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Articles in press have been peer-reviewed and accepted, which are not yet assigned to volumes/issues, but are citable by Digital Object Identifier (DOI).
Genome Characteristics and Evolution of Pseudorabies Virus Strains in Eastern China from 2017 to 2019
Xiaofeng Zhai, Wen Zhao, Kemang Li, Cheng Zhang, Congcong Wang, Shuo Su, Jiyong Zhou, Jing Lei, Gang Xing, Haifeng Sun, Zhiyu Shi, Jinyan Gu
doi: 10.1007/s12250-019-00140-1
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Since late 2011, outbreaks of pseudorabies virus (PRV) have occurred in southern China causing major economic losses to the pig industry. We previously reported variant PRV may from recombination in China that could be the source of continued epidemics. Here, we analyzed samples from intensive pig farms in eastern China between 2017 and 2019, and sequenced the main glycoproteins (gB, gC, gD, and gE) to study the evolution characteristics of PRV. Based on the gC gene, we found that PRV variants belong to clade 2 and identified a founder effect by the PRV epidemic process. In addition, we detected inter- and intra-clade recombination, in particular, inter-clade recombination in the gB gene of strains FJ-ZXF and FJ-W2, which were recombinants with clade 1 strains. Specific amino acid changes and positively selected sites possibly associated with functional changes were also observed. The characteristics of PRV emergence in China necessitate continuous monitoring and development of vaccines capable of providing immunity against specific PRV variants.

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Perspective
Boosting Global Yellow Fever Vaccine Supply for Epidemic Preparedness: 3 Actions for China and the USA
Daniel R. Lucey, Kristen R. Kent
2019, 34(3): 235-239.  doi: 10.1007/s12250-019-00129-w
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We advocate for three actions to be taken by the USA and China to help boost global supply of YF vaccine and increase epidemic preparedness and response. First, both nations would work with the WHO to have their YF vaccines prequalified. Thus, their vaccines could be used outside their own borders as part of the international stockpile of YF vaccine as traditionally administrated by the WHO, UNICEF and other members of the multi-partner International Coordinating Group. Second, production of this vaccine would be sharply scaled-up by both countries. These vaccines could be made available nationally, regionally, and globally through the ICG, e.g., to help stop and prevent epidemics anywhere in the world. Third, clinical studies would be coordinated with the WHO to assess the efficacy and safety of fractional dosing using 1/5 normal dose and if needed, even a 1/10 dose.
Cetacean morbillivirus: A Land-to-Sea Journey and Back?
Giovanni Di Guardo, Sandro Mazzariol
2019, 34(3): 240-242.  doi: 10.1007/s12250-019-00128-x
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Cetacean morbillivirus (CeMV), the most relevant pathogen impacting the health and conservation of several already threatened cetacean populations worldwide (Van Bressem et al. 2014), has shown in recent years an apparently increased tendency to cross "interspecies barriers" (Jo et al. 2018a), thereby giving rise to disease and mortality outbreaks in free-ranging dolphins and whales (Mazzariol et al. 2016, 2017; Jo et al. 2018b). Additional cases of infection have been also reported in aquatic mammals with a mixed aquatic-terrestrial ecology like common seals (Phoca vitulina) (Mazzariol et al. 2013) and Eurasian otters (Lutra lutra) (Padalino et al. 2019), increasing the overall concern and attention towards this Morbillivirus genus member. Within such context, the demonstrated ability of the dolphin morbillivirus (DMV) strain to utilize both dolphin and seal SLAM/CD150 as host cell receptors (Jo et al. 2018b) is biologically relevant and supports cross-species viral transmission events.
Research Article
Decreases in Both the Seroprevalence of Serum Antibodies and Seroprotection against Japanese Encephalitis Virus among Vaccinated Children
Ran Wang, Lyu Xie, Na Gao, Dongying Fan, Hui Chen, Peigang Wang, Hongning Zhou, Jing An
2019, 34(3): 243-252.  doi: 10.1007/s12250-019-00099-z
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The incidence of Japanese encephalitis (JE) has significantly decreased in China due to JE vaccines. In this study, we investigated the post-JE vaccination seroprevalence and protection provided by vaccinated sera against Japanese encephalitis virus (JEV) to elucidate the persistence and waning of antibodies to JEV among JE-SA14-14-2-vaccinated children. A total of 300 serum samples were collected from vaccinated children aged 3–10 years in Zhaotong, Yunnan, China. The seroprevalence of anti-JEV antibodies was determined by enzyme-linked immune sorbent assay and plaque reduction neutralization test. The highest seropositivity of 82% was observed in vaccinated children during the first 0.5–1.5 years after booster vaccination. Then, the seropositivity began to decline and remained lower than the original level observed in the 0.5–1.5-year group. An association was found between the waning of seroprevalence and elapsed time of the post-booster vaccination. Similarly, the neutralizing antibody (nAb) titres gradually decreased over time, and the levels showed a positive correlation with the protective efficacy in mice. This finding suggests that nAbs play an important role in the antiviral process and that the nAb titre is an adequately credible parameter for evaluating the protective efficacy induced by the JE vaccine. Our results provide data that clarify the persistence and waning of antibodies to JEV, which may help elucidate the pathogenesis of JE.
Early Pattern of Epstein-Barr Virus Infection in Gastric Epithelial Cells by "Cell-in-cell"
Wenxing Yue, Meijuan Zhu, Lielian Zuo, Shuyu Xin, Jing Zhang, Lingzhi Liu, Shen Li, Wei Dang, Siwei Zhang, Yan Xie, Fanxiu Zhu, Jianhong Lu
2019, 34(3): 253-261.  doi: 10.1007/s12250-019-00097-1
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Epstein-Barr virus (EBV) is an important human dsDNA virus, which has been shown to be associated with several malignancies including about 10% of gastric carcinomas. How EBV enters an epithelial cell has been an interesting project for investigation. "Cell-in-cell" infection was recently reported an efficient way for the entry of EBV into nasopharynx epithelial cells. The present approach was to explore the feasibility of this mode for EBV infection in gastric epithelial cells and the dynamic change of host inflammatory reaction. The EBV-positive lymphoblastic cells of Akata containing a GFP tag in the viral genome were co-cultured with the gastric epithelial cells (GES-1). The infection situation was observed under fluorescence and electron microscopies. Real-time quantitative PCR and Western-blotting assay were employed to detect the expression of a few specific cytokines and inflammatory factors. The results demonstrated that EBV could get into gastric epithelial cells by "cell-in-cell" infection but not fully successful due to the host fighting. IL-1β, IL-6 and IL-8 played prominent roles in the cellular response to the infection. The activation of NF-κβ and HSP70 was also required for the host antiviral response. The results imply that the gastric epithelial cells could powerfully resist the virus invader via cell-in-cell at the early stage through inflammatory and innate immune responses.
The Saffold Virus-Penang 2B and 3C Proteins, but not the L Protein, Induce Apoptosis in HEp-2 and Vero Cells
Yishi Xu, Carla Bianca Luena Victorio, Tao Meng, Qiang Jia, Yee-Joo Tan, Kaw Bing Chua
2019, 34(3): 262-269.  doi: 10.1007/s12250-019-00116-1
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Our previous work has shown that Saffold virus (SAFV) induced several rodent and primate cell lines to undergo apoptosis (Xu et al. in Emerg Microb Infect 3:1–8, 2014), but the essential viral proteins of SAFV involved in apoptotic activity lack study. In this study, we individually transfected the viral proteins of SAFV into HEp-2 and Vero cells to assess their ability to induce apoptosis, and found that the 2B and 3C proteins are proapoptotic. Further investigation indicated the transmembrane domain of the 2B protein is essential for the apoptotic activity and tetramer formation of the 2B protein. Our research provides clues for the possible mechanisms of apoptosis induced by SAFV in different cell lines. It also opens up new directions to study viral proteins (the 2B, 3C protein), and sets the stage for future exploration of any possible link between SAFV, inclusive of its related uncultivable genotypes, and multiple sclerosis.
The Susceptibility of Primary Dermis Fibroblasts from the Chinese Tree Shrew to Human Cytomegalovirus Infection
Shu-Wei Dong, Ling-Shuai Jiao, Ming Yang, Yi-Bo Chen, Ying-Liang Duan, Fei Zhao, A-Mei Zhang, Li Liu, Min-Hua Luo, Xue-Shan Xia
2019, 34(3): 270-277.  doi: 10.1007/s12250-019-00106-3
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As a universal pathogen leading to neonatal defects and transplant failure, human cytomegalovirus (HCMV) has strict species specificity and this has prevented the development of a suitable animal model for the pathogenesis study. The mechanism of cross-species barrier remains elusive and there are so far no non-human cell culture models that support HCMV replication. The Chinese tree shrew (Tupaia belangeri chinensis) is a small laboratory animal and evolutionary closely related with primates. We investigated the susceptibility of primary tree shrew dermis fibroblasts (TSDF) to HCMV infection. Infection with a GFP-expressing HCMV virus resulted in green fluorescence in infected cells with the expression of IE1, UL44 and pp28. The titers of cell-free viruses reached 103 PFU/mL at 96 hpi, compared to titers of 104 PFU/mL observed in primary human foreskin fibroblasts. Our results suggested that TSDF was semi-permissive for HCMV infection. The TSDF model could be further used to investigate key factors influencing cross-species multiplication of HCMV.
HSV-2-encoded miRNA-H4 Regulates Cell Cycle Progression and Act-Dinduced Apoptosis in HeLa Cells by Targeting CDKL2 and CDKN2A
Yang Zhao, Jingjing Yang, Yan Liu, Jianyong Fan, Huilan Yang
2019, 34(3): 278-286.  doi: 10.1007/s12250-019-00101-8
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MicroRNAs (miRNAs) encoded by latency-associated transcript are associated with both latent and acute stages of herpes simplex virus 2 (HSV-2) infection. In this study, miRNA-H4-5p and miRNA-H4-3p were ectopically expressed in HeLa cells to explore potential cellular targets of viral miRNAs and demonstrate their potential biological functions. The results showed that miRNA-H4-5p could reverse apoptosis induced by actinomycin D (Act-D) and promote cell cycle progression, but miRNA-H4-3p had no such obvious functions. Bioinformatics analysis, luciferase report assay, quantitative reverse transcription polymerase chain reaction (qRT-PCR), and Western blotting demonstrated that miRNA-H4-5p could bind to the 3'-untranslated region (UTR) of cyclin-dependent kinase inhibitor 2A (CDKN2A) and cyclin-dependent kinase-like 2 (CDKL2) to negatively regulate their expression. We verified that these two targeted genes were associated with cell apoptosis and cell cycle. Furthermore, in HeLa cells infected with HSV-2, we detected significantly reduced expression of CDKN2A and CDKL2 and demonstrated the negative regulation effect of miRNA-H4-5p on these two target genes. Our findings show that viral miRNAs play a vital role in regulating the expression of the host's cellular genes that participate in cell apoptosis and progression to reshape the cellular environment in response to HSV-2 infection, providing further information on the roles of encoded herpesvirus miRNAs in pathogen–host interaction.
Bacteriophages Isolated in China for the Control of Pectobacterium carotovorum Causing Potato Soft Rot in Kenya
Peter Muturi, Junping Yu, Alice Nyambura Maina, Samuel Kariuki, Francis B. Mwaura, Hongping Wei
2019, 34(3): 287-294.  doi: 10.1007/s12250-019-00091-7
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Soft rot is an economically significant disease in potato and one of the major threats to sustainable potato production. This study aimed at isolating lytic bacteriophages and evaluating methods for and the efficacy of applying phages to control potato soft rot caused by Pectobacterium carotovorum. Eleven bacteriophages isolated from soil and water samples collected in Wuhan, China, were used to infect P. carotovorum host strains isolated from potato tubers showing soft rot symptoms in Nakuru county, Kenya. The efficacy of the phages in controlling soft rot disease was evaluated by applying individual phage strains or a phage cocktail on potato slices and tubers at different time points before or after inoculation with a P. carotovorum strain. The phages could lyse 20 strains of P. carotovorum, but not Pseudomonas fluorescens control strains. Among the 11 phages, Pectobacterium phage Wc5r, interestingly showed cross-activity against Pectobacterium atrosepticum and two phage-resistant P. carotovorum strains. Potato slice assays showed that the phage concentration and timing of application are crucial factors for effective soft rot control. Phage cocktail applied at a concentration of 1×109 plaque-forming units per milliliter before or within an hour after bacterial inoculation on potato slices, resulted in ≥ 90% reduction of soft rot symptoms. This study provides a basis for the development and application of phages to reduce the impact of potato soft rot disease.
Cleavage of the Babuvirus Movement Protein B4 into Functional Peptides Capable of Host Factor Conjugation is Required for Virulence
Jun Zhuang, Wenwu Lin, Christopher J. Coates, Pengxiang Shang, Taiyun Wei, Zujian Wu, Lianhui Xie
2019, 34(3): 295-305.  doi: 10.1007/s12250-019-00094-4
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Banana bunchy top virus (BBTV) poses a serious danger to banana crops worldwide. BBTV-encoded protein B4 is a determinant of pathogenicity. However, the relevant molecular mechanisms underlying its effects remain unknown. In this study, we found that a functional peptide could be liberated from protein B4, likely via proteolytic processing. Site-directed mutagenesis indicated that the functional processing of protein B4 is required for its pathogenic effects, including dwarfism and sterility, in plants. The released protein fragment targets host proteins, such as the large subunit of RuBisCO (RbcL) and elongation factor 2 (EF2), involved in protein synthesis. Therefore, the peptide released from B4 (also a precursor) may act as a non-canonical modifier to influence host–pathogen interactions involving BBTV and plants.
Localization Analysis of Heterophilic Antigen Epitopes of H1N1 Influenza Virus Hemagglutinin
Chun-Yan Guo, Hai-Xiang Zhang, Jun-Jun Zhang, Li-Jun Sun, Hui-Jin Li, Dao-Yan Liang, Qing Feng, Yan Li, Yang-Meng Feng, Xin Xie, Jun Hu
2019, 34(3): 306-314.  doi: 10.1007/s12250-019-00100-9
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Previous studies have indicated that two monoclonal antibodies (mAbs; A1-10 and H1-84) of the hemagglutinin (HA) antigen on the H1N1 influenza virus cross-react with human brain tissue. It has been proposed that there are heterophilic epitopes between the HA protein and human brain tissue (Guo et al. in Immunobiology 220:941–946, 2015). However, characterisation of the two mAbs recognising the heterophilic epitope on HA has not yet been performed. In the present study, the common antigens of influenza virus HA were confirmed using indirect enzyme-linked immunosorbent assays and analysed with DNAMAN software. The epitopes were localized to nine peptides in the influenza virus HA sequence and the distribution of the peptides in the three-dimensional structure of HA was determined using PyMOL software. Key amino acids and variable sequences of the antibodies were identified using abYsis software. The results demonstrated that there were a number of common antigens among the five influenza viruses studied that were recognised by the mAbs. One of the peptides, P2 (LVLWGIHHP191–199), bound both of the mAbs and was located in the head region of HA. The key amino acids of this epitope and the variable regions in the heavy and light chain sequences of the mAbs that recognised the epitope are described. A heterophilic epitope on H1N1 influenza virus HA was also introduced. The existence of this epitope provides a novel perspective for the occurrence of nervous system diseases that could be caused by influenza virus infection, which might aid in influenza prevention and control.
Inhibition of Herpes Simplex Virus-1 Replication by Natural Compound Honokiol
Shuai Liu, Long Li, Lingbing Tan, Xiaozhen Liang
2019, 34(3): 315-323.  doi: 10.1007/s12250-019-00104-5
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Honokiol is a pleiotropic natural compound isolated from Magnolia and has multiple biological and clinically relevant effects, including anticancer and antimicrobial function. However, the antiviral activity of honokiol has not yet been well studied. Here we showed that honokiol had no effect on herpes simplex virus-1 (HSV-1) entry, but inhibited HSV-1 viral DNA replication, gene expression and the production of new progeny viruses. The combination of honokiol and clinical drug acyclovir augmented inhibition of HSV-1 infection. Our results illustrate that honokiol could be a potential new candidate for clinical consideration in the treatment of HSV-1 infection alone or combination with other therapeutics.
Interferon as a Mucosal Adjuvant for an Influenza Vaccine in Pigs
Lirong Liu, Wenhui Fan, He Zhang, Shuang Zhang, Liang Cui, Meng Wang, Xiaoyuan Bai, Wenxian Yang, Lei Sun, Limin Yang, Wenjun Liu, Jing Li
2019, 34(3): 324-333.  doi: 10.1007/s12250-019-00102-7
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Interferon, a natural protein that is produced by a variety of cells during viral infection, activates the transcription of multiple functional genes in cells, regulates synergy among various signaling pathways, and mediates many biological functions such as antiviral activity, immune regulation, and cell growth. However, clinical research on interferon in livestock is lacking. In this study, recombinant porcine interferon (PoIFNα) was used as an adjuvant, in combination with inactivated influenza virus, to vaccinate 6-week-old pigs via nasal infusion. The transcription of target genes was then monitored and the functions of PoIFNα were determined with respect to the activation of mucosal immunity. We found that a combination of low-dose PoIFNα and inactivated influenza virus could significantly up-regulate the expression of immunoregulatory cytokines such as IL-2, IL-18, IFN-γ, IL-6, and IL-10 by real-time PCR, suggesting the induction of a strong mucosal innate immune response after administration. In addition, low-dose PoIFNα can significant enhancing the transcription of genes encoding homing factors including CCR9 and CCR10 (P < 0.001), thereby resulting in the induction of higher levels of HA-specific antibodies (P < 0.05), which can be determined by ELISA and IFA. Post-immunization challenges with H1N1 virus demonstrated that PoIFNα, combined with inactivated influenza virus, could alleviate clinical signs in pigs during the early stages of viral infection. These studies reveal low-dose PoIFNα as a potential mucosal adjuvant for influenza virus in pigs.
Letter
Identification of a Novel Universal Potential Epitope on the Cytoplasmic Tail of H7N9 Virus Hemagglutinin
Xi Liu, Li Ding, Jing Yuan, Jian Liao, Lian Duan, Wenfei Wang, Weiguo Tan, Weiye Yu, Boping Zhou, Xinchun Chen, Zheng Yang
2019, 34(3): 334-337.  doi: 10.1007/s12250-019-00110-7
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This study aimed to identify a novel antigenic epitope on H7N9 virus HA7. In total, 37 patients with H7N9 infection admitted to Shenzhen Third People's Hospital between December 2012 and March 2015 were included. A library of 108 peptides covering the full-length HA7 was synthesized except for P9 and P18. Plasma samples from 11 H7N9-infected patients were tested for reactivity with the 108 synthetic peptides by peptide microarray ELISA. Results suggested that the HA7-CT epitope is a potentially antigenic epitope that could be recognized by the plasma of patients infected with influenza A virus and may drive a strong immune response after the onset of virus infection.
Reverse Transcription Recombinase Polymerase Amplification Assays for Rapid Detection of Tick-Borne Encephalitis Virus Infection
Jia Jia, Yuchang Li, Xiaoyan Wu, Sen Zhang, Yi Hu, Jing Li, Tao Jiang, Xiaoping Kang
2019, 34(3): 338-341.  doi: 10.1007/s12250-019-00105-4
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In summary, we established an RT-RPA assay for TBEV. The LOD of RT-RPA was determined to be 10 copies and 0.1 PFU/reaction. Although the sensitivity of RT-RPA for TBEV was lower than that of rRT-PCR, the assay was easy to perform, and amplification was much more rapid than that in rRT-PCR (RT-RPA, 15 min; rRT-PCR, 2 h); thus, our RT-RPA assay may be useful for detection of TBEV infection under field conditions.
Rift Valley Fever Virus and Yellow Fever Virus in Urine: A Potential Source of Infection
Meng Li, Beibei Wang, Liqiang Li, Gary Wong, Yingxia Liu, Jinmin Ma, Jiandong Li, Hongzhou Lu, Mifang Liang, Ang Li, Xiuqing Zhang, Yuhai Bi, Hui Zeng
2019, 34(3): 342-345.  doi: 10.1007/s12250-019-00096-2
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Yellow fever virus (YFV) and Rift Valley fever virus (RVFV) are important vector-borne pathogens, which all originated in Africa. Now both viruses spread globally and pose a wider threat to public health. Recent studies have shown that YFV was detected in serum and urine samples and viable YFV was isolated from urine samples. In contrast, RVFV has been detected in urine samples, but the isolation of variable virus has not been reported. Here, we report results from a comparative analysis of the detection window for YFV in patient sera and urine samples. Moreover, viable YFV and RVFV were isolated from clinical samples, verified by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and sequencing. YFV persisted in urine until 32 days after disease onset, which was longer than other reports on natural YFV infections. Overall, our data demonstrated that urine may be considered as an alternative non-invasive source of samples for clinical detection of YFV, RVFV, and other vector-borne pathogens, which should improve diagnosis, help with surveillance efforts against the spread of emerging or re-emerging arboviruses, and reduce the risk of spreading viruses through the urine.