Pathogenic analysis of coxsackievirus A10 in rhesus macaques

Abstract

Coxsackievirus A10 (CV-A10) is one of the etiological agents associated with hand, foot and mouth disease (HFMD) and also causes a variety of illnesses in humans, including pneumonia, and myocarditis. Different people, particularly young children, may have different immunological responses to infection. Current CV-A10 infection animal models provide only a rudimentary understanding of the pathogenesis and effects of this virus. The characteristics of CV-A10 infection, replication, and shedding in humans remain unknown. In this study, rhesus macaques were infected by CV-A10 via respiratory or digestive route to mimic the HFMD in humans. The clinical symptoms, viral shedding, inflammatory response and pathologic changes were investigated in acute infection (1-11 day post infection) and recovery period (12-180 day post infection). All infected rhesus macaques during acute infection showed obvious viremia and clinical symptoms which were comparable to those observed in humans. Substantial inflammatory pathological damages were observed in multi-organs, including the lung, heart, liver, and kidney. During the acute period, all rhesus macaques displayed clinical signs, viral shedding, normalization of serum cytokines, and increased serum neutralizing antibodies, whereas inflammatory factors caused some animals to develop severe hyperglycemia during the recovery period. In addition, there were no significant differences between respiratory and digestive tract infected animals. Overall, all data presented suggest that the rhesus macaques provide the first non-human primate animal model for investigating CV-A10 pathophysiology and assessing the development of potential human therapies.

Keywords: Coxsackievirus A10 (CV-A10); Hand, Foot and mouth disease (HFMD); Non-human primate model; Pathogenic analysis; Rhesus macaque.

Fig. 1

Clinical manifestations in CV-A10-infected rhesus macaques. Eleven 3- to 4-month-old rhesus macaques were labeled and grouped as follows: A1, A2, A3, and A4 in the respiratory tract group, labeled RI; B1, B2, B3, and B4 in the digestive tract group, labeled DI; and C1, C2, and C3 in the control group, labeled CG. Rhesus macaques in RI and DI groups were infected with CV-A10 (10⁵ CCID₅₀/monkey) via respiratory tract or digestive tract respectively. The three rhesus monkeys in GC group were not treated. A Ulcerated blisters with red, swollen lesions on the hands and feet and in the mouth of an infected rhesus macaque at 3–6 ​d.p.i., which are typical symptoms associated with HFMD. B Observed changes in the body weights of rhesus macaques. C Hematological changes, including leukocytes (WBC), lymphocytes (LYMPH), monocytes (MONO) and neutrophils (NEUT), in CV-A10-infected rhesus macaques were monitored using the flow cytometry (FCM) method.

Fig. 2

Dynamic distribution of viruses in CV-A10-infected rhesus macaques. Rhesus macaques in RI and DI groups were infected with CV-A10 (10⁵ CCID₅₀/monkey) via respiratory tract or digestive tract respectively. The three rhesus monkeys in GC group were not treated. The viral loads in blood and swab samples were monitored to evaluate viral replication kinetics in rhesus macaques at the scale of copies/200 ​μL or copies/100 ​mg qRT-PCR based on the TaqMan probe method was performed after extracting viral RNA to determine the CV-A10 RNA load at specific time points. A Detection of viral RNA in blood. B Detection of viral RNA in pharyngeal samples. C Detection of viral RNA in fecal samples. D On day 7 following infection, the homology of one randomly chosen RT-PCR product from rhesus monkey blood, a pharyngeal swab sample, and feces was compared to the reference sequence. The viral copy number was quantified based on in vitro synthesized 5′UTR protein RNA by the formula [(micrograms of RNA/μL)/(molecular weight)] ​× ​Avogadro's number ​= ​viral copy number/μL. At each time point, the values are represented as the average of two measurements.

Fig. 3

Etiological and pathologic observation of macaque infected CV-A10 via digestive tract. A Changes in body temperature and body weight at 0–10 ​d.p.i.; B viral load in blood at 0–10 ​d.p.i.; C viral load in throat swab samples at 0–10 ​d.p.i.; D dynamic changes in viral load in feces at 0–10 ​d.p.i.; E viral copy number in tissues from 24 different anatomical sites at 0–10 ​d.p.i., showing nucleic acid positivity in the brain, visceral, immune, and intestinal tissues, clearly indicating that the multiple tissues were subjected to viral infection. F H-E staining of lung and heart tissues. G H-E staining of liver and spleen tissues. H immunohistochemical staining analysis of CV-A10 in lung, liver and pancreas tissues. Red arrows represent inflammatory granulomas, yellow arrows represent foam-like cells, green arrows represent lymphocytic infiltrates, and black arrows represent inflammatory cell infiltrates. I immunohistochemical staining analysis of CV-A10 in heart, spleen and kidney tissues. The arrow shows the expression of CVA10-positive antigen. The black scale bar indicates 50 ​μm.

Fig. 4

Prolonged observation of and clinical symptoms in CV-A10-infected rhesus macaques. A Clinical symptoms ofrhesus macaques at 12–15 ​d.p.i.; B viral loads in throat swab and fecal samples of seven rhesus macaques at 11–50 ​d.p.i.; C levels of inflammatory cytokines in the serum of rhesus macaques after infection. D Blood glucose levels at 3, 6, and 9 months after infection. E Changes in serum neutralizing antibodies in rhesus macaques after infection.