Citation: Plourde JR, Pyles JA, Layton RC, Vaughan SE, Tipper JL, et al. (2012) Neurovirulence of H5N1 Infection in Ferrets Is Mediated by Multifocal Replication in Distinct Permissive Neuronal Cell Regions. PLoS ONE 7(10): e46605. doi:10.1371/journal.pone.0046605
Editor: Stephen Mark Tompkins, University of Georgia, United States of America
Received: May 9, 2012; Accepted: September 3, 2012; Published: October 8, 2012

Abstract

Highly pathogenic avian influenza A (HPAI), subtype H5N1, remains an emergent threat to the human population. While respiratory disease is a hallmark of influenza infection, H5N1 has a high incidence of neurological sequelae in many animal species and sporadically in humans. We elucidate the temporal/spatial infection of H5N1 in the brain of ferrets following a low dose, intranasal infection of two HPAI strains of varying neurovirulence and lethality. A/Vietnam/1203/2004 (VN1203) induced mortality in 100% of infected ferrets while A/Hong Kong/483/1997 (HK483) induced lethality in only 20% of ferrets, with death occurring significantly later following infection. Neurological signs were prominent in VN1203 infection, but not HK483, with seizures observed three days post challenge and torticollis or paresis at later time points. VN1203 and HK483 replication kinetics were similar in primary differentiated ferret nasal turbinate cells, and similar viral titers were measured in the nasal turbinates of infected ferrets. Pulmonary viral titers were not different between strains and pathological findings in the lungs were similar in severity. VN1203 replicated to high titers in the olfactory bulb, cerebral cortex, and brain stem; whereas HK483 was not recovered in these tissues. VN1203 was identified adjacent to and within the olfactory nerve tract, and multifocal infection was observed throughout the frontal cortex and cerebrum. VN1203 was also detected throughout the cerebellum, specifically in Purkinje cells and regions that coordinate voluntary movements. These findings suggest the increased lethality of VN1203 in ferrets is due to increased replication in brain regions important in higher order function and explains the neurological signs observed during H5N1 neurovirulence.

Introduction 

Highly pathogenic avian influenza A (HPAI), subtype H5N1, has infected humans in 12 countries and has been associated with approximately a 60% mortality rate since 1997 (http://www.who.int/influenza/human_anima​l_interface/EN_GIP_20111010CumulativeNum​berH5N1cases.pdf). Severe disease of H5N1 includes fast-progressing pneumonia, acute respiratory distress syndrome (ARDS), diarrhea, central nervous system (CNS) clinical signs, and multi-organ failure. Death often occurs within ten days of symptom onset [1]–[3]. Studies to identify virulence factors contributing to these phenotypes have been the focus of many recent investigations [4]–[8]. However the mechanisms leading to increased pathogenesis by H5N1, particularly non-pulmonary events, remain to be elucidated.

ARDS is a common manifestation of pulmonary influenza infection; however H5N1 has been atypically shown to also infect and damage the CNS. De Jong and colleagues reported acute encephalitis in brains of humans infected with H5N1. These patients did not present with respiratory illness but had severe diarrhea, with early onset of seizures and coma, and death occurring within one to five days post hospital admittance [9]. Murine infection models have illustrated that neurotropic H5N1 strains exhibit higher lethality than those that do not replicate efficiently in the brain [10]. Several groups have investigated possible routes of viral entry into the brain, including the olfactory system as a major route into the brain of experimentally infected ferrets [10]–[12]. Studies by Park et al. suggested that, in addition to the olfactory nerves, HPAI enters the CNS through the vagal, trigeminal, and sympathetic nerves [12]. Furthermore, the dissemination of H5N1 through the bloodstream is plausible due to the presence of virus in organs such as the spleen apart from the site of initial infection.
While these possible routes of infection in the brain have been identified, little is known regarding HPAI dissemination within the CNS and its contribution to clinical signs and lethality. Therefore, delineating neurotropic features of H5N1 infection in the ferret model could lead to a better understanding of mechanisms responsible for widespread infection throughout the central nervous system.
Herein, we compare two strains of H5N1 with distinct neurotropism and lethality in ferrets to elucidate the temporal-spatial neuroinvasion leading to death. We show that VN1203 resulted in wider dissemination in the brain and associated with higher morbidity and clinical signs of neurological involvement. By comparison, HK483 infection resulted in low mortality, no viable virus recovered from the brain, and a low incidence of brain lesions limited solely to the olfactory system. Furthermore, we identify brain regions and cell types susceptible to VN1203 that explain the myriad of neurological signs during lethal infection. These findings broaden our understanding of the neurovirulence of H5N1 viruses and support further investigation into therapies leading to CNS protection.

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Continued:  http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0046605