Dr David Lowry: Coronacrisis: there is a lot more going on in China on virology research than the UK media reports

Letter sent to The Times, 19 April 2020:

In their joint article (“US investigates laboratory leak claim,” 18 April 2020) your diplomatic and defence editors report that “Britain is also [in addition to the US] examining the theory [that the coronavirus leaked from the Chinese State national Virology research laboratory in Wuhan] and has not ruled it out."

So, ministers are apparently keeping an open mind, unlike your cold war specialist writer, Ben Macintyre, who regards this thesis as “political propaganda.” (“Operation Infektion is spreading again,” Apr 18)

However, it seems UK Government ministers have become more inquisitive than they were last month over the origin of the furtive virus.

When Lord Alton of Liverpool asked the Department of Health and Social Care what

"assessment they have made of the (1) number, and (2) location, of microbiology laboratories in China that handle advanced viruses such as the Wuhan coronavirus; and what assessment they have made of the role any such laboratories may have had in the initial spread of the Wuhan coronavirus?" (HL1839), he was given the curt brush-off by Lord Bethell – who was made a minister only on 9 March – in a written reply on

10 March, stating: “We do not hold this information."

(https://www.parliament.uk/business/publications/written-questions-answers-statements/written-question/Lords/2020-02-25/HL1839/)

Shortly after, the youngest MP in Parliament, Nadia Whittome, representing Nottingham East, was told by DH&SC minister, Jo Churchill, in a written answer on 26 March “ We have no plans to authorise research into the security control of viruses under investigation at the Wuhan State Institute of Virology…We are concentrating on the stages that we have set out in paragraph 3.9 of the COVID-19 action plan. These stages are: contain, delay, research and mitigate.”

To be sure it is good news now ministers are now opening their eyes to this possibility, but why did they keep them closed for several months?

China lab seeks patent on use of Gilead's coronavirus treatment

https://www.channelnewsasia.com/news/business/china-lab-patent-gilead-coronavirus-treatment-12396008

Singapore, 5 Feb 2020 03:50PM

BEIJING:　A state-run Chinese research institute has applied for a patent on the use of Gilead Sciences' experimental US　antiviral drug, which scientists think could provide treatment for the coronavirus that has killed hundreds and infected thousands.

The Wuhan Institute of Virology of the China Academy of Sciences, based in the city where the outbreak is believed to have originated, said in a statement on Tuesday (Feb 4)　　it applied to patent the use of Remdesivir, an antiviral drug developed by Gilead, to treat the virus.

A study published in the New England Journal of Medicine last week reported a coronavirus patient in the United States was found to show an improvement after taking Remdesivir, which is also used to treat infectious diseases such as Ebola.

The Wuhan Institute of Virology did not respond to Reuters' request for comment.

"Even if the Wuhan Institute's application gets authorised, the role is very limited because Gilead still owns the fundamental patent of the drug," said Zhao Youbin, a Shanghai-based intellectual property counsel at Purplevine IP Service Co.

"Any exploitation of the patent must seek approval from Gilead."

Gilead did not immediately respond to request for comment but last week said it was working with China to test Remdesivir for use in a small number of patients with the coronavirus.

The application was submitted jointly with the Military Medicine Institute of the People's Liberation Army Academy of Military Science, according to the Wuhan Institute of Virology.

Scientists from both institutes said in a paper published in Nature's Cell Research on Tuesday that they found both Remdesivir and Chloroquine, which is used to treat malaria, to be an effective way to inhibit the coronavirus.

The Wuhan-based laboratory said in its statement that the patent application was filed on Jan　21 and aimed at protecting China's national interests. However, it said it would temporarily drop its patent claims if the opportunity arose to collaborate with foreign pharmaceutical firms to fight the epidemic.

Source: Reuters/nr

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The journal, 'Genomic characterization and infectivity of a novel SARS-like coronavirus in Chinese bats', actually found the bat-SL-CoVZC45 was "more distant than that observed previously for bat SL-CoVs in China" to human/civet SARS CoVshttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC6135831/　…

1 reply 9 retweets 17 likes

Galileo Cheng‏　@galileocheng Jan 22

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Why such rumour? Since the research was led by researcher from Department of Epidemiology, Research Institute for Medicine of Nanjing Command, Nanjing and Third Military Medical University, Chongqing, and funded by Army Logistics Scientific Research Projects.

7:28 PM - 22 Jan 2020

1 reply 5 retweets 14 likes

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Galileo Cheng‏　@galileocheng Jan 22

And Wuhan got BSL-4 Biosafety lab - Wuhan Institute of Virology of the Chinese Academy of Sciences, the first, only and visible BSL-4 laboratory in China. People then tried to link all these together and rumour mill took advantage of this.

2 replies 5 retweets 16 likes

Galileo Cheng‏　@galileocheng Jan 22

If you compare the 4 #nCoV2019 BetaCoV/Wuhan sample from patients & compare them to bat-SL-CoVZC45, it's 89% similar in 95% of query in @NIH GeneBank, Mythbusted. However, the high similarity to SARS CoV is still a warning.pic.twitter.com/Ha66a22yQm

https://www.ncbi.nlm.nih.gov/pubmed/30209269

Emerg Microbes Infect. 2018 Sep 12;7(1):154. doi: 10.1038/s41426-018-0155-5.

Genomic characterization and infectivity of a novel SARS-like coronavirus in Chinese bats.

Hu D^1,2, Zhu C², Ai L², He T², Wang Y³, Ye F², Yang L², Ding C², Zhu X², Lv R², Zhu J², Hassan B⁴, Feng Y⁵, Tan W⁶, Wang C^7,8.

Abstract

SARS coronavirus (SARS-CoV), the causative agent of the large SARS outbreak in 2003, originated in bats. Many SARS-like coronaviruses (SL-CoVs) have been detected in bats, particularly those that reside in China, Europe, and Africa. To further understand the evolutionary relationship between SARS-CoV and its reservoirs, 334 bats were collected from Zhoushan city, Zhejiang province, China, between 2015 and 2017. PCR amplification of the conserved coronaviral protein RdRp detected coronaviruses in 26.65% of bats belonging to this region, and this number was influenced by seasonal changes. Full genomic analyses of the two new SL-CoVs from Zhoushan (ZXC21 and ZC45) showed that their genomes were 29,732 nucleotides (nt) and 29,802 nt in length, respectively, with 13 open reading frames (ORFs). These results revealed 81% shared nucleotide identity with human/civet SARS CoVs, which was more distant than that observed previously for bat SL-CoVs in China. Importantly, using pathogenic tests, we found that the virus can reproduce and cause disease in suckling rats, and further studies showed that the virus-like particles can be observed in the brains of suckling rats by electron microscopy. Thus, this study increased our understanding of the genetic diversity of the SL-CoVs carried by bats and also provided a new perspective to study the possibility of cross-species transmission of SL-CoVs using suckling rats as an animal model.

PMID:

30209269

PMCID:

PMC6135831

DOI:

10.1038/s41426-018-0155-5

[Indexed for MEDLINE]

Author information

Department of Epidemiology, College of Preventive Medicine, Third Military Medical University, Chongqing, 400038, China.

Department of Epidemiology, Research Institute for Medicine of Nanjing Command, Nanjing, 210002, China.

Jiangsu Institute of Parasitic Diseases, Wuxi, Jiangsu Province, 214064, P.R. China.

Stony Brook University, Stony Brook, 11794, USA.

Department of Pathogen Biology & Microbiology and Department of General Intensive Care Unit of the Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, 310058, China. fengyj@zju.edu.cn.

Department of Epidemiology, Research Institute for Medicine of Nanjing Command, Nanjing, 210002, China. njcdc@163.com.

Department of Epidemiology, College of Preventive Medicine, Third Military Medical University, Chongqing, 400038, China. science2008@hotmail.com.

Department of Epidemiology, Research Institute for Medicine of Nanjing Command, Nanjing, 210002, China. science2008@hotmail.com.

Emerg Microbes Infect. 2018 Sep 12;7(1):154. doi: 10.1038/s41426-018-0155-5.

Genomic characterization and infectivity of a novel SARS-like coronavirus in Chinese bats.

Hu D^1,2, Zhu C², Ai L², He T², Wang Y³, Ye F², Yang L², Ding C², Zhu X², Lv R², Zhu J², Hassan B⁴, Feng Y⁵, Tan W⁶, Wang C^7,8.

Abstract

PMID:

30209269

PMCID:

PMC6135831

DOI:

10.1038/s41426-018-0155-5

[Indexed for MEDLINE]

Author information

Department of Epidemiology, College of Preventive Medicine, Third Military Medical University, Chongqing, 400038, China.

Department of Epidemiology, Research Institute for Medicine of Nanjing Command, Nanjing, 210002, China.

Jiangsu Institute of Parasitic Diseases, Wuxi, Jiangsu Province, 214064, P.R. China.

Stony Brook University, Stony Brook, 11794, USA.

Department of Epidemiology, Research Institute for Medicine of Nanjing Command, Nanjing, 210002, China. njcdc@163.com.

Department of Epidemiology, College of Preventive Medicine, Third Military Medical University, Chongqing, 400038, China. science2008@hotmail.com.

Department of Epidemiology, Research Institute for Medicine of Nanjing Command, Nanjing, 210002, China. science2008@hotmail.com.

Introduction

Coronaviruses (CoVs) are a family of RNA viruses belonging to the Coronaviridae family and the Coronavirinae subfamily and are the largest group of positive-sense single-stranded RNA viruses. From an academic perspective, CoV can be divided into four genera, namely Alphacoronaviruses, Betacoronaviruses, Gammacoronaviruses, and Deltacoronaviruses. The alphacoronaviruses and betacoronaviruses are usually found in mammals, while the gammacoronaviruses and deltacoronaviruses are mainly associated with birds¹^,². SARS-CoV is the causative agent of the severe acute respiratory syndrome (SARS) outbreak that occurred in 2002–2003. This SARS outbreak was the first human pandemic to break out since the beginning of the 21st century, and it resulted in nearly 8000 cases of infection and 800 deaths worldwide³^,⁴. SARS-CoV belongs to the Betacoronavirus genus, and its genomic sequence exhibits low levels of similarity with the previously identified human CoVs-OC43 and 229E. Thus, we hypothesized that SARS-CoV underwent a long and independent evolutionary process. The SARS-CoV genome usually encodes four structural proteins: the spike protein (S), envelope protein (E), membrane protein (M), and nucleocapsid protein (N). Among them, the S protein is a trimeric, cell-surface glycoprotein that consists of two subunits (S1 and S2), whereas the S1 subunit is responsible for receptor binding. Variations in the S protein, to a large extent, are responsible for the tissue tropism and host ranges of different CoVs⁵^,⁶.

The origin of SARS-CoV has always been a focus of research. Palm civets were initially considered the natural reservoir of SARS-CoV due to the isolation of several strains of SARS-CoV from palm civets that were traded in the wet markets of the Guangdong province of China in 2003⁷. However, subsequent studies showed that the virus was detected only in palm civets of market origin that were tested prior to culling, but not in those tested later; palm civets captured from the wild also tested negative for the virus. This finding suggested that palm civets served only as an intermediate reservoir and are therefore not a natural reservoir for SARS-CoV⁸^,⁹. Recently, bats have captured our attention due to their ability to act as natural reservoirs for a wide variety of viruses, including many important zoonotic viruses that are associated with several severe forms of emerging infectious diseases, such as Ebola virus, Nipah virus, Hendra virus, and Marburg virus¹⁰^,¹¹. In 2005, teams from Hong Kong and Mainland China almost simultaneously discovered the presence of SL-CoVs in wild Chinese horseshoe bats (Rhinolophus sinicus) from China. These findings suggested that the bats were the natural hosts of SARS-CoV¹². Notably, during longitudinal surveillance of the Rhinolophus sinicus colony in the Yunnan Province of China over the past few years, a Chinese research team successfully isolated a live SL-CoV sample from Vero E6 cells that were incubated in the bat feces in 2013¹³. The isolated virus showed more than 95% genome sequence identity with human and civet SARS-CoVs. Further studies on these indicated that the SL-CoV from bats may directly infect humans and does not require an intermediate host. SL-CoV, similar to SARS-CoVs, possesses the ability to infiltrate cells using its S protein to combine with angiotensin-converting enzyme 2 (ACE2) receptors¹⁴. This observation indicated that SARS-CoV originated from Chinese horseshoe bats and that SL-CoV isolated from bats therefore poses a potential threat to humans.

In recent years, many novel SL-CoVs have been identified in a variety of bat species throughout the world, including Asia, Europe, Africa, and America. Most SL-CoVs were discovered in rhinolophids from China, Slovenia, Bulgaria, and Italy¹⁵^–¹⁷, while novel beta-coronaviruses related to SARS-CoV have been detected in Hipposideros and Chaerophon species from Kenya and Nigeria¹⁸^,¹⁹.

However, analysis of the RNA-dependent RNA polymerase (RdRp) amino acid sequence showed that the genomic sequences of these bat SL-CoV samples obtained from different parts of the world shared 80–90% identity among themselves and exhibited 87–92% identity with the SARS-CoVs extracted from human or civet sources²⁰^,²¹.

These findings indicated that SARS-CoV likely evolved in bats over longer periods of time. Previous research conducted by our group revealed that bats found in Southeast China have high carrying capacities for SL-CoVs²². After conducting an epidemiological survey on the bats carrying CoVs, two novel SL-CoVs were identified in the Rhinolophus pusillus specimens from Zhoushan city, Zhejiang Province, China; subsequently, a rat infection model was utilized to assess the cross-species transmission potential of the viruses.

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Results

Sampling

Between 2015 and 2017, 334 bats were sampled from Zhoushan, China. These bats belonged to the species Rhinolophus pusillus as determined by the sequences of the mitochondrial cytochrome b gene in their muscle tissues²³. All 334 bat samples were screened for CoV RNA using a pan-coronavirus reverse transcription (RT)-PCR assay. The overall prevalence of the virus was 26.65% (89/334, bats; Table 1). Additionally, a higher prevalence was observed in samples collected in July (66.7% in 2015) than in those collected in October (21% in 2016) or February (13% in 2017). A phylogenetic tree was constructed according to the 440-bp RdRp partial sequences, and the positive samples were classified into Alphacoronaviruses and Betacoronaviruses. As shown in Fig. S1, 89 amplicons were grouped into five clades with 66–100% nucleotide identities between them, and they shared 94–100% identities with the viruses that were extracted from Hong Kong, Guangdong, and Hainan in China as well as those from Spain.

Summary of the bat-CoVs detection in bats from the Zhejiang province of China

Time

Locus

Sample number

Bat species

CoV positive

July, 15

Dinghai, Zhoushan city (ZXC)

45

Rhinolophus sinicus

66.7% (30/45)

January, 16

Dinghai, Zhoushan city (Z2)

120

Rhinolophus sinicus

25% (30/120)

October, 16

Daishan, Zhoushan city (DXC)

84

Rhinolophus sinicus

21% (18/84)

February, 17

Dinghai, Zhoushan city (ZC)

85

Rhinolophus sinicus

13% (11/85)

Total

334

26.65 (89/334)

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Full genomic sequence comparison and recombination analyses

To further explore the evolution of SL-CoV from Zhoushan, two complete genomic sequences of the representative bat-derived CoVs were generated by sequencing several overlapping amplicons. Specifically, sequences were generated from the following samples: SL-CoV ZXC21 (MG772934) bat that was extracted from a sample procured in July 2015, and SL-CoV ZC45 (MG772933) bat that was extracted from a sample procured in February 2017. The full genomes of ZXC21 and ZC45 consisted of 29,732 nt and 29,802 nt, respectively. The genomic organization in both cases was similar to that of the most well-known bat-SL-CoVs. Using the RDP program, the potential recombinant events between ZXC21, ZC45 and other representative strains of 13 human/civet and bat SARS-like CoVs were initially predicted. The results did not identify any potential recombination events. The genomic sequence similarity among the five bat-SL-CoVs and the SARS-CoV SZ3 strain was examined by Simplot analysis (Fig. 1). The results showed that the genomes had 38.9% GC content and had 13 open reading frames (ORFs) similar to the HKU3-1 strain. The two new bat SL-CoVs shared 97% genomic sequence identity among themselves. The overall nucleotide sequence identity of these two genomes with civet SARS-CoV (SZ3 strain) was 81%, which was lower than the previously reported observations associated with bat SL-CoVs collected from China (88–92%). From homology analyses of different ORFs, ORF8 fragments showed the lowest homology with the reported SL-CoV homology data²⁴, presenting a shared identity of only 60% with its closest relatives.

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Fig. 1

A gene map of the two novel SL-CoVs and the recombination analysis of novel SL-CoVs with other SL- CoVs.

Similarity plots were conducted with SARS CoV SZ3 as the query and bat SL-CoVs, including Rs3367, Longquan-140, and HKU3-1, as potential parental sequences. The analysis was performed using the Kimura model, with a window size of 2000 base pairs and a step size of 200 base pairs

The S protein is responsible for the entry of the virus and is functionally divided into two domains, S1 and S2. The bat SL-CoV Rs3367 is the most closely related virus to the human SARS-CoV and has 89.9% amino acid sequence identity to the SARS-CoV with respect to the whole spike protein. Comparatively speaking, the S proteins of ZXC21 and ZC45 identified in this study were slightly more different than their counterpart in SARS-CoV, which showed 77% identity at the amino acid level. Phylogenetic analyses based on the S protein suggested that the S proteins of ZXC21 and ZC45 represented a separate clade related to the lineage B CoVs (Fig. 2b). The highest amino acid sequence identity shared with the Rs806 strain was only 83%. Like other bat-SL-CoVs, the S1 domain of the bat SARS-like CoVs exhibited a very low nucleotide similarity with SARS CoV, and there are several key deletions and mutations in most of the variable regions within the receptor-binding domain (RBD) (Fig. 2a).

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Fig. 2

Characterization of S1 domains of the SARS CoV and SL-CoVs.

a Amino acid sequence comparison of the S1 subunit. The receptor-binding domain (aa 318–510) of SARS-CoV. b A phylogenetic analysis of the entire S1 amino acid sequences based on the neighbor-joining method. The SARS-CoV-GD01, BJ302, and GZ02 strains were isolated from patients of the SARS outbreak in 2003. The SARS-CoV SZ3 was identified from civets in 2003. Other bat-SL-CoVs were identified from bats in China.The sequences of SL-CoVs in this study are marked as filled triangles

Rat infection and virus detection test

Despite the failed isolation of the infectious virus from PCR-positive samples in Vero E6 cells, we attempted to isolate the virus from suckling rats by infecting them with tissue samples that were positive for the coronavirus. After 15 days, pathological analysis showed that the tissues and organs of the infected rats exhibited varying degrees of inflammation, and the inflammatory reaction in the brain tissues was most evident. Of the ten suckling rats, four showed clinical symptoms, including drowsiness, slow action, and mental depression. The new suckling rats infected with the diseased brain tissue still had irregular onset, whereas five of the 11 suckling rats in one nest had clinical symptoms. Numerous apoptotic neurons were seen in the focal areas of the brain tissue, and the chromatin in the nuclei was condensed and unclear. The lung tissues were well structured, but the alveolar cavities were partly fused together and showed clear signs of mild emphysema. Intestinal tissue analysis showed a loss in the structure of the intestinal mucosa; the mucous membranes were thin, the crypts were shallow, the intrinsic glands were reduced, and the stroma showed a dispersed inflammatory infiltrate (Fig. 3). Subsequently, the viral load of different tissues was detected by quantitative PCR, and the viral loads of the lung tissues remained the highest, showing approximately 10⁴ viral genome copies per 1 μl of tissue suspension (data not shown).

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Fig. 3

Light microscopy observations of rat tissues infected with bat-SL-CoVs:

Sectioned brain, intestine, lung and liver tissues were sampled from rats infected with bat-SL-CoV ZC45

Suspected viral particles were observed in the nuclei of denatured neurons in the brain tissues of the rats using transmission electron microscopy (TEM). These viral particles presented the typical coronavirus morphology and were approximately 100 nm in size with apparent surface spikes (Fig. 4). Simultaneously, various viral RT-PCR tests were conducted on the tissues to detect viral particles. The tissues were tested for the presence of viral particles associated with a wide variety of viruses, such as CoVs, henipaviruses, respiroviruses, avulaviruses, rubulaviruses, and the influenza-A virus of the Orthomyxoviridae family, using previously published methods²⁵^,²⁶. The test results revealed that the tissues were positive only for CoV.

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Fig. 4

Transmission electron micrographs of infected rat brain tissues.

a, b CoV-like particles are considered SL-CoVs ZC45 in different locations of the infected rat brain tissues

Analysis of the N protein antigen and western blotting

Similar to other CoVs, the nucleocapsid protein is one of the core components of the SARS-CoV. The N protein is one of the most predominantly expressed proteins during the early stages of SARS-CoV infection and has been an attractive diagnostic tool due to the initiation of strong immune response against it. Evolutionary analyses have shown that the homology between the N protein and its counterparts in the well-known SARS-CoV and bat SL-CoV ranged from 89 to 91%. The antigenic analysis was based on the amino acid sequence of the N protein (Fig. 5), and the results suggested that the two alternative antigenic peptides, including KHD2016288-1:KDKKKKADELQALPQ and KHD2016288-2:QQQGQTVTKKSAAEA, were selected for peptide synthesis

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Fig. 5

Prediction of the antigenicity of the bat SL-CoV N protein.

a The predicted antigenicity for the N protein. b Amino acid sequence of the N protein. The high antigenicity portion is indicated in the red circle. The two synthesized polypeptides are indicated in red

To further characterize the antigenic reactivity of the virus in infected murine tissues with ZC45-specific antibodies compared to that of ZC45, polyclonal antibodies against the polypeptides (KHD2016288-1:KDKKKKADELQALPQ) derived from ZC45 N proteins were generated and then subjected to western blotting analysis (Fig. 6). The anti-polypeptides were derived from the ZC45 N protein antibodies from six different sources of N proteins (50 kDa), including the intestinal tissues, brain tissues and lung tissues of infected rats.The results indicated that the polypeptide antigen was synthesized correctly, and the polyclonal antibodies produced against this polypeptide could react with the N proteins of the bat SL-CoV. The polyclonal antibodies reacted specifically with the infected rat tissues, but not with the rat tissues derived from the control specimens.

These results indicated that the virus can circulate and proliferate in infected rats.

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Fig. 6

Detection of N protein expression in infected rat tissues by western blotting.

Proteins from the following tissues were analyzed: rat brain from the control specimen (lane 1), intestinal tissue from bat ZC45 (lane 2), intestinal tissue from the infected rat (lane 3,6), lung tissue from the infected rat (lane 4,7), and brain tissue from the infected rat (lane 5,8)

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Discussion

Since the first report on the origin of SL-CoVs from bats in 2005, CoVs have been found in ten different bat species within six families from more than ten countries, including China, Africa, and Europe²¹^,²⁷. Our 2-year longitudinal surveillance of bats in Zhoushan indicated that all 334 bats that were collected belonged to the species Rhinolophus sinicus, suggesting that it was the dominant bat species found in our study and has been shown to be the natural reservoir of SARS-CoV. Nested PCR amplification of the conserved region of RdRp showed that the CoV carrying rate associated with this species of bat was much higher than that reported previously²⁸^,²⁹. At the same time, the summer carrying rate was higher than that associated with the other seasons due to the influence of seasonal distribution. In this region, there were two clades of Alphacoronaviruses and three clades of Betacoronaviruses identified, indicating that a wide variety of CoVs circulate in the bats of the Zhoushan area, and these CoVs were the most widely transmitted in the bat colonies found in this region.

To explore the possibility of CoV transmission from bats in this area, two full-length samples of bat-SL-CoVs were procured from the viral-infected bats. These two bat SL-CoVs were obtained from the same location but during different seasons; a genomic sequence identity of 88–99% was presented among them, indicating that the bats are the natural reservoirs of these SL-CoVs and that these SL-CoVs can circulate within single colonies. Meanwhile, there was a great difference between the two viruses described in this study and the viruses described in earlier studies, especially with respect to the hypervariability of the S1 domain³⁰^,³¹. It was noted that the gene encoding the S protein showed a high degree of variability. The S protein is responsible for viral entry and is functionally divided into two domains, namely, S1 and S2. The S1 domain is involved in receptor binding, while the S2 domain is involved in cellular membrane fusion. The S1 domain can be functionally subdivided into two domains, an N-terminal domain (S1-NTD) and a C-terminal domain (S1-CTD), and both can bind to host receptors, hence functioning as RBDs³². ZXC21 and ZC45 showed huge diversities with the previously reported CoVs of bats associated with the S1 region, and the highest level of shared identity was only 83%. An attempt was made to perform a recombination analysis during the course of this study. In our study, no potential recombination events could be identified. This could be because the two strains originated from an unsampled SL-CoV lineage residing in a bat species that is phylogenetically closer to ZXC21 and ZC45 than all other known bat SL-CoV samples. Then, we used simplot to analyze the sequence similarity of five bat-SL-CoVs and the SARS-CoV SZ3. The Longquan-140 strain is the most homologous to ZC45 and ZXC21, the Rs3367 is the closest strain of bat origin to the human pathogenic SARS coronavirus, and SZ3 is the representative strain of civetorigin.

In this study, a suckling rat model was initially used to study the possibility of the proliferation of bat-derived CoVs in other animals. Previously, only one report had shown promising results associated with the isolation of live SL-CoVs from the fecal samples of bats with Vero E6 cells¹³. The live SL-CoV cultured in Vero E6 cells presented a typical CoV morphology and has the ability to use ACE2 from humans, civets, and Chinese horseshoe bats for cell entry³³. An attempt to isolate the virus with Vero E6 cells was unsuccessful, which was likely due to a low viral load or a lack of compatibility with Vero E6 cells. This study found that the SL-CoVs derived from bats could replicate successfully in suckling rats, and pathological examination showed the occurrence of inflammatory reactions in the examined organs of the suckling rats.

This result indicated that the virus can proliferate in rats and has the potential of cross-species transmission.

When CoV particles procured from the infected brain tissues of the rats were studied by electron microscopy, the morphology of the particles was found to be identical to the typical coronavirus particles, as described in previous studies³⁴. However, the typical spikes could not be visualized by electron microscopy. This observation can be partially explained by the hypothesis that the S1 and S2 domains of the S protein (which are not well-connected) were easily detached from the virion using excessive freeze-thawing or ultracentrifugation⁶. Thus, there was a loss of S1 domains, which likely occurred during the preparation of the samples for electron microscopy. Meanwhile, the infected rat tissues could react with the polyclonal antibodies associated with the ZC45 N protein, according to the results from the western blotting assay, indicating that the virus can circulate in rats. Despite the negative western blotting results in the intestinal tissues of rat and the positive results of western blotting in the brain and lung tissues, we considered that these differences may be caused by different viral loads in different tissues.

In conclusion, based on the early detection of a high carrying rate for SL-CoVs, which originated from the bats in Zhoushan, China, this study involved continuous surveillance of the SL-CoVs that originated from the bats of this region. Diverse bat SL-CoVs were identified in this region, and the SL-CoVs in this region remained stable and could be transmitted to each other. Although there were several differences between the SARS-CoVs and the bat-SL-CoVs procured from this region based on the two-full-length samples obtained in this study, especially pertaining to the S protein region, this strain could still cause infection in neonatal rats. This observation highlights the possibility of cross-species transmission of these viruses. These findings strongly suggest the need for continued surveillance of viruses originating from wild animals andpromote further research to study the possibility of cross-species transmission of these viruses.

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Materials and methods

Ethics statement

The procedures for sampling of bats were reviewed and approved by the Administrative Committee on Animal Welfare of the Institute of Zhejiang CDC Veterinary (Laboratory Animal Care and Use Committee Authorization). All live bats were maintained and handled according to the Principles and Guidelines for Laboratory Animal Medicine (2006), Ministry of Science and Technology, China. All animal experiments were approved by the Ethics Committee of the Research Institute for Medicine, Nanjing Command. All methods were performed in accordance with the relevant guidelines and regulations (Approval number: 2015011).

Sampling

Overall, 334 adult bats were captured live at the mountain cave with mist nets at four separate times from July 2015 to February 2017 in Zhoushan city (including Dinghai and Daishan), Zhejiang Province, China. All bats appeared healthy and had no obvious clinical signs at capture. After completion of collection from each sample site, all bats were immediately dissected, and bat details are shown in Table 1. Each sample (approximately 1 g of intestinal tissues) was immediately transferred into viral transport medium (Earle’s balanced salt solution, 0.2% sodium bicarbonate, 0.5% bovine serum albumin, 18 g/l amikacin, 200 g/l vancomycin, 160 U/l nystatin), stored in liquid nitrogen prior to transportation to the laboratory, and ultimately stored at −80 °C.

RNA extraction and RT-PCR screening

All specimens were pooled and subjected to nested RT-PCR analysis as reported in the previous study²². Briefly, each intestinal sample (approximately 0.1 g) was homogenized in a glass grinder with ten volumes of SM buffer (50 mM Tris, 10 mM MgSO4, 0.1 M NaCl, pH 7.5). The homogenate was centrifuged at 12,000 g for 10 min at 4 °C, but only the supernatant was used. The supernatant of each sample was passed through 0.22 μm Pellicon II filters (Millipore, Billerica, MA) to filter out the ruptured tissues, bacteria, and other impurities. The viral RNA was extracted with a Viral RNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s recommendations. RNA was eluted in 35 μl RNase-free H₂O and stored at −80 °C. Reverse transcription was carried out using the first cDNA synthesis kit (TaKaRa, Dalian, China) according to the manufacturer’s protocol with double-distilled water (ddH₂O) as a negative control. All samples were amplified by a nested PCR that targeted a 440-nt fragment in the gene RdRp of all known alpha and betacoronaviruses³⁵^,³⁶. For the first round PCR, the 20 μl reaction mix contained 18 μl of PCR reaction solution (Takara), 10 pmol of each primer and 1 μl of the DNA template. The amplification was performed under the following conditions: 94 °C for 3 min; 40 cycles at 94 °C for 30 s, 52 °C for 30 s and 72 °C for 1 min for 40 cycles of in-house reaction; and extension at 72 °C for 10 min. For the second round PCR, the 20 μl reaction mix contained 18 μl of PCR reaction buffer, 10 pmol of each primer, and 1 μl product of the first round PCR. The amplification was performed under the following conditions: 94 °C for 3 min followed by 30 cycles consisting of 94 °C for 30 s, 52 °C for 30 s, 72 °C for 30 s, and a final extension of 72 °C for 10 min with ddH₂O as a negative control. Positive PCR products were sequenced in both directions by an ABI 3730 DNA Analyzer (Invitrogen, Beijing, China).

Sequencing of full-length genomes

To obtain the full genomic sequences of ZXC21 and ZC45, 19 degenerated PCR primer pairs were designed by multiple alignment of available SARS-CoV and bat SL-CoV sequences deposited in GenBank, targeting almost the full length of the genome. Primer sequences are available upon request. Sequences of 5′ and 3′ genomic ends were obtained by 5′ and 3′ RACE (Takara), respectively. PCR products with expected size were gel-purified and directly subjected to sequencing. The sequences of overlapping genomic fragments were assembled to obtain the full-length genome sequences, with each overlapping sequence longer than 600 bp.

Phylogenetic analysis of amplicons

All 440-bp-long amplicons were aligned with their closest phylogenetic neighbors in GenBank using ClustalW v.2.0. Representatives of different species in the genera of Alphacoronavirus and Betacoronavirus as well as some unapproved species were included in the alignment. Phylogenetic trees based on nucleotide sequences were constructed using the neighbor-joining method using MEGA v.7 with the Maximum Composite Likelihood model and a bootstrap value of 1000³⁷.

The aligned full sequences were initially scanned for recombination events using the Recombination Detection Program (RDP)³⁸. The potential recombination events between ZXC21, ZC45, Rs3367 (KC881006), Longquan-140 (KF294457.1), and HKU3-1 (DQ022305.2), as suggested by RDP with strong P values (<10⁻²⁰), were investigated further by similarity plot and bootscan analyses using SimPlot v.3.5.1³⁹.

Suckling rat infecting assay

To test the pathogenicity of the ZC45 agent, infection experiments were performed in suckling rats. 3-day-old suckling BALB/c rats (SLAC, China) were intracerebrally inoculated with 20 μl of volume grinding supernatant of ZC45 intestinal tissue. Animal housing care and all animal experiments were performed in a biosafety level 3 (BSL-3) facility and were approved by the local ethics committee. After 14 days, the brain, lungs, intestine, and liver tissues from infected rats were selected to prepare pathological sections. Briefly, the tissues were fixed in 10% (vol/vol) neutral-buffered formalin. After routine tissue processing, including dehydration by graded alcohol solutions, washing, and incubation in paraffin, 4 µm thick sections were cut and stained with hematoxylin and eosin (H&E). Approximately 2 h later, the prepared tissue sections were imaged using optical microscopy (Olympus, Japan).

TEM was utilized to obtain more detailed pathological information responsible for the major symptoms. The tissue samples were fixed in 2.5% (vol/vol) dialdehyde for 2 h, postfixed in 1% (vol/vol) osmium tetroxide for 1 h, dehydrated in graded ethanol, and embedded in Epon-812 epoxy resin. Then, 70 nm ultrathin sections were produced and quickly stained in aqueous uranyl acetate and Reynolds’ lead citrate. Finally, the generated tissue sections were examined using a JEM-1200 TEM (Jeol Ltd. Tokyo, Japan).

Quantitative RT-PCR was performed using tissue suspensions of rats positive for SL-CoV by RT-PCR. cDNA was amplified in SYBR Green I fluorescence reactions (Roche) using specific primers (5′-TGTGACAGAGCCATGCCTAA-3′ and 5′-ATCTTATTACCATCAGTTGAAAGA-3′)¹². A plasmid with the target sequence for generating the standard curve was used. At the end of the assay, PCR products (280-bp fragment of pol) were subjected to melting curve analysis (65–95 °C, 0.1 °C/s) to confirm the specificity of the assay.

Preparation of rabbit antiserum against two peptides

To obtain the polyclonal antibody of bat SL-CoV ZC45 N protein, two partial peptides with 15-amino acid residues of N protein were synthesized (Sangon Biotech, Shanghai, China) after a homology search according to the bioinformatics analysis and prediction of signal peptide (SignalIP-4.1), hydrophilicity and antigenicity of N protein. New Zealand White rabbits (2–2.3 kg) were injected subcutaneously using 0.6 mg of two peptides in 1 ml phosphate-buffered saline (PBS) emulsified with 1 ml Freund’s complete adjuvant (Sigma). Animals were boosted twice by the same route at 2-week intervals with approximately 0.3 mg of two peptides in 1 ml of PBS emulsified with 1 ml of Freund’s incomplete adjuvant (Sigma). One week after the last booster immunization, blood samples were collected, and sera were isolated for biological activity assays. The antibody titer was tested by indirect enzyme-linked immunosorbent assay. Preimmune rabbit serum was collected before the first injection.

Determination of virus infectivity by western blotting assay

Western blotting was performed to characterize the antigenic reactivity of infected rat tissue with N protein antibody of bat SL-CoV-ZC45. Infected intestine, lung and brain tissue samples were homogenized and lysed in RIPA buffer supplemented with proteinase inhibitors. Equal amounts of proteins (40 μg) were loaded and separated on 8% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) gel. Following electrophoresis, the proteins were transferred onto a PVDF (polyvinylidene difluoride) membrane, blocked with 5% (w/v) milk, and incubated with primary and secondary antibodies. Blots were developed and detected by enhanced chemiluminescence (GE Healthcare, Little Chalfont, UK). Rat tissues from the control specimens and intestinal tissues from bat ZC45 were used as negative and positive controls, respectively.

Nucleotide sequence accession numbers

All amplicon sequences and the full genomes of ZXC21 and ZC45 generated in this study have been deposited in GenBank under accession numbers MG772844 through MG772934.

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Supplementary Material

Supplementary Figure S1:

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Supplementary Information:

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Notes

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Conflicts of interest

All authors declare that they have no conflicts of interest.

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Electronic supplementary material

Supplementary Information accompanies this paper at (10.1038/s41426-018-0155-5).

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Notes

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Acknowledgements

This study was supported by National Major Infectious Diseases (2017ZX10303401-007), National Natural Science Foundation of China (U1602223), Army Logistics Scientific Research Projects (BWS14C051), Jiangsu Province Science and Technology Support Program Project (BE2017620), National Postdoctoral Special Aid (2016T91011), and Jiangsu Postdoctoral Fund (1501147C).

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Virome analysis for identification of novel mammalian viruses in bats from Southeast China

https://www.ncbi.nlm.nih.gov/pubmed/28883450

Hu D^1,2, Zhu C², Wang Y^1,2, Ai L², Yang L², Ye F², Ding C², Chen J², He B³, Zhu J², Qian H⁴, Xu W⁴, Feng Y⁵, Tan W⁶, Wang C^7,8.

Author information

1

Department of Epidemiology, College of Preventive Medicine, Third Military Medical University, Chongqing, 400038, China.

2

Department of Epidemiology, Research Institute for Medicine of Nanjing Command, Nanjing, 210002, China.

3

Key Laboratory of Jilin Province for Zoonosis Prevention and Control, Institute of Military Veterinary, Academy of Military Medical Sciences, Changchun, Jilin, China.

4

Key Laboratory of Laboratory Medicine of Jiangsu Province, School of Medicine, Jiangsu University, Zhenjiang, 212013, Jiangsu, China.

5

College of Animal Sciences, Zhejiang University, Hangzhou, 310058, Zhejiang, China.

6

Department of Epidemiology, Research Institute for Medicine of Nanjing Command, Nanjing, 210002, China. njcdc@163.com.

7

Department of Epidemiology, College of Preventive Medicine, Third Military Medical University, Chongqing, 400038, China. science2008@hotmail.com.

8

Department of Epidemiology, Research Institute for Medicine of Nanjing Command, Nanjing, 210002, China. science2008@hotmail.com.

Abstract

Bats have been shown as important mammal resevoirs to carry a variety of zoonotic pathogens. To analyze pathogenic species in bats from southeast coastal regions of China, we performed metagenomic sequencing technology for high throughput sequencing of six sentinels from southeast coastal area of China. We obtained 5,990,261 high quality reads from intestine and lung tissue of 235 bats, including 2,975,371 assembled sequences. 631,490 reads predicted overlapping sequences for the open reading frame (ORF), which accounts for 2.37% of all the sequences (15,012/631,490). Further, the acquired virus sequences were classified into 25 viral families, including 16 vertebrate viruses, four plant viruses and five insect viruses. All bat samples were screened by specific PCR and phylogenetic analysis.

Using these techniques, we discovered many novel bat viruses and some bat viruses closely-related to known human/animal pathogens, including coronavirus, norovirus, adenovirus, bocavirus, astrovirus, and circovirus.

In summary, this study extended our understanding of bats as the viral reservoirs. Additionally, it also provides a basis for furher studying the transmission of viruses from bats to humans.

PMID:

28883450

PMCID:

PMC5589946

DOI:

10.1038/s41598-017-11384-w

China 'appoints its top military bio-warfare expert to take over secretive virus lab in Wuhan', sparking conspiracy theories that coronavirus outbreak is linked to Beijing's army

Chen Wei, 54, has reportedly taken the helm of Wuhan Institute of Virology

Her appointment prompted claims that the classified lab is run by the army

One theory suggests Beijing could be making bioweapons in the Wuhan lab

It alleges the virus could have been created there and leaked by accident

A director at the lab has denied the allegations and called for investigation

Coronavirus symptoms: what are they and should you see a doctor?

By Billie Thomson

Mail on Line : 11:39, 14 February 2020

https://www.dailymail.co.uk/news/article-8003713/China-appoints-military-bio-weapon-expert-secretive-virus-lab-Wuhan.html

27 comments

China has reportedly appointed its top military biological weapon expert to take over a secretive virus laboratory in Wuhan after the outbreak of a new coronavirus, sparking conspiracy theories that the health crisis could be connected to the army.

Chen Wei, a Major General of the People's Liberation Army, was flown in to Wuhan by the central government late last month before officially taking the helm of Wuhan Institute of Virology, according to a report.

The 54-year-old's designation prompted some people to speculate that the epidemic could have been spawned in the little-known lab and that the lab is run by Beijing's military.

Chen Wei , a Major General of the People's Liberation Army, was flown in to Wuhan late last month after the coronavirus broke out there, according to Chinese state-run media

Chen is also a leading specialist in genetic engineering vaccines in China. She developed a medical spray during the SARS outbreak in 2003, preventing around 14,000 medical workers from contracting the virus, said another state-media report

Wuhan Institute of Virology (pictured) has been the centre of conspiracy theories after the coronavirus epidemic started. One theory claims that the virus was a biological weapon engineered by China and was leaked from the lab by accident - to which China denied

Chen and her team were already developing a quicker way to screen the COVID-19 coronavirus from a tent in the epicenter on January 30, according to an official report from China.

Chen, also a leading specialist in genetic engineering vaccines in China, developed a medical spray during the SARS outbreak in 2003. The product prevented around 14,000 medical workers from contracting the virus, said another state-media report.

She is also known in the country as the 'terminator of Ebola' for leading a team to create a vaccine against the fatal virus.

Speaking of fighting the novel coronavirus, Chen said: 'The epidemic is like a military situation. The epicentre equals to the battlefield.'

Chen and her team were already developing a quicker way to screen the COVID-19 coronavirus from a tent in the epicenter on January 30, according to an official report from China. She is pictured being interviewed by to a reporter from CCTV outside the mobile laboratory in Wuhan

China's central government has sent at least 2,600 military doctors to Wuhan in a bid to curb the epidemic. Members of a military medical team are pictured heading for Wuhan Jinyintan Hospital, where most of the coronavirus patients are being looked after, on January 26

Several of Wuhan's major hospitals as well as two newly built coronavirus hospitals are now being managed by the People's Liberation Army. A military medical worker is pictured taking over the work from a medical worker at Wuhan Jinyintan Hospital on January 26

Although China's official media had little information on where Chen was working from in Wuhan, Radio France Internationale last Saturday claimed that she had already taken the leadership of Wuhan Institute of Virology.

The lab opened in November, 2018, and is classified as P4, the highest level in bio-safety.

The report cited a post on Chinese forum Douban as its source and claimed that the move revealed the possible relation between the lab and the army.

'This kind of connection shows that the previous [speculation] suggesting that the Chinese troops were developing biological weapons in Wuhan P4 did not come out of thin air,' it said.

The article was referring to an earlier theory, which claims that the COVID-19 virus was a biological weapon engineered by China and was leaked from the lab by accident.

The new coronavirus has killed at least 1,383 people and infected more than 64,460 globally

The claims came from a report byThe Washington Times, citing a former Israeli military intelligence officer named Dany Shoham. It suggested that the coronavirus originated in the lab which was engaging in a biowarfare programme.

Chinese authorities have denied the allegations.

Shi Zhengli, a director at Wuhan Institute of Virology, said earlier this month: 'The 2019 novel coronavirus is nature's punishment for humans' uncivilised life habits. I, Shi Zhengli, use my life to guarantee, [the virus] has no relation with the lab.'

Shi urged the Chinese authorities to launch an official investigation into the matter.

She told Chinese news outlet Caixin: 'Conspiracy theorists don't believe in science. I hope our country's professional departments can come to investigate and prove our innocence.'

Xu Bin (second from the right) from Beijing Chaoyang An'yuan Hospital affiliated to Capital Medical University, talks to journalists while a young family is discharged from the hospital in China's capital after all members recovered from the COVID-19 coronavirus on Friday

A separate conspiracy theory alleges that the COVID-19 virus was created by the United States which released it on purpose.

The theory proposes that the virus was used by Washington as part of a multi-pronged war against China, said a columnist at South China Morning Post, citing Hong Kong-based YouTube influencer Jonathan Ho Chi-kwong.

The author criticised the conspiracy theory, saying that it had been refuted by experts.

'Experts have pointed out that as a bioweapon, the new virus is pretty useless. It appears to kill just 2 per cent of victims and each patient spreads it to an average of only 2.2 people,' said the op-ed.

Medical workers check on the conditions of patients in Wuhan's Jinyintan Hospital on Thursday. The hospital has been designated to treat critical sufferers of the COVID-19 virus

Medical staff work in the negative-pressure isolation ward in Jinyintan Hospital on Thursday

China has reported another sharp rise in the number of people infected with the killer coronavirus, with the death toll now nearing 1,400.

The National Health Commission said 121 more deaths were recorded yesterday, as well as 5,090 new confirmed cases.

The number of reported cases has been rising more quickly after the hardest-hit province changed its method of counting them.

There are now almost 64,000 confirmed cases in mainland China, of which 1,380 have died, according to the national body.

Hubei province is now including cases based on a physician's diagnosis and before they have been confirmed by lab tests.

The acceleration in the number of cases does not necessarily represent a sudden surge in new infections of the SARS-CoV-2 virus.

Globally, the COVID-19 virus has so far killed at least 1,383 people and infected more than 64,460.

Read more:

中国首席生化武器专家陈薇少将接管武汉P4病毒实验�

mp.weixin.qq.com...

m.junshi.china.c...

Coronavirus link to China biowarfare program possible, analyst says - Washington Times

独家|石正丽回应质疑专家一致认为新冠病毒非人造_财新网_财新�

Was the new coronavirus created by China or the United States? | South China Morning Post

BELOW GIVES A GREAT LIST OF CHINESE CONTACTS IN EXPERT VIROLOGY RESEARCH -DL

The 9th Japan-China International Conference of Virology

June 12-13, 2012, Sapporo Japan

https://web.sapmed.ac.jp/hygiene/images/Final%20Program.pdf

Chairperson of the Conference

Prof. Koichi Yamanishi (Japan) Prof. George Fu Gao (China)

Honorary Chairperson of the Conference

Prof. Guanfu Zhu (China)

Scientific Board Members

Japan: Hiroshi Ushijima, Kazuyoshi Ikuta, Toshio Hattori, Kimiyasu Shiraki, Yasuo Suzuki,

Nobumichi Kobayashi, Jiro Arikwa

China: Ting Zhang, Zhenghong Yuan, Yuanyang Hu, Yuan Qian, Xiaoyan Zhang, Fengmin

Zhang

Organizing Committee

Chairperson:

Prof. Jiro Arikawa (Japan), Prof. Nobumichi Kobayashi (co-chairperson, Japan)

Prof. Fengmin Zhang (China)

Members:

Japan: Kumiko Yoshimatsu, Kenta Shimizu, Motoko Takashino,

China: Zhaohua Zhong, Xu Teng, Yong Fang

Sponsors

Japanese Society for Virology

Committee on Virology, Chinese Society for Microbiology

Co-sponsors

The Research Foundation for Microbial Diseases of Osaka University

Harbin Medical University,

China State Key Laboratory for Infectious Disease Prevention and Control,

China State Key Laboratory for Pathogen & Biosecurity,

China State Province Key Laboratories of Biomedicine Pharmaceutics of China

Preface

Distinguised participants and guests, on behalf of the Japan-China International

Conference of Virology and Japanese side Local Organizing Committee, I sincerely welcome

you to the 9th Japan-China International Conference of Virology, in Sapporo.

The Japan-China International Conference of Virology has been held every 4 or 2 years

since its first conference, held in Beijing in year 1992, when the 20th Anniversary of

normalization of Sino-Japanese relations. It is our great pleasure to be able to have 40 years

of Anniversary of normalization of Sino-Japanese relations, as well as the first 20 years

Anniversary in the Japan-China International Conference of Virology in 2012.

This conference seeks to provide a good platform for exchanging ideas and information

among virologists in various fields. The internet now unable us to communicate with each

other without time. However, if we do not have real friendship with mutual confidence, the

new communication system will not assist for our real collaboration. In this sense,

establishment of friendship through face-to-face communication is still a very important. I

believe that this conference provides good opportunity for scientists in both countries,

particularly young researchers to meet together and make friendship which continues to next

generations.

On March 11th, 2011, an unprecendented earthquake and tsunami hit the Tohoku region

in Japan. We would like to express our sincere appreciation for contribution and warm

messages from Chinese Government and Chinese people to our difficult experience.

I would like to convey all our best wishes for the 9th Japan-China International

Conference of Virology to be great scientific and collaborative for every one and to be very

successful. Enjoy your stay in Sapporo.

Koichi Yamanishi, M.D., Ph.D.

Chairperson of the 9th Japan-China International Conference of Virology

Preface

We would like to take this opportunity to express our sincere thanks to the Japanese

Society of Virology for your warm hospitality and great effort to organize the 9th China-Japan

International Conference of Virology. And, on behalf of the Committee on Virology, Chinese

Society for Microbiology, we would be very happy to extend our cordial welcome to all

participants attending this meeting.

In the past year, China still experienced great challenges in virus related emerging and

re-emerging infectious diseases, such as influenza, avian influenza, hand-foot-mouth disease,

hepatitis, as well as HIV/AIDS. Chinese government input more and more on scientific

researches, especially the infectious diseases prevention and control mega science and

technology projects in the "Eleventh Five-Year" plan. With this support, Chinese scientists

gained lots of progress and would be happy to share with scientists around the world and to

look for more opportunities to cooperate with international scientists, including Japanese

virologists. The conference will provide a good platform for virologists from China and Japan

to share their research progress and development in the field of virology, viral immunology,

and public health.

Lastly, we wish the 9th China-Japan International Conference of Virology satisfactory

and fruitful. Hopefully, this conference will facilitate the understanding of new progress in

virus related fields and promote more collaboration among scientists from China and Japan.

George F Gao, DPhil

Chairperson, the 9th China-Japan International Conference of Virology

Deputy Chairman, Committee on Virology, Chinese Society for Microbiology

Yiming Shao, M.D., Ph.D.

Chairman, Committee on Virology, Chinese Society for Microbiology

1

PROGRAM

Venue: “Furate” Hall, the alumni hall at the Hokkaido University Graduate School of

Medicine (Kita-15, Nishi-7, Sapporo 060-8638)

June 11, (Mon) 2012

14:00 ~ 18:00 Registration “Furate” Hall Lobby

19:00 ~ 20:00 Meeting of Panel Members

June 12 (Tue) 2012

9:00 ~ 9:30 Opening Ceremony “Furate” Hall

Opening Speech by Professor Koichi Yamanishi

Professor George Fu Gao

Professor Jiro Arikawa

9:30 ~ 10:30 Keynote lecture

Chairperson: Jiro Arikawa, George Fu Gao

For the control of highly pathogenic avian Influenza

Hiroshi Kida

Member of the Japan Academy

Specially Appointed Professor, Graduate School of Veterinary Medicine

Head, Research Center for Zoonosis Control

Head, OIE Reference Laboratory for Avian Influenza

Head, WHO Collaborating Centre for Zoonoses Control

Hokkaido University

Recognition of HLA-A*2402 restricted HIV-peptide by an αβTCR using Vδ1 segment

Yi Shi, Ai Kawana-Tachikawa, Chuansheng Liu, Jia Gao, Aikichi Iwamoto, George F.

Gao

CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of

Microbiology, Chinese Academy of Sciences

2

10:30 ~ 11:00 Group photo and coffee break

11:00 - 12:36 Session 1: Orthomyxoviruses

Chairpersons: Yasuo Suzuki, Kun Yao

1. Applicability of a sensitive duplex real-time PCR assay for identifying B/Yamagata and

B/Victoria lineages of influenza virus from clinical specimens

Shisong Fang1, Ting Wang2, Jianxiong Li3, , Cunyou Zhao4, Xin Wang1, Xing Lv1, Chunli

Wu1, Renli Zhang1, Jinquan Cheng1, Hong Xue4, Xiaowen Cheng1

1 Shenzhen Centre for disease control and prevention, Shenzhen, PR China, 2 School of

Public Health, Sun Yat-Sen University, Guangzhou, PR China, 3 Jiangxi province Center

for disease control and prevention, Beijing, PR China, 4 Department of biochemistry,

Hong Kong University of Science and Technology, Hong Kong, China

2. Influenza surveillance in Shenzhen, the biggest migratory metropolitan city of China,

2006-2009

X. Wang, C. L. Wu, X. Lv, S. S. Fang, H. W. Ma, J. F. He, X. Xie, S. J. Mei, Y. Li, J. Q.

Cheng, X. W. Cheng

Shenzhen Center for Disease Control and Prevention, Shenzhen, China

3. A cross-sectional serological study on the prevalence of antibodies to influenza A (H1N1)

2009 virus in residents of Shenzhen

Lu Xing, Charles Farthing, Wang Xin, Wu Chunli, Fang Shisong, Mou Jin, Zhao Jin,

Cheng Xiaowen, Zhang Renli

Shenzhen Center for Disease Control and Prevention, Shenzhen, China

4. Clinical and Molecular Characteristics of 2009 Pandemic Influenza H1N1 Infections with

Severe or Fatal Disease from 2009 to 2011 in Shenzhen, China

Chunli Wu, Xiaowen Cheng, Xin Wang, Xing Lv, Fan Yang, Tao Liu, Shisong Fang,

Renli Zhang and Jinquan, Cheng

Centers for Disease Control and Prevention, Shenzhen, China

5. Japanese apricot fruit juice concentrate contains anti-influenza compound, mumefural

Nongluk Sriwilaijaroen1,2, Akio Kadowaki3, Yuriko Onishi3, Nobuki Gato3, Makoto

3

Ujike4, Takato Odagiri5, Masato Tashiro5, Yasuo Suzuki2,6

1Thammasat University, Pathumthani 12120, Thailand, 2Health Science Hills, College of

Life and Health Sciences, Chubu University, Aichi, 487-8501, Japan, 3Food Science Res.

Lab. Nakano BC Co. Ltd., Wakayama 642-0034, Japan, 4Nippon Vet. Life Aci. Univ.,

Japan, 5Influenza virus Res. Center, National Inst. Infect. Dis., Tokyo 208-0011, Japan,

6Global COE Program, Univ. of Shizuoka, Shizuoka, Japan

6. Preparation of HuMAb against influenza virus and the evaluation of effectiveness and

safety

Mayo Yasugi1,4, Yuta Kanai1, Ritsuko Kubota-Koketsu2,4, Norihito Kawashita1,

Naphatsawan Boonsathorn3, Yoshinobu Okuno2, Takaaki Nakaya1, and Kazuyoshi Ikuta1,

4

1Institute for Microbial Diseases, Osaka University; 2Kanonji Institute, The Research

Foundation for Microbial Diseases of Osaka University, 3Ministry of Public Health,

Thailand; 4JST/JICA, Science and Technology Research Partnership for Sustainable

Development (SATREPS)

7. Heterosubtypic binding activity of hemagglutinin-specific antibodies induced by

inoculation of inactivated influenza virus in mice

Mieko Muramatsu, Reiko Yoshida, Ayato Takada

Division of Global Epidemiology, Research Center for Zoonosis Control, Hokkaido

University, Sapporo, Japan

8. Neutralizing antibody response in nasal mucus and serum of healthy adults after intranasal

vaccination with inactivated whole influenza virus vaccine

Akira Ainai1,2, Shin-ichi Tamura2, Tadaki Suzuki2, Elly van Riet1, Ryo Ito2, Takato

Odagiri1, Masato Tashiro1, Takeshi Kurata2, and Hideki Hasegawa2

1Influenza Virus Research Center and 2Department of Pathology, National Institute of

Infectious Diseases, Tokyo, Japan

12:36 ~ 14:00 Lunch

4

14:00 - 14:36 Session 2: retrovirus, bornavirus and bocavirus

Chairpersons: Toshio Hattori, Yuanyang Hu

9. Cross-Subtype Neutralizing Antibodies in Treatment-naive HIV-1-infected Individuals in

China and characteristics of viral envelope derived from broad neutralizers

Hong Ling1, Ping Zhong2, Caiyun Ren1, Haotong Yu1, Song Liu1,Yan Li1, Min Zhuang1

Guochao Wei1, Jiaye Wang2, Zhijie Chen3, Feng Sun3, Wei Liu4, Shujia Liang4

1Harbin Medical University, Heilongjiang province, 2Shanghai Municipal Center for

Disease Control and Prevention, Shanghai, 3Yili Prefecture CDC, Xinjinag province,

4Guangxi CDC, Guangxi province, China

10. CD56+ T Cells Inhibit HIV-1 Infection of Macrophages

Yong Feng, Ni Zhu, Li Li, Hai-Rong Xiong, Fan Luo, Zhan-Qiu Yang, and Wei Hou

State Key Laboratory of Virology/Institute of Medical Virology, School of Basic Medical

Science, Wuhan University, Wuhan, China

11. Prevalence of Extraordinary low level of HIV-1 infection and HIV-1 specific T cell

response in Beijing homosexual cohort

Li Ren1, Quanbi Zhao1, Meiling Zhu1, Haiying Zhu2, Hao Wu3, Tuofu Zhu2, Yiming

Shao1

1 Division of Virology and Immunology, National Center for AIDS/STD Control and

Prevention, China CDC, Beijing, PR China

2 Department of Microbiology, University of Washington, Seattle, USA

3 Center for Infectious Diseases, Beijing You-An Hospital, Capital Medical University,

Beijing, China

12. MAVS-mediated apoptosis is negatively regulated by X protein of Borna disease virus

Yujun Li12, Wuqi Song1,2, Jing Wu1, Qingmeng Zhang1, Aimei Li1, Wenping Kao1,

Junming He1, Yunlong Hu1, Aixia Zhai1, Jun Qian1, Fengmin Zhang1,2

1 The Heilongjiang Key Laboratory of Immunity and Infection, Pathogenic Biology,

Department of Microbiology, Harbin Medical University, Harbin, Heilongjiang, China

2 Key Laboratory of Bio-Pharmaceutical, Harbin Medical University, Ministry of

Education, Harbin, Heilongjiang, China

5

13. Regulation of miR-155 in the Homeostasis between Persistent Infection with Borna

Disease Virus and Host Innate Immunity

Aixia Zhai1, Jun Qian1, Wenping Kao1, Aimei Li1, Yujun Li1,2, Qingmeng Zhang1, Wuqi

Song1,2, Yingmei Fu1, Jing Wu1, Xiaobei Chen1, Hui Li1, Zhaohua Zhong1, Hong Ling1,

Fengmin Zhang1,2

1 Department of microbiology, Harbin Medical University; Key Laboratory for Immunity

and infection, Pathogenic biology, Heilongjiang Province, China

2 Bio-pharmaceutical Key Laboratory, Harbin Medical University, Ministry of Education,

China

14. Anti-BDV N protein antibody inhibits Borna disease virus replication in the chronic

fatigue syndrome patient and persistently infected oligodendrocytes

Yang Chen 1, Jun Qian 1, Qingmeng Zhang 1, Yujun Li 1,2, Aixia Zhai 1, Wuqi Song 1,

Xiaobei Chen 2, Jizi Zhao1,2, Yunlong Hu 1,2, Junming He1,2, Fengmin Zhang 1,2

1 Department of microbiology, Harbin Medical University; Key Laboratory for Immunity

and infection, Pathogenic biology, Heilongjiang Province, China

2 Bio-pharmaceutical Key Laboratory, Harbin Medical University, Ministry of Education,

China

15. Detection of human bocavirus 1-4 from nasopharyngeal swab samples collected from

patients with respiratory tract infections

Naoko Koseki1, Shinobu Teramoto1, Miki Kaiho1, Rika Endo (Gomi)2, Tadashi Ariga1,

and Nobuhisa Ishiguro1

1Department of Pediatrics, 2Department of Microbiology, Hokkaido University Graduate

School of Medicine, Sapporo, Japan

16. Molecular characterization of human bocavirus isolated from children with acute

gastroenteritis in Japan and Thailand

Pattara Khamrin1, Niwat Maneekarn1, Aksara Thongprachum2, Dinh Nguyen Tran2,

Satoshi Hayakawa3, Shoko Okitsu3, Hiroshi Ushijima3

6

11 Department of Microbiology, Faculty of Medicine, Chiang Mai University, Chiang Mai,

Thailand, 2 Department of Developmental Medical Sciences, Institute of International

Health, Graduate School of Medicine, the University of Tokyo, Tokyo, Japan, 3 Division

of Microbiology, Department of Pathology and Microbiology, Nihon University School of

Medicine, Tokyo, Japan

15:36 ~ 16:00 Coffee break

16:00 - 17:36 Session 3: Flavivirus

Chairpersons: Kazuyoshi Ikuta, Zishu Pan

17. Etiological Study of a Local Dengue Fever Outbreak and Molecular Characterization of

the Dengue Virus Isolated in Shenzhen

Yang F1, Ma HW1, Guo GZ2, Chen JQ1, Ma HW1, Liu T1, Huang DN1, Yao CH3, Zhang

L3, Zhang RL1

1 Shenzhen Centre for Disease Control and Prevention, China, 2 Department of

Pathogenic Organism, Fourth Military Medical University, Xian, China, 3 Laboratory of

Cell and Molecular Biology, Palmer Center for Chiropractic Research – Florida campus,

Palmer College of Chiropractic Florida, USA

18 The Study of Molecular Epidemiological of an local Dengue Fever Outbreak in Shenzhen

for the first time

YANG Fan, ZHANG Renli, CHEN Simin, XIONG Ying, LIU Tao, HUANG Dana, WU

Weihua, LI Yue

Shenzhen Center for Disease Prevention and Control, Shenzhen, China

19. Inhibitory Effect of Glutathione on Oxidative Liver Injury Induced by Dengue Virus

Serotype 2 Infections in Mice

Juan Wang, Yanlei Chen, Na Gao, Yisong Wang, Yanping Tian, Jiangman Wu, Junping

Zhu, Dongying Fan, Jing An

Department of Microbiology, School of Basic Medical Sciences, Capital Medical

University, Beijing, China

7

20. Identification of a novel inhibitor against dengue virus NS2B/NS3 protease by a

structure-based study

Takeshi Kurosu1, Sabar Pambudi1, Norihito Kawashita1,2, Promsin Masrinoul1, Kriengsak

Limkittikul3, Teruo Yasunaga1, Tatsuya Takagi1,2, Kazuyoshi Ikuta1

1 Research Institute for Microbial Diseases, Osaka University, Osaka, Japan, 2 Graduate

School of Pharmaceutical Sciences, Osaka University, Osaka, Japan, 3 Department of

Tropical Pediatrics, Faculty of Tropical Medicine, Mahidol University, Bangkok,

Thailand

21. Suppressive Effects on the Immune Response and Protective Immunity to a JEV DNA

Vaccine by Co-administration of a GM-CSF-Expressing Plasmid in Mice

Hui Chen, Na Gao, Dongying Fan, Jiangman Wu, Junping Zhu, Jieqiong Li, Juan Wang,

Yanlei Chen, Jing An

Department of Microbiology, School of Basic Medical Sciences, Capital Medical

University, Beijing, China

22. Chimeric classical swine fever (CSF)-Japanese encephalitis (JE) viral particles as a

non-transmissible bivalent marker vaccine candidate against CSF and JE infections

Zishu Pan1, Zhenhua Yang1, Rui Wu1, Ruangang Pan, Xiufen Zou2

1 State Key Laboratory of Virology, College of Life Sciences, Wuhan University, 2 School

of Mathematics and Statistics, Wuhan University, Wuhan, China

23. Molecular diagnosis and analysis of imported chikungunya virus strains, Japan,

2006-2011.

Chang-Kweng Lim, Meng Ling Moi, Akira Kotaki, Masayuki Saijo, Ichiro Kurane and

Tomohiko Takasaki

Depertment of Virology I, National Institute of Infectious Diseases, Tokyo, Japan

24. Rapid, Simple and Sensitive Detection of Q fever by Loop-Mediated Isothermal

Amplification of the htpAB Gene

8

Lijuan Zhang1, Lei Pan1, Desheng Fan2, Xiuchun Zhang3, Hong Liu4, Qunying Lu5, Qiyi

Xu2, Weihong Li3, Yonglin Shi4, Liping Jiang5, Yonggen Zhang4, Qiang Yu1, Lina Tian1,

Jianguo Xu1

1Dept.of Rickettsiology, China ICDC, Beijng, China, 2 YiLi Prefecture CDC, YiLi, China;

3 Beijing CDC, Beijing, China, 4Anhui provincial CDC, Hefei China, 5 Zhejiang CDC,

Hangzhou, China

18:30 - 20:30 Welcome party

Hokkaido University Faculty House Restaurant “En-re-i so”

June 13 (Wed) 2012

8:30 - 10:06 Session 4: Paramyxovirus and reovirus

Chairpersons: Nobumichi Kobayashi, Fengmin Zhang

25. Increase of Matrix Metalloproteinase-10 in human nasal epithelial cells during respiratory

syncytial virus infection

Satoshi Hirakawa, Takashi Kojima, Kazuhumi Obata, Kazuaki Nomura, Tomoyuki

Masaki, Akira Takasawa, Tetsuo Himi, Norihito Sawada, Hiroyuki Tsutsumi

Departments of 1Pediatrics, 2Pathology, 3Otolaryngology, and 4Microbiology, Sapporo

Medical University, School of Medicine, Sapporo, Japan

26. IPS-1-dependent innate immune response is indispensable for limiting the SARS-CoV

propagation in airway epithelial cell

Tomoki Yoshikawa1, 2, Shuetsu Fukushi1, 2, Clarence J. Peters1, 3, 4, and Chien-Te K Tseng1,

4

1 Departments of Microbiology and Immunology, 3 Pathology, and 4 Center for

Biodefense and Emerging Infectious Disease, University of Texas Medical Branch,

Galveston, Texas, 2 Department of Virology I, National Institute of Infectious Diseases,

Tokyo, Japan

27. Study on M gene based measles virus detection method by Real-Time PCR

Zhuo Fei

9

Shenzhen Luohu center for disease control and prevention, Shenzhen, China

28. Study on the characteristic of the current measles wild-type strains after continuous

passage

Fu Yan, Xu Chang-ping, Feng Yan, Zhong Su-ling, Lu Yi-yu

Zhejiang Provincial Center for Disease Control and Prevention, China

29. Comparison of neutralization capacity of Measles virus vaccine strain and epidemic

strains to different types of human serum

Feng Yan, Lu Yi-yu, Xu Chang-ping, Shi Wen, Jiang Xiao-hui, Li Zhen.

Zhejiang Provincial Center for Disease Control and Prevention, China

30. Investigation for rotavirus and adenovirus in stool specimens from hospitalized children

with diarrhea during 2010-2011 in Beijing, China

Liu Li-Ying, Zhang You, Qian Yuan, Jia Li-Ping, Deng Jie, Dong Hui-Jin

Laboratory of Virology, Capital Institute of Pediatrics, Beijing, China

31. Whole genomic analysis of a rare human G1P[9] rotavirus strain

Souvik Ghosh 1, Tsuzumi Shintani 1, Koki Taniguchi 2, Nobumichi Kobayashi 1

1 Department of Hygiene, Sapporo Medical University School of Medicine, Sapporo,

Japan. 2 Department of Virology and Parasitology, School of Medicine, Fujita Health

University, Toyoake, Japan

32. Full Genome Analysis of Rotavirus P[23] Collected from Piglets with Diarrhea in

Thailand, 2006-2008

Shoko Okitsu1,2, Pattara Khamrin3, Aksara Thongprachum2, Masashi Mizuguchi2, Satoshi

Hayakawa1, Niwat Maneekarn3, Hiroshi Ushijima1

1Division of Microbiology, Department of Microbiology and Immunology, Nihon

University School of Medicine, Tokyo, Japan, 2Department of Developmental Medical

Sciences, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan,

3Department of Microbiology, Faculty of Medicine, Chiang Mai University, Chiang Mai,

Thailand

10

10:06~ 10:20 Coffee break

10:20 - 11:56 Session 5: Bunyavirus, filovirus and hepatitis

Chairpersons: Kumiko Yoshimatsu, Zhaohua Zhong, Jing An

33. Isolation and characterization of hantaviruses from wild rodents and epidemiology of

hemorrhagic fever with renal syndrome in Russia

Hiroaki Kariwa1, Takahiro Seto1, Keisuke Yoshikawa1, Evgeniy A. Tkachenko2,

Vyacheslav G. Morozov3, Leonid I. Ivanov4, Raisa Slonova5, Tatyana A. Zakharycheva6,

Yoichi Tanikawa1, Takahiro Sanada1, Saasa Ngonda1, Ichiro Nakamura7, Kumiko

Yoshimatsu8, Jiro Arikawa8, Kentaro Yoshii1, Ikuo Takashima1

1 Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Japan, 2

Chumakov Institute of Polyomyelitis and Viral Encephalitidis, Moscow, Russia, 3 Medial

Company “Hepatolog” Incorporated, Samara, Russia, 4 Plague Control Station of

Khabarovsk, Russia, 5 Research Institute of Epidemiology and Microbiology, Siberian

Branch of Russian Academy of Medical Sciences, Vladivostok, Russia, 6 Far Eastern

State Medical University, Khabarovsk, Russia, 7 Research Center for Zoonosis Control,

Hokkaido University, Sapporo, Japan, 8 Graduate School of Medicine, Hokkaido

University, Sapporo, Japan

34. Development of immunochromatographic test strips for the detection of HFRS and HPS

hantavirus antibody in the human and rodent serum

Takako Amada1, Kumiko Yoshimatsu1, Shumpei P. Yasuda1, Takaaki Koma1, Kenta

Shimizu1, Rie Isozumi1, Nobuhito Hayashimoto2, Akira Takakura2, Jiro Arikawa1

1Dept.of Microbiology, Graduate School of Medicine, Hokkaido University, Sapporo,

Japan, 2Central Institute for Experimental Animals, Kawasaki, Japan

35. Persistence of Seoul virus in natural host (Rattus norvegicus)

Kumiko Yoshimatsu1, Shumpei P. Yasuda1, Kenta Shimizu1, Takaaki Koma1, Takako

Amada1, Tetsu Yamashiro2, Futoshi Hasebe3, Nguyen Thuy Hoa4, Le Thi Quynh Mai4,

Jiro Arikawa1

11

1 Department of Microbiology, Graduate School of Medicine, Hokkaido University,

Japan, 2 Center for Infectious Disease Research in Asia and Africa, Nagasaki University,

Japan, 3 Center of International Collaborative Research, Nagasaki University, Japan, 4

National Institute of Hygiene and Epidemiology, Vietnam

36. Analysis of humoral immune response among cynomolgus monkeys naturally infected

with Reston ebolavirus during 1996 outbreak in the Philippines

Satoshi Taniguchi1,2, Yusuke Sayama1, Noriyo Nagata1, Tetsuro Ikegami3, Mary E.

Miranda4, Shumpei Watanabe2, Itoe Iizuka1, Shuetsu Fukushi1, Tetsuya Mizutani1,

Yoshiyuki Ishii2, Masayuki Saijo1, Hiroomi Akashi2, Yasuhiro Yoshikawa2, Shigeru

Kyuwa2, and Shigeru Morikawa1

1 National Institute of Infectious Diseases, Japan, 2 University of Tokyo, Japan, 3 The

University of Texas Medical Branch, Galveston, Texas, USA, 4 Veterinary Public Health

Specialist, Aralia, Silang, Philippines

37. Analysis of filovirus glycoprotein-induced steric shielding effect against host proteins

Osamu Noyori, Keita Matsuno, Masahiro Kajihara, Ayato Takada

Division of Global Epidemiology, Research Center for Zoonosis Control, Hokkaido

University, Sapporo, Japan

38. Application of Allele-specific RNAi in Hepatitis B virus lamivudine resistance

Xu Teng, Di Li, Hong-Xi Gu*

Department of Microbiology, Harbin Medical University; Heilongjiang Provincial Key

Laboratory for Infection and Immunity; Key Lab of Heilongjiang Province Education

Bureau for Etiology, China

39. Antigenicity and infectivity of rat hepatitis E viruses

Tian-Cheng Li1, Kumiko Yoshimatsu4, Shumpei P. Yasuda5, Jiro Arikawa4, Michiyo

Kataoka2, Yasushi Ami3, Yuriko Suzaki3, Koji Ishii1, Naokazu Takeda6 and Takaji Wakita1

1 Department of Virology II, 2 Department of pathology, 3 Division of Experimental

Animals Research, National Institute of infectious Diseases, 4 Department of

Microbiology, Graduate School of Medicine, Hokkaido University, 5 The Tokyo

12

Metropolitan Institute of Medical Science. 6 Research Institute for Microbial Diseases,

Osaka University

40. Epidemiology of rat hepatitis E virus infection in human and rodents in Vietnam

Kenta Shimizu1, Tian-Cheng Li2, Shumpei P Yasuda1, Kumiko Yoshimatsu1, Takaaki

Koma1, Futoshi Hasebe3, Tetsu Yamashiro4, Nguyen Thuy Hoa5, Le Thi Quynh Mai5,

Koya Ariyoshi6, Jiro Arikawa1

1Department of Microbiology, Hokkaido University Graduate School of Medicine,

Hokkaido University, Japan

2Department of Virology II, National Institute of Infectious Diseases, Japan

3Center for Infectious Disease Research in Asia and Africa, Nagasaki University, Japan

4Center of International Collaborative Research, Nagasaki University, Japan

5National Institute of Hygiene and Epidemiology, Vietnam

6Department of Clinical Medicine, Institute of Tropical Medicine, Nagasaki University,

Japan

11:56 ~ 13:30 Lunch

13:30 - 15:06 Session 6: Pox, herpes and papilomavirus

Chairpersons: Masayuki Saijo, Hong Ling

41. The Research of Investigation and controlling to Ecthyma contagiosa in Guizhou Province

of China

Yang Mao-sheng, Xu Jin-e, Yu Bo, Shi Kai-zhi, Wu Wei-hen, Yang Li

Institute of Animal Science and Veterinary Medicine, Guiyang, China

42. Development of virus-Specific CD4+ and CD8+ Regulatory T Cells induced by Human

Herpesvirus-6 Virus Infection

Fang Wang2, Jing Chi1, Guangyong Peng3, Jinfeng Wang1, Lingyun Li4, Feng Zhou1 ,Bin

Gu1, Kun Yao1

1 Department of Microbiology and Immunology, Nanjing Medical University, Nanjing,

Jiangsu Province, China, 2 Department of Laboratory Medicine, the First Affiliated

Hospital of Nanjing Medical University, Jiangsu Province, China, 3 Division of

Infectious Diseases, Allergy & Immunology and Department of Internal Medicine, Saint

13

Louis University, St. Louis, Missouri, USA, 4 Department of Developmental Genetics,

Nanjing Medical University, Jiangsu Province, China

43. The role of MAPK in CD4(+) T cells toll-like receptor 9-mediated signaling following

HHV-6 infection

Yao Kun

Department of Microbiology and Immunology, Nanjing Medical University, Nanjing,

China

44. Analysis and Mapping of a 3’-Coterminal Transcription unit derived from Human

Cytomegalovirus Open Reading Frames UL30 through UL32

Yanping Ma, Ning Wang, Mali Li, Shuang Gao, Lin Wang, Bo Zheng, Ying Qi and Qiang

Ruan

Virus Laboratory, the Affiliated Shengjing Hospital, China Medical University, Shenyang,

China

45. Drug-resistant herpes simplex virus type 1 infections in children

Masayuki Saijo and Satuki Kakiuchi

Department of Virology 1, National Institute of Infectious Diseases, Tokyo, Japan

46. Administration of Acyclovir for Acute Lymphadenopathy Reduces Duration of

Hospitalization and Febrile Period

Yugo Ashino, Osamu Usami, Hiroki, Saitoh, and Toshio Hattori

1Department of Emerging Infectious Diseases, Tohoku University School of Medicine,

Sendai, Japan

47. Tobacco exposure results in increased DNA damage and mutation rates in cervical cells

maintaining oncogenic episomal human papillomavirus 16 genomes

Lanlan Wei1,2, Hongxi Gu1, Yan Wang1, Anastacia M. Maldonado2, Michelle A. Ozbun2

1 Department of Microbiology, Harbin Medical University, Harbin, Heilongjiang China,

2 Department of Molecular Genetics and Microbiology, The University of New Mexico

School of Medicine, Albuquerque, NM USA

14

48. Variations of human papillomavirus type 58 E6, E7 and L1 genes in strains from women

with cervical lesions in Liaoning province, China

Jian-hua Liu, Gui-li Wang, Wei-qiang Zhou, Chao Liu, Lian-xia Yang, Qiang Ruan and

Zheng-rong Sun

Virus Laboratory, The Affiliated Shengjing Hospital, China Medical University. China

15:06~ 15:20 Coffee break

15:20 - 16:32 Session 7: Picornavirus

Chairpersons: Hiroshi Ushijima, Maosheng Yang, Lijuan Zhang

49. Novel Picornaviruses in Children and Adults with Diarrhea, Thailand

Hiroshi Ushijima1, Pattara Khamrin2, Aksara Thongprachum3, Dinh Nguyen Tran3,

Satoshi Hayakawa1, Shoko Okitsu1, Niwat Maneekarn2

1 Division of Microbiology, Department of Pathology and Microbiology, Nihon

University School of Medicine, Tokyo, Japan,2Department of Microbiology, Faculty of

Medicine, Chiang Mai University, Chiang Mai, Thailand, 3 Department of Developmental

Medical Sciences, Institute of International Health, Graduate School of Medicine, the

University of Tokyo, Tokyo, Japan

50. In vivo bioluminescence imaging of enterovirus 71 infection by monitoring the 3C

protease activity

Zhi-Wei Guo1, Shuo Wu1, Ye-Lu Han1, Ying Qin1, Yang Chen1, Tian-Ying Wang1, Yan

Wang1, Le-Xun Lin1, Lei Tong1, Feng-min Zhang1,Wen-Ran Zhao2, Zhao-Hua Zhong1

1 Department of Microbiology, 2 Department of Cell Biology, Harbin Medical University,

Harbin, China

51. Blood selenium of low-level associated with development of hand- foot-mouth disease

Zhang Dongxiao 1, Yang Fan1, Wang Bing1, Liu Tao 1, Zhang Renli1

Shenzhen Centre for Diseases Control and Prevention, Shenzhen, China

52. Characterization of Ectropis obliqua virus 3C-like Protease Processing Activities

15

Shan Ye, Hongjie Xia, Congyi Zheng, Jiamin Zhang, Xi Zhou and Yuanyang Hu

State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan,

Hubei, China

53. Multiple suppression of RNA silencing by B2 protein from Wuhan Nodavirus in

Drosophila Cells

Nan Qi, Zhaowei Wang, Congyi Zheng, Jiamin Zhang, Xi Zhou and Yuanyang Hu

State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan,

Hubei, China

54. A Single Amino Acid at the Hemagglutinin Cleavage Site Contributes to the

Pathogenicity and Neurovirulence of H5N1 Influenza Virus in Mice

Yi Zhang1, Yipeng Sun1, Honglei Sun1, Juan Pu1, Xishan Lu2, Yi Shi2, Jing Li3, Qingyu

Zhu3, George F. Gao2, Hanchun Yang1, and Jinhua Liu1

1 Key Laboratory of Animal Epidemiology and Zoonosis, Ministry of Agriculture，College

of Veterinary Medicine, China Agricultural University, Beijing, 100193, China, 2 Chinese

Academy of Sciences Key Laboratory of Pathogenic Microbiology and Immunology

(CASPMI), Institute of Microbiology, Chinese Academy of Sciences, Beijing, China, 3

State Key Laboratory of Pathogens and Bio-security, Academy of Military Medical

Sciences, Beijing, China

16:32 - 16:45 Closing ceremony

China 'appoints military bio weapon expert to take over secretive virus lab in Wuhan'

https://www.dailymail.co.uk/news/article-8003713/China-appoints-military-bio-weapon-expert-secretive-virus-lab-Wuhan.html

China 'appoints its top military bio-warfare expert to take over secretive virus lab in Wuhan', sparking conspiracy theories that coronavirus outbreak is linked to Beijing's army

Chen Wei, 54, has reportedly taken the helm of Wuhan Institute of Virology

Her appointment prompted claims that the classified lab is run by the army

One theory suggests Beijing could be making bioweapons in the Wuhan lab

It alleges the virus could have been created there and leaked by accident

A director at the lab has denied the allegations and called for investigation

Coronavirus symptoms: what are they and should you see a doctor?

By Billie Thomson For Mailonline

Published: 11:39, 14 February 2020 | Updated: 18:41, 14 February 2020

Dr David Lowry

Monday 20 April 2020

Coronacrisis: there is a lot more going on in China on virology research than the UK media reports

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Time	Locus	Sample number	Bat species	CoV positive
July, 15	Dinghai, Zhoushan city (ZXC)	45	Rhinolophus sinicus	66.7% (30/45)
January, 16	Dinghai, Zhoushan city (Z2)	120	Rhinolophus sinicus	25% (30/120)
October, 16	Daishan, Zhoushan city (DXC)	84	Rhinolophus sinicus	21% (18/84)
February, 17	Dinghai, Zhoushan city (ZC)	85	Rhinolophus sinicus	13% (11/85)
Total		334		26.65 (89/334)