Viral control with endosymbiotic bacteria - the dengue story

No sooner did we hear that we have effectively caused the extinction of one important virus from the earth, we get news of the dramatically increasing incidence of mosquito-borne Dengue virus (DENV) infection worldwide with the numbers doubling over the last decade (two-fifths of the global population now at-risk estimated at 2.5 billion individuals). Dengue is a textbook example of an emerging disease although has been largely ignored in most developed countries as it is mostly found in urban districts of tropical/sub-tropical countries. It is now considered the most important arthropod-borne viral disease and developed countries should take note as DENV is predicted to expand its range with a changing climate.

Fig.1. WHO stats on DENV distribution 2005

Dengue viruses cause what is known as a severe “flu-like” illness including symptoms such as rash, mild/high fever, headache and muscle pain. Although it is rarely fatal, in some instances it can develop into Dengue Haemorrhagic Fever (DHF), a potentially deadly complication. There are generally considered four sub-types (DENV-1 to 4) of Dengue virus due to the sufficient antigenic differences exhibited between them leading to little or no effective cross-neutralisation making it difficult to develop a ‘universal’ Dengue vaccine or treatment. The viruses are spread by certain Aedes species of mosquito passing infectious virus on during feeding by the females and due to the lack of treatments, the inhibition of mosquito activity has been seen as key to Dengue prevention.

A number of ‘vector control’ methods are being carried out to limit mosquito-human viral transmission which focus on disrupting the ecological niche in which the vector requires to breed – in this case stagnant water pools found in urban areas in which larva are found (e.g. rainwater filled tyres and cups). One major strategy is the biological control of Aedes populations including the use of mosquito larvae preying fish and invertebrates, release of Aedes infecting viruses or by genetic modification of mosquito genomes. An attractive means of control has come from the realisation that certain bacterial species (Wolbachia) - and only a specific strain of it - which naturally infects mosquitoes and other insects could inhibit dengue virus transmission thus negating the need for transgenic mosquitoes in the environment. These obligate, intracellular parasites which survive in the host cell’s cytoplasm are passed maternally from generation to generation. The bacteria can affect the mosquitoes and dengue virus in a number of ways, both general and specific for example: reduced lifespan of Aedes hosts; reduced dengue virus replication; increased antiviral immunity; and spatial exclusion of virus from the cytoplasm.

Fig.2. DENV transmission in Aedes species http://activity.ntsec.gov.tw/lifeworld/english/content/images/en_dis_c10.jpg

Frentiu et al report their investigations into the mechanism of reduced viral (dengue virus serogroup 2) replication at the cellular level paralleling earlier observations of whole organism Wolbachia infection. Their transition to a cell-culture experimental system may facilitate easier study of viral-host interactions and the group documents significantly reduced dengue virus replication in Wolbachia infected cell lines compared to non-infected controls possibly being related to an increased bacterial density within the cytoplasm. A bacterial ‘priming’ of the insect immune system may also contribute to decreased replication. They also show evidence that Wolbachia infected mosquitoes may display a fitness benefit compared to those not infected when challenged with dengue virus. This predicted increased fitness in the wild may aid the use of this biological control technique in a natural ecosystem. Measures such as these will benefit from the large, field trials already planned to study Aedes-Wolbachia interactions.

Fig.3. Wolbachia (Green) within a drosophila embryo imaged by confocal microscopy. http://www.genetics.org/content/vol178/issue4/cover.dtl

Dengue virus is an important emerging arthropod-borne pathogen worldwide and is predicted to further increase its range into more temperate regions. Currently there is no effective vaccine or treatment and much research has focused on the interactions between virus and Aedes mosquito including the use of intracellular bacterial pathogens to limit viral replication and transmission. A number of strategies have been studied and seem attractive on a wider scale in endemic countries however the mechanisms of DENV/Wolbachia interactions and the effects on arthropods in the wild are understudied. Frentiu et al highlight the importance of a mechanistic understanding of dengue control and the development of novel control strategies.

Of owls and viruses

Viral Research in Brazilian Owls (Tyto alba and Rhinoptynx clamator) by Transmission Electron Microscopy

^*Catroxo, M. H. B.; ^*Taniguchi, D. L.; ^*Melo, N. A.; ^**Milanelo, L.; ^***Petrella, S.; ^**Alves, M.; ^*Martins, A. M. C. R. P. F. & ^*Rebouças, M. M.

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SUMMARY: The barn-owl (Tyto Alba) and striped-owl (Rhinoptynx clamator) belong respectively to the families Tytonidae and Strigidae. Avian paramyxoviruses have been isolated from a variety of species of wild and domestic birds wordlwide causing diverse clinical symptoms and signs. Paramyxoviruses belong to the family Paramyxoviridae and Avulovirus genus, including nine serotypes (APMV 1 to 9). The lymphoid leukosis is a retrovirus-induced neoplasia. The avian retroviruses belong to the Retroviridae family and to the Alpharetrovirus genus. Coronaviruses can cause respiratory and enteric disease in several species of birds. They belong to the Coronaviridae family and to the groups 3a e 3c. In this study, we describe the presence of viruses in four owls, two barn owls (Tyto alba) and two striped owls (Rhinoptynx clamator), rescued from tree-lined streets of Sao Paulo, Brazil and sent to the Recovery Center of Wild Animals of the Tietê Ecological Park, where the animals died. Fragments of lung, liver and small intestine of these birds were processed for transmission electron microscopy utilizing negative staining (rapid preparation), immunoelectron microscopy and immunocitochemistry techniques. Under the transmission electron microscopy paramyxovirus particles, pleomorphic, roughly spherical or filamentous, measuring 100 to 500 nm of diameter containing an envelope covered by spikes, an herring-bone helical nucleocapsid-like structure, measuring 15 to 20 nm in diameter, were visualized in the samples of lung, liver and small intestine of all owls. In small intestine samples of the two striped-owl (owls 3 and 4) it was detected pleomorphic coronavirus particles with a diameter of 75-160 nm containing a solar corona-shaped envelope, with projections of approximately 20 nm of diameter. In liver fragments of one striped-owl (owl 4) pleomorphic particles of retrovirus with a diameter of 80-145 nm containing an envelope with short projections and diameter of 9 nm were observed. The presence of aggregates formed by antigen-antibody interaction, characterized the positive result obtained during the immunoelectron microscopy technique for paramyxovirus, retrovirus and coronavirus. In the immunocytochemistry technique, the antigen-antibody interaction was strongly enhanced by the dense colloidal gold particles over these viruses.

Fig.1 Barn owl on the left. http://curiousanimals.net/funnies-bunnies/night-queens-owls/ and Striped owl on the right. http://www.freewebs.com/gahoolejake/owlpics.htm

It is said that for every species there is at least one virus that infects it – no organisms are exempt from these obligate intracellular parasites. Viral infection can be a serious problem in not just individual hosts but for whole ecosystems, with pathogenesis leading to fatalities with knock-on effects for local communities especially in those species that are already endangered.

Avian species are not resistant to these problems and a number of diverse viruses are known to infect these animals causing a range of debilitating pathologies. Notable viral pathogens causing significant disease are Avian paramyxoviruses including Newcastle disease virus (Avian paramyxovirus-1) which can be fatal, oncogenic retroviruses and the coronaviruses (remember SARS?) which can lead to severe respiratory illness. Detailed knowledge of the number and type of viral infections is thus a principle element in preventing pathogenesis in both wild and farmed avian species.

In order to generate valuable data on this subject, Catroxo et al publish in the International Journal of Morphology the detection of a number of viruses from two owl species - The barn-owl (Tyto Alba) and striped-owl (Rhinoptynx clamator) - from a small area of Brazil. The used electron microscopy (see video) and immunohistochemical techniques to detect the presence of viral particles in tissue samples (lung, liver and small intestine) taken from four necropsied owls – a limited sample size - who were recovered from the streets of Sao Paulo and later died in care. Symptoms displayed when found were also recorded.

Fig.2. Electron microscopy pictures of Paramyxovirus (top), Retrovirus (middle) and Coronavirus (bottom) virions from tissues of infected owls

Using the morphological characteristics of virions and antibody binding, they discovered viruses which had features of and resembled paramyxoviruses, coronaviruses and retroviruses and were able to relate these to known symptoms described. Although limited in detection of distinct types of virus (no genetic methods were used), these results further extend the list of species susceptible to paramyxoviruses which is important in determining the effects of wild bird movements of disease spread. The chance of detecting a novel, unknown virus is possible as no matching up of virions to distinct viral species was carried out. The ability to match up viral infection to symptoms in a diverse range of species allows us to study the variation in the development of pathogenesis.

This study highlights the importance of virus tracking in a diverse range of species across the world. Viruses infecting avian species can have a profound impact on wild populations and communities as well as our farmed poultry (and could also impact directly on our health like Influenza viruses). Even though the use of morphological detection of viruses has its limits when compared to the more detailed genome based analyses like PCR and sequencing, it can still readily inform us about the general range of pathogens in a sample. This is important in preventing viral disease worldwide and in local populations.