Figure 2.

Schematic representation of BRSV replication, images of its cytopathic effect in cell culture, and histological changes in an infected lung. (A) Viral entry and replication cycle. (1) Viral attachment and viral entry through (2a) direct fusion to the host cell membrane mediated by F;or (2b) through virions that are internalized via macropinocytosis and clathrin- or caveolin-mediated endocytosis, with fusion taking place in an endosome. (3) Upon fusion, the genome is released in the cytoplasm. (4) Transcription of mRNAs. (4a): Transcription occurs in an obligatorily sequential, polarized manner to generate 10 sub-genomic mRNAs following a gradient in relation to the order in which the genes appear in the ssRNA (genes located closer to the 3′ end of the ssRNA molecule are transcribed at higher levels than genes located towards the 5′ end). (5) The antigenome is replicated for progeny generation. (6) mRNA from step (4) is translated into viral proteins, except for G, F, and SH. (7) Glycoproteins G, F, and SH are synthesized in the rough endoplasmic reticulum (RER). (8) Glycoproteins G, F, and SH undergo maturation, modification, and packaging in the Golgi complex and are transported in vesicles to the cell membrane. (9) Viral assembly: The N protein binds to the newly synthesized viral RNA genome, forming a ribonucleoprotein (RNP) complex, which also includes other genome-associated proteins (P, L, and M2-1). The matrix (M) protein interacts with the RNP complex and assists its transport to the plasma membrane, where assembly occurs. The M protein bridges the RNP complex with the inner surface of the cell membrane, where the G, F, and SH proteins are embedded. (10) Newly formed particles bud off or stay associated with the host cell membrane. (B) Immunofluorescence staining of BRSV (green) in bovine turbinate infected cells displaying characteristic syncytia formation (white arrows). Cells were infected with BRSV (375) (MOI: ~0.5) and staining was performed 48 h post-infection using 0.01% BRSV anti-serum (VMRD^TM, Pullman, WA, USA) and 0.4% Alexa Fluor 488 (SouthernBiotech^TM Birmingham, AL, USA). Cell nuclei (blue) were counterstained with DAPI (4′,6-diamidino-2-phenylindole). (C) Bronchioles and alveolar spaces contain small to moderate amounts of edema, necrotic cellular debris, foamy macrophages, and fibrin. Rare syncytial cells are within bronchioles or alveolar spaces (arrowhead) (40×, H&E). (D) Histological findings in a naturally infected calf showing bronchointerstitial pneumonia associated with BRSV. This animal also tested positive for Histophilus somni. Alveoli and bronchioles contain degenerate neutrophils, foamy macrophages, edema, and fibrin. Alveoli in most severely affected areas are indistinct. Bronchioles contain abundant cellular exudate (arrow) (10×, H&E). (Image (B) is courtesy of Dr. Mayara Maggioli; images (C,D) are courtesy of Dr. Alexandra K. Ford). Abbreviations: NS1 and NS2 = nonstructural proteins 1 and 2; N = nucleoprotein; P = phosphoprotein; M = matrix protein; SH = small hydrophobic protein; G = major attachment glycoprotein; F = fusion protein; M2 = matrix protein 2; L = large polymerase protein; mRNA = messenger RNA; RER = rough endoplasmic reticulum. Figure 2A was created using BioRender (BioRender.com/e26l126).