Enterovirus 71 (EV71) infection causes severe mortality involving multiple possible mechanisms, including cytokine storm, brain stem encephalitis, and fulminant pulmonary edema. Gamma interferon (IFN-␥) may confer anti-EV71 activity; however, the claim that disease severity is highly correlated to an increase in IFN-␥ is controversial and would indicate an immune escape initiated by EV71. This study, investigating the role of IFN-␥ in EV71 infection using a murine model, showed that IFN-␥ was elevated. Moreover, IFN-␥ receptor-deficient mice showed higher mortality rates and more severe disease progression with slower viral clearance than wild-type mice. E nterovirus 71 (EV71) is a single-stranded RNA virus in the Picornaviridae family. The EV71 genome encodes four structural proteins, VP1 to VP4, and seven nonstructural proteins, 2A to 2C and 3A to 3D (1). Numerous studies have investigated the functions of viral proteins in viral replication and virulence (2). During EV71 infection, capsid VP proteins mediate virus entry by binding to cellular receptors, human scavenger receptor class B and P-selectin glycoprotein ligand 1 (3). Additionally, VP proteins participate in the assembly of viral particles (4). The 3C protein, a chymotrypsin-like protease, reduces host cell transcription dramatically by inhibiting cell polyadenylation (5) and induces caspase-regulated neural cell apoptosis (6). To develop specific anti-EV71 drugs, a number of small molecules targeting viral proteins have been designed, such as the 3C inhibitor rupintrivir and the 3D inhibitor aurintricarboxylic acid (ATA) (7-9).EV71 infection typically causes mild, self-limiting hand-footand-mouth disease; however, patients sometimes have significant morbidity and mortality resulting from hemorrhagic pulmonary edema following acute central nervous system-related cardiopulmonary failure and brain stem encephalitis (2,10,11). In addition to the direct cytotoxicity caused by EV71 infection (12-16) and the resultant virulence factors (6, 17), host factors such as the aberrant production of cytokines that is detected during EV71-associated pulmonary edema can also lead to disease. In infected

Enterovirus 71 (EV71) infection has induced fatal encephalitis in thousands of young children in the Asia-Pacific region over the last decade. EV71 infection continues to cause serious problems in areas with outbreaks, because vaccines and antiviral therapies are not available. Lymphocytes are present in the brains of infected patients and mice, and they protect mice from infection by decreasing the viral burden. The chemokines responsible for recruiting lymphocytes to infected organs are yet to be identified. Among the lymphocyte chemokines detected, high levels of interferon-gamma-inducible protein-10 (IP-10) are found in the plasma and cerebral spinal fluid of patients with brainstem encephalitis as compared with the levels of a monokine induced by gamma interferon (Mig). Using a murine model to investigate the induction of IP-10 by EV71 infection, we observed that EV71 infection significantly enhanced IP-10 protein expression in the serum and brain, with kinetics similar to viral titres in the blood and brain. Brain neurons of infected mice expressed IP-10. Using wild-type mice and IP-10 gene knockout mice to investigate the role of IP-10 in EV71 infection, we found that IP-10 deficiency significantly reduced levels of Mig in serum, and levels of gamma interferon and the number of CD8 T cells in the mouse brain. Absence of IP-10 significantly increased the mortality of infected mice by 45 %, with slow virus clearance in several vital tissues. Our observations are consistent with a model where EV71 infection boosts IP-10 expression to increase gamma interferon and Mig levels, infiltration of CD8 T cells, virus clearance in tissues and the survival of mice.

Enterovirus 71 (EV71) has induced fatal encephalitis in hundreds of thousands of infants and young children in the Asia-Pacific region since the past decade. Lymphocyte and antibody responses have been suspected to aggravate EV71-induced neurological symptoms, so anti-inflammatory agents have been used to treat patients with neurological symptoms. In the present study, we found that mice deficient in CD4+ T cells were resistant to EV71 infection as wild-type mice, whereas mice deficient in B cells were highly susceptible to viral infection. Compensation of CD4 T-cell function by other immune cells was not likely, because wild-type mice depleted of CD4+ T cells were also resistant to viral infection. Infected CD4 T-cell-deficient mice produced virus-specific neutralizing antibodies, IgM and IgG. Moreover, adoptive transfer of the virus-specific antibody produced by infected CD4 T-cell-deficient mice protected B-cell-deficient mice from infection by reducing tissue viral loads. Collectively, our results show that the CD4 T-cell-independent antibody response promotes the survival of EV71-infected mice and suggest great potential for the use of vaccines and neutralizing antibodies to reduce fatal symptoms in patients.

Herpes simplex virus 1 (HSV-1) infects the majority of the human population and can induce encephalitis, which is the most common cause of sporadic, fatal encephalitis. An increase of microglia is detected in the brains of encephalitis patients. The issues regarding whether and how microglia protect the host and neurons from HSV-1 infection remain elusive. Using a murine infection model, we showed that HSV-1 infection on corneas increased the number of microglia to outnumber those of infiltrating leukocytes (macrophages, neutrophils, and T cells) and enhanced microglia activation in brains. HSV-1 antigens were detected in brain neurons, which were surrounded by microglia. Microglia depletion increased HSV-1 lethality of mice with elevated brain levels of viral loads, infected neurons, neuron loss, CD4 T cells, CD8 T cells, neutrophils, interferon (IFN)-β, and IFN-γ. In vitro studies demonstrated that microglia from infected mice reduced virus infectivity. Moreover, microglia induced IFN-β and the signaling pathway of signal transducer and activator of transcription (STAT) 1 to inhibit viral replication and damage of neurons. Our study reveals how microglia protect the host and neurons from HSV-1 infection.

Herpes simplex virus 1 (HSV-1) establishes latency in neural tissues of immunocompetent mice but persists in both peripheral and neural tissues of lymphocyte-deficient mice. Thymidine kinase (TK) is believed to be essential for HSV-1 to persist in neural tissues of immunocompromised mice, because infectious virus of a mutant with defects in both TK and UL24 is detected only in peripheral tissues, but not in neural tissues, of severe combined immunodeficiency mice (T. Valyi-Nagy, R. M. Gesser, B. Raengsakulrach, S. L. Deshmane, B. P. Randazzo, A. J. Dillner, and N. W. Fraser, Virology 199:484 -490, 1994, https://doi.org/10.1006/viro .1994.1150). Here we find infiltration of CD4 and CD8 T cells in peripheral and neural tissues of mice infected with a TK-negative mutant. We therefore investigated the significance of viral TK and host T cells for HSV-1 to persist in neural tissues using three genetically engineered mutants with defects in only TK or in both TK and UL24 and two strains of nude mice. Surprisingly, all three mutants establish persistent infection in up to 100% of brain stems and 93% of trigeminal ganglia of adult nude mice at 28 days postinfection, as measured by the recovery of infectious virus. Thus, in mouse neural tissues, host T cells block persistent HSV-1 infection, and viral TK is dispensable for the virus to establish persistent infection. Furthermore, we found 30-to 200-fold more virus in neural tissues than in the eye and detected glycoprotein C, a true late viral antigen, in brainstem neurons of nude mice persistently infected with the TK-negative mutant, suggesting that adult mouse neurons can support the replication of TK-negative HSV-1.IMPORTANCE Acyclovir is used to treat herpes simplex virus 1 (HSV-1)-infected immunocompromised patients, but treatment is hindered by the emergence of drugresistant viruses, mostly those with mutations in viral thymidine kinase (TK), which activates acyclovir. TK mutants are detected in brains of immunocompromised patients with persistent infection. However, answers to the questions as to whether TKnegative (TK Ϫ ) HSV-1 can establish persistent infection in brains of immunocompromised hosts and whether neurons in vivo are permissive for TK Ϫ HSV-1 remain elusive. Using three genetically engineered HSV-1 TK Ϫ mutants and two strains of nude mice deficient in T cells, we found that all three HSV-1 TK Ϫ mutants can efficiently establish persistent infection in the brain stem and trigeminal ganglion and detected glycoprotein C, a true late viral antigen, in brainstem neurons. Our study provides evidence that TK Ϫ HSV-1 can persist in neural tissues and replicate in brain neurons of immunocompromised hosts.