Evaluation of a novel antiviral compound

177 Pages·2013·1.15 MB·English

by  George Crandell

Evaluation of a novel antiviral compound effective
against multiple pestivirus species.

by

Benjamin Wendell Newcomer

A dissertation submitted to the Graduate Faculty of
Auburn University
in partial fulfillment of the
requirements for the Degree of
Doctor of Philosophy

Auburn, Alabama
May 7, 2013

Keywords: bovine viral diarrhea virus; antiviral; pestivirus; cattle

Copyright 2013 by Benjamin Wendell Newcomer

Approved by

M. Dan Givens, Chair, Professor of Pathobiology,
Interim Associate Dean of Academic Affairs
Misty Edmondson, Associate Professor of Clinical Sciences
Julie Gard, Associate Professor of Clinical Sciences
John Neill, Research Microbiologist, USDA National Animal Disease Center
Paul Walz, Associate Professor of Pathobiology
Abstract

Bovine viral diarrhea virus (BVDV) is a viral pathogen of cattle and other wild
and domestic ungulates that is capable of affecting multiple body systems, most notably
the gastrointestinal, reproductive, immune and respiratory systems. Aside from causing
clinical concern, BVDV is a significant contaminant in laboratory systems and biological
products due to its often noncytopathic nature. The virus is also widely used as a
surrogate viral model to study Hepatitis C infection in humans. Despite its significance to
multiple clinical and research fields, practical methods of BVDV control are limited
largely to management protocols and biosecurity safeguards. Specific therapeutics or
decontaminants for BVDV are not currently available for use in the farm or the
laboratory.
Previous research identified a family of antiviral compounds that were safe and
effective when used to treat or prevent BVDV infection in various cell lines. This
research continued exploration of the identified compound with the highest therapeutic
index (DB772) and its potential to be used as an antiviral preventive or therapeutic in
vivo as well as it effectiveness against other viruses closely related to BVDV. In the
initial study, calves persistently infected with BVDV were treated intravenously with
either the antiviral compound or the drug diluent every eight hours over a six day period.
In addition to other parameters, viral titers in white blood cells were compared to pre-
treatment levels. Treatment with DB772 was associated with a rapid drop in viral titers.
ii
However, with one exception, the titers rebounded to near pre-treatment levels during or
immediately following the treatment period.
Further study revealed that virus isolated from the treated calves with rebound
titers was resistant to DB772 whereas pre-treatment isolates were susceptible to the
compound. Full genome sequencing was performed on the isolates and revealed single or
multiple mutations in the region encoding NS5b, which forms the RNA-dependent RNA
polymerase. These mutations have not been described in wild-type isolates and
consequently the growth curves of wild-type and mutant isolates were studied but
significant changes were not detected.
In a second study, calves naïve to BVDV were again treated with either the
compound or diluent intravenously before intranasal challenge with BVDV. Treatment
with DB772 successfully prevented infection in calves although these same calves were
shown to be susceptible to BVDV following treatment after the serum concentration of
DB772 was calculated to have fallen below in vitro protective levels.
Thirdly, the in vitro efficacy of DB772 against BVDV and closely related viruses
was examined by infection of cells with BVDV, border disease virus, HoBi virus,
pronghorn virus and Bungowannah virus. Infected cells were incubated in in medium
containing 0, 0.006, 0.01, 0.02, 0.05, 0.1, 0.2, 0.39, 0.78, 1.56, 3.125, 6.25, 12.5 or 25 μM
DB772 and subsequently assayed for the presence of virus by virus isolation and titration
or polymerase chain reaction. Cytotoxicity of the compound for different cell types used
in the study was evaluated using a commercially available cell counting kit. The
compound was shown to effectively inhibit all pestiviruses studied at concentrations >
0.20 μM. Cytotoxicity was not evident until DB772 concentrations exceeded 25 μM.
iii
Acknowledgments

The author would like to thank M. Daniel Givens for his mentorship, direction,
teaching, support and encouragement as chair of the graduate committee. Paul Walz
offered sound clinical mentorship and research advice. Collaboration with John Neill
provided unexpected learning opportunities that continue to yield fruit. Julie Gard and
Misty Edmondson offered continuous support both on the clinic floor and in the research
arena. The efforts of M. Shonda Marley in the instruction of research design and
laboratory conduct cannot be overstated and are much appreciated. The assistance of Kay
Riddell, Pat Galik, Yijing Zhang and Kathy McMullen in the laboratory is gratefully
acknowledged. Special recognition is afforded to Sara-Louise Newcomer, for her endless
support and sacrifice during the doctoral program. The parents of the author, Ben and
Mary Jane Newcomer also deserve mention for their nurturing, discipline and
unconditional support. Finally, eternal thanks are due to Father God who has blessed the
author immeasurably more than can be asked or imagined.

iv
Table of Contents

Abstract ............................................................................................................................. ii
Acknowledgments............................................................................................................ iv
List of Tables .................................................................................................................. vii
List of Illustrations ......................................................................................................... viii
List of Abbreviations ....................................................................................................... ix
Introduction ..................................................................................................................... 1
Literature Review ............................................................................................................ 3
Bovine viral diarrhea virus .................................................................................. 3
Introduction ............................................................................................... 3
Taxonomy ................................................................................................. 4
Genomic Structure .................................................................................... 6
Biotypes .................................................................................................. 16
Viral Structure ......................................................................................... 19
Viral Replication ..................................................................................... 20
Viral Host Range..................................................................................... 23
Clinical Disease ...................................................................................... 25
Diagnosis................................................................................................. 31
Antiviral agents in cattle ................................................................................... 37
Introduction ............................................................................................. 37
v
Hepatitis C .............................................................................................. 38
Interferons ............................................................................................... 40
Aromatic Cationic Compounds............................................................... 43
Nucleosidic Compounds ......................................................................... 44
Polyprotein Processing Molecules .......................................................... 46
Antisense Strategies ................................................................................ 48
Vegetal Extracts ...................................................................................... 49
Miscellaneous Compounds ..................................................................... 51
Summary ................................................................................................. 52
Statement of Research Objectives ................................................................................ 53
Antiviral Treatment of Calves Persistently Infected with Bovine
Viral Diarrhea Virus ......................................................................................... 54
Effect of Treatment with a Cationic Antiviral Compound on Acute Infection
with Bovine Viral Diarrhea Virus ..................................................................... 75

Efficacy of an Antiviral Compound to Inhibit Multiple Pestivirus Species ................. 97
Mutations Induced in the NS5B Gene of Bovine Viral Diarrhea Virus by Antiviral
Treatment Convey Resistance to the Compound ............................................ 105
Summary and Conclusions ......................................................................................... 122
References ................................................................................................................... 126

vi
List of Tables

Table 1. Results of virus isolation of BVDV-2 from calves inoculated with BVDV-2
aerosol inoculation with or without administration of DB772 ....................................... 88
Table 2. Results of inhibitory testing of several pestiviruses with different
concentrations of DB772. ............................................................................................. 102
Table 3. Sequencing results of wild-type and mutant BVDV isolates obtained from
persistently infected calves treated with DB772 following five passages in
white blood cells after co-infection with fixed ratios...……………………………….116

vii
List of Illustrations

Figure 1. Microscope photographs of Madin-Darby Bovine Kidney cell cultures
after infection with a noncytopathic or cytopathic strain of BVDV ............................... 17
Figure 2. Organization of the bovine viral diarrhea virus genome ............................................. 22
Figure 3. Serum DB772 concentrations after intravenous administration after
single or repeated dosages of DB772.............................................................................. 64
Figure 4. Number of cell culture infectious doses /mL (CCID50) of nasal swab media
50
in treated and untreated calves persistently infected with BVDV .................................. 68
Figure 5. Number of viral RNA copies/μL of serum in treated and untreated calves ................ 70
Figure 6. Average relative lymphocyte counts in treated and untreated calves after intranasal
challenge with BVDV ..................................................................................................... 84
Figure 7. Average rectal temperature readings in treated and untreated calves challenged
with BVDV ..................................................................................................................... 86
Figure 8. Serum neutralization titers to BVDV in treated and untreated miniature calves ........ 89
Figure 9. Growth curves for wild-type and mutant BVDV isolates cultured in the presence and
absence of DB772 ......................................................................................................... 114

viii
List of Abbreviations

ACE antigen capture ELISA
ACM aromatic cationic molecule
AI artificial insemination
AUMC area under the curve and its first moment
BCoV bovine coronavirus
BDV border disease virus
BHV-1 bovine herpesvirus 1
BHV-4 bovine herpesvirus 4
BLV bovine leukemia virus
BoIFN bovine interferon
BRSV bovine respiratory syncytial virus
BRV bovine rotavirus
BVDV bovine viral diarrhea virus
CBC complete blood count
CCID 50% cell culture infective dose
50
Cl clearance
C maximal serum concentration
max
CSFV classical swine fever virus
CP cytopathic
CPE cytopathogenic effects
ix
DB606 2-(4-[2-imidazolinyl]phenyl)-5-(4-methoxyphenyl)furan
DB772 2-(2-benzimidazolyl)-5-[4-(2-imidazolino)phenyl]furan dihydrochloride
dsRNA double-stranded ribonucleic acid
E1 primary envelope glycoprotein
E2 secondary envelope glycoprotein
Erns ribonuclease soluble envelope glycoprotein
FMDV foot-and-mouth disease virus
GGT gamma-glutamyltransferase
HCV hepatitis C virus
HPLC high performance liquid chromatography
HuIFN human interferon
IFN interferon
IHC immunohistochemistry
IL interleukin
IRES internal ribosome entry site
IVF in vitro fertilization
Jiv J-domain protein interacting with viral protein
MD mucosal disease
MDBK Madin-Darby Bovine Kidney (cells)
MEM minimum essential medium
MOI multiplicity of infection
mRNA messenger RNA
MRT mean residence time
x