African horsesickness (AHS) is one of the most deadly diseases of horses, with a
mortality rate of over 90% in horses that have not been exposed to any African
horsesickness virus (AHSV) serotype previously (Howell, 1960; Darpel et al., 2011). The
Orbiviruses, African horsesickness virus (AHSV) and Bluetongue virus (BTV), are
primarily transmitted to their mammalian hosts through certain haematophagous midge
vectors (Culicoides spp.) (Erasmus, 1973). The selective cleavage of BTV and AHSV VP2
by trypsin-like serine proteases (Marchi et al., 1995) resulted in the generation of
subsequent infectious sub-viral particles (ISVP) (Marchi et al., 1995; van Dijk & Huismans,
1982). It is believed that this cleavage affects the ability of the virus to infect cells of the
mammalian and vector host (Darpel et al., 2011). Darpel et al (2011) identified a trypsinlike
serine protease in the saliva of Culicoides sonorensis (C. sonorensis), which also
cleaves the serotype determinant viral protein 2 (VP2) of BTV. And, a similar cleavage
pattern was also observed by van Dijk & Huismans (1982) and Marchi et al (1995) with
the use of trypsin and chymotrypsin. Manole et al (2012) recently determined the structure
of a naturally occurring African horsesickness virus serotype 7 (AHSV7) strain with a
truncated VP2. Upon further investigation, this strain was also shown to be more infective
than the AHSV4 HS32/62 strain, since it outgrew AHSV4 in culture (Manole et al., 2012).
Therefore, through proteolytic cleavage of these viral particles, the ability of the adult
Culicoides to transmit the virus might be significantly increased (Dimmock, 1982; Darpel
et al., 2011). Based on these findings, it is important to investigate the factors that
influence the capability of arthropod-borne viruses to infect their insect vectors,
mammalian hosts and their known reservoirs.
In this study, we postulated that one of the vectors for AHSV, Culicoides imicola (C.
imicola), has a protease similar to the 29 kDa C. sonorensis trypsin-like serine protease
identified by Darpel et al (2011). Proteins in the total homogenate of C. imicola were
separated on SDS-PAGE and yielded several protein bands, one of which also had a
molecular mass of around 29 kDa. Furthermore, proteolytic activity was observed on a
gelatin-based sodium dodecyl sulfate polyacryamide gel electrophoresis (SDS-PAGE) gel.
The activity of the protein of interest was also confirmed to be a trypsin-like serine
protease with the use of class-specific protease inhibitors. A recombinant trypsin-like
serine protease of C. sonorensis was generated using the pColdIII bacterial expression
vector. The expressed protein was partially purified with nickel ion affinity
chromatography. Zymography also confirmed proteolytic activity. With the use of the protease substrates containing fluorescent tags and class specific protease inhibitors, the
expressed protein was classified as a serine protease. It was also proposed that
incubation of purified AHSV4 with the recombinant protease would result in the cleavage
of AHSV4 VP2, resulting in similar VP2 digestion patterns as observed in BTV by Darpel
et al (2011) or the truncated VP2 of AHSV7 by Manole et al (2012). BHK-21 cell cultured
AHSV4 was partially purified through Caesium chloride gradient ultracentrifugation after
which the virus was incubated with the recombinant protease. Since not enough virus
sample was obtained, the outcome of VP2 digestion was undetermined.
In the last part of this study, it was postulated that C. imicola and C. sonorensis have the
same trypsin-like serine protease responsible for the cleavage of VP2 based on the
protease activity visualised in the whole midge homogenate. Since the genome of C.
imicola is not yet sequenced, the sequence of this likely protease is still unknown.
Therefore, we attempted to identify this C. imicola protease through polymerase chain
reaction (PCR) amplification. Total isolated ribonucleic acid (RNA) of C. imicola was used
to synthesize complementary deoxyribonucleic acid (cDNA). The cDNA was subjected to
PCR using C. sonorensis trypsin-like serine protease-based primers. An 830 bp DNA
fragment was amplified. However, sequence alignment and the basic local alignment
software tool (BLAST), revealed that DNA did not encode with any other known proteins
or proteases.
From the literature it seems that there is a correlation between the proteases in the vector
and the mammalian species that succumb to AHS (Darpel et al., 2011, Wilson et al.,
2009, Marchi et al., 1995). Based on the work performed in the study, a proteolytically
active protein similar to the 29 kDa protein of C. sonorensis is present in C. imicola. The
29 kDa protease of C. sonorensis can also be expressed in bacteria which could aid in
future investigations on how proteolytic viral modifications affect infectivity between
different host species. / MSc (Biochemistry), North-West University, Potchefstroom Campus, 2014
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:nwu/oai:dspace.nwu.ac.za:10394/10742 |
| Date | January 2014 |
| Creators | Van Vuuren, Lihandra Jansen |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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