to immunocompromised individuals and pregnant women. Virus-like particles (VLPs) are empty particles composed merely of viral proteins, which can serve as a safe and valuable tool for clinical prevention and treatment strategies. In this study, we used a new strategy to produce ZIKV VLPs based on the baculovirus expression system and demonstrated the feasibility of their use as a vaccine candidate. The pre-membrane (prM) and envelope (E) proteins were co-expressed in insect cells and selfassembled into particles similar to ZIKV. We found that the ZIKV VLPs could be quickly and easily prepared in large quantities using this system. The VLPs were shown to have good immunogenicity in immunized mice, as they stimulated high levels of virus neutralizing antibody titers, ZIKV-specific IgG titers and potent memory T cell responses. Thus, the baculovirus-based ZIKV VLP vaccine is a safe, effective and economical vaccine candidate for use against ZIKV.

Evaluation of Epstein-Barr Virus Salivary Shedding in HIV/AIDS Patients and HAART Use: A Retrospective Cohort Study

Yan Yan, Yong Ren, Renfang Chen, Jing Hu, Yongjia Ji, Junyang Yang, Jiayin Shen, Lvyin Hu, Hao Pei, Jun Wang, Yuanwang Qiu, Hongzhou Lu, Lihua Huang

2018, 33(3): 227 doi: 10.1007/s12250-018-0028-z

Received: 25 October 2017 Accepted: 09 March 2018 Published: 13 April 2018
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Little data is available on the evaluation of the occurrence rates of Epstein-Barr virus (EBV) in saliva and relationship with highly active antiretroviral therapy (HAART) use in HIV/AIDS patients in China. We conducted a retrospective cohort study of EBV serological tests for HIV/AIDS patients who were treated in the hospitals for infectious diseases in Wuxi and Shanghai, China from May 2016 to April 2017. The EBV-seropositive samples were identified by ELISA. EBV-specific primers and probes were used for the quantitative detection of viralDNAfrom saliva via quantitativereal-time polymerase chainreaction. CD4 cell counts of the HIV/ AIDS patientswere detected by a flow cytometry.A total of 372HIV/AIDS patients were ultimately selected and categorizedfor this retrospective cohort study. For EBV IgG and IgM, the HIV/AIDS HAART use (H) and non-HAART use (NH) groups had significantly higher seropositive rates than the HIV-negative control group. The HIV/AIDS (NH) group had the highest seropositive rate (IgG, 94.27%; IgM, 68.98%) and the highest incidence of EBV reactivation or infection. For salivary EBV DNA-positive rates and quantities, the HIV/AIDS (H) (73.69%) and the HIV/AIDS (NH) (100%) groups showed significantly higher values than the HIV-negative control group(35.79%,> twofold).Further,the salivaryEBVDNA-negative population hadsignificantly higher CD4 cell counts than the EBV DNA-positive population in the HIV/AIDS (H) group and the HIV/AIDS (NH) groups. Thus, HAART use is beneficial in decreasing the EBV salivary shedding in HIV/AIDS patients and indirectly decreases EBV transmission risk.

Carrier Cells for Delivery of Oncolytic Measles Virus into Tumors: Determinants of Efficient Loading

Chun Xu, Mao Xia, Gang Meng, Chunyan Li, Aiqin Jiang, Jiwu Wei

2018, 33(3): 234 doi: 10.1007/s12250-018-0033-2

Received: 16 November 2017 Accepted: 11 April 2018 Published: 16 May 2018
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Oncolytic measles virus (OMV) is a promising antitumor agent. However, the presence of anti-measles neutralizing antibodies (NAbs) against the hemagglutinin (H) protein of OMV is a major barrier to the therapeutic application of OMV in clinical practice. In order to overcome this challenge, specific types of cells have been used as carriers for OMV. Differential loading strategies appear to result in different therapeutic outcomes; despite this, only few studies have reported practical ex vivo loading strategies required for effective treatment. To this end, we systematically evaluated the antitumor efficacy of OMV using different loading strategies; this involved varying the in vitro loading duration and loading dose of OMV. We found that improved oncolysis of carrier cells was achieved by a prolonged loading duration in the absence of NAbs. However, the enhanced oncolytic effect was abrogated in the presence of NAbs. Further, we found that the expression of H protein on the surface of carrier cells was predominantly determined by the loading duration rather than the loading dose. Finally, we showed that NAbs blocked viral transfer by targeting H protein prior to the occurrence of cell-to-cell interactions. Our results provide comprehensive information on the determinants of an effective loading strategy for carrier cell-based virotherapy; these results may be useful for guiding the application of OMV as an antitumor agent in clinical practice.

Exhaustive Exercise Does Not Affect Humoral Immunity and Protection after Rabies Vaccination in a Mouse Model

Lun Xia, Minrui Li, Yajing Zhang, Juncheng Ruan, Jie Pei, Jiale Shi, Zhen F. Fu, Ling Zhao, Ming Zhou, Dayong Tian

2018, 33(3): 241 doi: 10.1007/s12250-018-0026-1

Received: 17 December 2017 Accepted: 31 January 2018 Published: 28 March 2018
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Rabies is one of the most dangerous and widespread zoonosis and is characterized by severe neurological signs and a high case-mortality rate of nearly 100%. Vaccination is the most effective way to prevent rabies in humans and animals. In this study, the relationship between exhaustive exercise and the humoral immune response after immunization with inactivated rabies vaccine was investigated in a mouse model with one-time exhaustive exercise. It was found that compared with the mice with no exercise after vaccination, no significant differences were found in those with exhaustive exercise after vaccination on body-weight changes, virus-neutralizing antibody (VNA) titers, antibody subtypes and survivor ratio after lethal rabies virus (RABV) challenge. This study indicated that exhaustive exercise does not reduce the effects of the rabies inactivated vaccine.

Porcine FcεRI Mediates Porcine Reproductive and Respiratory Syndrome Virus Multiplication and Regulates the Inflammatory Reaction

Peidian Shi, Lilin Zhang, Jiashun Wang, Dong Lu, Yi Li, Jie Ren, Menglu Shen, Lei Zhang, Jinhai Huang

2018, 33(3): 249 doi: 10.1007/s12250-018-0032-3

Received: 25 December 2017 Accepted: 02 April 2018 Published: 14 May 2018
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Porcine reproductive and respiratory syndrome virus (PRRSV) shows characteristic antibody-dependent enhancement (ADE) of infection and causes porcine systemic inflammation, which is similar to a type I allergic reaction; however, the role of porcine FcεRI in ADE is still unclear. In this study, the expression of different Fc receptors (FcRs) on macrophages was investigated in a PRRSV 3D4/21 cell infection model in the presence or absence of PRRSV antibody. The transcription level of FcγII and FcεRI was significantly up-regulated under PRRSV-antibody complex infection. Internalization and proliferation of PRRSV were promoted by the ADE mechanism when FcεRI was expressed in permissive 3D4/21 cells and the non-permissive cell line HEK 293T. Transcriptome sequencing data showed that the expression levels of AKT, ERK and other signal molecules in the anti-inflammatory pathway were significantly increased, especially in the cells infected with the PRRSV-antibody immune complex. Inflammatory regulatory molecules such as PLA2G6, LOX, TRPM8 and TRPM4 were significantly up-regulated following PRRSV infection but significantly down-regulated in the cells infected with the PRRSV-antibody immune complex. Our results demonstrated that FcεRI could be involved in PRRSV ADE, the antigen presenting process and regulation of the inflammatory response during PRRSV infection, which provides new insights into PRRSV infection mediated by FcεRI and the PRRSV-antibody immune complex.

HIV-1 Protein Tat1–72 Impairs Neuronal Dendrites via Activation of PP1 and Regulation of the CREB/BDNF Pathway

Yu Liu, Deyu Zhou, Jiabin Feng, Zhou Liu, Yue Hu, Chang Liu, Xiaohong Kong

2018, 33(3): 261 doi: 10.1007/s12250-018-0031-4

Received: 09 February 2018 Accepted: 02 April 2018 Published: 08 May 2018
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Despite the success of combined antiretroviral therapy in recent years, the prevalence of human immunodeficiency virus (HIV)-associated neurocognitive disorders in people living with HIV-1 is increasing, significantly reducing the health-related quality of their lives. Although neurons cannot be infected by HIV-1, shed viral proteins such as transactivator of transcription (Tat) can cause dendritic damage. However, the detailed molecular mechanism of Tat-induced neuronal impairment remains unknown. In this study, we first showed that recombinant Tat (1-72 aa) induced neurotoxicity in primary cultured mouse neurons. Second, exposure to Tat1-72 was shown to reduce the length and number of dendrites in cultured neurons. Third, Tat1-72 (0-6 h) modulates protein phosphatase 1 (PP1) expression and enhances its activity by decreasing the phosphorylation level of PP1 at Thr320. Finally, Tat1-72 (24 h) downregulates CREB activity and CREB-mediated gene (BDNF, c-fos, Egr-1) expression. Together, these findings suggest that Tat1-72 might impair cognitive function by regulating the activity of PP1 and the CREB/BDNF pathway.

Micro-droplet Digital Polymerase Chain Reaction and Real-Time Quantitative Polymerase Chain Reaction Technologies Provide Highly Sensitive and Accurate Detection of Zika Virus

Yuan Hui, Zhiming Wu, Zhiran Qin, Li Zhu, Junhe Liang, Xujuan Li, Hanmin Fu, Shiyu Feng, Xiaoen He, Weizhi Lu, Weiwei Xiao, Qinghua Wu, Bao Zhang, Wei Zhao

2018, 33(3): 270 doi: 10.1007/s12250-018-0037-y

Received: 07 March 2018 Accepted: 23 May 2018 Published: 14 June 2018
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The establishment of highly sensitive diagnostic methods is critical in the early diagnosis and control of Zika virus (ZIKV) and in preventing serious neurological complications of ZIKV infection. In this study, we established micro-droplet digital polymerase chain reaction (ddPCR) and real-time quantitative PCR (RT-qPCR) protocols for the detection of ZIKV based on the amplification of the NS5 gene. For the ZIKV standard plasmid, the RT-qPCR results showed that the cycle threshold (Ct) value was linear from 101 copy/μL to 108 copy/μL, with a standard curve R2 of 0.999 and amplification efficiency of 92.203%; however, a concentration as low as 1 copy/μL could not be detected. In comparison with RT-qPCR, the ddPCR method resulted in a linear range of 101 to 104copy/μL and was able to detect concentrations as low as 1 copy/μL. Thus, for detecting ZIKV from clinical samples, RT-qPCR is a better choice for high-concentration samples (above 101 copy/μL), while ddPCR has excellent accuracy and sensitivity for low-concentration samples. These results indicate that the ddPCR method should be of considerable use in the early diagnosis, laboratory study, and monitoring of ZIKV.
Letter

Histopathological Features and Viral Antigen Distribution in the Lung of Fatal Patients with Enterovirus 71 Infection

Zhenli Liang, Hongbo Pan, Xijing Wang, Yinchuan Zhu, Yiwu Dang, Xiaohui Fan, Lingxi Gao, Zengfeng Zhang

2018, 33(3): 278 doi: 10.1007/s12250-018-0029-y

Received: 29 November 2017 Accepted: 09 March 2018 Published: 19 April 2018
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Enterovirus 71 (EV71) infection causes hand-foot-and-mouth disease (HFMD) in children and might be accompanied by severe neurological complications. It has become one of the most important pathogens of central nervous system infection. To explore the causes of lung injury by EV71, the distribution of EV71 receptors, SCARB2 and PSGL-1, in human lung tissues was examined. Our results revealed that SCARB2 was positively distributed in the bronchial and bronchiolar epithelial cells, alveolar cells and macrophages, while PSGL-1 was positively scattered in bronchial and bronchiolar epithelial cells and macrophages, and negatively distributed in alveolar cells. The pathological changes of fatal lung with EV71 infection demonstrated intrapulmonary bronchitis and bronchiolitis, diffuse or focal infiltration of inflammatory cells, such as T cells and B cells in the wall and surrounding tissues, widened alveolar septum, capillaries in the septum with highly dilated and congested, and infiltrated inflammatory cells, showing different degrees of protein edema with fibrin exudation in the alveolar cavity, as well as obvious hyaline membrane formation in some alveolar cavities. The EV71 antigen in lung tissues was detected, and the viral antigen was positive in lung bronchial and bronchiolar epithelial cells, and positively scattered in the alveolar cells and macrophages. Therefore, in addition to the complications of central nervous system injury, the lung remains the main target organ for virus attack in severe EV71 infected patients. Lung injury was mainly caused by neurogenic damage and/or direct invasion of the virus into the lungs in critically serious children, and the lesions were mainly pulmonary edema and interstitial pneumonia.

Emergence of Eurasian Avian-Like Swine Influenza A (H1N1) Virus from an Adult Case in Fujian Province, China

Jian-Feng Xie, Yan-Hua Zhang, Lin Zhao, Wen-Qiong Xiu, Hong-Bin Chen, Qi Lin, Yu-Wei Weng, Kui-Cheng Zheng

2018, 33(3): 282 doi: 10.1007/s12250-018-0034-1

Published: 24 May 2018
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The Eurasian avian-like swine influenza A (H1N1) viruses (EAS-H1N1) have circulated in pigs since 1979, and human sporadically infected with EAS-H1N1 have been reported in several countries. In October 2016, the EAS-H1N1 infection in an adult hospitalized patient with severe pneumonia was identified in Fujian Province in China. The viral RNA was extracted form chicken embryo culture inoculated with throat swab, and complete genome sequences were obtained by RT-PCR and sequencing. Phylogenetic analysis showed that the virus was a novel triple-reassortant H1N1 virus containing genes from the Eurasian avian-like swine (HA, NA), A (H1N1) pdm09 (PB2, PB1, PA, NP, M) and classical swine (NS) lineages, termed influenza A/Fujian-cangshan/SWL624/2016 (FJ/624/16) virus. Complete sequences of FJ/624/16 virus were 94.3%-98.2% and 91.2%-95.4% identical in all 8 gene segments with A/Hunan/42443/2015 virus and A/swine/Guangdong/1/2010 virus respectively. In this study, the isolate contained E190D and D225E substitution within HA, suggesting the preference to α-2,6-linked sialosides and increase in the binding force of H1N1 virus in human upper respiratory tract. The virus was sensitive to neuramidinase inhibitors, but resistant to adamantanes. Several amino acid residues of FJ/624/16 virus might increase viral virulence or adaptation in mammals. In order to find the virulence and human-to-human transmission ability of viruses timely, further epidemiological and virological surveillance in swine and human ought to be enhanced as a part of an overall pandemic preparedness plan.

N-Terminal Myristoylated VP5 is Required for Penetrating Cell Membrane and Promoting Infectivity in Aquareoviruses

Qingxiu Chen, Hong Guo, Fuxian Zhang, Qin Fang

2018, 33(3): 287 doi: 10.1007/s12250-018-0036-z

Received: 16 March 2018 Accepted: 10 May 2018 Published: 05 June 2018
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Nonenveloped animal viruses must disrupt the host cell membrane to initiate infection. Although myristoylated outer capsid protein is known to be responsible for membrane penetration in nonenveloped viruses, the mechanisms underlying membrane penetration to enter the host cytoplasm remain poorly understood, compared to the membrane fusion mechanisms of enveloped viruses. Therefore, we aimed to directly visualize the permeation of fluorescently labeled N-terminal myristoylated VP5 (myr-VP5N) peptides of grass carp reovirus (GCRV) and investigate the molecular mechanism of GCRV infection initiation. We found that myr-VP5N peptides quickly permeate live cell membrane in a cell type-independent manner. Moreover, pretreatment with myr-VP5N promoted the viral activity in infected cells. These results provide a foundation for further studies focused on investigated the molecular events occurring during host cell entry of aquareoviruses.
PERSPECTIVE

Non-Chinese Immigrants: Challenge Faced by Yunnan of China to Achieve the 90–90–90 Goals

Xin Chen, Lin Duo, Mei Ye, Chiyu Zhang, Yong-Tang Zheng

2018, 33(3): 291 doi: 10.1007/s12250-018-0038-x

Received: 02 March 2018 Accepted: 17 May 2018 Published: 11 June 2018
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Yunnan is one of the worst hit regions by HIV/AIDS in China. In last decade, Yunnan has made great achievements in prevention and control of HIV/AIDS. However, Yunnan has a large number of non-Chinese immigrants. HIV prevalence and spreading in this risk population is a huge challenge for Yunnan to achieve the 90-90-90 goals by 2020 and eradicate AIDS epidemic by 2030. Here, we analyzed current status and problems of the prevention and control of HIV/AIDS among non-Chinese immigrants and discussed strategic needs for preventing new HIV infections associated with non-Chinese immigrants.
Correction

Correction to: Molecular detection of viruses in Kenyan bats and discovery of novel astroviruses, caliciviruses and rotaviruses

Cecilia Waruhiu, Sheila Ommeh, Vincent Obanda, Bernard Agwanda, Francis Gakuya, Xing-Yi Ge, Xing-Lou Yang, Li-Jun Wu, Ali Zohaib, Ben Hu, Zheng-Li Shi

2018, 33(3): 294 doi: 10.1007/s12250-018-0035-0

Published: 28 May 2018
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