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2019 Vol.34(4)

Mitochondria are multifunctional eukaryotic organelles with diverse roles, including energy production and distribution, apoptosis, innate immunity, metabolic regulation, cell-cycle control and signal transduction. The interactions between viruses and the host mitochondria are crucial for virus replication and pathogenicity. Yang Yang et al. found that Enterovirus A71 (EV-A71) infection resulted in a perinuclear redistribution of the mitochondria, and the microtubule skeleton and the dynein motor complex were required for this redistribution. They further revealed that the interaction between EV-A71 2BC protein and mitochondrial Rho GTPase 1 played a crucial role in the regulation of mitochondrial motility, and the mitochondria redistribution was required for EV-A71 replication and proliferation. Their study reports a novel function of the EV-A71 2BC protein and provides an insight into the regulation of organelle transport in the EV-A71-infected cells. Please see page 397–411 for details.

Review

Specific and Selective Bacteriophages in the Fight against Multidrugresistant Acinetobacter baumannii

Natalia Bagińska, Anna Pichlak, Andrzej Górski, Ewa Jończyk-Matysiak

2019, 34(4): 347 doi: 10.1007/s12250-019-00125-0

Received: 05 December 2018 Accepted: 29 March 2019 Published: 15 May 2019
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Acinetobacter baumannii causes serious infections especially in immunocompromised and/or hospitalized patients. Several A. baumannii strains are multidrug resistant and infect wounds, bones, and the respiratory tract. Current studies are focused on finding new effective agents against A. baumannii. Phage therapy is a promising means to fight this bacterium and many studies on procuring and applying new phages against A. baumannii are currently being conducted. As shown in animal models, phages against multidrug-resistant A. baumannii may control bacterial infections caused by this pathogen and may be a real hope to solve this dangerous health problem.
Research Article

Prevalence of HIV Indeterminate Western Blot Tests and Follow-up of HIV Antibody Sero-Conversion in Southeastern China

Shouli Wu, Min Gao, Jian Zheng, Pingping Yan, Wei Wang, Xiaoli Lu, Yuefeng Qiu, Yansheng Yan

2019, 34(4): 358 doi: 10.1007/s12250-019-00130-3

Received: 07 December 2018 Accepted: 30 May 2019 Published: 12 June 2019
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HIV-indeterminate Western blotting (WB) results are typically obtained in WB confirmatory assays, and the number of indeterminate samples may increase with the detection of HIV infections, which will present considerable challenges for the management of HIV/AIDS. Nucleic acid detection has been used as a laboratory test for screening suspected or indeterminate samples. However, the effectiveness of these assays for the differential diagnosis of HIV-indeterminate WB samples remained undetermined. In this study, 210 subjects with HIV-indeterminate WB results were detected from 6360 positive HIV screening samples between 2015 and 2016 in southeastern China, in which HIV-indeterminate WB results accounted for 3.30%. The highest proportion of indeterminate results was observed in pregnant and lying-in women receiving physical examinations (16.67%), followed by that in voluntary blood donors (8.82%). The most common WB band patterns were p24, gp160 and p24, and gp160. The follow-up study revealed that the highest negative and positive conversion rates of HIV antibodies were in samples with a single p24 band (80.28%), and with gp160 and p24 bands (86.21%), respectively. Among the Env, Gag, and Pol antibodies, samples with a Gag band showed the highest negative conversion rate (81.25%), whereas the highest positive conversion rate was observed in samples with an Env band (56.76%). In addition, quantitative and qualitative HIV nucleic acid testing exhibited the highest sensitivity (96.3%) and specificity (97.85%), respectively. Our results indicate a lower proportion of HIV indeterminate WB results in southeastern China compared to previous reports, and the follow-up re-examination of patients with HIV indeterminate results should be performed. Nucleic acid testing facilitates the identification of HIV infections.

Cross-sectional Seroprevalence and Genotype of Hepatitis E Virus in Humans and Swine in a High-density Pig-farming Area in Central China

Yilin Shu, Yameng Chen, Sheng Zhou, Shoude Zhang, Qin Wan, Changcai Zhu, Zhijiang Zhang, Hailong Wu, Jianbo Zhan, Ling Zhang

2019, 34(4): 367 doi: 10.1007/s12250-019-00136-x

Received: 12 December 2018 Accepted: 29 March 2019 Published: 01 July 2019
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Hepatitis E virus (HEV) infection is a common public health problem in developing countries. However, the current prevalence of HEV and the relationship of HEV genotype between swine and human within high-density pig-farming areas in central China are still inadequately understood. Here, cross-sectional serological and genotypic surveys of HEV among the 1232 general population, 273 workers occupationally exposed to swine, and 276 pigs in a high-density pig-breeding area, were undertaken by ELISA and nested RT-PCR methods. Anti-HEV IgG was detected in 26.22% of general population and 48.35% of occupational workers. The prevalence of swine serum HEV-Ag was 6.52%. The prevalence of anti-HEV IgG was significantly higher among the workers occupationally exposed to swine than among the general population. An increased HEV seropositivity risk among the general population was associated with either being a peasant or male and was very strongly associated with the increase of age. Among the occupationally exposed group, the prevalence of anti-HEV IgG antibodies increased with age and working years. Among the 30 HEV-IgM-positive people, the infection rates of clerks in the public, peasants, pork retailers, and pig farmers were higher than those of others. A phylogenetic analysis revealed that all the isolates belonged to subgenotype 4d, and four people and four pigs shared 97.04%–100% sequence homology. This study revealed a high HEV seroprevalence among the general population and workers occupationally exposed to swine in the Anlu City, and supports the notion that swine are a source of human HEV infection.

Complementation of Wild-Type and Drug-Resistant Hepatitis B Virus Genomes to Maintain Viral Replication and Rescue Virion Production under Nucleos(t)ide Analogs

Chunchen Wu, Baolin Li, Xiaoyong Zhang, Kaitao Zhao, Yingshan Chen, Yifei Yuan, Yan Liu, Rongjuan Chen, Dongping Xu, Xinwen Chen, Mengji Lu

2019, 34(4): 377 doi: 10.1007/s12250-019-00143-y

Received: 19 November 2018 Accepted: 14 May 2019 Published: 19 June 2019
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As the open reading frames of hepatitis B virus (HBV) genomes are overlapping, resistance mutations (MTs) in HBV polymerase may result in stop codon MTs in hepatitis B surface proteins, which are usually detected as a mixed population with wild-type (WT) HBV. The question was raised how the coexistence of nucleos(t)ide analogs (NAs) resistance MTs and WT sequences affects HBV replication. In the present study, HBV genomes with frequently detected reverse transcriptase (RT)/surface truncation MTs, rtA181T/sW172*, rtV191I/sW182* and rtM204I/sW196*, were phenotypically characterized alone or together with their WT counterparts in different ratios by transient transfection in the absence or presence of NAs. In the absence of NAs, RT/surface truncation MTs impaired the expression and secretion of HBV surface proteins, and had a dose-dependent negative effect on WT HBV virion secretion. However, in the presence of NAs, coexistence of MTs with WT maintained viral replication, and the presence of WT was able to rescue the production of MT HBV virions. Our findings reveal that complementation of WT and MT HBV genomes is highly effective under drug treatment.

Effect of Loss-of-function of the Herpes Simplex Virus-1 microRNA H6-5p on Virus Replication

Rongquan Huang, Xusha Zhou, Shuqi Ren, Xianjie Liu, Zhiyuan Han, Grace Guoying Zhou

2019, 34(4): 386 doi: 10.1007/s12250-019-00111-6

Received: 07 December 2018 Accepted: 28 February 2019 Published: 24 April 2019
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To date, 29 distinct microRNAs (miRNAs) have been reported to be expressed during herpes simplex virus infections. Sequence analysis of mature herpes simplex virus-1 (HSV-1) miRNAs revealed five sets of miRNAs that are complementary to each other: miR-H6-5p/H1-3p, miR-H6-3p/H1-5p, H2-5p/H14-3p, miR-H2-3p/H14-5p, and miR-H7/H27. However, the roles of individual miRNAs and consequences of this complementarity remain unclear. Here, we focus on two of these complementary miRNAs, miR-H6-5p and miR-H1-3p, using loss-of-function experiments in vitro and in a mouse model of infection using an miRNA sponge approach, including tandem multiplex artificial miRNA-binding sequences that do not match perfectly to the target miRNA inserted downstream of a green fluorescent protein reporter gene. Infection with recombinant virus expressing the miR-H6-5p sponge reduced viral protein levels and virus yield. Decreased accumulation of viral proteins was also observed at early stages of infection in the presence of both an miR-H6-5p inhibitor and plasmid-expressed miR-H1-3p. Moreover, establishment of latency and reactivation did not differ between the recombinant virus expressing the miR-H6-5p sponge and wild-type HSV-1. Taken together, these data suggest that miR-H6-5p has an as-yet-unidentified role in the early stages of viral infection, and its complement miR-H1-3p suppresses this role in later stages of infection. This report extends understanding of the roles of miRNAs in infection by herpes simplex viruses, supporting a model of infection in which the production of virus and its virulent effects are tightly controlled to maximize persistence in the host and population.

Mitochondria Redistribution in Enterovirus A71 Infected Cells and Its Effect on Virus Replication

Yang Yang, Haolong Cong, Ning Du, Xiaodong Han, Lei Song, Wenliang Zhang, Chunrui Li, Po Tien

2019, 34(4): 397 doi: 10.1007/s12250-019-00120-5

Received: 21 December 2018 Accepted: 25 March 2019 Published: 08 May 2019
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Enterovirus A71 (EV-A71) is one of the main causative agents of hand, foot and mouth disease (HFMD) and it also causes severe neurologic complications in infected children. The interactions between some viruses and the host mitochondria are crucial for virus replication and pathogenicity. In this study, it was observed that EV-A71 infection resulted in a perinuclear redistribution of the mitochondria. The mitochondria rearrangement was found to require the microtubule network, the dynein complex and a low cytosolic calcium concentration. Subsequently, the EV-A71 non-structural protein 2BC was identified as the viral protein capable of inducing mitochondria clustering. The protein was found localized on mitochondria and interacted with the mitochondrial Rho GTPase 1 (RHOT1) that is a key protein required for attachment between the mitochondria and the motor proteins, which are responsible for the control of mitochondria movement. Additionally, suppressing mitochondria clustering by treating cells with nocodazole, EHNA, thapsigargin or A23187 consistently inhibited EV-A71 replication, indicating that mitochondria recruitment played a crucial role in the EV-A71 life cycle. This study identified a novel function of the EV-A71 2BC protein and provided a potential model for the regulation of mitochondrial motility in EV-A71 infection.

Trans Complementation of Replication-defective Omsk Hemorrhagic Fever Virus for Antiviral Study

Qiuyan Zhang, Na Li, Chenglin Deng, Zherui Zhang, Xiaodan Li, Kentaro Yoshii, Hanqing Ye, Bo Zhang

2019, 34(4): 412 doi: 10.1007/s12250-019-00109-0

Received: 16 January 2019 Accepted: 28 February 2019 Published: 04 April 2019
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Omsk hemorrhagic fever virus (OHFV) is a tick-borne flavivirus classified as a biosafety level-4 (BSL4) pathogen. Studies of OHFV are restricted to be conducted within BSL4 laboratories. Currently, no commercial vaccines or antiviral drugs are available against OHFV infection. In this study, we recovered a replication-deficient OHFV with an NS1 deletion (OHFV-ΔNS1) and reporter virus replacing NS1 with the Gaussia luciferase (Gluc) (OHFV-ΔNS1-Gluc). Both the defective OHFV-ΔNS1 and OHFV-ΔNS1-Gluc virus could only replicate efficiently in the BHK21 cell line expressing NS1 (BHK21NS1) but not in naïve BHK21 cells. The Gluc reporter gene of OHFV-ΔNS1-Gluc virus was maintained stably after serial passaging of BHK21NS1 cells and was used to surrogate the replication of OHFV. Using NITD008, OHFV-ΔNS1-Gluc virus was validated for antiviral screening, and high-throughput screening parameters were optimized in a 96-well plate format with a calculated Z′ value above 0.5. The OHFV-ΔNS1-Gluc reporter virus is a powerful tool for antiviral screening as well as viral replication and pathogenesis studies in BSL2 laboratories.

In Vitro Infectious Risk Assessment of Heliothis virescens ascovirus 3j (HvAV-3j) toward Non-target Vertebrate Cells

Huan Yu, Yi-Yi Ou-Yang, Ni Li, Madoka Nakai, Guo-Hua Huang

2019, 34(4): 423 doi: 10.1007/s12250-019-00113-4

Received: 20 December 2018 Accepted: 06 March 2019 Published: 29 April 2019
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As specific pathogens of noctuid pests, including Spodoptera exigua, S. litura, Helicoverpa armigera, and Mythimna separata, ascoviruses are suitable for the development of bioinsecticides. In this study, the infectivity of Heliothis virescens ascovirus 3j (HvAV-3j) on insect and mammalian cells was evaluated. HvAV-3j infection induced drastic morphological changes in Sf9, HzAM1, SeFB, and HaFB cells, including swelling and detachment. Notably, the latter phenomena did not occur in HvAV-3j-inoculated mammalian cells (HEK293, 7402, HePG2, PK15, ST, and TM3). MTT assays indicated that HvAV-3j inhibited the growth of host insect cells from the 6th hpi, but no effects were detected in the HvAV-3j-inoculated mammalian cells. Furthermore, viral DNA replication, gene transcription, and protein expression were investigated, and the results consistently suggested that HvAV-3j viruses were not able to replicate their genomic DNA, transcribe, or express their proteins in the non-target vertebrate cells. The HvAV-3j genes were only transcribed and expressed in the four insect cell lines. These results indicated that HvAV-3j was infectious to cells derived from S. frugiperda, S. exigua, H. armigera, and H. zea but not to cells derived from human, pig, and mouse, suggesting that ascoviruses are safe to nontarget vertebrate cells.

Molecular Characterization and Expression Analysis of ftr01, ftr42, and ftr58 in Zebrafish (Danio rerio)

Wanmeng Liu, Ming Kuang, Ze Zhang, Yuanan Lu, Xueqin Liu

2019, 34(4): 434 doi: 10.1007/s12250-019-00112-5

Received: 25 December 2018 Accepted: 21 February 2019 Published: 15 April 2019
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Tripartite motif (TRIM) proteins were shown to play an important role in innate antiviral immunity. FinTRIM (ftr) is a new subset of TRIM genes that do not possess obvious orthologs in higher vertebrates. However, little is known about its function. In this study, we used bioinformatic analysis to examine the phylogenetic relationships and conserved domains of zebrafish (Danio rerio) ftr01, ftr42, and ftr58, as well as qualitative real-time PCR to examine their expression patterns in zebrafish embryonic fibroblast (ZF4) cells and zebrafish tissues. Sequence analysis showed that the three finTRIMs are highly conserved, and all contain a RING domain, B-box domain, and SPRY-PRY domain. In addition, ftr42 and ftr58 had one coiled-coil domain (CCD), whereas ftr01 had two CCDs. Tissue expression analysis revealed that the mRNA level of ftr01 was the highest in the liver, whereas those of ftr42 and ftr58 were the highest in the gill; the expression of these finTRIMs was clearly upregulated not in the eyes, but in the liver, spleen, kidney, gill, and brain of zebrafish following spring viremia of carp virus (SVCV) infection. Similarly, the expression of these three finTRIM genes also increased in ZF4 cells after SVCV infection. Our study revealed that ftr01, ftr42, and ftr58 may play an important role in antiviral immune responses, and these findings validate the need for more in-depth research on the finTRIM family in the future.

Construction of a One-Vector Multiplex CRISPR/Cas9 Editing System to Inhibit Nucleopolyhedrovirus Replication in Silkworms

Zhanqi Dong, Qi Qin, Zhigang Hu, Peng Chen, Liang Huang, Xinling Zhang, Ting Tian, Cheng Lu, Minhui Pan

2019, 34(4): 444 doi: 10.1007/s12250-019-00121-4

Received: 09 January 2019 Accepted: 25 March 2019 Published: 19 June 2019
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Recently the developed single guide (sg)RNA-guided clustered regularly interspaced short palindromic repeats/associated protein 9 nuclease (CRISPR/Cas9) technology has opened a new avenue for antiviral therapy. The CRISPR/Cas9 system uniquely allows targeting of multiple genome sites simultaneously. However, there are relatively few applications of CRISPR/Cas9 multigene editing to target insect viruses. To address the need for sustained delivery of a multiplex CRISPR/ Cas9-based genome-editing vehicle against insect viruses, we developed a one-vector (pSL1180-Cas9-U6-sgRNA) system that expresses multiple sgRNA and Cas9 protein to excise Bombyx mori nucleopolyhedrovirus (BmNPV) in insect cells. We screened the immediate-early-1 gene (ie-1), the major envelope glycoprotein gene (gp64), and the late expression factor gene (lef-11), and identified multiple sgRNA editing sites through flow cytometry and viral DNA replication analysis. In addition, we constructed a multiplex editing vector (PSL1180-Cas9-sgIE1-sgLEF11-sgGP64, sgMultiple) to efficiently regulate multiplex gene-editing and inhibit BmNPV replication after viral infection. This is the first report of the application of a multiplex CRISPR/Cas9 system to inhibit insect virus replication. This multiplex system can significantly enhance the potential of CRISPR/Cas9-based multiplex genome engineering in insect virus.

Improving Baculovirus Transduction of Mammalian Cells by Incorporation of Thogotovirus Glycoproteins

Liangbo Hu, Yimeng Li, Fei Deng, Zhihong Hu, Hualin Wang, Manli Wang

2019, 34(4): 454 doi: 10.1007/s12250-019-00133-0

Received: 20 January 2019 Accepted: 09 April 2019 Published: 14 June 2019
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Baculovirus can transduce a wide range of mammalian cells and is considered a promising gene therapy vector. However, the low transduction efficiency of baculovirus into many mammalian cells limits its practical application. Co-expressing heterologous viral glycoproteins (GPs), such as vesicular stomatitis virus G protein (VSV G), with baculovirus native envelope protein GP64 is one of the feasible strategies for improving virus transduction. Tick-borne thogotoviruses infect mammals and their GPs share sequence/structure homology and common evolutionary origins with baculovirus GP64. Herein, we tested whether thogotovirus GPs could facilitate the entry of the prototype baculovirus Autographa californica multiple multiple nucleopolyhedrovirus (AcMNPV) into mammalian cells. The gp genes of two thogotoviruses, Thogoto virus and Dhori virus, were inserted into the AcMNPV genome. Both GPs were properly expressed and incorporated into the envelope of the recombinant AcMNPVs. The transduction rates of recombinant AcMNPVs expressing the two thogotovirus GPs increased for approximately 4–12 fold compared to the wild type AcMNPV in six of the 12 tested mammalian cell lines. It seemed that thogotovirus GPs provide the recombinant AcMNPVs with different cell tropisms and showed better performance in several mammalian cells compared to VSV G incorporated AcMNPV. Further studies showed that the improved transduction was a result of augmented virus-endosome fusion and endosome escaping, rather than increased cell binding or internalization. We found the AcMNPV envelope protein GP64-mediated fusion was enhanced by the thogotovirus GPs at relatively higher pH conditions. Therefore, the thogotovirus GPs represent novel candidates to improve baculovirus-based gene delivery vectors.
Letter

Establishment of Novel Monoclonal Fabs Specific for Epstein-Barr Virus Encoded Latent Membrane Protein 1

Gaoxin Li, Ling Ding, Xiaojing Ma, Qiliang Cai, Tianlei Ying, Fang Wei

2019, 34(4): 467 doi: 10.1007/s12250-019-00103-6

Received: 22 November 2018 Accepted: 23 January 2019 Published: 04 April 2019
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Here we isolated the Fab clones 1-A11, 6-C6, 10-B2, and 15-H10 and confirmed their recognition of LMP1 expressed on the surface of EBV+ B95.8 cells. Recognition of LMP1 on the cell membrane by clone 15-H10 was verified through immunofluorescent assay, while other clones failed the recognition. One of the possible reasons could be the stringent washing in this assay that ruptured the low affinity Fab-LMP1 binding. EBV infection is related with multiple diseases, and conventional approaches used in laboratory diagnostic tests for EBV infection and pathogenesis have their limitations [(generate false positive results or lack the ability to localize the expression of EBV within target cells) (Young and Rickinson 2004)]. The LMP1-specific Fab clones reported here need further evaluation in both affinity and specificity in testing EBV+ patient samples, and hopefully it could have potential applications in EBV diagnostics and directly targeting EBV-related tumors in adoptive T cell therapy.

Genomic Characterization of the First Parechovirus in Bats

Chang Zhang, Jinliang Sheng, Zihan Zhao, Chao Yan, Changchun Tu, Biao He

2019, 34(4): 471 doi: 10.1007/s12250-019-00108-1

Received: 15 January 2019 Accepted: 05 March 2019 Published: 05 April 2019
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As important hosts for several pathogenic viruses, bats harbor viruses from almost all families of vertebrate viruses, including several genera of family Picornaviridae, including Hepatovirus, Kobuvirus, Crohivirus, and Sapelovirus (Drexler et al. 2015; Wu et al. 2016; Yinda et al. 2017). However, PeVs were not reported in bats until our recent viral metagenomic analysis of 122 adult healthy bats (Pipistrellus pipistrellus) obtained from two locations in Xinjiang (Xinyuan, n = 46; Qapqal, n = 76) in 2016, revealing thousands of reads related to PeV (Zhang et al. 2018). PCR-based screening revealed that 6.5% and 10.5% bats from Xinyuan and Qapqal, respectively, harbored this virus, and preliminary phylogenetic analysis of 396-nt-long amplicons targeting the VP1 region (GenBank accession numbers: MH921430–MH921443) revealed > 91.5% identities among each other and 63.7%–64.2% identity with their closest phylogenetic neighbor, FPeV (Smits et al. 2013; Zhang et al. 2018). This study reports the complete genomic characterization of the first bat PeV to better understand its evolutionary history.
Meeting Report

Virology at the Lorne Infection and Immunity Conference 2019

Sarah L. Londrigan, Gregor Ebert, Prasad N. Paradkar

2019, 34(4): 474 doi: 10.1007/s12250-019-00115-2

Received: 25 March 2019 Accepted: 27 March 2019 Published: 11 April 2019
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The Lorne Infection and Immunity Conference is one of five scientific meetings held during each month of February at the Cumberland resort in the picturesque seaside town of Lorne, on the Great Ocean Road in Victoria (Australia). The specific aim of the meeting is to bring together basic, clinical and translational researchers—those who examine microbes and their impact on the innate or adaptive immune response, researchers who study the mechanisms that regulate immune responses, and those who apply this knowledge to preventing and treating infectious and inflammatory diseases. The average number of attendees is 220, with registrants appreciative of the welcoming and relaxed atmosphere (Fig. 1).