Tissue tropism of the Musca domestica salivary gland hypertrophy virus

Abstract

The tissue tropism of Musca domestica salivary gland hypertrophy virus (MdSGHV) infecting adult house flies was examined by transmission electron microscopy (TEM) and quantitative real-time PCR. TEM demonstrated that characteristic MdSGHV-induced nuclear and cellular hypertrophy was restricted to the salivary glands. Both nucleocapsids and enveloped virions were present in salivary gland cells. In contrast, thin sections of midguts, ovaries, abdominal fat body, crops, air sacs and brains showed the presence of enveloped virions in vacuoles of tracheal cells associated with these tissues. However, no sites of viral morphogenesis were detected in the tracheal cells. Quantitative analysis of MdSGHV DNA and transcript titers revealed that viral DNA was present in all hemolymph and tissue samples collected from MdSGHV-infected flies. Average numbers of MdSGHV genome copies per 50 ng of DNA varied significantly between examined tissues and ranged from 3.83 × 10(8) (±3.75 × 10(7)) in salivary gland samples to 7.98 × 10(5) (±2.91 × 10(5)) in hemolymph samples. High levels of viral genome copies were detected in midgut, fat body and brain samples. Viral transcripts were present in all examined samples, and transcript abundance was also at the highest level in salivary glands and at the lowest level in hemolymph. However, over the range of different tissues that were analyzed, there was no correlation between estimated quantities of genome copies and viral transcripts. The function of viral transcripts in host tissues that do not show sites of viral morphogenesis remains to be elucidated.

Fig. 1

Light micrographs of thick sections through (A) healthy and (B) MdSGHV-infected salivary glands. Images were taken at identical magnification with bars equal to 50 μm. The salivary gland supports viral replication along its entire length. The relative number of cells in healthy and infected glands remains constant; the enlarged state of the infected gland is due to extensive cellular and nuclear hypertrophy induced by viral replication. Cp, cytoplasm; nu, nucleus.

Fig. 2

Transmission electron micrographs of hypertrophied salivary glands. (A) The enlarged nuclei were characterized by virogenic stroma within which the nucleocapsids (back arrows) were assembled and then (B) translocated to the nuclear membrane where they were observed to exit (black arrow) the nucleus via the nuclear pores and to be released (white arrow) into the region of the perinuclear cistern. (C) Virus particles (white arrow) appear to be associated with endoplasmic reticulum. (D) Both clusters (white arrow) and individual (black arrow) enveloped virions are found throughout the cytoplasm. (E) The enveloped virions (black arrow) migrate preferentially to the cell membrane (white arrow) and are released into the gland lumen via a budding process. (F) As the infection progresses the lumen becomes filled with enveloped virions (black arrows).

Fig. 3

Transmission electron micrographs of the MdSGHV-infected house fly midgut. (A) The food bolus enclosed by a well-developed peritrophic matrix contains numerous enveloped virions (see insert and black arrows). (B) Virions (see insert and black arrow) were detected in vesicles in the cytoplasm of the midgut columnar cells, note microvilli. Mv, microvilli; PM, peritrophic matrix.

Fig. 4

Electron micrographs of the MdSGHV (A) in tracheoles associated with the ovaries (see insert), (B) in the abdominal air sacs, and (C, D) the air sac in the head tagma that is closely opposed to the brain tissue. Black arrows in all figures indicate virions. White arrow in (B) indicates the basement membrane at the hemolymph face. AS, air sac; He, hemolymph; MF, muscle fibers; Tr, tracheoles.

Fig. 5

Average relative abundance of MdSGHV transcripts and genome copy numbers in different tissues of female house flies at 72 h post-infection. Abundance was estimated by real-time PCR and expressed relative to the levels in salivary glands. Numbers indicate the x-fold reduction of transcripts and genome copy numbers in each tissue sample relative to the abundance in salivary glands. For more details, refer to the methods section.