Discovery of two highly divergent negative-sense RNA viruses associated with the parasitic nematode, Capillaria hepatica, in wild Mus musculus from New York City

Abstract

Recent advances in high-throughput sequencing technology have led to a rapid expansion in the number of viral sequences associated with samples from vertebrates, invertebrates and environmental samples. Accurate host identification can be difficult in assays of complex samples that contain more than one potential host. Using unbiased metagenomic sequencing, we investigated wild house mice (Mus musculus) and brown rats (Rattus norvegicus) from New York City to determine the aetiology of liver disease. Light microscopy was used to characterize liver disease, and fluorescent microscopy with in situ hybridization was employed to identify viral cell tropism. Sequences representing two novel negative-sense RNA viruses were identified in homogenates of wild house mouse liver tissue: Amsterdam virus and Fulton virus. In situ hybridization localized viral RNA to Capillaria hepatica, a parasitic nematode that had infected the mouse liver. RNA from either virus was found within nematode adults and unembryonated eggs. Expanded PCR screening identified brown rats as a second rodent host for C. hepatica as well as both nematode-associated viruses. Our findings indicate that the current diversity of nematode-associated viruses may be underappreciated and that anatomical imaging offers an alternative to computational host assignment approaches.

Keywords: Amsterdam virus; Capillaria hepatica; Fulton virus; Mus musculus; New York City; bunyavirus; mononegavirus; nematode; virome; wild house mice.

Fig. 1.

Map of New York City with house mouse (green dot) and brown rat (red dot) trap locations.

Fig. 2.

Phylogenetic analysis and genome organizations of Fulton virus (a) and Amsterdam virus (b). Maximum-likelihood trees were constructed using RdRp protein domain sequences for mononegaviruses and bunyaviruses (pfam00946 and pfam04196, respectively). The scale bar represents substitutions per site and bootstrap support values are displayed when greater than 70 %. Representative viral genera are highlighted in blue. Viruses identified in this study are marked by a red star and the proposed genera are highlighted in orange. Genome illustrations indicate putative open reading frames (green arrows). Genome length is not to scale; numbers indicate nucleotide position. Terminal sequences (TSs) for Fulton virus are indicated with red boxes. Glycoproteins are labelled with hydrophobic domains (grey shading) and predicted cleavage sites (CS) with corresponding amino acid position. The Fulton virus N-terminal glycoprotein (Gn), non-structural movement protein (NSm) and C-terminal glycoprotein (Gc) are demarcated in blue.

Fig. 3.

Ventral abdominal cavity view demonstrating an enlarged liver (white arrows) with multifocal to coalescing areas of pallor from a mouse heavily infected with C. hepatica (left) compared to a normal liver (blue arrows) from a non-infected mouse (right).

Fig. 4.

Haematoxylin and eosin-stained section of liver from a mouse heavily infected with C. hepatica. Adult worms (blue arrow, ovum containing eggs; black arrow, intestine) and free eggs (white arrow) were located within the liver parenchyma and associated with multifocal to coalescent granulamatomous inflammation with central necrosis and multinucleated giant cells (orange arrow). This microscopic finding corresponded to the enlarged liver and areas of pallor depicted in Fig. 3. Insert: C. hepatica egg with characteristic bipolar plugs.

Fig. 5.

Fluorescent in situ hybridization demonstrating the presence of viral RNA within C. hepatica: white arrow, uninfected egg; red arrow, viral RNA-positive egg; grey arrow, air bubble; blue arrow, adult ovum; green arrow, adult intestine; orange arrow, AMSV RNA localized in adult nematode; (images from left to right) DAPI stain for cell nucleus, fluorescence ISH for viral RNA and overlay for co-localization. (a) Fulton virus RNA in nematode eggs; insert of higher magnification of virus-infected egg. (b) Amsterdam virus RNA in nematode egg; insert of higher magnification of virus-infected egg. (c) Amsterdam virus RNA in nematode adult and free egg.