UNIVERSITY OF CALGARY

Vaccine Induced Mucosal Disease

by

Adam Chernick

A THESIS

SUBMITTED TO THE FACULTY OF MEDICINE

IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE

DEGREE OF BACHELOR OF HEALTH SCIENCES HONORS

Bachelor of Health Sciences

Faculty of Medicine

University of Calgary

Calgary, AB

March 2012

© Adam Chernick, 2012

ii

Abstract

Bovine viral diarrhea virus (BVDV) is a member of the genus Pestivirus. Infection of

pregnant cows with BVDV during the first 125 days of gestation with a noncytopathogenic (ncp)

BVDV strain may result in persistently infected calves (PIs). Such animals act as virus reservoirs

within the herd, negatively impacting overall herd health. Superinfection of PIs with

cytopathogenic (cp) strains can result in the formation of mucosal disease (MD) characterized by

mucosal lesions and death. This superinfection may occur by infection with a cp virus but more

often through a variety of mutations within the NS2-3 gene of the existing ncp virus. These

mutations will lead to the cleavage of the NS2-3 protein resulting in the expression of the NS3

protein separately, the hallmark of cpBVDV. It is hypothesized that vaccination of PIs using a

cpBVDV strain antigenically similar to the existing ncp strain can induce MD. This project aims

to evaluate PIs that developed MD at feedlots to detect genetic similarities between vaccine and

field strains. The 5’UTR and NS2-3 regions of viral strains derived from vaccine and tissue

samples were sequenced following RT-PCR. Results indicate similarities between some vaccine

and animal isolates in these regions and that recombination may have occurred between these

strains.

Acknowledgments

The author would like to acknowledge the assistance of Frank van der Meer for his

supervision and advice, as well as Natalie Dow and Stephen Bonfield for their assistance in the

laboratory. Karin Orsel from UCVM and the veterinarians from Veterinary Agri-Health Services

in Airdrie are also thanked for their assistance with sample collection.

Contents

Abstract ........................................................................................................................................... ii

Acknowledgments........................................................................................................................... ii

List of Tables ................................................................................................................................. iii

List of Figures ................................................................................................................................ iii

Abbreviations ................................................................................................................................. iv

Introduction ..................................................................................................................................... 1

Methods........................................................................................................................................... 6

Results ........................................................................................................................................... 11

Discussion ..................................................................................................................................... 13

References ..................................................................................................................................... 18

Appendix ....................................................................................................................................... 20

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List of Tables

Table 1: Primer sequences used in RT-PCR reactions as outlined in methods. Note that Y

denotes a pyrimidine (C or T). Sequence pairs marked with a * were the ones used for all

sequences presented in the results.

Table 2: Sequencing primers proposed for evaluating the NS2-3 region (1).

Table 3: Viral strains identification based on BLAST search results. Note that two distinct strains

were detected in the sequencing of the BI vaccine in the 5’UTR.

Table 4: Percentage of identical bases in the alignment of the animal derived sequence noted and

the relevant sequence obtained from the vaccine that was administered to the animal. Values

marked as N/A were not available as the NS2-3 primers did not amplify an appropriate product

for these samples.

Table 5: Percentage of identical bases in the alignment of the vaccine and animal derived clone

sequences. Values marked as N/A were not available as the NS2-3 primers did not amplify an

appropriate product for these samples. * indicates that the BVDV type 2 sequences detected were

not included in this comparison. ** indicates that only one clone was available so no

comparisons could be made.

List of Figures

Figure 1: Genome organization of protein coding genes in pestiviruses (2).

Figure 2: Genotypes and subtypes for BVDV based on NPro

sequence data (3).

Figure 3: Examples of some mutations which may result in the formation of cpBVDV from

ncpBVDV (2).

Figure 4: 1.5% agarose gel electrophoresis of RT-PCR products using a primer pair which

amplified the entire NS2-3 region (1). The first lane is a 1kb plus ladder followed by the Novartis

vaccine, three animal samples, the BI vaccine, the Pfizer vaccine, and a water control.

Figure 5: 1.5% agarose gel electrophoresis of RT-PCR products using a primer pair which

amplified an approximately 400 base portion of the 5'UTR. Lanes 1-11 are animal derived

samples as follows: V0521, 6479, K1754, O8245, P5948, V1187, K1582, C1138, O3054,

O3042, and T2546. Lane 12 represents a product derived from the Pfizer vaccine while lane 13 is

a negative control.

Figure 6: 1.5% agarose gel electrophoresis of RT-PCR products using a primer pair which

amplified an approximately 400 base portion of the NS2-3 region. Lanes 1-10 are animal derived

samples as follows: V0521, 6479, K1754, O8245, P5948, V1187, K1582, C1138, O3054, and

T2546. Lane 11 represents a product derived from the Pfizer vaccine while lane 12 is a negative

control.

Figure 7: Sequence alignment of the 5'UTR from both vaccine and animal (6479) derived

sequences. Colored lines indicate mutations while breaks indicate insertions and deletions

relative to the reference sequence.

iv

Figure 8: Sequence alignment of the NS2-3 region from both vaccine and animal (6479) derived

sequences. Colored lines indicate mutations while breaks indicate insertions and deletions

relative to the reference sequence.

Figure 9: Sequence alignment of the 5'UTR from both vaccine and animal (K1754) derived

sequences. Colored lines indicate mutations while breaks indicate insertions and deletions

relative to the reference sequence.

Figure 10: Sequence alignment of the NS2-3 region from both vaccine and animal (K1754)

derived sequences. Colored lines indicate mutations while breaks indicate insertions and

deletions relative to the reference sequence.

Figure 11: Sequence alignment of the 5'UTR from both vaccine and animal (T2546) derived

sequences. Colored lines indicate mutations while breaks indicate insertions and deletions

relative to the reference sequence.

Figure 12: Sequence alignment of the NS2-3 region from both vaccine and animal (T2546)

derived sequences. Colored lines indicate mutations while breaks indicate insertions and

deletions relative to the reference sequence.

Abbreviations 5’UTR 5’ untranslated region

BI Boehringer Ingelheim

BVDV Bovine viral diarrhea virus

cDNA Complimentary DNA

cp Cytopathogenic

ELISA Enzyme linked immuosorbent assay

LB Luria-Bertani

MD Mucosal disease

MLV Modified live virus

ncp Noncytopathogenic

NS2-3 Non-structural 2-3

PBS Phosphate buffered saline

PCR Polymerase chain reaction

PI Persistently infected calf

RT-PCR Reverse transcriptase PCR

TI Transient infection

UV Ultraviolet

1

Introduction

Bovine viral diarrhea virus (BVDV) is a single stranded RNA virus; a member of the

genus Pestivirus, family Flaviviridae (4). Such viruses are enveloped with a genomic size of

approximately 12.3kb (2). The general layout of the viral genome is provided in Figure 1. The

genome includes structural and non-structural genes, as well as a 5’ untranslated region (5’UTR)

which has been used for typing of BVDV strains (5). Primary or acute infections, also called

transient infections (TI), of BVDV in cattle tend to result in low mortality but do have significant

implications for overall herd health. Symptoms generally include fever, diarrhea, and some

mucosal lesions (6). However, there have been cases reported where high virulence, BVDV type

2 strains have been detected and subsequent high mortality has been observed, including an

incident in Ontario in the early 1990’s (7). Such cases may be referred to as severe-acute or

haemorrhagic infections due to the bleeding observed. However, the primary concern with TIs of

BVDV is its ability to suppress immune responses in the host, thus allowing for secondary

infections to develop which will decrease animal growth and productivity (4).

Prevalence estimates provide a sense of how widespread BVDV infections are. Individual

cattle on dairy farms in Alberta are reported to be seropositive 28.4% and 8.9% of the time for

BVDV type 1 and BVDV type 2, respectively (8). The same study reported that 53.4% and

19.7% of all dairy herds tested had at least one animal seropositive for BVDV type 1 or BVDV

type 2, respectively. In Manitoba, 336 herds were tested and 5 were BVDV positive, resulting in

a herd prevalence of 1.5% (9). It should be noted that a seropositive test result does not

necessarily indicate an ongoing infection, only that the animal has developed immunity to a

certain strain of the virus at some point in its life. The estimated annual incidence of BVDV

2

infections in the United States is approximately 34% and produces cumulative economic losses

of around US$20 million per million calvings when it is assumed that a low virulence (non-

severe) strain predominates (10). Economic impact at the individual herd level can also be

substantial. In the Ontario outbreak of severe acute BVDV type 2 in 1993 mentioned previously,

the cost to individual dairy herds ranged from $40 000 to $100 000 (7). These costs reinforce the

need for effective control programs which may include elimination of PI animals as well as

routine vaccination.

BVDV isolates can be divided into two main genotypes, BVDV 1 and BVDV 2. This

distinction is often made based on 5’UTR sequences for diagnostic purposes, but has also been

based on other regions including the Npro

coding region (3, 5, 11). These genotypes can also be

broken down into further subgroups based on these sequences. For example, phylogenetic

analysis of a variety of sequences obtained from GenBank has indicated 16 distinct BVDV 1

subtypes (a through o) and three distinct BVDV 2 subtypes (a through c) have currently been

documented (3). Figure 2 shows a proposed organization of these types and subtypes with

relation to one another based on phylogenetic analysis of Npro

sequence data. Genotype also has

implications for the clinical manifestations of BVDV infection (7, 12). The frequency of specific

mutations which result in the transformation from noncytopathogenic (ncp) to cytopathogenic

(cp) strains also differs between genotypes (12).

The nonstructural 2-3 (NS2-3) region of the genome is of particular interest due to its

involvement in the production of cp strains from ncp strains of the virus (1, 2, 13, 14). In cell

culture, ncpBVDV strains produce no visible differences in cell growth and the formation of

plaques is not noted. cpBVDV strains produce extensive cytopathogenic effects and result in

plaque formation. While the difference between these two biotypes is obvious in cell culture, the

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differences are far less notable in animal infections. The distribution of the biotypes throughout

various tissues may differ, as may the way in which the immune system is activated (15, 16).

cpBVDV strains can be formed due to mutations in the NS2-3 region of a ncpBVDV virus.

These mutations may include the insertion of ubiquitin coding sequences between the NS2 and

NS3 genes resulting in post-translational cleavage, duplication of the NS3 gene alone, or other

changes as shown in Figure 3 (2). While ncp viruses will only express the entire NS2-3 protein,

cp viruses express both NS2-3 and NS3 independently of one another. Although not significant

in terms of the symptoms observed in acute infections, the difference between cpBVDV and

ncpBVDV biotypes becomes important when understanding the genesis of PIs and development

of mucosal disease (MD) in these animals.

PIs are animals born immunotolerant to a specific ncpBVDV strain. Due to an

intrauterine infection during the first 125 days of gestation, a calf may recognize a specific strain

of the virus as “self” (17). Abortion, malformation, or the birth of a normal calf may also result

depending largely on the timing of the transient infection of the pregnant cow. PIs will often

suffer from chronic respiratory infections, poor growth, and chronic enteritis (18). These animals

suffer from a 50% higher mortality rate in their first year of life and also increase the mortality

rate for non-PIs in the same herd by increasing their risk of acute BVDV infection (4). As PIs

remain BVDV positive and shed the virus for their entire lives, they act as a long term reservoir

of infection for the entire herd. As such, they pose a significant threat to overall herd health and

it is generally recommended that these animals be removed if they are identified (4). However,

these animals can be difficult to identify by clinical signs alone due to non-specific signs until

they develop MD (18). Even then animals may still be misdiagnosed at this stage due to some

variability in presentation of clinical signs. MD is a condition that develops only in PIs and is

4

due to a superinfection with a cpBVDV strain that is antigenically similar to the already

established, persistent, ncpBVDV infection (19). MD is characterized by bloody diarrhea as well

as extensive lesions on mucosal membranes. Typically it is fatal within two to three weeks of

presentation of symptoms and is often diagnosed during routine post-mortem examinations (18,

19).

Vaccination of cattle is routine practice in most beef and dairy operations. In feedlot

operations vaccination occurs during processing as animals arrive from auction. Among other

procedures, a vaccine against BVDV is administered. In 1999, over 94% of cattle on feedlots

were vaccinated against BVDV (20). Multivalent vaccines are those which contain a number of

viral and/or bacterial strains and can protect against a number of diseases. Programs using these

vaccines have been shown to be more effective by certain measures of health (final weight and

daily weight gain) as well as economically advantageous when compared to univalent vaccine

programs (21). Three multivalent vaccines commonly used on feedlots were evaluated in this

project. The Bovi-Shield® Gold 5 vaccine from Pfizer protects against multiple infections

including type 1 and type 2 BVDV. Both the type 1 and type 2 strains are modified live viruses

(MLVs), indicating that while the viruses are still potentially infective they should not cause any

symptoms associated with the original strains. The provided vaccine information does not

indicate whether the strains are cp or ncp. The Express® 5-PHM vaccine from Boehringer

Ingelheim (BI) also protects against multiple infections including BVDV type 1 and type 2. Also

containing MLVs, the BI vaccine information states that it is derived from the Singer type 1a cp

strain as well as the 296 type 2 cp strain. Starvac® 4 Plus by Novartis also contains MLV BVDV

types 1 and 2, but no further information is provided. While the extent of information available

5

regarding these vaccines as well as the specific strains used to manufacture them is variable,

most vaccines available today do contain cp strains of the virus (4).

The use of cpBVDV strains in the production of BVDV vaccines may have important

implications for PIs. As described earlier, PIs can develop MD if they are superinfected with a cp

strain that is antigenically similar to the existing ncp strain. Although these vaccines are based on

MLVs, which should not be infective in the same way wild type viruses are, it is unclear how

such viruses may impact PIs. If the cp strain present in a vaccine was antigenically similar to the

existing ncp strain in a PI or were to recombine with it, there exists the potential to induce MD.

Anecdotal evidence seems to support such a possibility. Veterinarians and feedlot owners report

a spike in MD cases on feedlots two to four weeks post-processing and vaccination. This spike

may be linked to the vaccination of PIs with the MLV cp strains found in most vaccines.

Additionally, observations recorded in a variety of publications indicate that this phenomenon is

occurring. Publications from as far back as 1967 describe a “MD-like syndrome” occurring only

10 to 20 days following the application of a BVDV vaccine (22). Although the precise

mechanism was not necessarily understood at the time, the vaccine was implicated as a cause.

More recent work has proposed that a recombination event between the cp vaccine strain and

persistent ncp strain can produce a new cp strain responsible for MD (23). This work noted a

unique insertion in a region encoding for a protein now referred to as NS2-3; a number of such

insertion/deletion events are described in Figure 3 (24). Additionally, the recombination of a

viral strain containing a partial ubiquitin coding sequence with an endogenous strain has been

implicated in MD development as well (25). Such concerns are strong enough for the vaccine

producer Boehringer Ingelheim to indicate that the use of its vaccine on PIs is contraindicated.

Documentation provided by Novartis and Pfizer make no such recommendations. The potential

6

implications of vaccination for PIs and the development of MD require further research to better

understand this relationship at a molecular level. The following hypothesis and aim target this

gap in the literature.

Hypothesis: Vaccination of a PI BVDV calf with a modified live cpBVDV vaccine that is

antigenetically similar to the existing ncpBVDV infection will result in the development of MD.

Aim: To determine the relationship between persistent infection with a ncp strain of BVDV,

vaccination with a homologous strain of cpBVDV vaccine, and the development of MD.

Methods

Multiple tissue samples from a number of animals were obtained from local feedlots

during routine post-mortem examinations. Tissue samples included tonsil, mesenteric lymph

node, ileum, blood, feces, urine, and any other obvious lesions noted during examination. Not all

tissues were collected from all animals. For example, some cadavers did not have enough urine

or feces present during the post-mortem examination to produce an adequate sample. In such

cases other tissue may have been collected including the urinary bladder or ovaries. Animals

were diagnosed as having MD based on veterinarian opinion, particularly the observation of

lesions in the esophagus and mouth and depletion of lymphoid tissues (18). While multiple

tissues were sampled, the mesenteric lymph node was chosen for analysis in this project due to

its ease of processing, reliable polymerase chain reaction (PCR) amplification, and that it was

available from all animals sampled. The implication of lymphoid tissue in disease progression

according to the veterinary reports also seemed indicative of virus being present in mesenteric

lymph node samples. If a sample was found to be negative for BVDV by PCR it was excluded

from further analysis regardless of the veterinarian’s diagnosis. All samples were stored at 4°C

7

during transport and frozen at -20°C while they were held by the veterinarians. Unfortunately,

this resulted in haemolysis of the blood samples making RNA extraction difficult later. For this

reason blood and various blood fractions could not effectively be evaluated.

Once the samples were returned to the lab they were partially thawed at room

temperature for aliquoting and homogenization. The large tissue portions were removed from

their storage tubes and cut into pieces of approximately 0.5-1cm3. If not homogenized

immediately, the pieces were then stored at -80°C in 1.5mL tubes. Care was taken to avoid the

external surfaces of the large tissue pieces due to concerns of cross contamination during

collection at the feedlots. Aliquoted samples were then homogenized using a scalpel in a Petri

dish. Although it was proposed that this would be accomplished using a motorized rotor-stator

style homogenizer concerns were noted regarding cross contamination. Due to difficulties in

cleaning the head of the mechanized homogenizer the disposable scalpel blades were preferred.

Once the tissue had been cut into a mostly homogenous paste, 200µL of phosphate buffered

saline (PBS) was added to the tissue. After vortexing for approximately 30 seconds the sample

was centrifuged at 14 000RPM for 2 minutes to separate the tissue particles from the supernatant.

The remaining fluid was recovered for use in the E.Z.N.A® Viral RNA Kit (Omega Bio-Tek™

Inc., Norcross, GA) as per the manufacturer’s instructions. This kit works by reversibly binding

RNA to a silica-based membrane. The RNA can then be eluted after a series of washes using

RNase-free water. While the name of the kit implies that it will separate viral RNA from other

RNA, it does not according to the provided supplemental information. As such, primers must be

tested for specificity with respect to bovine RNA sequences as well as to BVDV sequences.

Reverse transcriptase PCR (RT-PCR) was used to amplify regions of interest from the

RNA extracted from tissue samples. A two step protocol was originally used where a one step

8

RT-PCR reaction was performed followed by a second nested PCR to type the virus based on the

5’UTR. Primer sequences are provided in Table 1 (26). The first reaction was conducted with the

BluePrint™ One Step RT-PCR Kit (Takara Bio Inc. ™, Japan) The total reaction volume was

25µL consisting of 12.5µL 2x reaction buffer, 1µL enzyme mixture, 1µL of template RNA, 1µL

of each forward and reverse primer (each to a final concentration of 0.4µM), and 8.5µL double

distilled water. The thermal cycler parameters were 50°C for 1 hour and 94°C for 2 minutes

followed by 30 cycles of 94°C for 30 seconds, 50°C for 45 seconds, and 72°C for 1 minute. A

final extension at 72°C for 10 minutes was included. The second PCR substituted the

BluePrint™ components for traditional PCR reagents (no reverse transcriptase was added). All

volumes were the same except that the enzyme mix was replaced with an equal volume of Taq

DNA polymerase from Invitrogen™ (Burlington, ON). The thermal cycler parameters were as

follows: 94°C for 2 minutes and then thirty cycles of 94°C for 30 seconds, 50°C for 45 seconds,

and 72°C for 1 minute. An extension at 72°C for 7 minutes was included at the end. The

resulting products were run at 120V for 45 minutes on a 1.5% agarose gel and then visualized

under ultraviolet (UV) light. The first RT-PCR was designed to detect all BVDV strains while

the second round only detected type 2 viruses. This protocol was discarded due to inconsistent

results and the lengthy reaction time. An additional primer pair with a similar protocol was tested

but also discarded for similar reasons (27). The primer pair eventually selected for analysis

amplified a region of the 5’UTR which is around 278 bases long (5). The main reason for the use

of these primers was due to the reliability of results produced and the ease of thermal cycler

program optimization. This reaction used the BluePrint™ One-Step kit and the same volumes of

reagents as the first step of the initial primer pair tested. The thermal cycler protocol was as

follows: 50°C for 30 minutes and 94°C for 2 minutes, followed by 40 cycles of 98°C for 10

9

seconds, 52°C for 30 seconds, and 72°C for 1 minute. An extension at 72°C for 7 minutes was

also included. Primers were also tested for amplification and sequencing of the NS2-3 gene (see

Table 1 and Table 2). One pair provided a fragment 402bp in length after the following thermal

cycler program: 50°C for 30 minutes and 94°C for 2 minutes followed by 35 cycles of 95°C for 1

minute, 53°C for 1 minute, and 72°C for 2 minutes, then a single 7 minute extension at 72°C.

The BluePrint™ kit was used for these primers and the reagent volumes were also the same as

those used for the successful 5’UTR primer pair. This primer pair is being used to analyze the

NS2-3 junction (28). A published primer pair that spanned the entire NS2-3 region was also

tested and found to bind non-specifically to bovine sequences without amplifying any BVDV

sequences, see Figure 4 (1). Further refinements to protocols must be made for this primer pair to

be used effectively.

After successfully amplified 5’UTR and NS2-3 sequences were run on a 1.5% agarose

gel, the bands were cut out under UV light for extraction. The E.Z.N.A® Gel Extraction Kit

(Omega Bio-Tek™ Inc., Norcross, GA) was used to isolate the DNA fragments from the

agarose. This kit works in a similar manner as the RNA isolation kit. The agarose is melted in a

proprietary binding buffer and then run through a selective membrane which binds DNA. After

several washes the DNA was eluted for use in a ligation. Ligations were accomplished using the

pGEM®-T Easy Vector System I (Promega Corporation™, Madison, WI). In this reaction, the

purified PCR product was combined with the linearized vector and the enzyme T4 ligase. The

reaction was allowed to proceed overnight at 4°C, producing a vector containing the PCR

product. This vector allows for simplified downstream sequencing as it contains a T7 and an SP7

sites each on either side of the multiple cloning site. Following transformation of the vector, the

E. coli cells were grown on Luria-Bertani (LB)-agar prepared with ampicillin to a concentration

10

of 50µg/mL and previously plated with 40µL of 20mg/mL X-gal. There are both ampicillin

resistance genes as well as the lac operon built into the pGEM-T® Easy vector. The ampicillin

resistance gene allows for selection of transformed bacteria that contain any intact plasmid, while

the lacZ gene in the lac operon (which spans across the cloning site) allows for selection of

bacteria containing plasmids that contain a PCR product and have not closed upon themselves. If

the plasmid closed without a PCR product ligated into it, leaving the lacZ gene intact, the

substrate X-gal in the solid agar media would be metabolized into a blue precipitate and form a

blue colony. If a PCR product was ligated into the cloning site, the lacZ gene would be disrupted

and the X-gal substrate would not be metabolized, thus the colony would appear white in colour.

5 to 12 white colonies from each ligation and subsequent transformation were picked from the

plate and grown overnight in LB broth with ampicillin at 50µg/mL. By picking multiple colonies

it becomes possible to detect variation within the viral population as amplified by the selected

primers. Similar methods have been used to evaluate the diversity of HIV-1 quasi-species in the

human gut (29). This allows for the analysis of multiple strains using one PCR and one cloning

reaction. These overnight cultures were then processed using the E.Z.N.A® Plasmid Mini Kit I

from Omega. Following centrifugation and the application of a lysis-RNase buffer, the lysate

was run through a column which selectively binds plasmid DNA. Following a series of washes

the plasmid was eluted from the column. The purified plasmid concentrations were determined

using a NanoDrop™ (Thermo Scientific™, Wilmington, DE) and then diluted with double

distilled water accordingly to reach a final concentration between 100 and 200ng/µL as per

Eurofins MWG Operon’s (a commercial sequencing company, www.operon.com) suggestions.

Plasmids were sent for Sanger sequencing using a SP6 primer. Sequences were only read from

one direction since the size of the PCR fragments were small enough to allow for a single read.

11

Larger sequences such as the complete NS2-3 region would require reads from both directions

and/or the use of additional sequencing primers such as those noted in Table 2.

Geneious 5.5.6 (Biomatters™, Auckland, NZ) was used for analysis of sequence data.

Upon receipt of sequences the plasmid portion of the sequence was removed and all 5’UTR and

NS2-3 sequences were searched through the non-redundant nucleotide BLAST database

(http://www.ncbi.nlm.nih.gov/genbank/). Alignments were performed using the ClustalW

algorithm. All sequences from a single animal were aligned to all available vaccine sequences

for the both the 5’UTR and NS2-3 data. The reference sequence was set to that of the vaccine

strain administered based on the available health records. In the case of vaccine sequences only

one of the clones was selected for comparison to animal sequences since all clones shared a very

high percentage of identical bases (usually ≥99%). All sequences derived from animal samples

were retained in the alignment. The percentage of identical bases between vaccine and animal

derived sequences was also obtained based on the sequence alignments for comparison.

Results

The 5’UTR RT-PCR confirmed that this primer pair was effective in most cases as shown

in Figure 5. Unfortunately, no product was obtained for animals V0521 and C1138 and therefore

they were excluded from further analysis. 5’UTR products for both the BI and Novartis vaccines

were also obtained. The PCR results for the NS2-3 region are shown in Figure 6. Products of 400

bases in size are the desired product while bands around 100 bases are due to the primers non-

specifically binding to bovine genes. As is visible, PCR products were only obtained for three

animal samples (6479, K1754, and T2546) as well as from the Pfizer vaccine. We were able to

get a positive PCR signal for the Novartis vaccine virus (not shown). The NS2-3 sequences for

12

the BI vaccine were obtained from reference sequences (cp Singer (BVDV 1a) GenBank

accession number DQ088995.2 and cp 296 (BVDV 2a) GenBank accession number

AF268171.1) according to the provided supplemental information.

Viral strain train typing via BLAST searches for all sequences are provided in Table 3.

Only one type is noted for each group of clones as sequences were highly similar within each

group. The exception is for the BI vaccine strain which returned two distinct populations of virus

in the 5’UTR.

Sequence data indicates three potential outcomes at a genetic level following vaccination.

With respect to samples derived from animal 6479, comparisons were made between vaccine and

animal derived strains. In all alignments, the reference sequence was that of the vaccine the

animal received, in this case one of the BI vaccine strains. It was noted that the 5’UTR of both

groups of sequences shared between 97.9 and 100% identical bases in the alignment. When the

NS2-3 regions of both groups were compared it was found that 99.3-99.6% of bases were

identical (see Figure 7 and Figure 8). The same procedure was followed for sequences derived

from animal K1754 except the reference sequence used was from the Novartis vaccine strain.

Figure 9 shows the sequence alignment comparing sequences of the 5’UTR, while Figure 10

shows the sequence alignment comparing sequences of the NS2-3 region. Numerous mutations

are immediately apparent. Only 74.6-75.5% of bases in the 5’UTR were identical when

comparing the animal derived strains to the vaccine strain the animal received, while 74.4-74.6%

were identical within the NS2-3 region. BLAST search indicated the animal derived sequences

were type 2 while the Novartis strains were type 1. The third complete data set (where both the

5’UTR and NS2-3 sequences were available) was derived from animal T2546. The alignments

provided in Figure 11 and Figure 12 show few mutations between the vaccine (Novartis) and

13

animal derived strains in the 5’UTR, but many mutations with respect to the NS2-3 region.

When quantified it was found that 99.3-99.6% and 83.0-83.8% of bases were identical in the

comparisons of the 5’UTR and NS2-3 sequences, respectively.

For four of the animals samples only the 5’UTR sequence was obtained as the NS2-3

primers failed to amplify the appropriate product in these cases (see Figure 6). In three of these

cases the percentage of identical bases in the respective alignments ranged from only 75.0-76.7%

(K1582, O8245, and P5948), while the percentage in the fourth case was 99.3-99.6% (V1187).

The percentage of identical bases for all animal derived samples is summarized in Table 4.

With respect to the multiple clones performed for each animal based RT-PCR, there was

a high level of similarity between all clones. The 5’UTR primers pair produced clones that were

at least 95.7% identical to one another based on sequence alignments in all animals. Clones

derived from the NS2-3 RT-PCR demonstrated at least 98.9% identical bases when aligned

against one another in all animals. A similar trend was seen with respect to the vaccine derived

clones. Sequences in these cases were generally at least 91.6% identical. These values were

calculated by performing pairwise comparisons of all sequences obtained from each set of

clones. See Table 5 for the ranges of identical bases for the clones from all animals and vaccines.

Discussion

Pairwise comparisons of sequences derived from the multiple clones sequenced from

each animal indicate a very high degree of similarity between the clones. As mentioned in the

results, the most dissimilar pair of clones within a single animal was still 95.7% identical in the

5’UTR sequences and 98.9% identical in the NS2-3 sequences. Although this does leave room

for a small degree of variability in the population, it strongly indicates that within each

14

mesenteric lymph node sample used there is a single, dominant viral strain present. This finding

is based on the fact that even with as many as 12 colonies being picked there were no major

variations detected between the resulting sequences. Since a single viral strain is dominant within

the tissue, it becomes possible to infer some connections between the 5’UTR and NS2-3

sequence data even though they were obtained via separate RT-PCR and sequencing reactions.

Typically such comparisons can only be made when the sequence data is obtained in a single

sequencing run, which requires the regions to be within about 1500 bases of one another, or if

the viral strains are first isolated and purified via conventional and reverse plaque isolation

methods for cp and ncp strains, respectively (30). Although such methods present future

directions to solidify any conclusions made here, such conclusions are still reasonable

considering the lack of diversity of viral strains within the animal derived clone sequences.

These data represent some of the possible ways in which a MLV BVDV vaccine may

interact with endogenous strains already present in a PI. Of particular interest are the three

animals from which both 5’UTR and NS2-3 sequences were obtained. One such mechanism of

interaction is depicted by the alignments of animal 6479 derived sequences to the sequences

from the vaccine it received. The high degree similarity between both the 5’UTR and NS2-3

sequences in the animal and vaccine derived sequences indicates that the strain detected in the

animal likely originated from the vaccine itself. This is important to note as it means that the

vaccine strain is capable of becoming the dominant strain present following vaccination. This

presents two potential explanations regarding the development of MD in this case. First, as it is

known that the BI vaccine was developed based on two cpBVDV strains (Singer and 296 based

on the supplemental vaccine information), it is possible that one of these strains was

antigenically similar enough to the persistent ncpBVDV strain to allow MD to develop without

15

any interaction (such as recombination) between two strains. Second, it is also possible that

vaccination had no effect with respect to MD. MD may have developed via a mutation to the

persistent ncp strain that was independent of the vaccine strain. The evidence presented here

allows for these mechanisms but does not support them exclusively. However, as the vaccine

strain was not detected in the animal tissue it seems more likely that it was not involved in MD

development.

Alignments based on sequences from animal K1754 and the vaccine it received

(Novartis) indicate a different type of interaction than what was seen in animal 6479. The

greatest percentage of identical bases for either the 5’UTR or the NS2-3 region is 75.5%, far less

than what was observed in the alignments from animal 6479. BLAST results indicate that these

viral strains actually represent different genotypes entirely (see Table 3). As such, it appears that

the vaccine has not altered the dominant strain in any notable way detectable by these methods.

There does not appear to be any recombination events between these two regions either. As the

dominant strain detected in the animal is a different genotype than the vaccine strain, it seems

unlikely that the vaccine played a role in this incident of MD. In such a case it is possible that the

animal acquired the antigenically similar cp strain from the environment or through a

spontaneous mutation of the persistent ncp strain. It is also possible that the vaccine may have

recombined in a region of the genome not evaluated here. For example, the E2 structural protein

has been documented as an important site for antigenic recognition and could be a site of

recombination if the vaccine were involved in this case (31). However, the data presented here

does not provide evidence for either of these proposed mechanisms.

Alignments from animal T2546 present the most interesting proposed interaction found

here. The alignments for the 5’UTR show that the animal and vaccine derived sequences are

16

highly similar to one another (99.3-99.6%). However, the alignments for the NS2-3 regions show

a much lower level of similarity (83.0-83.8%). BLAST results indicate that all strain in this case

were type 1. In this case, the fact that the 5’UTR from the animal strains is almost the same as

that of vaccine strains while the NS2-3 regions are more dissimilar indicates that a recombination

event likely occurred between the vaccine strain and some endogenous strain between these two

regions of the genome. Such an interaction presents a plausible mechanism of how vaccination

could induce MD. By recombining with the persistent ncp strain, a cp vaccine strain would be

able to gain genetic material that would make it antigenically similar to the persistent ncp strain.

In this way the introduction of an otherwise antigenically dissimilar cp strain could result in the

formation of an antigenically similar cp strain by recombination with an endogenous ncp strain.

Although recombination between vaccine and persistent strains has been previously implicated

in the induction of MD, this was due to the introduction of a partial ubiquitin monomer coding

sequencing found in the vaccine strain into the persistent ncp strain (25). The mechanism

proposed here does not rely on the insertion of a vaccine derived ubiquitin sequence into the

persistent strain, rather upon the vaccine strain acquiring adequate antigenic similarity to and

from the persistent ncp strain via recombination with it. Although this data does not specifically

indicate the acquisition of the genes by the vaccine strain necessary to become antigenically

similar to the persistent strain, it does demonstrate that a recombination event has occurred.

Further work is needed to strengthen these conclusions. In all of the scenarios presented it

would be highly beneficial to isolate and sequence the ncp/cp viral pair responsible for the

development of MD. As only a single viral strain was identified in each animal it is not yet

possible to state whether or not the strain detected was a member of the ncp/cp viral pair. Whole

genome sequencing of plaque and reverse plaque isolated viruses would also allow for better

17

detection of recombination events without the need to guess and check which regions of the

genome should be sequenced using the methods presented here. Pending whole genome

sequencing, analysis of structural genes that are known to have antigenic properties would be

beneficial. Such regions include the E2 gene (31).

In conclusion, the data presented here indicates that a number of different interactions

may occur between the vaccine and the endogenous BVDV strains. A vaccine strain may

become the dominant viral strain present (as proposed for animal 6479) or it may have little or

no impact on the dominant strain (as proposed for animal K1754). Most interestingly, the

vaccine strain may recombine with the persistent ncp strain and produce an antigenically similar

cpBVDV strain (as may have occurred in animal T2546). This last mechanism presents a

plausible mechanism by which vaccination of a PI could induce MD. While further work is

clearly needed to solidify these conclusions, these data provide some initial insight into these

processes.

18

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Appendix

Tables

Table 1: Primer sequences used in RT-PCR reactions as outlined in methods. Note that Y

denotes a pyrimidine (C or T). Sequence pairs marked with a * were the ones used for all

sequences presented in the results.

Name Sequence (5’-3’) Target Gene Reference

Set A Sense GTA GTC GTG AGT GGT TCG 5’UTR (26)

Set A Antisense GCC ATG TAC AGC AGA GAT 5’UTR (26)

Set B Sense CGA CAC TCC ATT AGT TGA GG 5’UTR (26)

Set B Antisense GTC CAT AAC GCC ACG AAT AG 5’UTR (26)

UTR DL1 GCC ATG CCC TTA GTA GGA CTA GC 5’UTR (27)

UTR DL4 CAA CTC CAT GTG CCA TGT ACA GC 5’UTR (27)

First Round Left* CAT GCC CAT AGT AGG AC 5’UTR (5)

First Round Right* CCA TGT GCC ATG TAC AG 5’UTR (5)

NS2-3-F* GCA GAT TTT GAA GAA AGA CAC TA NS2-3 (28)

NS2-3-R* TTG GTG TGT GTA AGC CCA NS2-3 (28)

125AF GAG GGG CCG GTA GAA AAG AC NS2-3 (1)

125BR GCA TAY TGG AGG TGG GTK GTG T NS2-3 (1)

Table 2: Sequencing primers proposed for evaluating the NS2-3 region (1).

Name Sequence (5’-3’) Target Gene

AF2V1F TGG TTG GAG GAA TGG CAA GG NS2

3873F GAT CAC GTC CAT CGT AAT GG NS2

V5F TCT GAT GAT TGC CAC CCT AT NS2

4815R CTG GTT CCT AAC CTT GTG CT NS2

V6R AAT TGT CCC CTA GCG GCG TAT NS2

V7F GCC AAG AGG AAC CAC GCC AA NS3

V8R CTC AAA TAT GGG TAG ACC TG NS3

V9F CGC AAT GGT AGA GTA TTC CT NS3

V15R TTC CTA TGA CAG CCA ACT GC NS3

V10R TGC CTC TTC GCT GAG CCT GTT C NS3

CL3F AGG TAC GGA ATA GAG GAT GG NS3

21

Table 3: Viral strains identification based on BLAST search results. Note that two distinct strains

were detected in the sequencing of the BI vaccine in the 5’UTR.

5’UTR NS2-3

6479 Singer 1 Singer 1

K1754 2 2

T2546 Oregon 1a 1

K1582 NY 2 N/A

O8245 NY2 N/A

P5948 2 N/A

V1187 Oregon 1a N/A

Novartis Oregon 1a NADL/Singer 1

Pfizer NADL 1 Oregon 1a

BI Singer 1, NY 2 N/A

Table 4: Percentage of identical bases in the alignment of the animal derived sequence noted and

the relevant sequence obtained from the vaccine that was administered to the animal. Values

marked as N/A were not available as the NS2-3 primers did not amplify an appropriate product

for these samples.

5’UTR NS2-3

6479 97.9-100 99.3-99.6

K1754 74.6-75.5 74.4-74.6

T2546 99.3-99.6 83.0-83.8

K1582 75.2-75.5 N/A

O8245 75.3-76.7 N/A

P5948 75.0-75.6 N/A

V1187 99.3-99.6 N/A

22

Table 5: Percentage of identical bases in the alignment of the vaccine and animal derived clone

sequences. Values marked as N/A were not available as the NS2-3 primers did not amplify an

appropriate product for these samples. * indicates that the BVDV type 2 sequences detected were

not included in this comparison. ** indicates that only one clone was available so no

comparisons could be made.

5’UTR Clones NS2-3 Clones

6479 99.3-100 98.9-100

K1754 95.7-100 99.8-100

T2546 99.6-100 99.3-100

K1582 97.5-100 N/A

O8245 96.2-99.3 N/A

P5948 95.8-99.7 N/A

V1187 99.6-100 N/A

BI 99.3-99.6* N/A

Novartis 91.6-100 **

Pfizer 100 99.5-100

Figures

Figure 1: Genome organization of protein coding genes in pestiviruses (2).

23

Figure 2: Genotypes and subtypes for BVDV based on NPro

sequence data (3).

24

Figure 3: Examples of some mutations which may result in the formation of cpBVDV from

ncpBVDV (2).

25

Figure 4: 1.5% agarose gel electrophoresis of RT-PCR products using a primer pair which

amplified the entire NS2-3 region (1). The first lane is a 1kb plus ladder followed by the Novartis

vaccine, three animal samples, the BI vaccine, the Pfizer vaccine, and a water control.

Figure 5: 1.5% agarose gel electrophoresis of RT-PCR products using a primer pair which

amplified an approximately 400 base portion of the 5'UTR. Lanes 1-11 are animal derived

samples as follows: V0521, 6479, K1754, O8245, P5948, V1187, K1582, C1138, O3054,

O3042, and T2546. Lane 12 represents a product derived from the Pfizer vaccine while lane 13 is

a negative control.

26

Figure 6: 1.5% agarose gel electrophoresis of RT-PCR products using a primer pair which

amplified an approximately 400 base portion of the NS2-3 region. Lanes 1-10 are animal derived

samples as follows: V0521, 6479, K1754, O8245, P5948, V1187, K1582, C1138, O3054, and

T2546. Lane 11 represents a product derived from the Pfizer vaccine while lane 12 is a negative

control.

Figure 7: Sequence alignment of the 5'UTR from both vaccine and animal (6479) derived

sequences. Colored lines indicate mutations while breaks indicate insertions and deletions

relative to the reference sequence.

Figure 8: Sequence alignment of the NS2-3 region from both vaccine and animal (6479) derived

sequences. Colored lines indicate mutations while breaks indicate insertions and deletions

relative to the reference sequence.

27

Figure 9: Sequence alignment of the 5'UTR from both vaccine and animal (K1754) derived

sequences. Colored lines indicate mutations while breaks indicate insertions and deletions

relative to the reference sequence.

Figure 10: Sequence alignment of the NS2-3 region from both vaccine and animal (K1754)

derived sequences. Colored lines indicate mutations while breaks indicate insertions and

deletions relative to the reference sequence.

Figure 11: Sequence alignment of the 5'UTR from both vaccine and animal (T2546) derived

sequences. Colored lines indicate mutations while breaks indicate insertions and deletions

relative to the reference sequence.

Figure 12: Sequence alignment of the NS2-3 region from both vaccine and animal (T2546)

derived sequences. Colored lines indicate mutations while breaks indicate insertions and

deletions relative to the reference sequence.