Nectin-4 Interactions Govern Measles Virus Virulence in a New Model of Pathogenesis, the Squirrel Monkey (Saimiri sciureus)

Abstract

In addition to humans, only certain nonhuman primates are naturally susceptible to measles virus (MeV) infection. Disease severity is species dependent, ranging from mild to moderate for macaques to severe and even lethal for certain New World monkey species. To investigate if squirrel monkeys (Saimiri sciureus), which are reported to develop a course of disease similar to humans, may be better suited than macaques for the identification of virulence determinants or the evaluation of therapeutics, we infected them with a green fluorescent protein-expressing MeV. Compared to cynomolgus macaques (Macaca fascicularis) infected with the same virus, the squirrel monkeys developed more-severe immunosuppression, higher viral load, and a broader range of clinical signs typical for measles. In contrast, infection with an MeV unable to interact with the epithelial receptor nectin-4, while causing immunosuppression, resulted in only a mild and transient rash and a short-lived elevation of the body temperature. Similar titers of the wild-type and nectin-4-blind MeV were detected in peripheral blood mononuclear cells and lymph node homogenates, but only the wild-type virus was found in tracheal lavage fluids and urine. Thus, our study demonstrates the importance of MeV interactions with nectin-4 for clinical disease in the new and better-performing S. sciureus model of measles pathogenesis.IMPORTANCE The characterization of mechanisms underlying measles virus clinical disease has been hampered by the lack of an animal model that reproduces the course of disease seen in human patients. Here, we report that infection of squirrel monkeys (Saimiri sciureus) fulfills these requirements. Comparative infection with wild-type and epithelial cell receptor-blind viruses demonstrated the importance of epithelial cell infection for clinical disease, highlighting the spread to epithelia as an attractive target for therapeutic strategies.

Keywords: cellular receptors; immune suppression; measles virus; pathogenesis; primate models.

FIG 1

Infection levels associated with wild-type MeV infection in macaques and squirrel monkeys. Groups of 5 female macaques and 8 female squirrel monkeys were inoculated intranasally with 10^4.5 TCID₅₀ of wild-type MeV. Photographs of skin rash and fluorescent signals in skin and tissues in animals sacrificed on day 12. Photographs and fluorescent images of the skin were taken during necropsy. The red wedge is the necropsy cut line, and arrows indicate areas of intense rash. Fluorescent images of fresh spleen and lung tissue were photographed using an inverted fluorescence microscope at 100-fold magnification.

FIG 2

Wild-type and nectin-4-blind MeV infection levels in macaque and squirrel monkey tissues. (A and B) Paraffin-embedded sections from wild-type-MeV-infected macaques (A), wild-type-MeV-infected squirrel monkeys (B, left panels), and MeV-N4^blind-infected squirrel monkeys (B, right panels) harvested on day 12 after infection were stained with a nucleoprotein-specific antibody revealed with the brown chromogen DAB and counterstained with hematoxylin. Slides were photographed at ×400 magnification. (C) Quantification of MeV N protein-positive cells per visual field. For each group, MeV N protein-positive cells were counted in three separate visual fields on slides from the three animals sacrificed at that time point. The columns show the group means, the error bars indicate the standard deviations, and asterisks describe levels of statistical significance (***, P < 0.001).

FIG 3

Cell-associated viremia and immunosuppression levels in wild-type-MeV-infected macaques and squirrel monkeys infected with wild-type MeV or MeV-N4^blind. Groups of 8 and 6 female squirrel monkeys were inoculated intranasally with 10^4.5 TCID₅₀ of wild-type MeV or MeV-N4^blind, respectively. Data from a previously published study (10) with a group of 5 female cynomolgus macaques inoculated intranasally with 10^4.5 TCID₅₀ of the same stock of wild-type MeV are also shown. Levels of cell-associated viremia (A), white blood cell (WBC) counts (B), and in vitro lymphocyte proliferation activity levels (C) at the indicated days postinfection. Cell-associated viremia is expressed as log₁₀ TCID₅₀ per 10⁶ PBMCs, white blood cell count is expressed as 10³ cells per mm³, and proliferation index reflects the ratio of BrdU incorporation in PHA-stimulated and nonstimulated PBMCs. Each symbol represents one animal, thick horizontal black lines indicate the group means, and asterisks describe levels of statistical significance (*, P < 0.05; **, P < 0.01).

FIG 4

Rash in squirrel monkeys infected with wild-type MeV or MeV-N4^blind. Photographs of animals 12 days after infection with wild-type MeV or MeV-N4^blind.

FIG 5

Infection levels in lymph nodes and body fluids. Virus titers were quantified in mesenteric lymph nodes (A), tracheal lavage fluids (B), and urine (C) from animals sacrificed at different times after inoculation as indicated above the panels. Three animals of each group were sacrificed on days 7 and 12, and two animals infected with wild-type MeV were sacrificed on day 21. Titers were determined by limited dilution and are expressed as log₁₀ TCID₅₀/milligram of tissue or milliliter, respectively. nd, not done.

FIG 6

Susceptibility of human and nonhuman primate PBMCs to in vitro MeV infection. (A) PBMCs were isolated from humans, pig-tailed macaques, and squirrel monkeys and were infected with either wild-type MeV or MeV-N4^blind. Cells were harvested 24 h after infection and fixed in 2% PFA. The percentage of GFP-positive lymphocytes was then quantified by fluorescence-activated cell sorting. (B) SLAM protein alignments from humans, squirrel monkeys, and macaques. Residues 58, 59, 60, 61, and 63 (indicated by asterisks) are known to be directly involved in H protein binding. Accession numbers: human (Homo sapiens), NM_003037; squirrel monkey (Saimiri boliviensis), XM_003937956; macaque (Macaca fascicularis), XM_015454807.