Models of Drosophila virus infection

Insects are susceptible to highly diverse families of RNA and DNA viruses (Friesen and Miller, 2001). Interestingly, many members of these insect virus families have counterparts in vertebrates. This is the case for insect viruses that belong to the families Poxviridae, Reoviridae, Picornaviridae, or Parvoviridae, and this observation suggests that viruses of vertebrates and invertebrates have common origins. The unusual conservation of specific genes found in baculoviruses, entomopoxviruses, and orthomyxoviruses also suggests an evolutionary link between these diverse virus families. More than 25 viruses have been documented to infect Drosophila, and the effects of few of these have been studied recently (Huszar and Imler, 2008). Below we review briefly the main characteristics of the viruses that naturally infect Drosophila or which were isolated from other insects, but can infect Drosophila cells and be used to study antiviral reactions in flies.

4.2.1 Drosophila viruses

4.2.1.1 Sigma virus

Sigma virus (SIGMAV) is widespread in natural populations of Drosophila and is one of the best characterized of the viruses infecting fruit flies. SIGMAV is a member of the Rhabdoviridae, an important family of RNA viruses infecting both animals and plants. Insects play a central role in the horizontal transmission of these viruses to either plants or insects (Hogenhout et al, 2003). SIGMAV is atypical, in that it has no known vertebrate or plant hosts, and only infects Drosophila in which it is maintained through vertical transmission via germ cells (Table 4.1). Rhabdoviruses are enveloped RNA viruses with a single-stranded RNA genome of negative polarity (ss(-)RNA), meaning that it has to be transcribed in infected cells before viral proteins can be translated. Virions have a characteristic bullet shape, with a length of 180-200 nm and a diameter of 60-80 nm.

Flies infected with SIGMAV suffer few adverse effects, which include reduced viability of infected eggs and lower survival over winter. In fact, the parameter used in the laboratory to monitor infection is the sensitivity of exposure to pure CO2, a treatment used routinely for brief anaesthesia of the flies. This artificial treatment has a dramatic effect on SIGMAV-infected flies, which become irreversibly paralysed, possibly as a result of viral proliferation in the central nervous system (Tsai et al., 2008). Five host loci have been proposed to be involved in the control of SIGMAV infection. The best characterized is ref(2)P, a strongly polymorphic gene from the second chromosome (see below).

4.2.1.2 Drosophila C virus (DCV) DCV is the best-studied Drosophila virus. It was first reported in 1972 in a laboratory stock that exhibited unusually high and unexplained lethality. DCV is a non-enveloped RNA virus that resembles picor-naviruses by many aspects of its structural properties and replication cycle, and belongs to a new family of RNA viruses, the Dicistroviridae, order Picornavirales (Le Gall et al, 2008) (Figure 4.1). The virion is a non-enveloped, icosaedral particle with a diameter of 30 nm containing a single-stranded

Table 4.1 Characteristics of viruses infecting insects.

Characteristics

Family

Representative member

dsDNA, enveloped

Baculoviridae

Autographa californica nuclear polyhedrosis virus (AcNMPV)

Polydnaviridae

Campoletis sonorensis virus (CsV)

Poxviridae

Amsacta moorei entomopoxvirus (AmEPV)

dsDNA, non-enveloped

Iridoviridae

Chilo iridescent virus (CIV)

ssDNA, non-enveloped

Parvoviridae

Galleria mellonella densovirus (GmDNV)

dsRNA, non-enveloped

Reoviridae

Bluetongue virus (BTV), Drosophila F virus (DFV)

Birnaviridae

Drosophila X virus (DXV)

ssRNA, enveloped

Togaviridae

Sindbis virus (SINV)

Flaviviridae

Yellow fever virus (YFV), West Nile virus (WNV)

Rhabdoviridae

Sigma virus (SIGMAV)

Bunyaviridae

Sandfly fever virus (SFSV)

Errantiviridae

Gypsy virus (GypV)

ssRNA, non-enveloped

Nodaviridae

Flock House virus (FHV)

Dicistroviridae

Drosophila C virus (DCV)

ds, double-stranded; ss, single-stranded ds, double-stranded; ss, single-stranded positive strand RNA genome (ss(+)RNA). The viral replication cycle of DCV has been studied extensively in vitro and in vivo. Viral particles are internalized by clathrin-mediated endocytosis (Cherry and Perrimon, 2004). DCV then replicates on cellular vesicles derived from the Golgi apparatus, and translation of viral proteins is highly sensitive to the levels of ribosomes in the cells (Cherry et al., 2006). The mechanism of assembly of the viral particles after replication and translation is still poorly characterized.

The outcome of the infection varies strikingly depending on the infection route. DCV is extremely pathogenic when injected intrathoraci-cally into adult flies, replicating to high levels in multiple tissues. By contrast, natural infection (by the oral route or also possibly the respiratory tract) does not lead to major symptoms of infection, and is almost non-pathogenic. DCV is not transmitted vertically and infection occurs exclusively between individuals, either at the larval or adult stages. Overall, these findings point to the existence of a complex network of interactions between DCV and its host (reviewed in Huszar and Imler, 2008).

4.2.1.3 Drosophila X virus (DXV) DXV was first identified as a contaminant in a series of experiments with SIGMAV. It was later found in many Drosophila cell lines, although it has never been found in wild populations of flies. The name DXV reflects the enigmatic origin of this virus.

DXV belongs to the Birnaviridae family. These viruses are characterized by a double-stranded (ds) RNA genome, and owe their name to their bipartite genome. The virions are non-enveloped, icosahedral particles with a diameter of 70 nm. In vivo interactions between DXV and Drosophila are poorly characterized. Adult flies injected with a suspension of DXV die 10-20 days after the injection, depending on the inoculum's concentration

DCV

1a

n

Hel

n

Pro

n

RdRP

(dsrm)

75% 1

53%

68% 1

CrPV

A

Hel

Pro

RdRP

©

ALPV

Hel

Pro

RdRP

TSV

u

Hel

u

Pro

u

RdRP

Figure 4.1 Schematic representation of the genome structure of members of the Dicistroviridae family. The genome is a single-stranded RNA molecule of positive polarity, with a covalently attached viral protein (VPg) at the 5' end and a poly(A) tail at the 3' end. It contains two open reading frames (ORFs) encoding non-structural (ORF1) and capsid (ORF2) proteins. Translation of viral proteins is regulated by two internal ribosome entry sites (IRES). The size of the three non-structural proteins encoded by DCV ORF1 is indicated, as well as the percentage of identity with the corresponding sequences in cricket paralysis virus (CrPV) ORF1. The variable domain at the N-terminal end of the polyprotein encoded by ORF1 in Drosophila C virus (DCV) and cricket paralysis virus suppresses host defence. APV, avian polyoma virus; BIR, baculovirus inhibitor of apoptosis repeat; DSRM, double-stranded RNA-binding motif; Hel, helicase; PRANC, Pox proteins repeats of ankyrin-C-terminal; Pro, protease; RdRP, RNA-dependent RNA polymerase; TSV, Taura syndrome virus.

(Zambon et al, 2005). As described above for SIGMAV, one symptom of infection by DXV is sensitivity to anoxia, which becomes apparent 5-7 days after infection. Viral particles in dead flies are found in the brain but also in several other organs (reviewed in Huszar and Imler, 2008).

4.2.1.4 Other Drosophila RNA viruses Other RNA viruses, belonging to the Reoviridae family, or as yet unclassified, have been reported in Drosophila. Drosophila F virus (DFV) has been identified as a latent virus in laboratory stocks and natural populations of D. melanogaster, and is also present in Drosophila tissue-culture cells. As a typical Reovirus, it is a non-enveloped virus with dsRNA genome. DFV virions are spherical particles of 60-70 nm diameter, with a capsid composed of two layers comprising eight polypeptides. These polypeptides are encoded by 10 segments of dsRNA. The replication cycle of DFV in Drosophila cells has not been studied.

Drosophila P virus (DPV) has been recovered from laboratory stocks and wild populations of flies, mostly from tropical areas. It has a single-stranded coding (positive-polarity) RNA genome and small (27-30 nm diameter) non-enveloped capsids, but differs significantly from DCV on the basis of serology, pathogenesis, and/or physico-chemical properties. The virus is much less virulent than DCV, and can be transmitted vertically through the female germ line. DPV has not been characterized molecularly at this stage, nor is its replicative mechanism known (reviewed in Huszar and Imler, 2008).

Finally, Nora virus causes persistent infection in D. melanogaster and is present in both laboratory stocks and wild populations, at a titre varying between 104 and 1010 genome copies per fly. It does not cause any obvious pathological effect. The viral particles are non-enveloped, with a diameter of about 30 nm, and contain a polyade-nylated positive-sense single-stranded RNA genome. Unlike other picorna-like viruses, the 11 879 nt RNA genome of Nora virus exhibits four open reading frames (ORFs) instead of one or two. Only the largest of these, ORF2, bears significant sequence similarity with picornavirus-like genes, and includes sequences coding an RNA helicase, a protease, and an RNA-dependent RNA polymerase (Habayeb et al., 2006).

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