MdSGHV-infected salivary glands dissected from adult female house flies at different times post-infection (pi). Day 1: infected salivary glands are indistinguishable from control glands. Day 2, Day 3: individual cells in glands appear somewhat swollen. Day 4, Day 5: glands display diagnostic hypertrophy.

Light micrographs of sections through the healthy (top) and hypertrophied (bottom) salivary glands. MdSGHV-infected cells and nuclei are both swollen relative to control cells. The massive swelling of infected cells does not result in cell lysis, and hypertrophy symptoms are retained throughout the life of the host fly. Infected insects do not recover from viral infection.

Epifluorescent micrograph of control and infected salivary glands stained with Alexa-Fluor phalloidin (green) and counterstained with DAPI (blue).

TEM of a nucleus from an MdSGHV-infected host salivary gland cell. Note the formation of numerous nucleocapsids.

Series of TEM micrographs depicting viral nucleocapsids being released from the host cell nucleus and acquiring an envelope in the cytoplasm. Enveloped virions migrate to, and are released into, the lumen (adapted from Geden et al. 2008).

Ovaries excised from 7-day-old female house flies. Fully developed ovaries of a healthy female (left) contain approx. 60 eggs per ovary, whereas the ovaries of an infected female (right) are halted in the pre-vitellogenic stage (adapted from Lietze et al. 2007).

Virus Biology

Pathology

Replication

Transmission

Impact on Reproduction

Replication

In all three fly hosts, the SGH virus replicates in the salivary gland tissue of both male and female adult flies.  Infected adults do not exhibit any external disease symptoms, although overt infections can occasionally be  diagnosed using a dissecting microscope by the presence of whitish, swollen salivary glands underlying the intersegmental regions of the ventral abdomen. The development of hypertrophy symptoms in host flies may occur as early as two days after infection, and is marked by the gradual swelling of the gland pair. In the case of Musca domestica, the gland pair extends the length of the host body. Each salivary gland is comprised of about 4,000 cells, all of which appear to be susceptible to MdSGHV.

The cell hypertrophy induced by MdSGHV infection does not induce either nuclear or cellular proliferation; approximately 4,000 cells/gland are detected both in healthy glands and in glands at the late infection stage.

Transmission electron microscopy (TEM) of infected salivary glands has revealed that nucleocapsid morphogenesis takes place in the host cell nucleus.  Mature nucleocapsids migrate through pores in the nuclear membrane, acquire an outer membrane in the cytoplasm, and directionally translocate to the luminal surface.  At the luminal cell membrane, numerous enveloped virus particles have been observed to bud out and accumulate in the lumen of the gland.

The SGHVs are believed to replicate in other tissues in addition to those in the salivary gland. In tsetse flies, the GpSGHV replicates in the reproductive tissues, including the milk glands (the modified female accessory glands that provide nutrition to the larval stages during their intra-uterine development). Replication in the milk gland provides an avenue for the vertical transmission of this virus to progeny flies.  Electron microscopy and RT-PCR of infected housefly tissues has provided evidence that, like the GpSGHV, the MdSGHV is able to infect, and undergo partial transcription in, non-salivary-gland tissues (Lietze et al., in preparation).

The virus particles produced in salivary secretions accumulate in the crop and are released onto food substrates during feeding. Using quantitative real-time PCR with virus gene specific primers, Lietze et al. (2009) have recently calculated that over one million virus particles are released per feeding event. Progeny virus in salivary secretions is detected as early as 2 days pi, and after 4 days pi, ~ 1 x 106 copies per feeding event are released. This value remains relatively constant over the lifespan of the infected house fly (up to 21 days).

A series of assays measuring aminopeptidase, esterase, lipase, and carbohydrase activities have not revealed any significant differences in enzyme concentrations between healthy and infected glands dissected at five days pi. An azocasein feeding assay was conducted on both male and female hosts to examine if viral infection inhibited the capability of house flies to ingest and digest a protein-rich food source. Analysis of consumption data demonstrated that infected females ingested more food than did healthy male and female flies.  However, based on the levels of p-nitroaniline released in the gut lumen, both male and female healthy flies had ~ 30% more proteolytic activity than their infected counterparts (Lietze et al. 2007).

Consumption and digestion of azocasein-laced food by healthy and MdSGHV-infected house flies (adapted from Lietze et al. 2007). Different letters indicate statistically significant differences.

Transmission

Consumption of contaminated substrates has been shown to be the major mode of horizontal transmission  for MdSGHV. An infected house fly can deposit 106 infectious virus particles within a single droplet of salivary secretion. Virus particles have also been detected in the fecal deposits of infected flies. The MdSGHV has been experimentally transmitted to adult house flies by feeding individual uninfected flies the salivary secretions of infected flies, by maintaining healthy flies in confined mixed populations with infected flies, and by introducing healthy flies into cages that had previously housed large groups of infected flies (Lietze et al. 2009, Geden et al. 2008). In feral populations, a significant positive correlation between fly density and MdSGHV infection rates has been detected (Geden et al. 2008). However, mating experiments between healthy and early-stage infected house flies provide no evidence for vertical transmission of the MdSGHV (Lietze et al. 2007).

By contrast, transmission of the GpSGHV within wild populations of hematophagous tsetse flies is believed to be vertical, from mother to offspring. In this case, virus transfer may either occur transovarially to the embryo or viviparously through the infected milk gland to the developing larvae (Jura et al. 1989, Sang et al. 1996, 1998). In laboratory-reared colonies of tsetse flies, the GpSGHV can also be transmitted horizontally when infected and healthy flies feed on a common blood-containing membrane (Abd-Alla et al. 2007).

MdSGHV Impacts on Reproduction

SGHV-infection has a major inhibitory impact on host fecundity. In house flies, the MdSGHV sterilizes females by inhibiting vitellogenesis (Lietze et al. 2007). Female flies infected with the MdSGHV before the onset of the first gonadotropic cycle will never develop eggs. A mated female infected after developing a full complement of eggs will be able to fertilize and deposit her eggs, which then hatch and develop into healthy adults. However, the subsequent gonadotropic cycles of the female fly will be completely inhibited. Reproductive inhibition is accompanied by reduced levels of yolk protein and female-specific hexamerin in the hemolymph of infected females.

Infection also alters the complex courtship behavior of house flies (Lietze et al. 2007). Late-stage infection renders female flies unresponsive to male mating attempts. By contrast, MdSGHV-infected male house flies are able to inseminate healthy females with viable sperm. However, at a late stage of infection, the ardor of males to induce courtship is significantly reduced.

The GpSGHV reduces the reproductive potential of both genders of the tsetse host. Viral replication in gonadal tissues often leads to ovarian abnormalities or to testicular degeneration in the infected female or male tsetse fly, respectively (Jura et al. 1988, Feldmann et al. 1992). Infected females produce significantly fewer progeny than healthy females (Sang et al. 1998). Only 5% of experimentally infected male tsetse flies were shown to form a fully functional spermatophore, which is necessary to successfully inseminate female flies (Sang et al. 1999). However, despite their sterility, virus-infected tsetse males retain a normal mating efficiency (Jura and Davies-Cole 1992).