Characterization of Macaque Pulmonary Fluid Proteome during Monkeypox Infection

Understanding viral pathogenesis is challenging because of confounding factors, including nonabrasive access to infected tissues and high abundance of inflammatory mediators that may mask mechanistic details. In diseases such as influenza and smallpox where the primary cause of mortality results from complications in the lung, the characterization of lung fluid offers a unique opportunity to study host-pathogen interactions with minimal effect on infected animals. This investigation characterizes the global proteome response in the pulmonary fluid, bronchoalveolar lavage fluid, of macaques during upper respiratory infection by monkeypox virus (MPXV), a close relative of the causative agent of smallpox, variola virus. These results are compared and contrasted against infections by vaccinia virus (VV), a low pathogenic relative of MPXV, and with extracellular fluid from MPXV-infected HeLa cells. To identify changes in the pulmonary protein compartment, macaque lung fluid was sampled twice prior to infection, serving as base line, and up to six times following intrabronchial infection with either MPXV or VV. Increased expression of inflammatory proteins was observed in response to both viruses. Although the increased expression resolved for a subset of proteins, such as C-reactive protein, S100A8, and S100A9, high expression levels persisted for other proteins, including vitamin D-binding protein and fibrinogen . Structural and metabolic proteins were substantially decreased in lung fluid exclusively during MPXV and not VV infection. Decreases in structural and metabolic proteins were similarly observed in the extracellular fluid of MPXV-infected HeLa cells. Results from this study suggest that the host inflammatory response may not be the only facilitator of viral pathogenesis, but rather maintaining pulmonary structural integrity could be a key factor influencing disease progression and mortality. Molecular & Cellular Proteomics 9:2760–2771, 2010. Monkeypox virus (MPXV) is a member of the Orthopoxviridae family and is closely related to variola virus, the causative agent of smallpox (1). Endemic to the rain forests of Central and West Africa, MPXV can cause potentially fatal smallpoxlike disease in both human and non-human primates (NHPs) (2). Human infection typically stems from zoonotic sources such as rodents and NHPs, although occasional human-tohuman transmission occurs (3). Awareness of the potential impact of this pathogen on global populations increased following the first incidence of human MPXV infection in the United States in 2003 (4, 5). An attenuated strain of vaccinia virus (VV), a closely related orthopoxvirus that serves as an effective vaccine for smallpox, can prevent disease caused by MPXV and variola. However, worldwide eradication of smallpox led to the discontinuation of routine vaccination for variola in the 1970s. As a result, there is growing concern regarding the reemergence of pathogenic orthopoxviruses such as MPXV introduced from either a zoonotic source or from bioterrorism (2). The replication cycle of all poxviruses occurs exclusively within the cytoplasm of an infected cell in which the virus hijacks cellular transcriptional and translational machinery to assemble viral factories, referred to as viroplasm (7), resulting in a general shift away from cellular protein expression toward viral mRNA and protein synthesis (8, 9). Importantly, the ability of poxviruses to replicate within a particular cell type is believed to typically be governed through postentry events, such as the proper recruitment of host cell machinery required for viral processes and attenuation of the innate immune response (10). Furthermore, the ability to regulate both the innate and adaptive immune responses is an extremely important aspect of poxvirus biology that governs viral replication and disease development in vivo. Some mechanisms