MicrobiologyBytes

Bluetongue is a severe disease of ruminants, notably sheep and cattle. The causal agent, the dsRNA Bluetongue virus, is spread by an insect vector and occurs in its vector’s habitat in temperate climates throughout much of the world. BTV is the type member of genus Orbivirus in the family Reoviridae, with 26 known serotypes. When bluetongue first broke out in the United Kingdom in autumn of 2007, the disease was already rapidly spreading throughout continental Europe, causing high mortality rates in sheep and having a detrimental effect on the livestock trade through trade restrictions and loss of stock. The only effective weapon against the disease is control of the spread of BTV through rigorous vaccination programmes. Currently available commercial vaccines are based on both inactivated virus and live, attenuated strains and protect against a single serotype or multiple serotypes when provided as a cocktail. However, the possibility of recombination between the live vaccine strain(s) and wild-type virus in infected animals, leading to the emergence of new infectious strains has motivated efforts to develop safer vaccines.

One approach in the development of an inherently safe vaccine has been the production of Bluetongue virus-like particles (VLPs). BTV has a nonenveloped icosahedral structure, with four main structural proteins (VP3, VP7, VP5 and VP2) arranged in concentric shells around the segmented double-stranded RNA genome and minor structural and nonstructural proteins involved in virus replication. French et al. have shown that these four structural proteins, expressed in insect cells using a baculovirus expression system, assemble into virus-like particles devoid of nucleic acid.

This paper describes plant-based high-level expression of assembled subcore-, core- and virus-like particles of BTV serotype 8. Purified preparations of the VLPs, consisting of all four structural proteins, elicited an immune response in sheep and provided protective immunity against challenge with a South African BTV-8 field isolate. This demonstrates that plant expression provides an economically viable method for producing complex VLPs, such as those of BTV, with the desired biological properties. It represents a significant advance in the use of plant-based systems for the production of complex biopharmaceuticals. The methods employed could also be applied to other situations where the expression of multiple proteins is required, such as the reconstruction of metabolic pathways.

A method for rapid production of heteromultimeric protein complexes in plants: assembly of protective bluetongue virus-like particles. Plant Biotechnol J. 06 May 2013 doi: 10.1111/pbi.12076
Plant expression systems based on nonreplicating virus-based vectors can be used for the simultaneous expression of multiple genes within the same cell. They therefore have great potential for the production of heteromultimeric protein complexes. This work describes the efficient plant-based production and assembly of Bluetongue virus-like particles (VLPs), requiring the simultaneous expression of four distinct proteins in varying amounts. Such particles have the potential to serve as a safe and effective vaccine against Bluetongue virus (BTV), which causes high mortality rates in ruminants and thus has a severe effect on the livestock trade. Here, VLPs produced and assembled in Nicotiana benthamiana using the cowpea mosaic virus-based HyperTrans (CPMV-HT) and associated pEAQ plant transient expression vector system were shown to elicit a strong antibody response in sheep. Furthermore, they provided protective immunity against a challenge with a South African BTV-8 field isolate. The results show that transient expression can be used to produce immunologically relevant complex heteromultimeric structures in plants in a matter of days. The results have implications beyond the realm of veterinary vaccines and could be applied to the production of VLPs for human use or the coexpression of multiple enzymes for the manipulation of metabolic pathways.

First it was foot and mouth virus.
Then it was bluetongue virus.
Now it is Schmallenberg virus.
So here’s 10 things you didn’t know about Schmallenberg virus:

1. Schmallenberg virus was first isolated in Schmallenberg, Germany, in November 2011.
2. Schmallenberg virus is a Bunyavirus, one of a large group of of negative-stranded RNA viruses.
3. Why should I care? In cows, Schmallenberg virus causes fever and a drastic reduction in milk production. In sheep it causes congenital malformations and stillborn lambs (also stillborn calves in cows).
4. Schmallenberg virus was first identifed in the UK on 23rd January 2012.
5. Like Bluetongue, Schmallenberg virus is transmitted by midges (Culicoides spp.), which means we will be unlikely to be able to eradicate it – vaccination of anaimals is the only likely effective response.
6. Where did Schmallenberg virus come from? The virus genome is most closely related to sequences of a different Orthobunyavirus called Shamonda virus which belongs to the so-called Simbu serogroup known to infect ruminants and be transmitted by midges. In other words, it has form. But whether it is newly evolved (unlikely) or just newly discovered we don’t yet know.
7. How did Schmallenberg virus reach the UK? We don’t know. It could have been due to animal movements, but since it was first identifed in eastern England, it’s possible that it arrived in midges travelling under their own steam.
8. Is Schmallenberg virus going to spread to other parts of the UK and other countries? Yes, you can bet on that (just like bluetongue did).
9. Can I catch Schmallenberg virus? Honest answer: We don’t know. Possibly, but there have been no reports of human illness from areas where the virus is known to exist, so I wouldn’t worry too much.
10. Where can I find the latest news about Schmallenberg virus? Right here.
11. OK, one last time, why should I care? Because Schmallenberg virus is going to cost European and probably worldwide ecomonies millions of pounds. And that will affect you.

Tweet

Culicoides biting midges are widely distributed throughout the world and are vectors of internationally important livestock viruses, including bluetongue virus (BTV), African horse sickness virus (AHSV), Akabane virus and Epizootic haemorrhagic disease virus (EHDV). Bluetongue disease (BT) has gained considerable notoriety in recent years because of an unprecedented globalisation and climate change-mediated expansion of its range in Europe, resulting in BTV reaching areas with no historical record of the disease. The economic impact of outbreaks of BTV in these areas has been considerable as a result of both indirect costs (e.g. the restrictions placed on movement of infected ruminants) and direct losses from disease in both sheep and cattle. In addition, whilst vaccination campaigns across northern Europe eventually controlled outbreaks in this region,  it took approximately eighteen months from the initial incursion in 2006 to the deployment of vaccine in the field. During this lag period, attempts to control the spread of BTV were limited to the restriction of animal movement and the application of methods to control Culicoides midges (primarily through the use of pour-on pyrethroid insecticides to vulnerable stocks).

BTV is an arbovirus and therefore depends almost entirely on the occurrence of farm-associated populations of competent Culicoides biting midges for transmission to its ruminant hosts. As a period of extrinsic replication is required within these vectors, control measures directed at adults have the potential to reduce the spread of midge-transmitted diseases through shortening or interrupting their lifespan. Indeed, epidemiological transmission models of vector-borne diseases show that the adult lifespan is the single most important factor affecting risk of transmission. At present, the majority of approaches to control populations of biting midges are based upon the application of insecticides (primarily synthetic pyrethroids) which in northern Europe are most commonly applied to livestock, although systematic testing of compounds to date has demonstrated equivocal results. Wide scale larvicidal or adulticidal use of these compounds against Culicoides has not been considered sustainable because of the paucity of knowledge surrounding larval habitats and adult resting places, combined with increasing restrictions within the EU on untargeted use of pyrethroid insecticides. An alternative insecticide, Ivermectin, is effective in killing Culicoides species when applied intradermally or subcutaneously and also toxic to midge larvae when excreted in faeces (a potential breeding site) but has also been shown to be harmful to beneficial insects such as dung beetles. Farmers are therefore caught between the need to control populations of biting midges and the diminishing number of chemical insecticides as they are withdrawn because of their perceived risk to humans and the environment.

Entomopathogenic Fungus as a Biological Control for an Important Vector of Livestock Disease: The Culicoides Biting Midge. 2011 PLoS ONE 6(1): e16108. doi:10.1371/journal.pone.0016108
The recent outbreak of bluetongue virus in northern Europe has led to an urgent need to identify control measures for the Culicoides (Diptera: Ceratopogonidae) biting midges that transmit it. Following successful use of the entomopathogenic fungus Metarhizium anisopliae against larval stages of biting midge Culicoides nubeculosus Meigen, we investigated the efficacy of this strain and other fungi (Beauveria bassiana, Isaria fumosorosea and Lecanicillium longisporum) as biocontrol agents against adult C. nubeculosus in laboratory and greenhouse studies.
Exposure of midges to ‘dry’ conidia of all fungal isolates caused significant reductions in survival compared to untreated controls. Metarhizium anisopliae strain V275 was the most virulent, causing a significantly decrease in midge survival compared to all other fungal strains tested. The LT50 value for strain V275 was 1.42 days compared to 2.21–3.22 days for the other isolates. The virulence of this strain was then further evaluated by exposing C. nubeculosus to varying doses (108–1011 conidia m−2) using different substrates (horse manure, damp peat, leaf litter) as a resting site. All exposed adults were found to be infected with the strain V275 four days after exposure. A further study exposed C. nubeculosus adults to ‘dry’ conidia and ‘wet’ conidia (conidia suspended in 0.03% aq. Tween 80) of strain V275 applied to damp peat and leaf litter in cages within a greenhouse. ‘Dry’ conidia were more effective than ‘wet’ conidia, causing 100% mortality after 5 days.
This is the first study to demonstrate that entomopathogenic fungi are potential biocontrol agents against adult Culicoides, through the application of ‘dry’ conidia on surfaces (e.g., manure, leaf litter, livestock) where the midges tend to rest. Subsequent conidial transmission between males and females may cause an increased level of fungi-induced mortality in midges thus reducing the incidence of disease.

I’ve written quite a lot on MicrobiologyBytes about the growing threat from Bluetongue virus. New figures from the European Commission now show that BTV 8, the epidemic strain of the virus, has been virtually eradicated from mainland Europe after an extensive vaccine campaign.

Tens of thousands of cases of bluetongue, predominantly the BTV8 strain, were identified across Europe in 2007 and 2008. The numbers dropped significantly in 2009 and are on course for a further significant decline this year.

The latest figures from the European Commission show incidence of BTV 8 has declined to just two recorded cases, while rare European cases of other strains are also on the way down.

Bluetongue virtually eradicated in Europe. Farmer’s Guardian, 2 November 2010

Related:

• Bluetongue virus
• Midges and the emergence of bluetongue virus in northern Europe
• How does bluetongue virus survive the winter?

The replication cycle of viruses involves entry into host cells, synthesis of virus genes and proteins, assembly of progeny virus particles and their subsequent release. Along with the plasma membrane, viruses also have to interact with the endosomal and vesicular membranes during their replication in host cells. All cellular membranes are composed of lipids and proteins that are usually arranged in various micro domains. During infection of cells by enveloped viruses, the lipids present in both viral and cellular membranes mediate fusion and fission reactions to facilitate virus entry and release. Since non-enveloped viruses do not have a lipid envelope, it is generally believed that their entry mechanism does not involve membrane fusion activity and that these viruses are mainly released by cell lysis. Usually, non-enveloped viruses enter the cells by penetrating the membrane barrier, either via the endocytic pathway using clathrin-coated vesicles, or by the formation of a pore at the cell surface. Recent data obtained from biochemical and structural studies indicate that the overall mechanisms of both entry (Reoviridae) and release of certain non-enveloped viruses (e.g., members of the Picornaviridae and Reoviridae) are analogous to that of enveloped viruses, and that the capsid proteins can function in these activities in a similar manner to the membrane viral proteins. This review discusses the role of lipids in the entry, maturation and release of non-enveloped viruses, focusing mainly on Bluetongue virus (BTV).

Role of Lipids on Entry and Exit of Bluetongue Virus, a Complex Non-Enveloped Virus. Viruses 2010 2 (5) 1218-1235. doi:10.3390/v2051218

Related:

• Bluetongue virus
• Lipid Membranes in Poxvirus Replication
• How the antiviral protein viperin works

There is strong evidence to suggest that climate change has, and will continue to affect the occurrence, distribution and prevalence of livestock diseases in Great Britain (GB). This paper reviews how climate change could affect livestock diseases in GB. Factors influenced by climate change and that could affect livestock diseases include the molecular biology of the pathogen itself; vectors (if any); farming practice and land use; zoological and environmental factors; and the establishment of new microenvironments and microclimates. The interaction of these factors is an important consideration in forecasting how livestock diseases may be affected. Risk assessments should focus on looking for combinations of factors that may be directly affected by climate change, or that may be indirectly affected through changes in human activity, such as land use (e.g. deforestation), transport and movement of animals, intensity of livestock farming and habitat change. A risk assessment framework is proposed, based on modules that accommodate these factors. This framework could be used to screen for the emergence of unexpected disease events.

The effect of climate change on the occurrence and prevalence of livestock diseases in Great Britain: a review. J Appl Microbiol. (2009) 106(5): 1409-1423

Related:

• Bluetongue virus
• MicrobiologyBytes: Climate Change

Bluetongue is a disease of ruminants caused by Bluetongue virus (BTV) and it is transmitted by biting midges. In sheep, clinical signs of BTV infection can include fever, vasodilation, swelling and, in severe cases, death, although the severity of symptoms varies with the breed of sheep, the individual animal and the strain of virus involved. Cattle are a major reservoir host for BTV infection, primarily because of the less obvious clinical signs in these animals. After an exhaustive search for a natural agent of transmission, Culicoides midges were shown to be the vectors for Bluetongue virus (Culicoides and the emergence of bluetongue virus in northern Europe. Trends Microbiol 2009 17(4): 172-178).

Although BTV infection was initially centred in Africa, the virus was first detected in Greece in 1989, from where it has spread steadily north. Since the disease is spread exclusively by insects, and because the virus is quite specific about which midge species can be used as vectors, predictions about the spread of the disease were based on known ranges of different midge species, which in turn depends on climate. However, midges can sometimes be carried over very long distances by weather systems, or by ships or aircraft.

Despite widespread speculation regarding the exact origin of BTV-8 as the strain of the virus found in northern Europe, no single convincing hypothesis has been proposed. Although future full-genome sequencing might assist this task (as was the case in the incursion of West Nile virus into North America), the small number of reference strains of BTV-8 from areas of potential origin collected before the incursion into northern Europe makes it unlikely that this approach will provide unambiguous evidence. As long as our understanding of the potential routes of virus introduction remains poor, we will be unable to accurately estimate the potential for future introductions of BTV, as has been illustrated by the more recent detection of BTV-6 in Europe, or of other midge-borne arboviruses, such as African horse sickness virus (AHSV).

Subscribe to podcasts (free):
[iTunes] Enhanced podcasts & videos
[RSS] mp3 podcasts (audio only)
Play this episode: Enhanced version | Audio only

Although the technology to produce safe, effective, inactivated vaccines existed, no coordinated action was taken by any Member State of the European Union (EU) to begin production of a BTV-8 vaccine until late 2007, when the full damage began to become evident. This was in part due to the assumption that the virus would not overwinter under northern European conditions (despite the fact that BTV had been documented overwintering successfully in other areas with far cooler winter temperatures). In the absence of an available vaccine, knowledge concerning the entomology of the insects involved in BTV transmission became paramount.

The spread of BTV has provided a severe test of the way in which the movement of vector-borne pathogens is predicted, identified and controlled in Europe. There are many arobovirus diseases (spread by arthropod vectors such as midges, mosquitos and ticks), affecting human as well as animal health. Whether BTV represents a herald for future incursions by other arboviruses into Europe remains difficult to know. It is clear that there exists a similar potential for emergence of other insect-borne pathogens on grounds of climate alone, but where different vectors are used – for example, in the case of AHSV – the dynamics of the current BTV outbreak cannot easily be used to estimate risk. What has been shown by this outbreak is that arbovirus–vector relationships are highly dynamic and extremely difficult to combat. Unless regions that are potentially at risk of transmission are prepared to invest the resources required to provide adequate information regarding vectors and suitable control methods, this will remain the case.

Related:

• Bluetongue virus
• How does bluetongue virus survive the winter?
• Bluetongue virus polymerase

Bluetongue is a vector-borne virus disease of ruminants that is endemic in tropical and subtropical countries. Since 1998 the virus has also appeared in Europe. Partly due to the seriousness of the disease, bluetongue virus (BTV), a member of genus Orbivirus within the family Reoviridae, has been a subject of intense molecular study for the last three decades and is now one of the best understood viruses at the molecular and structural levels. Viruses of the family Reoviridae, including BTV and other orbiviruses, are characterized primarily by their genome of 10–12 segments of linear, double-stranded RNA (dsRNA). Almost all of these separate segments represent single genes, generating a total of 10–13 virus proteins. The virions are non-lipid-containing icosahedral capsid structures, usually with an outer capsid layer surrounding an inner capsid or core that contains the genome. Shortly after cell entry, this outer capsid is removed to release the inner capsid within which the genome remains sequestered from the cellular triggers of innate immunity. Cores must necessarily, therefore, carry all the transcription machinery of the virus, synthesizing and extruding multiple-capped positive-sense RNAs from each genomic segment into the host cell cytoplasm. Current models for the transcription of the dsRNA genome are based on the polymerase complex contacting the template RNA and the nascent transcript being directed out of the core particle through a pore on its surface. This requires the efficient co-ordination of some half-a-dozen enzyme activities, including helicase, polymerase and RNA capping activity. Considerable advances have been made in recent years in understanding the replicase complexes of these viruses, including BTV. In some cases, 3D structures have complemented this analysis to reveal the fine structural detail of these proteins. The combined activities of the core enzymes produce infectious transcripts necessary and sufficient to initiate BTV infection. Such infectious transcripts can now be synthesized wholly in vitro and, when introduced into cells by transfection, lead to the recovery of infectious virus. Future studies hold the possibility of analysing the consequence of mutation in a replicating virus system.

Bluetongue virus: dissection of the polymerase complex. J Gen Virol 2008 89: 1789-1804

Related:

• MicrobeTalk Podcast on this paper and more information from SGM
• Bluetongue virus

In 2006, Bluetongue virus – which infects livestock – reached Northern Europe for the first time. Some people thought that the outbreak would be limited to that particular year, as winter was expected to kill off the midges that host and spread the disease, bringing the threat of infection to an end. In actuality, the disease escalated in the following year, spreading to the UK. So, how did the virus survive the winter? The answer to this question is of great practical importance, as it will affect both national and international trade of ruminants, the livestock susceptible to infection, and will dictate trade rules for a long time even after the infection has passed. The answer is also relevant to how we can deal with bluetongue and other unpleasant midge-transmitted diseases in the future.

Although the major mechanism of bluetongue virus spread is undoubtedly that of Culicoides midges feeding on infected ruminants, growing the virus and then transmitting it to further susceptible animals, other mechanisms may also be at work. These may assume greater importance during the midge-free season (winter), such as we in northern latitudes experience. Evidence to date does not support the winter survival of bluetongue virus in the eggs of Culicoides midges. An alternative hypothesis is that, in mild winters such as that of 2006-07 in northern Europe, sufficient infected midges might survive until they become active again in spring. The midges may enter livestock barns to overwinter. Two other possibilities for disease endurance during winter are that bluetongue is spread by some susceptible species of long-lived ticks and/or by simple mechanical transmission by Melophagus ovinus, a wingless parasite that lives in the fleece of sheep. Additionally, there is evidence from Australia that bluetongue virus can survive in midges and in a small proportion of infected cattle for three to four months, which would be long enough for winter to come and go without killing the virus. Closer to home, the recent outbreaks of bluetongue in northern Europe have provided evidence for a different overwinter route – transplacental infections; the virus spreading from an infected pregnant animal to its fetus, a phenomenon also demonstrated by experiment. This phenomenon might be particularly important in cattle, where the long gestation period of nine months (four for sheep) means that the virus can grow and survive within a fetus, at just the right temperature, throughout the coldest of winters. There is also circumstantial evidence that cattle could become infected orally if they eat the afterbirth of an infected offspring from another cow. Experiments have revealed a toolbox of possible mechanisms, with the potential to interact with and complement one another.

Where does bluetongue virus sleep in the winter? PLoS Biol 2008 6(8): e210