A Neonatal Murine Model of Coxsackievirus A6 Infection for Evaluation of Antiviral and Vaccine Efficacy

Abstract

Hand, foot, and mouth disease (HFMD) is a global health concern. Family members, particularly enterovirus A71 (EVA71) and coxsackievirus A16 (CVA16), are the primary etiological agents of HFMD; however, a third enterovirus A species, CVA6, has been recently associated with epidemic outbreaks. Study of the pathogenesis of CVA6 infection and development of antivirals and vaccines are hindered by a lack of appropriate animal models. We have developed and characterized a murine model of CVA6 infection that was employed to evaluate the antiviral activities of different drugs and the protective efficacies of CVA6-inactivated vaccines. Neonatal mice were susceptible to CVA6 infection via intramuscular inoculation, and the susceptibility of mice to CVA6 infection was age and dose dependent. Five-day-old mice infected with 10 50% tissue culture infective doses of the CVA6 WF057R strain consistently exhibited clinical signs, including reduced mobility, lower weight gain, and quadriplegia with significant pathology in the brain, hind limb skeletal muscles, and lungs of the infected mice in the moribund state. Immunohistochemical analysis and quantitative reverse transcription-PCR (qRT-PCR) analyses showed high viral loads (11 log/mg) in skeletal muscle, and elevated levels of interleukin-6 (IL-6; >2,000 pg/ml) were associated with severe viral pneumonia and encephalitis. Ribavirin and gamma interferon administered prophylactically diminished CVA6-associated pathology , and treatment with IL-6 accelerated the death of neonatal mice. Both specific anti-CVA6 serum and maternal antibody play important roles in controlling CVA6 infection and viral replication. Collectively, these findings indicate that this neonatal murine model will be invaluable in future studies to develop CVA6-specific antivirals and vaccines. Although coxsackievirus A6 (CVA6) infections are commonly mild and self-limiting, a small proportion of children may have serious complications, such as encephalitis, acute flaccid paralysis, and neurorespiratory syndrome, leading to fatalities. We have established a mouse model of CVA6 infection by inoculation of neonatal mice with a CVA6 clinical isolate that produced consistent pathological outcomes. Here, using this model of CVA6 infection, we found that high levels of IL-6 were associated with severe viral pneumonia and encephalitis, as in an evaluation of antiviral efficacy , IL-6 had no protective effect and instead accelerated death in neonatal mice. We demonstrated that, as antiviral drugs, both gamma interferon and ribavirin played important protective roles in the early stages of infection, with increased survival in treated neonatal mice challenged with CVA6. Moreover, active and passive immunization with the inactivated vaccines and anti-CVA6 serum also protected mice against homologous challenge infections.

Protective Efficacies of Formaldehyde-Inactivated Whole-Virus Vaccine and Antivirals in a Murine Model of Coxsackievirus A10 Infection

“…In the present study, a high level of IL-6 in the peripheral blood of neonatal mice not only did not inhibit the replication of CVA10, but was associated with more severe clinical signs, with an average survival time 2 days shorter than that of the control group. Similar phenomena have also been observed in the CVA6 mouse model (17). In contrast, administration of anti-IL-6 neutralizing antibodies after the onset of clinical symptoms successfully improved the survival rates and reduced the clinical scores of the EVA71-infected mice (21).…”

“…The development of inactivated EVA71 vaccines is based on neonatal mouse and cynomolgus monkey models of viral infection (14)(15)(16). We have also shown that inactivated whole-virus vaccines were able to protect neonatal mice against coxsackievirus A6 infection (17). Therefore, inactivated vaccines have become the preferred choice for the prevention and control of CVA10-associated hand, foot, and mouth disease (HFMD) epidemics.…”

Divergent Pathogenic Properties of Circulating Coxsackievirus A6 Associated with Emerging Hand, Foot, and Mouth Disease

“…8 and 9). In addition, data from several studies, including the present one, indicated a close relationship between variations of the viral 2C protein and the lethality of CV-A6, at least in a mouse model (29,30). The 2C protein may benefit CV-A6 by causing cell death and promoting the release of the virion progeny, which would shorten the infection cycle of the virus (Fig.…”

“…Recombination is a common feature of enteroviruses, especially those associated with HFMD. Recently, many studies have reported that multiple HEV-A group viruses, especially non-EV-A71 and non-CV-A16 infections, could coinfect an individual, cocirculate in the same geographical area, and give the viruses the opportunity to undergo recombination (16,20,29). Our previous studies have also revealed that circulating CV-A16 and EV-A71 were recombinants based on their prototype viruses (7)(8)(9).…”

“…To further confirm 2C as a contributor to the pathogenicity of CV-A6, we introduced two more 2C protein sequences from CV-A6 strains with reported lethality. Weifang/ SD/CHN/2014/was effective in killing 5-day-old mice (29) and shared amino acids at positions 324, 339, and 556 with the lethal strains ( Fig. 8).…”