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2018 Vol.33(6)

PERSPECTIVE

On the Centenary of the Spanish Flu: Being Prepared for the Next Pandemic

William J. Liu, Yuhai Bi, Dayan Wang, George F. Gao

2018, 33(6): 463 doi: 10.1007/s12250-018-0079-1

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Influenza is one of the oldest infectious diseases affecting humans. Every influenza pandemic in history has ended with disastrous outcomes regarding public health and the social economy. This year marks the 100th anniversary of the Spanish flu (H1N1) outbreak of 1918, which is recognized as the most lethal natural event in recent history. In spite of limited travel and transportation at that time, the Spanish flu pandemic of 1918 reached peak activity on multiple continents simultaneously within several months after its emergence in late 1917 from different hypothesized origins, such as US military camps, the state of Kansas, or the troop staging and hospital camp in étaples, France (Patterson and Pyle 1991; Oxford et al. 2005; Shanks 2016). However, in some islands of the Pacific region, such as in New Caledonia, the pandemic’s lethal effects lasted for over 3 years, until July 1921 (Shanks et al. 2018). The pandemic flu is estimated to have infected more than 500 million people, causing between 50 and 100 million deaths globally (Patterson and Pyle 1991; Guan et al. 2010). The evidence suggests that most pandemicrelated deaths were not caused by the direct pathological effects of the influenza virus, but rather by the lethal effects of secondary bacterial pneumonia (Chien et al. 2009).

The Era of Immune Checkpoint Therapy: From Cancer to Viral Infection—A Mini Comment on the 2018 Medicine Nobel Prize

Jia Liu, Wen Pan, Dongliang Yang

2018, 33(6): 467 doi: 10.1007/s12250-018-0077-3

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The 2018 Medicine Nobel Prize was awarded jointly to two immunologists, James P. Allison at the University of Texas MD Anderson Cancer Center in Houston and Tasuku Honjo at Kyoto University in Japan, who pioneered a new way to treat cancers (Ledford et al. 2018). Both Laureates have shown how so called "immune checkpoints" on T cells can be used to manipulate the immune responses so that T cells can efficiently attack cancer cells. Using the immune system to fight cancers has been investigated for more than a 100 years. Recent advances in cancer immunotherapy, particularly immune checkpoint blockade therapy have dramatically changed the therapeutic strategy against advanced cancers. Through inhibiting negative immune regulation, these approaches have demonstrated improved overall survival for patients with advanced cancers. Importantly, for some of the patients treated with such strategies, their tumors seem to totally disappear.
REVIEW

Reverse Genetics for Peste des Petits Ruminants Virus: Current Status and Lessons to Learn from Other Non-segmented Negative-Sense RNA Viruses

Alfred Niyokwishimira, Yongxi Dou, Bang Qian, Prajapati Meera, Zhidong Zhang

2018, 33(6): 472 doi: 10.1007/s12250-018-0066-6

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Peste des petits ruminants (PPR) is a highly contagious transboundary animal disease with a severe socio-economic impact on the livestock industry, particularly in poor countries where it is endemic. Full understanding of PPR virus (PPRV) pathobiology and molecular biology is critical for effective control and eradication of the disease. To achieve these goals, establishment of stable reverse genetics systems for PPRV would play a key role. Unfortunately, this powerful technology remains less accessible and poorly documented for PPRV. In this review, we discussed the current status of PPRV reverse genetics as well as the recent innovations and advances in the reverse genetics of other non-segmented negative-sense RNA viruses that could be applicable to PPRV. These strategies may contribute to the improvement of existing techniques and/or the development of new reverse genetics systems for PPRV.
Research Article

Genetic Evidence of Middle East Respiratory Syndrome Coronavirus (MERS-Cov) and Widespread Seroprevalence among Camels in Kenya

Sheila Ommeh, Wei Zhang, Ali Zohaib, Jing Chen, Huajun Zhang, Ben Hu, Xing-Yi Ge, Xing-Lou Yang, Moses Masika, Vincent Obanda, Yun Luo, Shan Li, Cecilia Waruhiu, Bei Li, Yan Zhu, Desterio Ouma, Vincent Odendo, Lin-Fa Wang, Danielle E. Anderson, Jacqueline Lichoti, Erick Mungube, Francis Gakuya, Peng Zhou, Kisa-Juma Ngeiywa, Bing Yan, Bernard Agwanda, Zheng-Li Shi

2018, 33(6): 484 doi: 10.1007/s12250-018-0076-4

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We describe the first genome isolation of Middle East respiratory syndrome coronavirus (MERS-CoV) in Kenya. This fatal zoonotic pathogen was first described in the Kingdom of Saudi Arabia in 2012. Epidemiological and molecular evidence revealed zoonotic transmission from camels to humans and between humans. Currently, MERS-CoV is classified by the WHO as having high pandemic potential requiring greater surveillance. Previous studies of MERS-CoV in Kenya mainly focused on site-specific and archived camel and human serum samples for antibodies. We conducted active nationwide cross-sectional surveillance of camels and humans in Kenya, targeting both nasal swabs and plasma samples from 1,163 camels and 486 humans collected from January 2016 to June 2018. A total of 792 camel plasma samples were positive by ELISA. Seroprevalence increased with age, and the highest prevalence was observed in adult camels (82.37%, 95% confidence interval (CI) 79.50–84.91). More female camels were significantly seropositive (74.28%, 95% CI 71.14–77.19) than male camels (Porf4b in African viruses. None of the human plasma screened contained neutralizing antibodies against MERS-CoV. This study confirms the geographically widespread occurrence of MERS-CoV in Kenyan camels. Further one-health surveillance approaches in camels, wildlife, and human populations are needed.

Influence of Human Papillomavirus E7 Oncoprotein on Maturation and Function of Plasmacytoid Dendritic Cells In Vitro

Rui Han, Yin-Jing Song, Si-Yuan Sun, Qiang Zhou, Xian-Zhen Chen, Qiao-Li Zheng, Hao Cheng

2018, 33(6): 493 doi: 10.1007/s12250-018-0069-3

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The major difficulties of human papillomavirus (HPV) treatment are its persistence and recurrence. The HPV E7 oncoprotein-loaded dendritic cells have been evaluated as cellular vaccine in previous reports. Plasmacytoid dendritic cells (pDCs) play an essential role of connecting the innate immune response and adaptive immune response in the immune system. But they function in HPV E7 loading is unclear. To investigate whether loading of the HPV type 6b, 11, and 16 E7 proteins affects the activity of pDCs, human peripheral blood-separated pDCs and mouse bone marrow-derived pDCs were pulsed with the HPV E7 proteins. The expression levels of CD40, CD80, CD86, and MHC II were significantly upregulated in pDCs upon HPV 6b/11 E7 protein pulse. The secretion and gene expression of type I IFN and IL-6 were both upregulated by HPV 6b/11 E7 proteins, more significant than HPV 16 E7 protein. The expression of essential factors of TLR signaling pathway and JNK/p38 MAP kinase signaling pathway were all increased in HPV 6b/11 E7 proteins pulsed pDCs. Our results suggest that HPV E7 proteins could promote the differentiation and maturation of pDCs and activate the TLR and MAPK pathway to induce host innate immune response. It might be conducive to explore novel immunotherapy targeting HPV infection with HPV E7 loaded pDC.

Divergent Primary Immune Responses Induced by Human Immunodeficiency Virus-1 gp120 and Hepatitis B Surface Antigen Determine Antibody Recall Responses

Li Yuan, Wen-Jiang Chen, Jia-Ye Wang, Yan Li, Dan Tian, Ming-Xia Wang, Hao-Tong Yu, Ying-Chu Xu, Di Li, Min Zhuang, Hong Ling

2018, 33(6): 502 doi: 10.1007/s12250-018-0074-6

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The development of a vaccine based on human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) that elicits potent protective antibodies against infection has been challenging. Recently, we compared the antibody production patterns of HIV-1 Env gp120 and hepatitis B virus surface antigen (HBsAg) to provide insights into how we may improve the protective efficacy of Env-based immunogens. Our previous study showed that HIV Env and HBsAg display different mechanisms of antibody elicitation and that T cells facilitate the responses to repeated immunizations. Here, to elucidate the detailed roles of primary immunization in immune memory response formation and antibody production, we immunized C57BL/6 mice with each antigen and evaluated the development of T follicular helper (Tfh) cells, germinal centers, and the memory responses involved in prime and boost immunizations. We found that after prime immunization, compared with HBsAg, gp120 induced higher frequencies of Tfh cells and programmed death (PD)-1+ T cells, greater major histocompatibility complex II expression on B cells, comparable activated B cells, but weaker germinal center (GC) reactions and memory B cell responses in the draining lymph nodes, accompanied by slower antibody recall responses and poor immune memory responses. The above results suggested that more PD-1+ T cells arising in primary immunization may serve as major contributors to the slow antibody recall response elicited by HIV-1 Env.

Japanese Encephalitis Virus NS1' Protein Antagonizes Interferon Beta Production

Dengyuan Zhou, Fan Jia, Li Qiuyan, Luping Zhang, Zheng Chen, Zikai Zhao, Min Cui, Yunfeng Song, Huanchun Chen, Shengbo Cao, Jing Ye

2018, 33(6): 515 doi: 10.1007/s12250-018-0067-5

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Japanese encephalitis virus (JEV) is a mosquito-borne virus and the major cause of viral encephalitis in Asia. NS1', a 52-amino acid C-terminal extension of NS1, is generated with a -1 programmed ribosomal frameshift and is only present in members of the Japanese encephalitis serogroup of flaviviruses. Previous studies demonstrated that NS1' plays a vital role in virulence, but the mechanism is unclear. In this study, an NS1' defected (rG66A) virus was generated. We found that rG66A virus was less virulent than its parent virus (pSA14) in wild-type mice. However, similar mortality caused by the two viruses was observed in an IFNAR knockout mouse model. Moreover, we found that rG66A virus induced a greater type I interferon (IFN) response than that by pSA14, and JEV NS1' significantly inhibited the production of IFN-β and IFN-stimulated genes. Taken together, our results reveal that NS1' plays a vital role in blocking type I IFN production to help JEV evade antiviral immunity and benefit viral replication.

Phenotypic Characterization of Porcine IFNγ-Producing Lymphocytes in Porcine Reproductive and Respiratory Syndrome Virus Vaccinated and Challenged Pigs

Xiangdong Li, Zengyang Pei, Yilin Bai, Lihua Wang, Jishu Shi, Kegong Tian

2018, 33(6): 524 doi: 10.1007/s12250-018-0073-7

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Porcine reproductive and respiratory syndrome (PRRS) continues to be one of the most important swine diseases worldwide. Interferon-c (IFNγ)-mediated type I cell-mediated immune response plays an important role in protection from, and clearance of, PRRS virus (PRRSV). Several lymphocyte subsets including T-helper, CTLs, Th/memory cells, and γδ T lymphocytes were previously reported to produce IFNγ during PRRSV infection. However, the proportion and phenotypic characterization of these IFNγ-secreting lymphocytes have not been explored. In this study, IFNγ producted by different lymphocyte subsets was assessed by multi-color flow cytometry after vaccination with PRRSV modified live vaccine (PRRSV-MLV) and challenge with homogeneous or heterogeneous PRRSV. The results showed that T-helper cells were the major IFNγ-secreting cell population after PRRSV-MLV vaccination and PRRSV challenge. Additionally, the proportion of IFNγ producing Th/memory cells and γδ T cells increased after PRRSV challenge. This difference was accounted for an enhanced ability to produce IFNγ in Th/memory cells and an enlarged quantity of γδ T cells. The results presented here could contribute to our understanding of the roles of IFNγ in protective immunity against PRRSV infection and may be useful for assessment of cell-mediated immunity in vaccine tests.

The D253N Mutation in the Polymerase Basic 2 Gene in Avian Influenza (H9N2) Virus Contributes to the Pathogenesis of the Virus in Mammalian Hosts

Jinfeng Zhang, Rong Su, Xiaoyun Jian, Hongliang An, Ronbing Jiang, Chris Ka Pun Mok

2018, 33(6): 531 doi: 10.1007/s12250-018-0072-8

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Mutations in the polymerase basic 2 (PB2) gene of avian influenza viruses are important signatures for their adaptation to mammalian hosts. Various adaptive mutations have been identified around the 627 and nuclear localization sequence (NLS) domains of PB2 protein, and these mutations contribute to the replicative ability of avian influenza viruses. However, few studies have focused on adaptive mutations in other regions of PB2. In this study, we investigated the functional roles of the D253N mutation in PB2 in an H9N2 virus. This mutation was found to affect an amino acid residue in the middle domain of the PB2 protein. The virus with the D253N mutation showed higher polymerase activity and transiently increased viral replication in human cells. However, the mutant did not show significant differences in viral replication in the respiratory tract of mice upon infection. Our results supported that the D253N mutation in the middle domain of PB2, similar to mutations at the 627 and NLS domains, specifically contributed to the replication of avian influenza viruses in human cells.

In Vitro Anti-hepatitis B Virus Activity of 2', 3'-Dideoxyguanosine

Pinghu Zhang, Shuo Zhai, Jinhong Chang, Ju-Tao Guo

2018, 33(6): 538 doi: 10.1007/s12250-018-0065-7

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2',3'-dideoxyguanosine (DoG) has been demonstrated to inhibit duck hepatitis B virus (DHBV) replication in vivo in a duck model of HBV infection. In the current study, the in vitro antiviral effects of DoG on human and animal hepadnaviruses were investigated. Our results showed that DoG effectively inhibited HBV, DHBV, and woodchuck hepatitis virus (WHV) replication in hepatocyte-derived cells in a dose-dependent manner, with 50% effective concentrations (EC50) of 0.3 ± 0.05, 6.82 ± 0.25, and 23.0 ± 1.5 lmol/L, respectively. Similar to other hepadnaviral DNA polymerase inhibitors, DoG did not alter the levels of intracellular viral RNA but induced the accumulation of a less-than-full-length viral RNA species, which was recently demonstrated to be generated by RNase H cleavage of pgRNA. Furthermore, using a transient transfection assay, DoG showed similar antiviral activity against HBV wild-type, 3TC-resistant rtA181V, and adefovirresistant rtN236T mutants. Our results suggest that DoG has potential as a nucleoside analogue drug with anti-HBV activity.

The Scorpion Venom Peptide Smp76 Inhibits Viral Infection by Regulating Type-Ⅰ Interferon Response

Zhenglin Ji, Fangfang Li, Zhiqiang Xia, Xingchen Guo, Minjun Gao, Fang Sun, Yuting Cheng, Yingliang Wu, Wenxin Li, Syed Abid Ali, Zhijian Cao

2018, 33(6): 545 doi: 10.1007/s12250-018-0068-4

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Dengue virus (DENV) and Zika virus (ZIKV) have spread throughout many countries in the developing world and infect millions of people every year, causing severe harm to human health and the economy. Unfortunately, there are few effective vaccines and therapies available against these viruses. Therefore, the discovery of new antiviral agents is critical. Herein, a scorpion venom peptide (Smp76) characterized from Scorpio maurus palmatus was successfully expressed and purified in Escherichia coli BL21(DE3). The recombinant Smp76 (rSmp76) was found to effectively inhibit DENV and ZIKV infections in a dose-dependent manner in both cultured cell lines and primary mouse macrophages. Interestingly, rSmp76 did not inactivate the viral particles directly but suppressed the established viral infection, similar to the effect of interferon (IFN)-β. Mechanistically, rSmp76 was revealed to upregulate the expression of IFN-β by activating interferon regulatory transcription factor 3 (IRF3) phosphorylation, enhancing the type-I IFN response and inhibiting viral infection. This mechanism is significantly different from traditional virucidal antimicrobial peptides (AMPs). Overall, the scorpion venom peptide Smp76 is a potential new antiviral agent with a unique mechanism involving type-I IFN responses, demonstrating that natural AMPs can enhance immunity by functioning as immunomodulators.
LETTER

An Assessment of Amplicon-Sequencing Based Method for Viral Intrahost Analysis

Ming Ni, Chen Chen, Di Liu

2018, 33(6): 557 doi: 10.1007/s12250-018-0052-z

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RNA virus features the high per-generation mutation rate, and thus usually exists as a population presenting intrahost diversity. By the aids of deep sequencing and bioinformatic techniques, recent studies have provided new insights into viral intrahost evolution and viral-host interaction. Currently, many studies have employed the approach of sequencing of viral amplicons (amplicon-seq) to investigate the diversity of viral population. However, there remains concerns about the discrimination of intrahost variations from amplification or sequencing errors, and thus requires assessments on the sensitivity and the specificity of the amplicon-seq approach applying on virus studies. In this study, we designed an assay for assessment of the amplicon-seq approach in the aspects of false negative rate (FNR) and false positive rate (FPR) of intrahost single nucleotide variation (iSNV) calling, accuracy of mutated allele frequency (MuAF) estimation, and MuAF bias between amplicons. We found that when samples had a relatively large viral load (~25 Ct value or ~ 1 × 105 copies/μL), an iSNV calling threshold of MuAF > 0.3% was applicable enables a zero FNR and a Ct value or ~ 100 copies/μL), and a higher threshold of MuAF > 0.7% could be used to provide comparable FNR and FPR. Therefore, Amplicon-seq, as an efficient and applicable method for clinical and field viral samples, could provide sufficient accuracy and sensitivity for viral intrahost analysis.

Mink Circovirus Can Infect Minks, Foxes and Raccoon Dogs

Yanling Yang, Yuening Cheng, Nan Li, Shipeng Cheng, Li Guo, Yucheng Zhou, Haiwei Zhang, Xinyuan Zhang, Linzhu Ren

2018, 33(6): 561 doi: 10.1007/s12250-018-0059-5

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An investigation was carried out to test for mink circovirus (MiCV) infection in samples from minks, foxes and raccoon dogs with diarrhea in China. The results demonstrated that the infection rates of MiCV in minks, foxes and raccoon dogs were very high on some fur farms in China. The fox was the most sensitive animal to the viral infection, followed by the mink and the raccoon dog. Furthermore, three isolates, MiCV-LN, Fox CV-JL, and Raccoon CV-HLJ, were closely related to the previously reported isolate MiCV-DL13, with 99 % homology. Moreover, concurrent infection with MiCV and other pathogens may contribute to the disease in minks, foxes and raccoon dogs, as the rate of coinfection of MiCV and E. coli was 9 % (7/78) and that of triple infection (MiCV+ E. coli+ MEV) was 1.3 % (1/78) among the samples.