canping-huang-phd-novel-virus-discovery-in-bat-isn-translation

noqreport

This document is the partial translation,

from Chinese into English, of the PhD thesis entitled

Novel Virus Discovery in Bat and the Exploration of

Receptor of Bat Coronavirus HKU9

PhD candidate Canping Huang submitted this dissertation

to the Chinese Center for Disease Control and Prevention in 2016.

A translation commissioned by

Independent Science News.

A partial translation into English of the PhD thesis: “Novel Virus Discovery in Bat and the Exploration of

Receptor of Bat Coronavirus HKU9” by Canping Huang, Chinese Center for Disease Control and Prevention,

2016. Translation completed for Independent Science News in March 2021.


Preface to the Translation

Independent Science News arranged this translation of the PhD thesis “Novel Virus

Discovery in Bat and the Exploration of Receptor of Bat Coronavirus HKU9” to support the

search for the origins of the SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2)

virus. This is the novel betacoronavirus associated with the COVID-19 pandemic. The original

dissertation was written, mainly in Chinese, by PhD candidate Canping Huang. His research

was supervised by Gao Fu (George Gao, Director of the Chinese Center for Disease Control

and Prevention since 2017).

The parts of the thesis translated were those considered potentially relevant to the

origins of SARS-CoV-2/COVID-19. For example, Chapter 3 describes virus discovery research

carried out by Canping Huang and others in the same mine where, in 2013, Zhengli Shi of the

Wuhan Institute of Virology (WIV) obtained the bat anal swab sample from which the viral

genome sequence most closely related to SARS-CoV-2 was isolated. This putative virus is

called RaTG13. To the best of our knowledge, the research described in Chapter 3 has not

been published in the scientific literature. Therefore, Chapter 3, titled “Initial investigation of the

causes of unexplained severe pneumonia incidents related to an abandoned mining cave found

in Mojiang Hani Autonomous County, Yunnan Province,” was translated in its entirety.

Thus, in this document, the following parts of the thesis are translated into English: the

Introduction; Chapter 1 sections 1.1.2.2 and 1.2.6; Chapter 2 section 2.2.2; All of Chapter 3; All

of Chapter 5, including the text of Table 5.1; Part of Chapter 6 (pages 109-112); the Full Text

Summary; and the Acknowledgements. In the English translation of the Table of Contents,

these translated sections are marked with an asterisk (*). Untranslated, and thus omitted, pages

and sections are noted in square brackets within the body of the translation.

Entire sections (e.g. section 1.1.2.2) of the PhD (but not necessarily entire pages) were

translated. The original figures are included, but only for the translated sections. As in the

original PhD, figures are followed by their figure legends; however, as is also true of the original,

figure legends are not always on the same page as the figure they describe.

For the pages where Canping Huang provided an English translation in the original PhD,

his original English text has been preserved [i.e. the English title page (unnumbered) and the

English abstract (p.3)]. For this translation, the Chinese originals for the following pages (some

unnumbered) have been preserved: the front page image of the original thesis; the title page;

the declaration page signed by Canping Huang and Gao Fu; the Table of Contents (pages i and

ii); the Chinese Abstract (p.1); the Abbreviation Index (p.7); and the Publications page (p.139).

Their order in the translation is the same as in the original. This document also includes a

translation of the signed declaration page and the Table of Contents. For these, the English

translation follows directly after the Chinese. Otherwise, throughout this translation, page

numbers correlate with those in the original Chinese PhD thesis.

Blank pages in the original Chinese PhD thesis:

Pages 84, 86, 98, and 108 are blank in the original PhD.

Errors noted in the text of the original:

The original Chinese PhD Thesis is missing page 6. Also, the original Chinese Table of

Contents does not list the subsections for Part 1, Original Research, sections 1.1.1 and 1.1.2,

A partial translation into English of the PhD thesis: “Novel Virus Discovery in Bat and the Exploration of

Receptor of Bat Coronavirus HKU9” by Canping Huang, Chinese Center for Disease Control and Prevention,

2016. Translation completed for Independent Science News in March 2021.


that are present in the body of the PhD thesis (e.g. missing from the Table of Contents is 1.1.2.2

Sample Collection, p.18). Finally, in the discussion of Chapter 3 (section 3.3, page 82), the

thesis states the outbreak of severe pneumonia cases at the Mojiang mine took place in

November 2012. The correct date of the outbreak was April 2012, as stated on p. 75 of the

thesis. Any other errors are marked with square brackets [ ] and an asterisk * and explained in a

footnote.

A note on the reproduction of the figures: In order to reduce the size of the PDF file of the

translation, a loss of figure quality was incurred. If better quality is needed, readers are referred

to the original Chinese PhD thesis. However, Figure 1.4 (page 39) was illegible in the original.

Citation for the original Chinese PhD thesis:

Huang C. Novel virus discovery in bat and the exploration of receptor of bat coronavirus HKU9

(PhD Thesis). National Institute for Viral Disease Control and Prevention, Beijing: China (2016).

The original PhD thesis is available online at:

http://eng.oversea.cnki.net/kcms/detail/detail.aspx?dbcode=CDFD&QueryID=11&CurRec=1&db

name=CDFDLAST2018&filename=1017118517.nh&UID=WEEvREcwSlJHSldTTEYzWEpEZkt

mRXB3Sm9JeHRKZExVOG5ySkJjK0xHMD0%3d%249A4hF_YAuvQ5obgVAqNKPCYcEjKens

W4IQMovwHtwkF4VYPoHbKxJw!!&autoLogin=0

Acknowledgements

We are grateful to Twitter user @TheSeeker268 for first finding the original Chinese-language

2016 PhD thesis. We thank Monali Rahalkar for initially providing a PDF of the original Chinese

PhD thesis. Independent Science News subsequently downloaded its own copy using the link

provided in the citation above. We are also grateful to Francisco de Asis de Ribera Martin for his

earlier partial English translation of the Canping Huang thesis and to Rossana Segreto for

making it available on ResearchGate. We also thank Alina Chan for advice on the translated

text. We were originally alerted to the PhD thesis by its mention in the preprints of the following

two publications:

Rahalkar, M. C., & Bahulikar, R. A. (2020). Lethal pneumonia cases in Mojiang miners (2012)

and the mineshaft could provide important clues to the origin of SARS-CoV-2. Frontiers in

public health, 8, 638.

Segreto, R., & Deigin, Y. (2021). The genetic structure of SARS-CoV-2 does not rule out a

laboratory origin: SARS-COV-2 chimeric structure and furin cleavage site might be the result of

genetic manipulation. BioEssays, 43(3), 2000240.

A partial translation into English of the PhD thesis: “Novel Virus Discovery in Bat and the Exploration of

Receptor of Bat Coronavirus HKU9” by Canping Huang, Chinese Center for Disease Control and Prevention,

2016. Translation completed for Independent Science News in March 2021.


A partial translation into English of the PhD thesis: “Novel Virus Discovery in Bat and the Exploration of

Receptor of Bat Coronavirus HKU9” by Canping Huang, Chinese Center for Disease Control and Prevention,

2016. Translation completed for Independent Science News in March 2021.


A partial translation into English of the PhD thesis: “Novel Virus Discovery in Bat and the Exploration of

Receptor of Bat Coronavirus HKU9” by Canping Huang, Chinese Center for Disease Control and Prevention,

2016. Translation completed for Independent Science News in March 2021.


A partial translation into English of the PhD thesis: “Novel Virus Discovery in Bat and the Exploration of

Receptor of Bat Coronavirus HKU9” by Canping Huang, Chinese Center for Disease Control and Prevention,

2016. Translation completed for Independent Science News in March 2021.


Declaration of Dissertation Originality

I declare that the thesis/dissertation herein is the result of my own research, designed and

carried out by myself under the guidance of my supervisor. Unless explicitly acknowledged

in the text, this thesis contains no unpublished or previously published writing, research or

data produced by someone else.

If any part of this dissertation is untrue, I am aware that I am responsible for all the legal

consequences.

The author of this thesis: Huang Canping

10/20/2016

Authorization for Use of the Dissertation

I fully understand the regulations of the Chinese Center of Disease and Prevention on the

collection, preservation and use of this dissertation. I agree to allow the Center or other

related government offices to preserve this dissertation and I will submit a written and

electronic version of this thesis. I also permit the sharing of this dissertation with

acknowledgement of the intellectual property rights of the author and the Chinese Center

of Disease and Prevention. Citations of this research must mention the first author and the

Chinese Center of Disease and Prevention.

(After the period of confidentiality, the dissertation will be used according to the

orders mentioned above.)

Author Signature: Huang Canping Date: 2017/05/24

Instructor Signature: Gao Fu Date: 2017/05/24

A partial translation into English of the PhD thesis: “Novel Virus Discovery in Bat and the Exploration of

Receptor of Bat Coronavirus HKU9” by Canping Huang, Chinese Center for Disease Control and Prevention,

2016. Translation completed for Independent Science News in March 2021.


A partial translation into English of the PhD thesis: “Novel Virus Discovery in Bat and the Exploration of

Receptor of Bat Coronavirus HKU9” by Canping Huang, Chinese Center for Disease Control and Prevention,

2016. Translation completed for Independent Science News in March 2021.


A partial translation into English of the PhD thesis: “Novel Virus Discovery in Bat and the Exploration of

Receptor of Bat Coronavirus HKU9” by Canping Huang, Chinese Center for Disease Control and Prevention,

2016. Translation completed for Independent Science News in March 2021.


Table of Contents:

Chinese Abstract…………………………………………………………………………………..…..................................... 1

Abstract……………………………………………………………………………………………………..………………...........3

Abbreviation Index…………………………………………………………………………..…………………………..........7

Introduction………………………………………………………………………………………………..……………............ 9*

Part One: Original Research………...……………………………………………...................................................11

Chapter 1: Isolation of Rousettus Bat Coronavirus GCCDC1 and Research on

the Origin and Function of its Unique p10 Gene………………………....................................................13

1.1 Materials and Methods…..………………………………………………………………….....................................15

1.1.1 Research Materials…………………………………………………………………………..………….15

1.1.2 Research Methods.………………………………………………………………………......................18 (1.1.2.2*)

1.2 Results...……………………………………………………………..………………………………………………….......29

1.2.1 Bat Sampling…...……………………………………………………….,…………………………..........29

1.2.2 Cell Culture and Virus Isolation.…………………………………….…………………….…........29

1.2.3 Virus Naming and Whole Genome Sequencing…………...………..….……......................29

1.2.4 Whole Genome Analysis……………………….………..………………….…….............................30

1.2.5 Phylogenetic Analysis……………………….………..…………………….…..................................36

1.2.6 Evidence of Genetic Recombination of the Reovirus p10 Gene into the

Ro-BatCoVGCCDC1 Genome…...……….………..……………………….........................................39*

1.2.7 Subgenomic Structures of Ro-BatCoV GCCDC1..…..……………….……………….……….42

1.2.8 The P10 Gene is a Functional Gene…………………….………..………………………….........44

1.3 Discussion……………………….………..…………………………………………….………………………………….48

Chapter 2: Putative Receptor Binding Domain of Bat Coronavirus HKU9 Spike

Protein: Evolution of the Betacoronavirus Receptor Binding Motif….………………….........53

2.1 Materials and Methods…………………………………………………………………………...............................55

2.1.1 Research Materials…..……………………….………..………………………..................................,55

2.1.2 Main Research Instruments……………………….………..………………………......................,56

2.1.3 Research Methods…………………….………..……………………….............................................,57

2.2 Results ……………………….………..………………………........………………………………………………...........,61

2.2.1 Expression and Purification of HKU9-RBD…………………………………….....................,.61

2.2.2 Characteristics of HKU9 Spike Protein Structure and HKU9-RBD

Sequence………………….……………………………………………………………………......................62*

2.2.3 Binding of HKU9-RBD to the SARS-CoV and MERS-CoV Receptors..........................63

2.2.4 Growth Conditions for HKU9-RBD Crystallization…………………………………...........66

2.2.5 Testing for Heavy Atom Binding……………………….……..…..…………….…………...........66

2.2.6 Crystal Structure of HKU9-RBD……………………….………..………………...........................66

2.2.7 Structural Conservation of the RBD Core Subdomain in Beta-

Coronaviruses……………………………………………………………………………………………….68

2.2.8 Homologous Interaction Mode Anchoring the External Subdomain to the Core

Subdomain..……………………….………..………………………..........................................................69

2.3 Discussion…………………………………………………………………………………………………………………..72

Chapter Three: Initial investigation of the causes of unexplained severe pneumonia

incidents related to an abandoned mining cave found in Mojiang Hani Autonomous

County, Yunnan Province ………………………………………………………………………………………......75*

3.1 Materials and Methods.……………………….………..………………………....................................................76*

3.1.1 Reagents and Materials.……………………….………..………………….....................................76*

3.1.2 Sample Collection…………………..……….………..…………………............................................76*

3.1.3 Sample Handling…………………………….………..…………………............................................76*

3.1.4 Nucleic Acid Extraction………….………….………..………………….........................................76*

i

A partial translation into English of the PhD thesis: “Novel Virus Discovery in Bat and the Exploration of

Receptor of Bat Coronavirus HKU9” by Canping Huang, Chinese Center for Disease Control and Prevention,

2016. Translation completed for Independent Science News in March 2021.


3.1.5 Primer Design………………….………..…………………............................................................................................77*

3.1.6 Analysis of Samples………………….………..…………………...............................................................78*

3.1.7 Sequence Homology and Phylogenetic Analysis……..………………….....................................78*

3.2 Results………………………………………………….………..…………………..................................................................78*

3.2.1 Sample Analysis………………….………..………..……………….............................................................78*

3.2.2 Sequence Homology and Phylogenetic Analysis……..………………….....................................80*

3.3 Discussion…………………………………………………..…..…………………..................................................................82*

Part Two: Literature Review……..………………………………………………….........................................................85

Chapter Four: Emerging Animal-Derived Viruses and their Pathogenicity to

Humans……..…………..…………............................................................................................................................................87

Section 1: Definition and Classification of Emerging Infectious Diseases……..……………….....................87

Section 2: Emerging Infectious Diseases in the past 30 Years……..…………………........................................87

Section 3: Impact of Environmental Health on the Frequency of New Infectious Outbreaks………...90

Section 4: The Role of Wildlife in Cross-Species Transmission of Emerging Infectious

Diseases……………...................................................................................................................................................................94

Chapter Five: Bats and Emerging Infectious Disease….…...…….………………...........................................99*

Section 1: Cross-Species transmission of Flu and Coronavirus………………………………………99*

Section 2: The Biology and Ecology of Bats.………………….…............................................................100*

Section 3: Important Pathogens Found in Bats…………...…………………........................................104*

Section 4: The Role of Bats in Disease Eradication Programs ……………………….....................106*

Chapter Six: Technology Used to Identify New Viruses.……………………..…………..............................109*

Section 1: Traditional Methods and Technology for Virus Discovery….………………………..109*

Section 2: Molecular Biology and New Virus Discovery Technology….…………......................111*

Degenerate Primer PCR Detection Technology …………………….............................112*

Subtractive Hybridization………………………………...……………………………………...115

DNase Treatment Sequence-Independent Single-Primer Amplification

Technolology (DNase-SISPA)…………………………………………………………………...116

Virus Discovery Based on cDNA Amplified Fragment-Length Polymorphism

(VIDISCA)……………………………………………………………………………………………….117

Section 3: Metagenomics and Next Generation Sequencing Technology……………………..119

References……………………………………………………………….……………………………………..……………………..121

Full Text Summary……………………………………………………………….……………………………………….....…….137*

Publications……………………………………………………………….………………………………………………………….139

Acknowledgements……………………………………………………………….…………....................................................140*

* Sections translated into English

ii

A partial translation into English of the PhD thesis: “Novel Virus Discovery in Bat and the Exploration of

Receptor of Bat Coronavirus HKU9” by Canping Huang, Chinese Center for Disease Control and Prevention,

2016. Translation completed for Independent Science News in March 2021.


A partial translation into English of the PhD thesis: “Novel Virus Discovery in Bat and the Exploration of

Receptor of Bat Coronavirus HKU9” by Canping Huang, Chinese Center for Disease Control and Prevention,

2016. Translation completed for Independent Science News in March 2021.


A partial translation into English of the PhD thesis: “Novel Virus Discovery in Bat and the Exploration of

Receptor of Bat Coronavirus HKU9” by Canping Huang, Chinese Center for Disease Control and Prevention,

2016. Translation completed for Independent Science News in March 2021.


A partial translation into English of the PhD thesis: “Novel Virus Discovery in Bat and the Exploration of

Receptor of Bat Coronavirus HKU9” by Canping Huang, Chinese Center for Disease Control and Prevention,

2016. Translation completed for Independent Science News in March 2021.


A partial translation into English of the PhD thesis: “Novel Virus Discovery in Bat and the Exploration of

Receptor of Bat Coronavirus HKU9” by Canping Huang, Chinese Center for Disease Control and Prevention,

2016. Translation completed for Independent Science News in March 2021.


[Translator’s Note: The original PhD thesis does not have a page

numbered 6]

A partial translation into English of the PhD thesis: “Novel Virus Discovery in Bat and the Exploration of

Receptor of Bat Coronavirus HKU9” by Canping Huang, Chinese Center for Disease Control and Prevention,

2016. Translation completed for Independent Science News in March 2021.


A partial translation into English of the PhD thesis: “Novel Virus Discovery in Bat and the Exploration of

Receptor of Bat Coronavirus HKU9” by Canping Huang, Chinese Center for Disease Control and Prevention,

2016. Translation completed for Independent Science News in March 2021.


A partial translation into English of the PhD thesis: “Novel Virus Discovery in Bat and the Exploration of

Receptor of Bat Coronavirus HKU9” by Canping Huang, Chinese Center for Disease Control and Prevention,

2016. Translation completed for Independent Science News in March 2021.


Introduction

Coronaviruses are enveloped viruses with positive sense, single-stranded RNA genomes.

According to the current naming rules of the International Committee on Taxonomy of

Viruses, there are four categories under Coronaviridae: Alpha, Beta, Gamma (these

correspond to the former 1, 2, 3 groups) and Delta. In addition, based on evolutionary

differences, Betacoronaviruses can be further categorized into four groups: A, B, C and D

(see the chart below).

Coronaviruses are present in nature and can infect birds, animals, and human beings. They

can cause symptoms that vary from airway, gastrointestinal, urinary, and liver infections to

neurological disease. They can also be asymptomatic. Since the first animal-related

coronavirus (an infectious airway virus) was isolated from chicken in 1937, researchers

have continued to identify a wide variety of mammalian and human coronaviruses. To date,

at least 90 different types of coronavirus have been identified from wild birds, bats,

humans, and other animal species. Among all the coronaviruses, at least six can infect

humans and cause disease. Among coronaviruses, Betacoronaviruses have the most

outstanding ability to jump species and the strongest human pathogenicity, which has

resulted in multiple global human pandemics. From 2002-2003, one of these human

9

A partial translation into English of the PhD thesis: “Novel Virus Discovery in Bat and the Exploration of

Receptor of Bat Coronavirus HKU9” by Canping Huang, Chinese Center for Disease Control and Prevention,

2016. Translation completed for Independent Science News in March 2021.


pathogens, the Betacoronavirus SARS-CoV, was first identified in China and quickly spread

to other countries. At least 8000 people were infected and 800 of them died. In 2012, a

different pathogenic human Betacoronavirus, MERS-CoV, was identified in Saudi Arabia.

Despite the global effort to control the transmission of the virus, it broke out in multiple

countries in the Middle East, Europe, North America and Asia. Up until July 3, 2014, there

were 941 infection cases and 347 deaths. Meanwhile, in 2006, Yuan Guo Yong and his

colleagues from Hong Kong University identified a human infectious coronavirus

HCoVHKU1 in a patient with airway disease. These unexpected outbreaks caused by

Betacoronaviruses have posed a threat to global public health.

Notably, the current evidence indicates most (not all) of the human coronaviruses come

from bats. In 2013, Ge and fellow researchers were able to isolate a live SARS-like

Coronavirus (bat SL-CoV-WIVI) from the feces of a Rhinolophus bat from Yunnan Province.

The results of this research are the strongest evidence so far indicating that SARS

coronavirus originated in bats. It also proves that there is no need of an intermediate host

for cross-species infection. Multiple pieces of evidence indicate that the dromedary plays a

role as an intermediate host in the transmission of MERS coronavirus and bats were more

likely to be the natural reservoir of MERS coronavirus. Therefore, when there is a new

outbreak of human or other animal infection or cross-species transmitted disease, bats

should be the first group studied. The infectious outbreaks caused by bat coronaviruses

suggest virologists should focus more on the genomic diversity of coronaviruses carried by

bats.

As the figure above shows, among the four groups of Betacoronavirus, viruses in the A, B

and C groups have been shown to cause human infection. It has not yet been determined

whether the bat-related coronavirus HKU9 in group D is infectious to humans. Systematic

evolutionary analysis suggests BatCoV HKU9 and SARS-CoV are related. BatCoV HKU9 is an

important Betacoronavirus. Data from monitoring bats indicate that BatCoV HKU9 is

widespread in bat populations. Therefore, it is necessary to research the possibility of its

cross-species transmission.

10

A partial translation into English of the PhD thesis: “Novel Virus Discovery in Bat and the Exploration of

Receptor of Bat Coronavirus HKU9” by Canping Huang, Chinese Center for Disease Control and Prevention,

2016. Translation completed for Independent Science News in March 2021.


[Translator’s Note: The sections starting with Part One: Original

Research (starting page 11) through section 1.1.2.1 Primer Design

(ending page 18) were not translated into English]

1.1.2.2 Sample collection

With the assistance of local CDC staff and villagers, we caught about 300 bats in a cave in

the Xishuangbanna area of Yunnan. They were identified as brown fruit bats by species.

From 200 of these bats we collected lung, liver, intestinal tissue and anal swabs after

dissection, from some we also collected brain tissues. From another 100 bats we only

collected anal swabs. All tissues were stored in 2 ml cell cryopreservation tubes. Anal

swabs were soaked in 1.5 ml containing various antibiotics (50,000 μg/ml vancomycin,

50,000 μg/ml amikacin, 10,000 units/ml nystatin) and 5% BSA in the virus transport

solution. All specimens were sorted and packed in gauze bags and immediately stored in

liquid nitrogen then shipped back to the laboratory in dry ice. They were stored in a -80°C

freezer until removed for testing. This study passed the review of the Ethics and Animal

Welfare and Use Committee of the Institute for Viral Disease Control and Prevention of the

Chinese Center for Disease Control and Prevention.

[Translator’s Note: The sections starting from 1.1.2.3 RNA Extraction

(starting page 18) through section 1.25 Phylogenetic Analysis (ending on

page 39) were not translated into English]

18

A partial translation into English of the PhD thesis: “Novel Virus Discovery in Bat and the Exploration of

Receptor of Bat Coronavirus HKU9” by Canping Huang, Chinese Center for Disease Control and Prevention,

2016. Translation completed for Independent Science News in March 2021.


1.2.6 Evidence of recombination of the p10 gene into the Ro-BatCov GCCDC1 genome

As mentioned above, coronaviruses have single-stranded positive sense RNA genomes,

while reoviruses are double stranded RNA viruses. It is rare to see orthoreovirus-related

genes inserted into coronavirus genomes in nature. Therefore it is important to address

concerns about the potential for errors introduced via DNA Polymerase or inaccurate

assembly of NGS data. Therefore, we further examined the original NGS data. We extracted

all the reads, including the upstream junction of gene N and p10 and the downstream

junction of gene N and NS7a. Then, we mapped these to the reference genome (Figure 1.4).

39

A partial translation into English of the PhD thesis: “Novel Virus Discovery in Bat and the Exploration of

Receptor of Bat Coronavirus HKU9” by Canping Huang, Chinese Center for Disease Control and Prevention,

2016. Translation completed for Independent Science News in March 2021.


Figure 1.4 The continuity and integrity of the P10 gene flanking sequence. Using the original NGS data,

we extracted all the reads that are related to the p10 sequence and mapped these to the reference genome.

Notably, there are a series of reads that cover the junction of gene N and p10. Similarly, downstream of gene

p10, there is a series of reads that cover the junction of the P10 gene and NS7a. All of these indicate that the

p10 gene is inserted into the genome.

At the same time, we designed two pairs of sequence specific primers to confirm the

continuity and integrity of the upstream and downstream sequences of the p10 gene. The

results of agarose gel electrophoresis showed PCR products of the expected length. We

cloned these full-length PCR products into the pMD18-T vector and sequenced them. These

Sanger sequencing results confirmed the NGS sequence data for the p10 gene. As shown in

the figure of the DNA sequence, the sequence obtained extends from the N gene, through

the intergenic region and the whole p10 gene. Then, it continues through the intergenic

region and TRS [Transcription Regulatory Sequence] and finally into NS7a (see Figure 1.5

a). Meanwhile, as shown in Figure 1.5b, the p10 gene TRS is located in the coding sequence

of the N gene. The core TRS sequence is 5’-ACAAAC-3’. This differs by one nucleotide from

the TRS core sequence of many other coronaviruses. The length of the spacer sequence

between the TRS of the p10 gene and the start codon is 97 amino acids. This distance is

shorter than that between of ORFlab [start codon] and its TRS. However, it is longer than

the other spacer sequences of other genes. Sequence analysis further indicates that the

distance between the downstream end of the N gene and the TRS also differs (Figure 1.6).

40

A partial translation into English of the PhD thesis: “Novel Virus Discovery in Bat and the Exploration of

Receptor of Bat Coronavirus HKU9” by Canping Huang, Chinese Center for Disease Control and Prevention,

2016. Translation completed for Independent Science News in March 2021.


Figure 1.5 The identification of the reorganized p10 gene and its TRS. (A) Confirmation of the “foreign”

p10 gene. The upstream sequence spanning the junction of the N gene and the p10 gene and the downstream

junction of the p10 gene and the Ns7a gene are shown in the figure. The length of the spacer sequence

between the N gene and the p10 gene is denoted by a number. The TRS of the NS7a gene is marked with a red

arrow located within the spacer sequence between the genes. (B) The identification of the p10 gene TRS.

The TRS of the p10 gene within the N gene coding sequence is shown in the figure. The number indicates the

distance between the start codon of the p10 gene and its TRS. Numbers indicate the length of the spacer

sequence between the N gene and its downstream gene. The TRS of the gene downstream from the N gene is

marked with a red arrow.

Figure 1.6 The length of the spacer sequence between the end of the N gene and the TRS of the gene

downstream from it, in different virus genomes. N represents the nucleocapsid gene. TRS means

transcription regulatory sequence. The number between N and TRS is the length of the spacer sequence.

Noticeably, the 3’ end of the N gene in Ro-BatCoV GCCDC1 is truncated (Figure 1.7). The

integration of the “foreign ” p10 gene disrupts the open reading frame of the N gene. As a

result, the 3’ end is missing 8 amino acids and two amino acids were deleted in the

neighboring area.

41

A partial translation into English of the PhD thesis: “Novel Virus Discovery in Bat and the Exploration of

Receptor of Bat Coronavirus HKU9” by Canping Huang, Chinese Center for Disease Control and Prevention,

2016. Translation completed for Independent Science News in March 2021.


Figure 1.7 Comparison of the nucleotides and amino acid sequences at the terminus of gene N. As the

result shows, the 3’ end of the N gene in Ro-BatCoV GCCDC1 is truncated.

[Translator’s Note: The sections starting from 1.2.7 Subgenomic

Structures of Ro-BatCoV GCCDC1 (starting p. 42) through section 2.2.1

Expression and Purification of of HKU9-RBD (ending p. 62) were not

translated into English]

42

A partial translation into English of the PhD thesis: “Novel Virus Discovery in Bat and the Exploration of

Receptor of Bat Coronavirus HKU9” by Canping Huang, Chinese Center for Disease Control and Prevention,

2016. Translation completed for Independent Science News in March 2021.


2.2.2 The Characteristics of the Structure of HKU9 Spike Protein and HKU9-RBD

Sequence

We used bioinformatics to analyze the sequence of the S protein of BatCoV HKU9. The

length of the S Protein is 1274 amino acids, and it has typical coronavirus S protein

characteristics (for example, the characteristic heptad repeats 1 and 2 of the S2 subunit),

though it is predicted a furin-like protease cleavage site cuts the S protein into S1/S2

subunits. Our comparisons of full-length S protein found limited amino acid sequence

identity between the S protein of BatCoV HKU9 and other betacoronaviruses (the identity

with MERS-CoV S, HKU4 S and SARS-CoV S is 27.9%, 28.0% and 30.4% respectively).

However, as shown in Figure 2.3, we can make a prediction about the location of the RBD

based on the signature L(+)-Cysteine residue within the core subdomain. According to the

RBD structure [37, 79, 80, 95] we obtained, these residues can form 3 conservative disulfide

bonds to stabilize the folding of the core. The HKU9-RBD was located in residues spanning

355-521 of the S protein. The length of the core subdomain of HKU9-RBD is comparable to

that of other RBD sequences.

62

A partial translation into English of the PhD thesis: “Novel Virus Discovery in Bat and the Exploration of

Receptor of Bat Coronavirus HKU9” by Canping Huang, Chinese Center for Disease Control and Prevention,

2016. Translation completed for Independent Science News in March 2021.


However, the external subdomain is greatly shortened.

Figure 2.3 The sequence characteristics of HKU9-RBD. (A) Diagram of BatCoV HKU9 S Protein

Structure. The confirmation of the elements in the domain structure was done through either sequence

comparison or bioinformatics. The SP [signal peptide], TM [transmembrane domain] and the heptad repeats

HR 1 and 2 were predicted with the SignalP 4.0 server, TMHMM server, and Learncoil-VMF program

respectively. Sequence comparison between the S protein NTD [N-terminal domain] in murine hepatitis virus

and MERS-RBD was used to predict the NTD and RBD. The S1/S2 site potentially cleaved by furin proteases

could not be determined, so was marked as a question mark. The sequence comparison (B, C) between HKU9-

RBD, SARS-RBD, MERS-RBD and HKU4-RBD was based on structure. The arrows and spiral lines represent

strands and helices respectively. The secondary structures are labeled as illustrated in Figure [2.3]*. The

conserved cysteines that form the three disulfide bonds are marked with numbers 1-3. The core subdomain is

conserved between the four RBDs, yet the external subdomain is irrelevant to the structure. Therefore, we

present the sequences separately. Black square boxes identify the two elements that anchor the external

subdomain to the core subdomain. (B) Sequence of the core subdomain (C) Sequence of the exterior

subdomain.

* This should say “The secondary structures are labeled as illustrated in Figure 2.6”, the error

is in the original PhD.

[Translator’s Note: the sections starting from 2.2.3 Binding of HKU9-RBD

to the SARS-CoV and MERS-CoV Receptors (starting p. 63) through section

2.3 Discussion (ending p. 74) were not translated into English]

A partial translation into English of the PhD thesis: “Novel Virus Discovery in Bat and the Exploration of

Receptor of Bat Coronavirus HKU9” by Canping Huang, Chinese Center for Disease Control and Prevention,

2016. Translation completed for Independent Science News in March 2021.

63


Chapter 3. Initial Investigation of the Causes of Unexplained Severe

Pneumonia Incidents Related to an Abandoned Mining Cave Found in

Mojiang Hani Autonomous County, Yunnan Province

Severe pneumonia is a disease caused by lung infection. Sometimes it can be fatal. Usually,

the disease develops rapidly, and the rate of death is 30%-50%.

The causes of the disease are complicated, most often originating from infection by certain

strains of bacteria, fungus, virus and so forth. Among bacteria these include Streptococcus

pnuemoniae, Legionella pneumophila, Mycoplasma pneumonia, Klebsiella pneumoniae,

Pseudomonas aeruginosa, Haemophilus parainfluenzae etc. Fungi include Histoplasma

capsulatum and others; Viruses that cause severe pneumonia include severe acute

respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome

coronavirus (MERS-CoV), human coronavirus NL63 (HCoV-NL63), human coronavirus

HKU1 (HCoV-HKU1), influenza viruses, adenoviruses, respiratory syncytial virus (RSV),

human bocavirus (HBoV), human metapneumovirus (hMPV) and others. The

orthoreoviruses that are carried by bats, such as Melaka virus [62] and Kampar virus [63] , can

also cause severe pneumonia in humans. These also deserve attention. In addition, in some

cases, immunodeficiency can lead to lung co-infection and severe pneumonia. These cases

result from infection by organisms that are usually of low pathogenicity or non-pathogenic.

Testing for bacteria or fungus usually can be done either by cloning conserved 16S RNA

and 18S RNA genes or through the use of specific tests. Either is helpful in determining the

initial diagnosis of the cause of the disease. However, there are many kinds of virus that can

cause severe pneumonia. Except with other viruses in the same family or genus, viruses do

not share common conserved sequences. Therefore, each virus must be tested for

individually. Because of clinical testing limitations, it can be difficult to quickly confirm the

causes of disease.

In April 2012, there was an outbreak with several cases of severe pneumonia in Mojiang

Hani Autonomous County, where a group of mining workers was cleaning an abandoned

mining cave. Six people were infected and three of those died. The cause of the outbreak

still remains unclear. In October 2014, we caught 87 bats and a large rat in this abandoned

mining cave. We collected tissue samples of multiple organs and tested for possible

pathogens.

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Receptor of Bat Coronavirus HKU9” by Canping Huang, Chinese Center for Disease Control and Prevention,

2016. Translation completed for Independent Science News in March 2021.


3.1 Materials and Methods

3.1.1 Reagents and materials

The reagents, materials and equipment are the same as in Chapter 1.

3.1.2 Sample Collection

With the assistance of the staff from Yunnan Centers for Disease Control and Prevention

and the local Center for Disease Control, we caught 87 bats in an abandoned mining cave at

Tongguan Town, Mojiang Hani Autonomous County (N 23°10'36 E 101°21'28"). Of all the

bats, 84 were Rhinolophus sinicus and 3 were Miniopterus fuliginosus. At night, we set up a

rat trap in the cave. The following day we caught a wild rat. After we euthanized all the bats,

we collected tissue samples from the intestine, lung, liver, and spleen. Because of the size of

the bats, we weren’t able to take anal swabs. We followed the same procedures with the

wild rat. All tissue samples were kept separately in 2 ml Cryogenic Tubes. These were kept

in individual gauze bags and stored in liquid nitrogen immediately. They were sent to the

laboratory of the local Center for Disease Control to be stored until used for testing. This

study passed the review of the Ethics and Animal Welfare and Use Committee of the

Institute for Viral Disease Control and Prevention of the Chinese Center for Disease Control

and Prevention.

3.1.3 Sample Handling

The samples were taken out from the liquid Nitrogen in batches and were lined up in

numerical order. After defrosting, we removed an appropriate amount of tissue and ground

it in a sterilized glass hand mill. Grinding was done on ice. A small amount of antibiotic

(50,000 µg/mL vancomycin, 50,000 µg/mL amikacin, 10,000 units/ mL nystatin) was

added to the culture medium. After grinding, culture medium was added up to 1 ml and the

sample was transferred to a centrifuge tube. After centrifugation at 4° C at high speed,

leaving behind the pellet, the supernatant was carefully transferred to a new centrifuge

tube. 100 ul of supernatant was taken for nucleic acid extraction. The remainder was put

back into the -80° C freezer for storage.

3.1.4 Nucleic Acid Extraction

Nucleic acid extraction was done using the Qiagen Nucleic Acid test kit. For the full

procedure, see chapter two. The extracted nucleic acid was immediately used for reverse

transcription testing or kept in a -80° C freezer for storage.

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Receptor of Bat Coronavirus HKU9” by Canping Huang, Chinese Center for Disease Control and Prevention,

2016. Translation completed for Independent Science News in March 2021.


3.1.5 Primer design

We downloaded all available sequences of Astroviridae, Bocavirus, Coronaviridae,

Filoviridae, Influenza virus A, Henipavirus, Morbillivirus, and Rubullavirus from GenBank

and developed and designed degenerate primers based on the most conserved parts of the

amino acid sequences. All available Orthoreovirus sequences were also downloaded and

primers were designed based on the most conserved region of the nucleic acid sequence,

after sequence alignment. The methods used to design degenerate primers from the

conserved regions of amino acid and nucleic acid sequences are found in Chapter 6. The

primer sequences are as follows:

Table 3.1 Pan-viral degenerate primer screening

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Receptor of Bat Coronavirus HKU9” by Canping Huang, Chinese Center for Disease Control and Prevention,

2016. Translation completed for Independent Science News in March 2021.


Explanation: All N’s were replaced with I (Hypoxanthine) when the primers were synthesized. This reduces

the degeneracy rate for the primer and can enhance the amplification and detection rate.

3.1.6 Sample Examination

PCR amplification was conducted as soon as cDNA was obtained by reverse transcription of

the extracted nucleic acid. Given the large number of viruses to be tested for, and the

limited number of [PCR] machines in the lab, optimization reagent was added to allow the

PCR reaction to adapt to a broader range of annealing temperatures. This was usually 5 %

DMSO. For the first round of PCR, we used five cycles, with an annealing temperature of

44°C, followed by 30 cycles, with an annealing temperature of 54°C. In the second round of

PCR, we used 5 cycles with an annealing temperature of 48°C, followed by 30 cycles with an

annealing temperature of 58°C. If the expected bands were obtained, we sent the product

to a company for sequence analysis. If the expected band did not amplify in the first round,

5 μl of the product of the first round of PCR was used as the template for the second round

of PCR amplification. If the expected band was produced in the second round, then it was

sent to the company for sequence testing.

All positive samples were retested individually to avoid false positives.

3.1.7 Sequence Homology and Phylogenetic Analysis

Sequences were compared using BLASTn to ensure the [amplified PCR] sequences were the

same as the targeted virus. Then, virus sequences were downloaded from GenBank to

conduct homology analysis. Finally, phylogenetic analysis was conducted. Sequence

comparisons were carried out using ClustalW. MEGA7 software was used for the

construction of phylogenetic trees. The methodology used was neighbor-joining and the

bootstrap was 1000.

3.2 Results

3.2.1 Sample Examination

The results of PCR and sequence examination indicate two samples were coronavirus

positive. One was a Rhinolophus sinicus bowel sample (109-2).

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2016. Translation completed for Independent Science News in March 2021.


The other was from a Miniopterus fuliginosus spleen (101-4x). (Figure 3.1)

Figure 3.1 Testing for Coronavirus and Agarose Gel Electrophoresis Analysis. The numbers above the

images are the order number used during testing. 109-2 and 101-4x are the actual sample numbers. As

indicated by the red arrow, the positive bands are the expected size. The DNA molecular weight standards are:

2000 bp, 1000 bp, 750 bp, 500 bp, 250 bp, and 100 bp.

Two samples appear to be bocavirus positive: One liver and one lung sample from

Rhinolophus sinicus (B13-3). The other was a spleen sample from Rhinolophus sinicus

(B122-4). (Figure 3.2)

Figure 3.2 Testing for Bocavirus and Agarose Gel Electrophoresis. The numbers above the images are the

order number used during testing. B13-3 and 122-4 are the sample numbers. As indicated by the red arrow,

the positive bands are the expected size. The DNA molecular weight standards are: 2000 bp, 1000 bp, 750 bp,

500 bp, 250 bp, and 100 bp.

[11]* samples appear to be astrovirus positive: five bowel samples (B110-2, B135-2, B12-2,

B124-2 and B129-2); three liver and lung samples (B12-3, B124-3 and B43-3) and one

spleen sample (B124-4) from Rhinolophus sinicus (Figure 3.3).

* This should say “9 samples appear to be astrovirus positive”, the error is in the original PhD.

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Receptor of Bat Coronavirus HKU9” by Canping Huang, Chinese Center for Disease Control and Prevention,

2016. Translation completed for Independent Science News in March 2021.


Figure 3.3 Testing for Astrovirus and Agarose Gel Electrophoresis. The numbers above the figures are

the order number used during testing. B110-2, B135-2, B12-2, B124-2, B129-2, B12-3, B124-3, B43-4 and

B124-4 are the sample numbers. As indicated by the red arrow, the positive bands are the expected size. The

DNA molecular weight standards are: 2000 bp, 1000 bp, 750 bp, 500 bp, 250 bp, and 100 bp.

3.2.2 Sequence Homology and Phylogenetic Analysis

Figure 3.4 Coronavirus Phylogenetic Analysis. Red circles indicate coronaviruses detected in this research.

Blue diamonds indicate evolutionarily related coronaviruses. Phylogenetic trees were constructed with

MEGA7 software using the neighbor-joining method and a bootstrap value of 1000.

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Receptor of Bat Coronavirus HKU9” by Canping Huang, Chinese Center for Disease Control and Prevention,

2016. Translation completed for Independent Science News in March 2021.


The results of sequence comparison and homology analysis indicate 101-4x and bat

coronavirus MsBtBoV/4039(KU 343194) and bat coronavirus HKU7 strain WCF88

(DQ666339) share high homology, 94.9% and 92.6% respectively. Similarly, 109-2 and

BtMf-AlphaCoV/FJ2012 (KJ473799) and Miniopterus bat coronavirus BtCoV/3728-2

(KP876522) and bat coronavirus HKU8 (NC_010438) share high homology, 92.6%, 92.5%

and 91.2 % respectively. Phylogenetic analysis further indicates that 101-4x and 109-2x

are related to these coronaviruses (Figure 3.4).

Results of sequence homology analysis indicate B13-3 and B122-4 are 100% identical and

the nearest homology to [viruses in] other bocavirus hosts is 80% (Porcine bocavirus

isolate swBoV CH437: KF360033). This demonstrates B13-3 and B122-4 may come from

the same virus isolate and could possibly be a new type of bocavirus. As the validated

sequence obtained through testing and amplification was short (130 bp), the bootstrap

value between the nodes of the phylogenetic tree was not ideal (Figure 3.5). Thus longer

sequences for these two virus isolates are needed to carry out further phylogenetic analysis.

Figure 3.5 Bocavirus phylogenetic analysis. Red circles represent bocaviruses detected in this study. Blue

diamonds represent evolutionarily related bat bocaviruses. Phylogenetic trees were constructed with MEGA7

software using the neighbor-joining method and a bootstrap value of 1000.

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2016. Translation completed for Independent Science News in March 2021.


Similarly, sequence homology results indicate B12-2, B12-3, B124-2, B124-3, B124-4,

B129-2 are the same virus strain, while B46-3, B110-2, B135-2 are independent strains.

Sequence comparisons with astroviruses from other hosts indicate these are potentially

novel astroviruses. Phylogenetic analysis further supports this conclusion. However, the

sequences amplified for this study were short (~280 bp) and the bootstrap value between

nodes in the phylogenetic tree is low. Therefore, future research should amplify additional

sequences from these strains, to provide sufficient sequence information for further

phylogenetic analysis.

Figure 3.6 Phylogenetic analysis of astroviruses. Red circles represent astroviruses detected in this

research. Phylogenetic trees were constructed with MEGA7 software using the neighbor-joining method and

a bootstrap value of 1000.

3.3 Discussion

In [November]* 2012, there was an outbreak of severe pneumonia cases in Tongguan Town,

Mojiang Hani Autonomous County, where a group of mine workers were cleaning an

abandoned mining cave. There were 6 people infected and three died. In order to analyze

the pathology and the cause of the disease, the Chinese Yunnan Centers for Disease Control

and Prevention, other medical centers and other research labs collected samples from the

patients, bats, rats and the environment. The blood samples of four cases showed that: four

patients’ throat swabs and whole blood samples were negative for SARS coronavirus,

epidemic hemorrhagic fever, Dengue fever (type 1-4), epidemic encephalitis type B,

flavivirus and alphavirus pathogen nucleic acid tests (Chengdu Military Region for Disease

and Control). The blood samples of four of the cases, and from four other people who had

been to the cave yet had no symptoms, all showed no abnormal results (Guangdong,

* The correct date of the outbreak was April 2012, see Preface to the translation.

A partial translation into English of the PhD thesis: “Novel Virus Discovery in Bat and the Exploration of

Receptor of Bat Coronavirus HKU9” by Canping Huang, Chinese Center for Disease Control and Prevention,

2016. Translation completed for Independent Science News in March 2021.

82


lab of Nan Shan Zhong); The bats in the mine were dissected, and bat feces were tested, and

no abnormality was found (Wuhan Institute of Virology, Chinese Academy of Sciences);

Blood sample examination of four cases showed: four people carried SARS virus IgG

antibody. Among them, the two discharged patients had higher antibody levels, whereas

two hospitalized patients had lower antibody levels (Wuhan Institute of Virology). Half a

year later, the Jin Qi lab went to the same cave to collect samples from bats and wild rats

and tested for pathogens. They obtained a whole genome sequence of a Henipa-like virus

from a wild rat sample [99] . However, a correlation between this virus and the outbreak is

not proven. Therefore, despite the effort and time put into investigation to date, the cause

of the outbreak remains unknown.

In October 2014, we collected 87 bats and 1 wild rat from this cave. After dissection, we

examined various organ tissues. Two of the samples were coronavirus positive, with

similarity to BatCoV HKU7 and BatCoV HKU8. According to current knowledge, these two

viruses are not contagious to humans [65,100] . According to the reviewed literature, their

receptors are not the common SARS-CoV ACE2 or MERS-CoV CD26 receptors, but may be

APN (Aminopeptidase N). Two other samples were bocavirus positive. Bocavirus was first

identified in samples from children with severe pneumonia. Meanwhile, nine samples

tested astrovirus positive. Astrovirus is a common pathogen of the digestive system.

Currently, there is no evidence linking astroviruses to pneumonia or other respiratory

diseases. Therefore, the relationship between the viruses found and the outbreak needs

further investigation.

In our laboratory, rapid pathogen diagnosis is still preliminary and needs further

development. Currently, we have designed 10 universal primers for only 10 families or

genera. We need to design and test more universal primers in the future. Hopefully, they

can identify additional viruses and will be useful during future outbreaks.

The outbreak in the abandoned mining cave in Mojiang is notable. Before the outbreak,

many people went into this cave, yet there were no disease outbreaks. It is a reminder of

the need to conduct year long or long term monitoring of certain specific ecological

environments in order to understand the change and evolution of pathogens in specific

environments or animal species, and the potential for cross-species transmission these

changes may cause.

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Receptor of Bat Coronavirus HKU9” by Canping Huang, Chinese Center for Disease Control and Prevention,

2016. Translation completed for Independent Science News in March 2021.


Part Two: Literature Review

85

[Translator’s Note: Pages 84, 86, and 98 are left blank in the original PhD

thesis. The sections starting from: Chapter 4: Emerging Animal-Derived

Viruses and their Pathogenicity to Humans (page 87) through Chapter

Four, Section 4: The Role of Wildlife in Cross-Species Transmission of

Emerging Infectious Diseases (ending on page 97) were not translated

into English]

A partial translation into English of the PhD thesis: “Novel Virus Discovery in Bat and the Exploration of

Receptor of Bat Coronavirus HKU9” by Canping Huang, Chinese Center for Disease Control and Prevention,

2016. Translation completed for Independent Science News in March 2021.


Chapter Five: Bats and Emerging Infectious Diseases

Emerging infectious diseases are a continuous international occurrence. They pose a

significant threat and result in great costs to public health [146] . Since 1980, at least ninety

kinds of novel pathogen have been identified. This looks likely to increase each year [138,147] .

An outbreak of emerging infectious disease is usually due to a pathogen of one host

crossing the species barrier to infect a novel host. There are two general causes of crossing

over [105, 140, 148] : environmental and man-made. First, the outbreak of vector-borne disease

is closely linked to climate change [115] . Global warming alters the distribution of

arboviruses, which can cause some new areas to become hotspots, for instance, outbreaks

of Dengue fever [116-118] and the migration of West Nile virus [119, 120] . Climate change can

also impact the breeding, mortality, density and numbers of Glires. Studies show that from

1993-1994 and 1998-1999, the outbreak of Hantavirus pulmonary syndrome in the U.S.

South was related to the El Niño-Southern Oscillation (ENSO). Ample rainfall speeds up the

growth of vegetation, which provides sufficient food for Glires and increases group density.

This increases the possibility of transmitting Hantavirus to humans [121,122] . Second,

regarding man-made factors, reclamation, intensive farming, wildlife hunting and eating,

and urbanization cause significant damage to the environment and to animals’ habitats. All

of these can also potentially cause pathogens to cross over the species barrier and can lead

to disease outbreaks. Increased land reclamation and the establishment of breeding farms

damaged the habitats of fruit bats and led to the outbreak of Nipah virus. The virus jumped

from bats to pigs and from pigs to workers at the breeding farm, which eventually led to an

outbreak [123, 124] . Close interaction between humans and wildlife in rainforests also

increases the risk of emerging infectious disease outbreaks [125-127] . Hunters, villagers, mine

workers, lumberjacks and tourists can invade areas that were untouched in the past and

thus increase the chances of human interaction with animals living in those areas.

Outbreaks of Marburg virus and Ebola virus were the result of interactions between

humans and unknown hosts [128] . HIV is possibly related to SIV from monkeys and apes. In

the 1930s, humans acquired the disease from chimpanzees and spread it all over the world

[129] .

Section 1: Cross-Species Transmission of Influenza and Coronavirus

Influenza virus and coronavirus are two typical pathogens known to transmit across

species. Especially, as we enter the 21 st century

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2016. Translation completed for Independent Science News in March 2021.


these two viruses have led to human infections and massive outbreaks domestically and

internationally. It is important to closely study these two viruses. The hosts of these two

viruses are numerous, including wild animals, domestic animals and humans.

Influenza virus is a multi-segment RNA virus and it mutates quickly. Reorganization and

recombination constantly occur when viruses from different species meet. Studies show

that influenza epidemics are closely related to the influenza viruses carried by animals. In

1918, H1N1 virus originated from bird flu in Europe. Eight sections of the virus were all

from bird pathogens [149] . The viruses of the 1957 Asian flu and the Hong Kong flu of 1968

both have parts of their genome originating from waterfowl pathogens [150,151] . The flu

pandemic of 2009 was related to H1N1 virus. The genome of the H1N1 influenza virus is

derived from reassorted pig, poultry and human pathogens. Gene PB2 and PA originated

from North American bird flu. Gene PB1 originated from human H3N2 influenza virus.

Genes HA, NP and NS originated from classic swine flu virus. Gene NA and M originated

from European “bird like” swine flu [152-154] .

Unlike influenza virus, cross-species transmission is not common in coronaviruses.

However, when they do jump from animals to humans, they are highly pathogenic to

humans. Among the coronaviruses known to be infectious to humans, 229E, OC43, NL63,

HKU1, SARS-CoV, human gastrointestinal tract coronavirus 4408 and the recently found

MERS-CoV, several are cross species diseases. 29 countries and regions suffered from the

outbreak of severe acute respiratory disease caused by SARS coronavirus in 2002-2003.

The outbreak lead to 8433 confirmed cases and 916 deaths. According to the initial

investigation, SARS coronavirus jumped from civet to human. However, based on studies of

farmed and wild civet, civets are not the natural hosts of the SARS coronavirus. In the

process of transmission, civets only play a role as an intermediate host, also known as a

virus replication host. According to reports in September 2012, MERS is also caused by a

coronavirus. Research indicates that MERS coronavirus is also present in camels. Camels

very likely play the role of intermediate host in the process of virus transmission. Recent

studies suggest that DPP4 (CD26), which is conserved in mammals, is the receptor for

MERS coronavirus. Another study demonstrated that MERS coronavirus is infectious to

various mammalian cells including human, pig, monkey, bat and so forth. This indicates

that MERS coronavirus can jump from one species to another one.

Section Two: The Biology of Bats and their Ecology

People are now paying attention to the role wildlife plays in the spread of disease and

disease outbreaks.

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2016. Translation completed for Independent Science News in March 2021.


A research review examined 335 types of emerging infectious disease from 1940-2004. It

found that 71.8% of emerging infectious diseases originated from wildlife [138] . Research on

human-infectious animal viruses [135,155] , and confirmed cases [105,140-145] where viruses have

jumped species, indicates shared pathogens between animals and humans are related to

carriers such as hoofed animals, carnivores, Glires, bats, non-human primates, birds,

marsupials and so forth. Among these, the three most likely to transmit diseases to humans

are: bats, Glires and primates.

Bats are Chiroptera. They are the only flying mammals. There are many species of

bat, roughly 1240 around the world. They comprise about 20% of all mammals and are the

second largest group of mammals in the world [146] . The biggest group is Glires. Chiroptera

is divided into two suborders: Megachiroptera and Microchiroptera or frugivorous bats

and insectivorous bats. There are 19 families and 175 genera. Pteropodidae is the only

family under Megachiroptera. There are 1 family, 5 genera and 7 species in China. There

are 18 families within the Microchiroptera and we have 6 families, 24 genera and 100

species in China [156] .

Table 5.1 Bat family, distribution and eating habits

Family Distribution Eating Habits

Myotis scotti (2) South Africa Insets

Kitti's hog-nosed bat (1) Thailand, Myanmar Insects and spiders

Emballonuridae (54) Widely spread in tropical areas Insects and fruits

Furipteridae (2) New world tropical areas Insects

Hipposideridae (9) Old world tropical areas and subtropical areas Insects

Megadermatidae (5) Old world tropical areas Arthropods and small Vertebrates

Miniopterus schreibersi (29) Old world tropical and subtropical areas Insects

Molossidae(113) Widely spread in tropical areas Insects

Acerodon Mystacinidae jubatus (2) 10) New world Zealand tropical areas Insects, Arthropods, nectar, and fruits

Myzopoda(2) Madagascar Insects

Natalidae (12) New world tropical areas Insects

Bulldog bat (2) New world tropical areas Insects and fish

Nycteridae (16) Old world tropical areas Insects, spiders, scorpion, and fish

Phyllostomidae (204) New world tropical areas Animals and plants

Pteropodidae(198) Old world tropical and subtropical areas Fruits, nectar, and pollen

Rhinolophus sinicus (97) Old world tropical area and subtropical areas Insects

Rhinopomatidae (6) Old world tropical areas Insects

Thyroptera (5) New world tropical areas Insects

Vespertilionidae (455) All over the world Insects, Arthropods, fish and birds

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2016. Translation completed for Independent Science News in March 2021.


Bats are one of the most ancient mammal lineages in the world. Bats can be traced back

52.5 million years (Mya, million years ago) in the fossil record [157] . Before the evolution of

most mammals, bats had evolved into many genera [158] . Compared to other mammals, bats

changed little during the process of evolution [159] . Analysis of retroposons found in

mammalian genomes and protein sequence comparisons between bats and other kinds of

animals suggest that bats are most closely related to horses and most likely share a

common ancestor [161] .

After humans, bats are the most widely distributed mammals. Most bat species live in

tropical and subtropical areas. Bats can live in both natural and artificial environments

such as caves, houses, and trees. About half of bat species choose forest, bamboo forest,

shrubland and so forth as roosting sites. Others live in caves, cleavages of rock, mining

wells, graveyards, bridges, and all types of constructions [162] . Because of hunting, breeding,

hibernating, and hiding needs, bats have low roost fidelity. Different species of bat choose

different sites to inhabit in different seasons [163] . Bats are social animals. It is common to

see many bat species living together. Bats live in high densities. For instance, in a cave in

south Texas, during breeding season, there will be around 20 million Tadarida brasiliensis

Mexicana living together. In each square meter, there are about 27,000 bats [164] .

Bats eat all kinds of food, most are insectivorous, carnivorous, piscivorous, sanguinivorous,

or frugivorous. Those that eat fruit are called frugivorous bats. Microbats have a small body

size and specialized body structures. Most microbats eat insects. Those that eat insects are

called insectivorous bats. Bats frequently fly long distances during seasonal migration.

According to one report, Pteropus scapulatus can travel up to 320 kilometers and Pteropus

giganteus can travel up to 2000 kilometers [165] .

One distinctive feature of some bats in temperate climates is the ability to go into torpor

and hibernate. When temperatures are high, they can go into a short daily sleep (daily

torpor), however, in winter, they enter a seasonal hibernation. Their body temperature can

drop to 2-5 O C and the body’s metabolism stays in an energy saving state. In mammals, life

expectancy is associated with the ratio between rate of metabolism and weight. Bats are

exceptional. Compared to other mammals of the same size and weight, the life expectancy

of bats is high. Some temperate zone Microchiropteran bats can live up to 25 years. The

record age of Pteropus scapulatus is 35 years.

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The bat immune system is still not clearly understood. Being one of the earliest mammals

to evolve, the immune reaction of bats, both innate and adaptive, is recognizably different

from those of Glires and primates. Researchers are interested in changes to the bat immune

system during torpor and hibernation. Based on research on other mammals, hibernation

impacts the innate and adaptive immune system. It is assumed that the immune system of

bats is weakened during rest. Immunosuppression is common during hibernation. It is

beneficial for host energy preservation. Meanwhile, as most viruses have difficulty

replicating at low temperatures, the possibility of infection is low. Currently, the study of

bat immunology mostly focuses on orthologous genes that are shared with humans and

small rodents. The role these genes play in the innate immune system is critical. They

include interferon [166-168] , cytokine [169] , Toll like receptor [170] , STAT1 protein [171] and so

forth. Some research also focuses on their structure and function [172] . Research indicates

that the immune response of bats and of other animals that evolved later share some

similarity. For instance, Immunoglobulin G (IgG antibody), IgA and IgM can be purified

from the serum of the great fruit-eating bat Artibeus lituratus [173] . Macrophages, B-

lymphocytes, T- lymphocytes, and cells expressing surface Ig were found in the bone

marrow of Pteropus giganteus. These results suggest the evolution of lymph in bats and

other mammals was similar [174, 175] . Despite the findings from these gene comparison

studies, there are still slight differences between bats and other mammals, thus it is too

early to come to firm conclusions.

Bats have some distinct characteristics from the perspective of biology and ecology. These

make them the ideal host for viruses in the wild: 1. Bats are the oldest mammals. Viruses

that evolve in the body of bats can make use of highly conserved cell receptors to enhance

the transmission of viruses to other mammals. 2. Bats are the most widely distributed

group of mammals except humans. This provides a large “breeding ground” for viruses.

Diverse roosting sites and food sources, and the ability to travel long distances (some bats

can migrate several hundred kilometers to a hibernation site), create many opportunities

for bats to interact with other species at numerous different locations. This increases the

chance of cross-species transmission. 3. Bats live in multi-species groups. These high

densities facilitate the jumping of viruses from one bat species to another. This promotes

both virus longevity and virus attenuation. 4. Bats have a long life expectancy. Some bats

can live up to 25-35 years, which prolongs the potential timeframe of viral transmission.

Some bats are able to hibernate in wintertime and undergo daily torpor in other seasons,

both of which can lower energy consumption, body temperature and rate of metabolism.

Low body temperature and rate of metabolism slow down the growth of viruses inside the

bats.

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The organs, therefore, do not have a strong immune response toward infecting viruses.

Consequently, this slows the speed of virus eradication within a group of bats.

Section Three: Important Pathogens in Bats.

In the 1920s, rabies virus was found in bats from Central and South America.

Researchers started to realize that bats could act as reservoir hosts for certain viruses that

infect humans. Soon afterwards, an increasing number of diseases were discovered to have

originated in bats, including Henipavirus, Nipah virus, filovirus (Ebola and Marburg virus)

and SARS Coronavirus that has attracted much attention. As the technology for virus

detection improved, the number of newly discovered bat-derived viruses has greatly

increased over the last 20 years. Currently, there are at least 173 kinds of viruses isolated

or found in the tissues of bats. They belong to 28 different virus families (Table 5.2) and at

least 61 of them are shared between animals and humans [176, 177] .

Table 5.2 Viruses Isolated from Bats

Virus Family

Amount

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Explanation: 1. Resource: The Database of Bat-associated Viruses (DBatVir); 2. The numbers may not

be exact.

Although current research indicates that water birds are the main natural reservoir hosts

for type A influenza virus, influenza viruses found in bats are starting to get similar

attention [178] . In 1979, a Russian study showed that influenza virus A/H3N2 was found in

the Common noctule from the Republic of Kazakhstan [179] . In 1981, serological testing

indicated that multiple bat species in India were infected by A/H3N2 influenza virus [180] .

These results indicated that bats are easily infected by influenza virus, however, they were

never deemed to be critical reservoir hosts in the past in the eyes of epidemiologists. In

2009, Tong et al. found a new influenza virus, H17N10, in the body of Sturnira lilium of

Guatemala. This new influenza virus has eight gene segments that are greatly different

from the known A type influenza virus [181] . In 2013, Tong et al. went back to Peru and found

a new flu-like virus H18N11 in Artibeus planirostris. Serologic studies with the recombinant

H18 protein indicated that multiple bat species were infected by H18N11 flu-like virus. Flulike

viruses in bats have mutated over time, based on the detailed examination of these two

subtypes of influenza virus [182] . It also demonstrated that bats are like water birds, they

can be potential hosts for influenza viruses in nature. A recent study looked at 100 serum

samples from Eidolon Helvum from Ghana by using recombinant H1-H18 hemagglutinin

protein chips. It showed that 30% of the tested serum was H9 influenza A virus positive.

Some of the sera had a certain degree of cross-reaction with H8 and H12 subtypes. Given

H9 influenza virus is infectious to human, we should pay more attention to the role bats

play in influenza epidemics.

Recent outbreaks of new coronaviruses have made people recognize the importance of bats.

They could be the natural host for coronaviruses that are infectious to humans. Currently, a

massive research effort has proven that human infectious coronaviruses 229E, OC43,

NL63, HKU1, SARS-CoV, enteric coronavirus 4408, and the recently found MERS-CoV

potentially originate from bats. The research shows that the most likely host for SARS

coronavirus is Rhinolophus sinicus [72,76] . SARS-like coronaviruses are diverse and are found

in different species of bats and at different locations. Phylogenetic analysis demonstrates

that these viruses are spread across different evolutionary groups [86, 183,184] .

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In 2013, a study by Ge et al. strongly suggested that Rhinolophus sinicus is the reservoir

host for SARS coronavirus. It also proves there is no need for the existence of a secondary

host in the transmission process [22] . Co-infection by two different types of coronavirus

provides an ecological basis for the production of recombinant viruses and the diversity of

coronaviruses [185] . In 2012, the outbreak of MERS in the Middle East was caused by the

MERS coronavirus. Phylogenetic analysis indicates that MERS coronavirus belongs to the

Betacoronavirus genus and is similar to bat coronavirus HKU4 of Tylonycteris pachypus and

HKU5 of Pipistrellus pipistrellus [27, 186] . Recently, a coronavirus that is highly similar to

MERS virus was found in the feces of South African bats [28] . In addition, research on the

bats of Saudi Arabia found that one of the samples shared 100% homology with a virus

gene segment of a patient [31] . This evidence demonstrates that bats are likely to be one of

the natural hosts for MERS coronavirus. Current research shows that the diversity of bat

coronaviruses is broader than for other animal coronaviruses. Therefore, it is necessary to

systematically detect and analyze the coronaviruses and influenza-like viruses that bats

may carry in our country, and to understand the potential status of bats as the natural

hosts for related viruses.

Section Four: The Role of Bats in Disease Eradication Programs

As the natural hosts for many diseases, bats could serve as potential residual disease pools

in human disease eradication programs. There is a need for close monitoring. In 1980, the

World Health Organization announced a plan to eradicate smallpox around the globe.

Smallpox became the first eradicated disease in the world. The eradication of smallpox was

impactful for the global public health industry. Thanks to this experience, WHO also put in

great effort to eradicate polio and Guinea worm disease. In 2008, the disease eradication

working group under the Carter International Center (within the WHO) announced five

other potential eradicable diseases and nine non-eradicable diseases. These nine diseases

could, however, be prevented by eliminating disease-promoting conditions. Measles,

mumps, rubella, hepatitis B, and rabies are caused by viruses. There are some similarities

shared among polio, measles virus, mumps virus and rubella virus: (1) No animal host and

only humans are infected. (2) A single serotype. (3) Vaccines are effective. Therefore, these

are the eliminable diseases. The ways that hepatitis B and rabies transmit are distinctive

and vaccines are effective. Therefore, they can be eradicated in certain regions and

countries.

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However, in the planning stage for disease eradication and elimination, there are two

conditions that need attention: (1.) Could the related viruses exist in other non-host

animals; (2.) The possibility of cross-species transmission that is infectious to humans in

the post-eradication period. Bats are very likely to be the natural reservoir host for these

viruses and related viruses. In 2012, using second generation sequence analysis, Drexler et

al. found several unknown measles viruses in bats. Among the spleen samples of 22 African

yellow hair fruit bats, 21 tested positive for a high concentration of measles-related viruses

[187] . It is still to be confirmed whether these bat associated measles-related viruses

(Morbilli-related virus) are infectious to humans and other species. Meanwhile, in the same

study, one virus was found in the spleen samples of fruit bats and insectivorous bats.

Examination and analysis of the genome sequence indicates that this virus shares 90 %

similarity with the mumps virus. They are the same type of virus. According to

immunofluorescence results and cross-reaction response analysis of the bats’ serum, this

bat-related mumps virus might belong to the same serum type as the human mumps virus

[187] . As for Hepatitis B, in 2013 He et al. identified a new hepatitis virus in bats from

Myanmar through the study of virus metagenomics. They found particles of the hepatitis

virus in the liver of the bats using a negative staining method. This virus shares 64%

homology with the known human Hepatitis B virus according to genome sequence analysis

[188] . The same year, Drexler et al. did a study on 11 families and 54 species of bats and

collected 3080 samples (from 7 different countries). Three Hepadnaviridae were isolated

from 10 bat samples from Panama. These bat viruses have liver tropism and cross-react

with antiserum to human Hepatitis B virus. According to the results of whole genome

sequencing and phylogenetic analysis, these viruses share a common ancestor with human

Hepatitis B virus [189] . Meanwhile, bats are also the natural hosts for rabies [190] . The

connection between vampire bats and rabies has been confirmed. There are some

occasional cases of rabies disease caused by bat bites in North and South America, Europe,

Australia and Africa, especially in rural areas. Therefore, screening for these viruses in bats

and other suspected animal species is crucial for effective disease eradication.

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2016. Translation completed for Independent Science News in March 2021.


Chapter Six: Technology Used to Identify New Viruses

Last century, traditional virology made significant contributions to the discovery and

identification of pathogens. For example, tissue/cell culture, electronic microscopy,

serology, and immunology are the foundations of these methods. Along with the

development of new technology, molecular biology methods such as PCR, subtractive

hybridization techniques and microarray play important roles in the practice of clinical

virology studies. New metagenomic methods can analyze genomes directly from clinical

samples, which eliminates the need for tissue culture and gene cloning. Also, there is no

need for background knowledge of the pathogen. Therefore, the methods are applicable to

pathogen identification. New next-generation sequencing (NGS) methods greatly increase

the speed and effectiveness of sequence analysis, which advanced the usage of

metagenomics in clinical virus identification. The development of new technology has sped

up the identification of pathogens and plays a critical role in disease control.

Section One: Traditional Methods and Technology for Virus Discovery

In 1907, Anatomist Ross Harrison invented cell culture methods. In 1909, Karl Landsteiner

and Erwin Popper successfully propagated poliovirus using these methods [191] . In the

early stages of virology, there weren’t many effective ways to identify and examine viruses.

Many viruses were detected by methods of cell culture propagation. However, virus

propagation in cell culture still is the gold standard for virus discovery, as it has been for

over a century [192] .

In virus discovery research, cell and tissue culture are the only methods available to get live

virus. Virus culturing is useful in the study of viruses’ morphology and biology, including

growth kinetics, specificity for cells and tissues, receptors, serology characteristics, hosts,

pathogenesis, animal models and so forth. These studies are helpful in the study of disease

tracing and causation. Meanwhile, successful virus propagation impacts diagnosis and

vaccine development [193] .

There are many advantages of virus culturing, yet it is still somewhat limited. The study of

human hepatitis C virus (HCV) is an example [194] . The nucleic acid of HCV was first found in

blood samples in 1898. However, it took more than 10 years to successfully culture the

virus [193] .

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Because the growth conditions of newly discovered viruses are unknown, it takes

continued trial and error to successfully isolate the virus from the samples and culture it.

The challenges of culturing are determined by whether the researchers can successfully

identify an appropriate cell line where the virus can invade and replicate. Usually, the

former requires an adequate receptor or co-receptor and the latter needs adequate host

cytokine production to support transcription, replication, assembly, and release [195] . Also,

in the process of virus culturing, there should not be any limiting factor.

When the organism is infected with a virus, different clinical symptoms are produced in

different organs. These expressed symptoms are critical in determining which kinds of cell

lines to choose for virus isolation [196] . However, in practice, the samples should be

inoculated into as many cell lines as possible. We usually choose at least six kinds of tissues

from different organs and a broad spectrum of hosts [196] . Sometimes even though the

adequate receptor or co-receptor was chosen for virus isolation, there are still other

limiting physical and chemical factors such as the surface of the cell not being ideal for

electrostatic interactions. Live viruses may not be able to adhere to cells in vitro, which

prevents binding to cell surface receptors [196, 197] . This problem can usually be solved by

precautionary measures such as using positively charged liposome and polymer in the

inoculum. Multiple experiments from different labs show that such methods can increase

the infectivity of some viruses, CPE and the size of the virus plaque [196,198,199] .

In virus isolation experiments, we often have to consider the issues of TPCK treatment or

the concentration of the pancreatin added to the culture. Treatment with tosyl

phenylalanyl chloromethyl ketone prevents pancreatin from self-degrading. The treated

pancreatin can activate, hydrolyze and remove glycoproteins on the surface of the viruses,

which greatly increases their infectivity [196] . For some of the RNA viruses, glycoprotein

removal is a prerequisite for cell binding and fusion. For instance, the spike protein on the

surface of a coronavirus can only be infectious when it is cut into S1 and S2 subunits. The

new Porcine delta-coronavirus populating the US and China has caused severe illness

among pigs. When isolating this virus, TPCK-pancreatin treatment of the samples is

frequently a prerequisite [200, 201] . Similarly, when isolating certain strains of influenza virus,

it is usually necessary to add a certain concentration of pancreatin to the culture

medium [202, 203] .

Noticeably, the inducement and activation of the innate immune response prevents the

transcription, replication and/or protein biosynthesis of the virus, which makes culturing

more difficult. To solve this problem, we can rely on specific functions of certain cell lines,

which help curb the innate immune molecules.

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For instance, Vero cells are interferon deficient. An infected Vero cell does not release alpha

and beta interferon. Therefore, it is useful for isolating many viruses [204] . To implement the

isolation of more viruses, we can try to establish additional deficient cells lines.

Given the difficulties in finding adequate culture conditions, 99% of microorganisms

(including bacteria, viruses, fungi and so forth) are hard to breed outside of the body. 3D (3

dimension) cell culture is the latest cell culture technology. It can simulate the morphology

and function of the body tissues where the cells naturally grow and differentiate. Thus, the

cell can live outside the body in the way it is grown within. The 3D cell culture method is

useful in propagating viruses that are difficult to culture. Meanwhile, it can provide

convenience for the study of the interaction mechanism between a virus and a cell and a

virus and a host [205] . Labs, domestically and internationally, successfully propagate human

bocavirus and human coronavirus HKU1 outside of the body by using 3D cell culture.

When cell culture attempts fail, the only way to propagate a virus is animal inoculation. A

common method is brain inoculation of nude mouse. There are several advantages of

animal inoculation: (1) No need for complicated equipment or instruments. (2) Simulates

the natural infection process. (3) Easy to replicate and has a substantial success rate. In an

easy to control and modulate animal model, if the inoculated virus can infect and replicate

within the organism, research on virus infection, species and tissue tropism, and

pathogenicity can easily be carried out [193] .

Section Two: Molecular Biology and New Virus Discovery Technology

With the advent of PCR and recombinant DNA technology, molecular methods for virus

identification have become widely used. These include cDNA cloning, degenerate PCR

assays, gene chips, subtractive hybridization, differential display assays (DD), DNasesequence-independent

single-primer amplification (DNase-SISPA), virus discovery based

on cDNA-amplified fragment length polymorphism (VIDISCA) and so forth. General and

degenerate primer PCR and gene chips are easy and sensitive, however, they rely on known

sequences of nucleic acid and can only identify viruses that are related to previously known

viruses; Subtractive hybridization does not rely on sequence knowledge but the process is

complicated; DNase-SISPA and VIDISCA

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also do not rely on knowing the sequence. Random amplification can be supplemented by

cloning and sequencing. To a certain degree, it is limited by the depth of sequencing, but it

has been proven to be effective in the discovery of some highly distinctive viruses.

Degenerate Primer PCR Detection Technique

In the process of amplifying and identifying protein-coding genes for known homologous

proteins, the technology of single degenerate primer PCR is frequently used. For instance,

when identifying a new protein in a protein family or a gene from a different species with

functional similarity, PCR is used. For novel virus discovery, this technology can also be

used to detect new viral genomes that are evolutionarily related to known viruses.

Therefore, in the identification of a new member of a specific family or genus, degenerate

primer PCR detection technology is very practical.

Degenerate primer PCR detection technology uses mixtures of primers. These primers are

mainly for the most conserved amino acid motif (conserved motif) in the virus and contain

most or all of the nucleotide sequences corresponding to the motif. When designing the

PCR analysis, the first step is to identify all the conserved proteins in the specific virus

family or genus. This step requires specific knowledge and experience of virology and full

understanding of the protein coding gene characteristics of the members of the virus family.

After identifying the most conserved protein, one needs to be able to do a sequence

comparison of all the obtained amino acid sequences and determine all the conserved

blocks of sequence. A conserved pentapeptide in the degenerate PCR primer design is ideal.

Degenerate nucleic acid sequences are back translated from the amino acid sequence,

including all possible sequences of the coding pentapeptide. When designing degenerate

primers, degeneracy (the number of unique sequences in the primer mix) is critical.

Degeneracy depends on the number of codons for each amino acid. There is only one codon

for methionine and tryptophan. However, there are 6 codon sequences for leucine, serine

and arginine. When designing primers, the conserved pentapeptide for Methionine and

Tryptophan should be prioritized over the sequence for leucine, serine and arginine. The

sensitivity of degenerate primer PCR is in reverse ratio with degeneracy. Therefore, to

ensure the sensitivity of the PCR assay, degeneracy should less than 128.

To increase the amplification rate of the PCR reaction, it is necessary to add a nondegenerate

clamp sequence to the 5’end of the primer when designing it. This is helpful for

annealing recognition between the primer and the PCR product. Two different strategies

are used to design of clamp sequences. When the online CODEHOP is used, the amino acid

sequence on the side of target sequence is degenerate [206, 207] ; When we manually design

the degenerate primers, the clamp sequence comes from consensus sequences in the

corresponding position of the virus gene.

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[Translator’s Note: Pages starting with page 113 and continuing through

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Full text summary

When an infectious disease outbreak occurs, public health researchers need to rapidly

identify the cause of the outbreak. Identification is critical in providing information and

technical support for disease prevention and control. Therefore, the rapid identification of

pathogens is of great significance for the field of public health. Pan-viral screening using

degenerate primer PCR is a rapid diagnostic technique. Our lab has designed general

primers for 10 virus families and genera. Using these primers, we found a new type of

coronavirus, one boca virus, four astroviruses and one mutated orthoreovirus from bat

samples. In order to cover a wider range of viruses, for virus detection and new virus

discovery, we plan to design more general primers for virus families and genera.

Recombination is common in members of the coronavirus family, and therefore

coronaviruses are very diverse. However, up to this point, most of the reported

recombination events happened within sequences of related viruses and were homologous.

In this research, we found a new bat coronavirus with a functioning p10 gene in its genome.

Many experiments indicate that this p10 gene is possibly from an ancient non-enveloped

orthoreovirus. This is the first reported case of a fusion-associated small transmembrane

(FAST) protein in an enveloped virus. At the same time, it is also the first reported

recombination between a single positive strand RNA virus and a double-stranded

segmented RNA virus. These findings further reveal mechanisms of virus evolution and

provide important information for understanding the process of heterologous

recombination in coronaviruses.

The replication of the coronavirus genome and the mechanism of RNA synthesis are unique.

The formation of a sub-genome is a distinctive characteristic of coronavirus replication. In

this research, we directly identified the sub-genome in the samples to further determine

whether the coronavirus replicated inside the cells of hosts. Also, to determine whether a

specific gene is transcribed during the lifecycle of the virus. Meanwhile, by manually

constructing sub-genomic sequences, we could test the function of certain genes. This is

our innovative contribution to coronavirus research methodology.

SARS-CoV and MERS-CoV are two representative Betacoronaviruses that can jump the

cross-species barrier and infect humans. In addition, according to a recent study, another

Betacoronavirus BatCoV HUK4 is already adapted to infect human cells. Therefore, it is

urgent to study the potential for cross-species transmission of other prevalent

Betacoronaviruses. BatCoV HKU9 is also a Betacoronavirus and is commonly detected in

bats. In our research, we analyzed and examined the structure and function of the Bat CoV

HKU9 putative structural domain for receptor binding. This structural domain has proven

to be a key factor for receptor recognition and cross-species transmission in other

Betacornaviruses. Through the analysis of the structure of BatCoV

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Receptor of Bat Coronavirus HKU9” by Canping Huang, Chinese Center for Disease Control and Prevention,

2016. Translation completed for Independent Science News in March 2021.


HKU9 RBD, we identified a conserved core subdomain in BatCoV HKU9 RBD and other

Betacoronavirus RBDs. Meanwhile, there is a distinctive exterior substructural domain.

Based on the structure of all the known coronavirus RBDs, we further determined the

amino acid interaction patterns between subdomains are homologous. This distinction

reveals the evolution of polymorphisms found in Betacoronavirus spike proteins and their

use in binding host receptors.

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Articles Published

A partial translation into English of the PhD thesis: “Novel Virus Discovery in Bat and the Exploration of

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2016. Translation completed for Independent Science News in March 2021.


Acknowledgements

Time flies. My years spent in the doctoral degree are about to end. It has been a

long journey, Nanjing, Beijing, domestically and internationally. The feelings are mixed. As I

am about to finish this thesis, I took the chance to reflect on the past. I am filled with

gratitude, especially toward my teachers who have helped me and gave me instructions,

classmates, friends and my supportive family.

First, I would like to thank my instructor, Professor Fu Gao. In the past four years,

Professor Gao inspired and encouraged me. I will never forget. Professor Gao has great

sensitivity to research topics. He thinks forward and works diligently. I admire his

persistence and passion toward science and in-depth knowledge. He is and will always be

my role model.

I want to thank all my teachers in college, particularly Wei Hua Mao of Nanjing

Agriculture University. Mr. Mao did me a big favor in a critical moment. I appreciate my

instructor, Pin Jiang Professor, during years in master school. Thanks to professor Bao

Xiang Cai, professor Zhen Xin Zhang, professor Ming Qiu Zheng, Dr. Virkam Misra, Dr.

Andrew Potter and Dr. Volker for all their patience and assistance over the years.

I am thankful for teachers Yong Qing Liu, Dr. Dao Yu Huang, Dr. Bin Yan, Dr. Yan Yun

Huang, Si Jing Fen, Zhong Qin Wu, Jian Zhong Zhu and my dearest classmate Iran Yousefei

for being there for me when I was abroad.

Thanks Ji Ming Chen, teacher Wei Xin Fan, Rong Wei, Yong Qiang Zhang, Suo Liu,

especially Lili Tian who helped me when things were difficult for me. Thanks to my fellow

classmates at the university. Thank you for your support and encouragement over the past

20 years.

Thanks Qian Song Hwang at Beijing, Qian Bo Hwang, Jing Ping Hwang, classmate

from middle school and high school: Jiu Jia Fei, Ru Tian Li, Xin Chuang Xu, Chen Yi Wang, Jie

Xiang and Li Qing Wang; classmate from college: Zhuo Wang and Zhe Xu; Classmates from

master degree: Xue Mei Zhou, Ting Li, Yan Ming Zhang, Ren Quan Yang, Yao Ling Huang,

Hua Long, Fu Yuan Lu, Feng Hai Zhang, Yong Xia Wang. Friends and colleagues in the past:

Dr. Dan Di Li, Zi Qian Xu, Dr. Yan Lin Hao, Fu Wang, Xue Qing Dang, Dr. Jing Ou Yang, Dr.

Gang Wang, Professor Xiao Bing Wu. I am thankful that we live in the same city. With your

greetings and kind words, I am not alone.

Thanks to my classmates in my doctoral career: Xiao Juan Guo, Wen Fei Zhu, Yan

Gao, Li Na Chen, Chen Yan, Jin Song Li, Yang Liu, Yang Yang, Xing Wang Xie, Song Tao Xu,

Mao Zhong Li (Virology study), Fan Zhang, Yi Fei Ou Yang, Hai Hong Han, Shi Jun Lu

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Xiang Nan Ren, Chun Feng Yun, Hai Tao Shen, Chen Xi Li, Kao Zhao, Hui Liu (Nutrition

study), Sha Liu, Xiao Na Shen, Ci Xiu Li, Yan Wan, Jie Liu, Shao Wei Sang, Shuo Zhang, Ma

Chao Li (Epidemic study), Hui Xin Liu, Lan Hua Li, Yan Tao Jin, Jin Lei Qi, Zhi Yun Gao, Hua

Mian Wei, Xiao Jing Fu, Hai Zhao, Yang Jiao, Wen Rong Yao, Xuan He (AIDS study), Xia Qiu,

Zhe Zhang, Kun Li, Bao Ying Zhang, Yun Yun Wu, Shuo Zhang (Environment, Radiation and

occupational health study), Hong Xin Li (Water supply study), De Lu Yin, Jian Xin Yu

(Center). Especially classmates from Virology study, I am glad that we met and spent three

years together.

Thanks for the friends that “play” retrieval with me for over ten years: Dr. Yun Hai

Fang, Dr. Pei Yuan Zhu, Dr. Yun Fei Cao, Liu Ming Jiang, Dr. Xu Guang Hong, Xong Chen, Ying

Chun Zhou, Gang Li, Dr. Zhi Hong Xin and teacher Jian Wei Lu. The technique of information

retrieval learned at the time is so practical for later on study.

Thanks to the friends I met on the “Yi Wang Qing Sheng” website and Biolover: Lin

Shen Zhang, Xing Zhong Guo, teacher Jian Xiong Meng, teacher Xiang Mao, Dr. You Yu He,

Xin Li, Dr. Zhen Wang, Dr. Yun Feng Li, Dr. Ke Zhang, Zhi Hwang, Dr. Xiao Hua Chen, Dr.

Feng Liu, Dr. Gao Jian Shao, and Qi Hui Liu. Even though I never met some of you in person,

I am grateful to meet a group of friends like you so that we can discuss professional

technology and application of new software in wechat group.

I appreciate the instruction and assistance of teacher Jin Hua Yan. I appreciate teacher Jian

Xun Yan’s effort in collecting crystal structure analysis. Thanks to Qi Hui Wang, Yan Li, Yan

Fang Zhang and teacher Wei Zhang’s support. Thanks to Dr. Liang Wang and professor Di

Liu for their assistance in phylogenetic analysis. Thanks to the assistance from professor

Guang Wen Lu, Professor Yi Shi and Dr. Edward C. Holmes in the process of thesis writing.

Thanks to Yan Wu, Su Fang Ma, Yin Wu, Fei Yuan, Yu Bai, Qun Yan, Meng Chen, Xue Li, Hong

Mei Yan, Cui Yan Guo, Xiao Ping Huang, Lan Qin Ma, Xing Zhao, Zhou Quan, Xiao Qu, Shi Hua

Li, Lian Pan Dai, Shu Guan Tan and Yu Hai Bi. They are teachers and staff of the lab. Thank

you for their contribution to the great working environment. Thanks to the support of Jun

Liu, Hong Lan Zhao and Yue Wang from the virology lab of the Chinese Disease Control and

Prevention center. Thanks to my labmates: Wei Ji, Chuan Song Quan, Zi Qian Xu, Hao Jia, Ke

Fang Liu and so forth. Thanks to the support on the testing and the usage of the

Fluorescence microscope from Dr. Jing Dong Song, Dr. Xiao Hui Chu, Dr. Xiao Juan Guo and

teacher Jian Guo Qu of the Hong Tao lab under Virology institute. Thanks to director

WenXu, teacher Hong Li, Yong Ming Zhou

141

A partial translation into English of the PhD thesis: “Novel Virus Discovery in Bat and the Exploration of

Receptor of Bat Coronavirus HKU9” by Canping Huang, Chinese Center for Disease Control and Prevention,

2016. Translation completed for Independent Science News in March 2021.


Yi Bing Xiang and teacher De Ming Ning from Center of disease control and prevention in

Yunnan Province. Thanks to director Xin Hua Liu, associate director Hong Hua Wen of

Center of Disease control and prevention in Meng La County. Thanks to director Yan Jun

Zhang, Dr. Hao Yan and Xiu Yu Lou of Center of Disease control and Prevention of Zhe Jiang

province.

Thanks to Han Wang, Shui Jun Zhang, Hao Song, Ru Chao Peng, Jian Wei Liu, Yao Hua

Zhu, Shuo Yang, Kun Xu, Qari, Fei Wang, An Li, Jin Xiao and Jia Ming Lan and Xiao Juan Guo

both from Virology study.

Thanks to Ming Zhao, Yuan Yuan, He Lin Zhang, Ying Xu, Xiao Ying Xu, Jiao Quan,

Wen Qian Duan, Li Li Chu, Wen Wei Yang, Zhen Nan Zhao, Ying Zi Cui, Ling Hui Li, Ming

Wang, Jing Gao, Lei Feng, Xiao Zhang, Yu Hua Wan, Chun Xia Wen, Dan Qing Chen, Li Li Pang,

Jian Wei Liu, Li Wei Ying, Chang Zhang, Hai Hai Jiang, Chao Su, Sheng Liu, Hai Feng Li, Zi

Fang Shang, Teng Fei Zhu, Jian Song, Wen Liang Zhang, Xu Dong Wang, Ling Niu, Joel

Haywood, Yemi Oladejo, Joseph Obameso, Moki, Hua Bing Yang, Hao Zhang, Jun Fu Li, Xu Le

Hu, Fan Li Yang, Tao Wan, Christopher John Vavricka, Boris Tefsen, Qing Hao Liu, Wen Bo

Liu, Hai Bang Hao and so forth gathered together in the lab.

Lastly, I want to thank my parents, family, ex in-laws and grandparents. Thank you

for always being so supportive!

Canping Huang,

2016/4/18

142

A partial translation into English of the PhD thesis: “Novel Virus Discovery in Bat and the Exploration of

Receptor of Bat Coronavirus HKU9” by Canping Huang, Chinese Center for Disease Control and Prevention,

2016. Translation completed for Independent Science News in March 2021.

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