A non-mouse-adapted enterovirus 71 (EV71) strain exhibits neurotropism, causing neurological manifestations in a novel mouse model of EV71 infection

Enterovirus 71 (EV71) is a neurotropic pathogen that has been consistently associated with the severe neurological forms of hand, foot, and mouth disease. The lack of a relevant animal model has hampered our understanding of EV71 pathogenesis, in particular the route and mode of viral dissemination. It has also hindered the development of effective prophylactic and therapeutic approaches, making EV71 one of the most pressing public health concerns in Southeast Asia. Here we report a novel mouse model of EV71 infection. We demonstrate that 2-week-old and younger immunodeficient AG129 mice, which lack type I and II interferon receptors, are susceptible to infection with a non-mouse-adapted EV71 strain via both the intraperitoneal (i.p.) and oral routes of inoculation. The infected mice displayed progressive limb paralysis prior to death. The dissemination of the virus was dependent on the route of inoculation but eventually resulted in virus accumulation in the central nervous systems of both animal groups, indicating a clear neurotropism of the virus. Histopathological examination revealed massive damage in the limb muscles, brainstem, and anterior horn areas. However, the minute amount of infectious viral particles in the limbs from orally infected animals argues against a direct viral cytopathic effect in this tissue and suggests that limb paralysis is a consequence of EV71 neuroinvasion. Together, our observations support that young AG129 mice display polio-like neuropathogenesis upon infection with a non-mouse-adapted EV71 strain, making this mouse model relevant for EV71 pathogenesis studies and an attractive platform for EV71 vaccine and drug testing.     

Mosquito saliva causes enhancement of West Nile virus infection in mice

West Nile virus (WNV) is transmitted to vertebrate hosts primarily by infected Culex mosquitoes. Transmission of arboviruses by the bite of infected mosquitoes can potentiate infection in hosts compared to viral infection by needle inoculation. Here we examined the effect of mosquito transmission on WNV infection and systematically investigated multiple factors that differ between mosquito infection and needle inoculation of WNV. We found that mice infected with WNV through the bite of a single infected Culex tarsalis mosquito exhibited 5- to 10-fold-higher viremia and tissue titers at 24 and 48 h postinoculation and faster neuroinvasion than mice given a median mosquito-inoculated dose of WNV (10(5) PFU) by needle. Mosquito-induced enhancement was not due to differences in inoculation location, because additional intravenous inoculation of WNV did not enhance viremia or tissue titers. Inoculation of WNV into a location where uninfected mosquitoes had fed resulted in enhanced viremia and tissue titers in mice similar to those in mice infected by a single infected mosquito bite, suggesting that differences in where virus is deposited in the skin and in the virus particle itself were not responsible for the enhanced early infection in mosquito-infected mice. In addition, inoculation of mice with WNV mixed with salivary gland extract (SGE) led to higher viremia, demonstrating that mosquito saliva is the major cause of mosquito-induced enhancement. Enhanced viremia was not observed when SGE was inoculated at a distal site, suggesting that SGE enhances WNV replication by exerting a local effect. Furthermore, enhancement of WNV infection still occurred in mice with antibodies against mosquito saliva. In conclusion, saliva from C. tarsalis is responsible for enhancement of early WNV infection in vertebrate hosts.     

Polygenic control of neuroinvasiveness in California serogroup bunyaviruses.

The pathogenesis of the California serogroup bunyaviruses includes both extraneural and intraneural replicative phases that can be separated experimentally. The present study dissects the viral genetic determinants of extraneural replication. We have previously described two attenuated reassortant clones of California serogroup bunyaviruses which exhibit reduced neuroinvasiveness after subcutaneous inoculation into suckling mice. Clone B1-1a bears an attenuated middle RNA segment (neuroinvasiveness phenotype v alpha v), and clone B.5 bears an attenuated large RNA segment (neuroinvasiveness phenotype alpha vv). We prepared reassortant viruses between these two strains and found that the two attenuated gene segments acted independently and additively, since reassortants bearing two attenuated RNA segments were more attenuated than the parental clones. Reassortants bearing no attenuated RNA segments were much more neuroinvasive than either parental clone, indicating that a neuroinvasive strain can be derived from two attenuated clones. Pathogenesis studies demonstrated that after injection of 10(3) PFU, the attenuated reassortant clones did not replicate in peripheral tissue, failed to reach the brain, and did not cause disease. At a dose of 10(6) PFU, attenuated clones failed to replicate to a significant level in peripheral tissue and produced only a minimal passive plasma viremia during the first 24 h but nevertheless reached high titers in the brain and killed mice. Because of this result, we investigated the possibility that neuroinvasion occurs via retrograde axonal transport, by determining whether sciatic nerve sectioning could protect against virus infection after hind leg footpad inoculation. We found that nerve sectioning had no effect on lethality, ruling out this mode of entry and suggesting that passive viremia is likely to be sufficient for invasion of the central nervous system.     

Alpha/beta interferon protects adult mice from fatal Sindbis virus infection and is an important determinant of cell and tissue tropism

Infection of adult 129 Sv/Ev mice with consensus Sindbis virus strain TR339 is subclinical due to an inherent restriction in early virus replication and viremic dissemination. By comparing the pathogenesis of TR339 in 129 Sv/Ev mice and alpha/beta interferon receptor null (IFN-alpha/betaR(-/-)) mice, we have assessed the contribution of IFN-alpha/beta in restricting virus replication and spread and in determining cell and tissue tropism. In adult 129 Sv/Ev mice, subcutaneous inoculation with 100 PFU of TR339 led to extremely low-level virus replication and viremia, with clearance under way by 96 h postinoculation (p.i.). In striking contrast, adult IFN-alpha/betaR(-/-) mice inoculated subcutaneously with 100 PFU of TR339 succumbed to the infection within 84 h. By 24 h p.i. a high-titer serum viremia had seeded infectious virus systemically, coincident with the systemic induction of the proinflammatory cytokines interleukin-12 (IL-12) p40, IFN-gamma, tumor necrosis factor alpha, and IL-6. Replicating virus was located in macrophage-dendritic cell (DC)-like cells at 24 h p.i. in the draining lymph node and in the splenic marginal zone. By 72 h p.i. virus replication was widespread in macrophage-DC-like cells in the spleen, liver, lung, thymus, and kidney and in fibroblast-connective tissue and periosteum, with sporadic neuroinvasion. IFN-alpha/beta-mediated restriction of TR339 infection was mimicked in vitro in peritoneal exudate cells from 129 Sv/Ev versus IFN-alpha/betaR(-/-) mice. Thus, IFN-alpha/beta protects the normal adult host from viral infection by rapidly conferring an antiviral state on otherwise permissive cell types, both locally and systemically. Ablation of the IFN-alpha/beta system alters the apparent cell and tissue tropism of the virus and renders macrophage-DC-lineage cells permissive to infection.     

Enterovirus 71 Virion-Associated Galectin-1 Facilitates Viral Replication and Stability

Enterovirus 71 (EV71) infection causes a myriad of diseases from mild hand-foot-and-mouth disease or herpangina to fatal brain stem encephalitis complicated with pulmonary edema. Several severe EV71 endemics have occurred in Asia-Pacific region, including Taiwan, and have become a serious threat to children’s health. EV71 infection is initiated by the attachment of the virion to the target cell surface. Although this process relies primarily upon interaction between viruses and cell surface receptors, soluble factors may also influence the binding of EV71 to host cells.Galectin-1 has been reported to participate in several virus infections, but is not addressed in EV71. In this study, we found that the serum levels of galectin-1 in EV71-infected children were higher than those in non-infected people. In EV71 infected cells, galectin-1 was found to be associated with the EV71 VP1 and VP3 via carbohydrate residues and subsequently released and bound to another cell surface along with the virus. EV71 propagated from galectin-1 knockdown SK-N-SH cells exhibited lower infectivity in cultured cells and less pathogenicity in mice than the virus propagated from parental cells. In addition, this galectin-1-free EV71 virus was sensitive to high temperature and lost its viability after long-term storage, which could be restored following supplement of recombinant galectin-1. Taken together, our findings uncover a new role of galectin-1 in facilitating EV71 virus infection.     

A new model for Hendra virus encephalitis in the mouse

Hendra virus (HeV) infection in humans is characterized by an influenza like illness, which may progress to pneumonia or encephalitis and lead to death. The pathogenesis of HeV infection is poorly understood, and the lack of a mouse model has limited the opportunities for pathogenetic research. In this project we reassessed the role of mice as an animal model for HeV infection and found that mice are susceptible to HeV infection after intranasal exposure, with aged mice reliably developing encephalitic disease. We propose an anterograde route of neuroinvasion to the brain, possibly along olfactory nerves. This is supported by evidence for the development of encephalitis in the absence of viremia and the sequential distribution of viral antigen along pathways of olfaction in the brain of intranasally challenged animals. In our studies mice developed transient lower respiratory tract infection without progressing to viremia and systemic vasculitis that is common to other animal models. These studies report a new animal model of HeV encephalitis that will allow more detailed studies of the neuropathogenesis of HeV infection, particularly the mode of viral spread and possible sequestration within the central nervous system; investigation of mechanisms that moderate the development of viremia and systemic disease; and inform the development of improved treatment options for human patients.     

Immunity to avirulent enterovirus 71 and coxsackie A16 virus protects against enterovirus 71 infection in mice

In this study, we sought to determine whether intratypic and intertypic cross-reactivity protected against enterovirus 71 (EV71) infection in a murine infection model. We demonstrate that active immunization of 1-day-old mice with avirulent EV71 strain or coxsackie A16 virus (CA16) by the oral route developed anti-EV71 antibodies with neutralizing activity (1:16 and 1:2, respectively). Splenocytes from both EV71- and CA16-immunized mice proliferated upon EV71 or CA16, but not coxsackie B3 virus (CB3), antigen stimulation. Immunized mice became more resistant to virulent EV71 strain challenge than nonimmunized mice. There was an increase in the percentage of activated splenic T cells and B cells in the immunized mice 2 days after EV71 challenge. The CA16 immune serum reacted with EV71 antigens in an enzyme-linked immunosorbent assay and neutralized EV71 but not CB3 or poliovirus at a titer of 1:4. Passive immunization with the CA16 immune serum reduced the clinical score, diminished the organ viral load, and increased the survival rate of mice upon EV71 challenge. CB3 neither shared in vitro cross-reactivity with EV71 nor provided in vivo protection after both active and passive immunization. These results illustrated that live vaccine is feasible for EV71 and that intertypic cross-reactivity of enteroviruses may provide a way to determine the prevalence of EV71.     

Retrograde transport of transmissible mink encephalopathy within descending motor tracts

The spread of the abnormal conformation of the prion protein, PrP(Sc), within the spinal cord is central to the pathogenesis of transmissible prion diseases, but the mechanism of transport has not been determined. For this report, the route of transport of the HY strain of transmissible mink encephalopathy (TME), a prion disease of mink, in the central nervous system following unilateral inoculation into the sciatic nerves of Syrian hamsters was investigated. PrP(Sc) was detected at 3 weeks postinfection in the lumbar spinal cord and ascended to the brain at a rate of approximately 3.3 mm per day. At 6 weeks postinfection, PrP(Sc) was detected in the lateral vestibular nucleus and the interposed nucleus of the cerebellum ipsilateral to the site of sciatic nerve inoculation and in the red nucleus contralateral to HY TME inoculation. At 9 weeks postinfection, PrP(Sc) was detected in the contralateral hind limb motor cortex and reticular thalamic nucleus. These patterns of PrP(Sc) brain deposition at various times postinfection were consistent with that of HY TME spread from the sciatic nerve to the lumbar spinal cord followed by transsynaptic spread and retrograde transport to the brain and brain stem along descending spinal tracts (i.e., lateral vestibulospinal, rubrospinal, and corticospinal). The absence of PrP(Sc) from the spleen suggested that the lymphoreticular system does not play a role in neuroinvasion following sciatic nerve infection. The rapid disease onset following sciatic nerve infection demonstrated that HY TME can spread by retrograde transport along specific descending motor pathways of the spinal cord and, as a result, can initially target brain regions that control vestibular and motor functions. The early clinical symptoms of HY TME infection such as head tremor and ataxia were consistent with neuronal damage to these brain areas.     

A single intranasal inoculation with a paramyxovirus-vectored vaccine protects guinea pigs against a lethal-dose Ebola virus challenge

To determine whether intranasal inoculation with a paramyxovirus-vectored vaccine can induce protective immunity against Ebola virus (EV), recombinant human parainfluenza virus type 3 (HPIV3) was modified to express either the EV structural glycoprotein (GP) by itself (HPIV3/EboGP) or together with the EV nucleoprotein (NP) (HPIV3/EboGP-NP). Expression of EV GP by these recombinant viruses resulted in its efficient incorporation into virus particles and increased cytopathic effect in Vero cells. HPIV3/EboGP was 100-fold more efficiently neutralized by antibodies to EV than by antibodies to HPIV3. Guinea pigs infected with a single intranasal inoculation of 10(5.3) PFU of HPIV3/EboGP or HPIV3/EboGP-NP showed no apparent signs of disease yet developed a strong humoral response specific to the EV proteins. When these animals were challenged with an intraperitoneal injection of 10(3) PFU of EV, there were no outward signs of disease, no viremia or detectable EV antigen in the blood, and no evidence of infection in the spleen, liver, and lungs. In contrast, all of the control animals died or developed severe EV disease following challenge. The highly effective immunity achieved with a single vaccine dose suggests that intranasal immunization with live vectored vaccines based on recombinant respiratory viruses may be an advantageous approach to inducing protective responses against severe systemic infections, such as those caused by hemorrhagic fever agents.     

A mouse-adapted enterovirus 71 strain causes neurological disease in mice after oral infection

A mouse-adapted enterovirus 71 (EV71) strain with increased virulence in mice, MP4, was generated after four serial passages of the parental EV71 strain 4643 in mice. Strain MP4 exhibited a larger plaque size, grew more rapidly, and was more cytotoxic in vitro than strain 4643. Although strains 4643 and MP4 both induced apoptosis of SK-N-SH human neuroblastoma cells, MP4 was more virulent than 4643 in 1-day-old mice (50% lethal doses, 10(2) and 10(4) PFU/mouse, respectively). Strain MP4 (5 x 10(6) PFU/mouse), but not 4643, could orally infect 7-day-old mice, resulting in rear-limb paralysis followed by death 5 to 9 days after inoculation with the virus. Histopathologically, neuronal loss and apoptosis were evident in the spinal cords as well as the brain stems of the infected mice. The limb muscles displayed massive necrosis. There was early and transient virus replication in the intestines, whereas the spinal cord, brain, and muscle became the sites of viral replication during the late phase of the infection. Virus transmission occurred among infected and noninfected cagemates, as demonstrated by the occurrence of seroconversion and the presence of viable viruses in the stool samples of the latter. Protection against EV71 challenge was demonstrated following administration of hyperimmune serum 1 day after inoculation with the virus. Nucleotide sequence analysis of the genome of EV71 strain MP4 revealed four nucleotide changes on the 5' untranslated region, three on the VP2 region, and eight on the 2C region, resulting in one and four amino acid substitutions in the VP2 and 2C proteins, respectively.     

Experimental studies on vertical infection of mice with Japanese encephalitis virus. IV. Effect of virus strain on placental and fetal infection

An experiment was carried out to examine the effect of an inoculated strain of Japanese encephalitis virus on the establishment of experimental vertical infection of mice with this virus. In it, closed-colony mice of the CFW strain were inoculated intravenously with seven strains of the virus at 7 days of pregnancy. After that, an attempt was made to recover the virus from placenta and fetus, so that the infection rate of each strain might be determined. As a result, the infection rate was high for both placenta and fetus in the case of the AS-6 and Sagara strains both of which had undergone three passages in the mouse brain. The placental infection rate was high and the fetal infection rate relatively low in the case of the JaGAr01 and Fuji strains which had undergone 7 and 150 passages, respectively, in the mouse brain. The infection rate was very low for both placenta and fetus in the case of the Nakayama-Yakken strain which had undergone more than 100 passages in the mouse brain. There was no difference in the severity of viremia after inoculation between the AS-6 and Fuji strains. Both placental and fetal infection rates were low in the case of the JaTH160 strain which had undergone passages in mice by intraperitoneal inoculation and which presented a strong peripheral infectivity and induced a severe viremia after inoculation. Neither placental nor fetal infection occurred in the case of the S- strain used as live virus vaccine. These results indicated that placental and fetal infection rates varied from one virus strain to another.(ABSTRACT TRUNCATED AT 250 WORDS)     

The leukemogenic potential of an enhancer variant of Moloney murine leukemia virus varies with the route of inoculation.

We previously showed that the Mo+PyF101 variant of Moloney murine leukemia virus (M-MuLV) is poorly leukemogenic when inoculated subcutaneously (s.c.) into neonatal mice. We recently found that intraperitoneal (i.p.) inoculation of neonatal mice with the same virus significantly enhanced its leukemogenicity. In this study, infections of neonatal mice by the two different routes of inoculation were compared. We studied replication of the virus in vivo to identify critical preleukemic events. These would be observed in mice inoculated i.p. by Mo+PyF101 M-MuLV but not when inoculation was s.c. Infectious center assays indicated that regardless of the route of inoculation, Mo+PyF101 M-MuLV showed delayed infection of the thymus compared with wild-type M-MuLV. On the other hand, i.p.-inoculated mice showed more rapid appearance of infectious centers in the bone marrow than did s.c.-inoculated animals. Thus, the enhanced leukemogenicity of i.p. inoculation correlated with efficient early infection of the bone marrow and not with early infection of the thymus. These results suggest a role for bone marrow infection for efficient leukemogenesis in Mo+PyF101 M-MuLV-infected mice. Consistent with this notion, if bone marrow infection was decreased by injecting 10- to 12-day-old animals i.p., leukemogenicity resembled that of s.c. inoculation. Thus, two cell types that are critical for the induction of efficient leukemia were implicated. One cell delivers virus from the site of s.c. inoculation (the skin) to the bone marrow and is apparently restricted for Mo+PyF101 M-MuLV replication. The second cell is in the bone marrow, and its early infection is required for efficient leukemogenesis.     

Direct entry of rabies virus into the central nervous system without prior local replication.

Rabies virus pathogenesis was studied in a mouse model by inoculation of the masseter muscle. At different intervals, the masseter muscle, trigeminal ganglia, and brain were analyzed for virus-specific RNA with a polymerase chain reaction assay, which revealed that as early as 18 h postinfection (p.i.), virus-specific RNA was present in the trigeminal ganglia, and at 24 h p.i., viral RNA was identified in the brain stem. Analysis of the masseter muscle demonstrated virus at 1 h p.i. but no virus-specific RNA between 6 and 30 h p.i., indicating that virus invaded the nerve ending directly, without prior replication in the muscle. At 36 h p.i., viral RNA was detected again in the masseter muscle. Selective amplification of plus- and minus-strand RNA isolated from the masseter muscle at 96 h p.i. revealed that the majority of the rabies virus-specific RNA was in the positive sense, suggesting virus replication in muscle tissue during late stages of infection.     

Viral neuroinvasion as a marker for BBB integrity following exposure to cholinesterase inhibitors

Exposure to the nerve agent soman, an irreversible cholinesterase (ChE) inhibitor, results in changes in blood-brain barrier permeability attributed to its seizure-induced activity. However, smaller BBB changes may be independent of convulsions. Such minor injury may escape detection. A nonneuroinvasive neurovirulent Sindbis virus strain (SVN) was used as a marker for BBB permeability. Peripheral inoculation of mice with 2 x 10(3) plaque forming units (PFU) caused up to 10(5) PFU/ml viremia after 24 hours with no signs of central nervous system (CNS) infection and with no virus detected in brain tissue. Intra-cerebral injection of as low as 1-5 PFU of the same virus caused CNS infection, exhibited 5-7 days later as hind limb paralysis and death. Soman (0.1-0.7 of the LD50) was administered at peak viremia (1 day following peripheral inoculation). Sublethal soman exposure at as low as 0.1 LD50 resulted in CNS infection 6-8 days following inoculation in 30-40% of the mice. High virus titer were recorded in brain tissue of sick mice while no virus was detected in healthy mice subjected to the same treatment. No changes in the level of viremia or changes in viral traits were observed in the infected mice. The reversible anticholinesterases physostigmine (0.2 mg/kg, s.c.) and pyridostigmine (0.4 mg/kg, i.m.) injected at a dose equal to 0.1 LD50, induced similar results. Thus, both central and peripheral anticholinesterases (anti-ChEs) induce changes in BBB permeability sufficient to allow, at least in some of the mice, the invasion of this otherwise noninvasive but highly neurovirulent virus. This BBB change is probably due to the presence of cholinesterases in the capillary wall. SVN brain invasion served here as a highly sensitive and reliable marker for BBB integrity.     

Mechanism of differences in pathogenicity between two variants of a laboratory strain of herpes simplex virus type 1

The mechanisms responsible for the difference in neurovirulence to inbred mice between two variants of the Miyama strain of herpes simplex virus type 1 (HSV-1) were studied. After intraperitoneal (i.p.) inoculation, the +GC (LPV) variant reached the spinal cord and the brain, and caused death. Conversely, the -GCr variant lacked the ability to gain access to the central nervous system (CNS) after the same route of infection and failed to kill susceptible mice. The initial virus growth after i.p. inoculation, as indicated by the number of infective centers (ICs) produced by the peritoneal exudate cells (PECs), was compared between these two variants. The virulent +GC (LPV) strain induced much more ICs than the attenuated -GCr variant. When the attenuated variant was preinoculated i.p. 24 hr before the challenge inoculation with the virulent variant by the same route, the production of ICs by the pathogenic variant was highly inhibited, and growth of this variant did not occur in the CNS. Thus, mice were protected from lethal infection by the virulent variant by preinoculation with the attenuated one. Moreover, the ability of mice to resist i.p. infection by HSV-1 was shown to be age-dependent.     

Enterovirus 71 Infection Causes Severe Pulmonary Lesions in Gerbils,Meriones unguiculatus,Which Can Be Prevented by Passive Immunization with Specific Antisera

Neurogenic pulmonary edema caused by severe brainstem encephalitis is the leading cause of death in young children infected by Enterovirus 71 (EV71). However, no pulmonary lesions have been found in EV71-infected transgenic or non-transgenic mouse models. Development of a suitable animal model is important for studying EV71 pathogenesis and assessing effect of therapeutic approaches. We had found neurological disorders in EV71-induced young gerbils previously. Here, we report severe pulmonary lesions characterized with pulmonary congestion and hemorrhage in a gerbil model for EV71 infection. In the EV71-infected gerbils, six 21-day-old or younger gerbils presented with a sudden onset of symptoms and rapid illness progression after inoculation with 1×10^5.5 TCID₅₀ of EV71 via intraperitoneal (IP) or intramuscular (IM) route. Respiratory symptoms were observed along with interstitial pneumonia, pulmonary congestion and extensive lung hemorrhage could be detected in the lung tissues by histopathological examination. EV71 viral titer was found to be peak at late stages of infection. EV71-induced pulmonary lesions, together with severe neurological disorders were also observed in gerbils, accurately mimicking the disease process in EV71-infected patients. Passive transfer with immune sera from EV71 infected adult gerbils with a neutralizing antibody (GMT=89) prevented severe pulmonary lesion formation after lethal EV71 challenge. These results establish this gerbil model as a useful platform for studying the pathogenesis of EV71-induced pulmonary lesions, immunotherapy and antiviral drugs.     

Neuroinvasion of fluorescein-positive monocytes in acute simian immunodeficiency virus infection

Monocytes and macrophages play a central role in the pathogenesis of human immunodeficiency virus (HIV)-associated dementia. They represent prominent targets for HIV infection and are thought to facilitate viral neuroinvasion and neuroinflammatory processes. However, many aspects regarding monocyte brain recruitment in HIV infection remain undefined. The nonhuman primate model of AIDS is uniquely suited for examination of the role of monocytes in the pathogenesis of AIDS-associated encephalitis. Nevertheless, an approach to monitor cell migration from peripheral blood into the central nervous system (CNS) in primates had been lacking. Here, upon autologous transfer of fluorescein dye-labeled leukocytes, we demonstrate the trafficking of dye-positive monocytes into the choroid plexus stromata and perivascular spaces in the cerebra of rhesus macaques acutely infected with simian immunodeficiency virus between days 12 and 14 postinfection (p.i.). Dye-positive cells that had migrated expressed the monocyte activation marker CD16 and the macrophage marker CD68. Monocyte neuroinvasion coincided with the presence of the virus in brain tissue and cerebrospinal fluid and with the induction of the proinflammatory mediators CXCL9/MIG and CCL2/MCP-1 in the CNS. Prior to neuroinfiltration, plasma viral load levels peaked on day 11 p.i. Furthermore, the numbers of peripheral blood monocytes rapidly increased between days 4 and 8 p.i., and circulating monocytes exhibited increased functional capacity to produce CCL2/MCP-1. Our findings demonstrate acute monocyte brain infiltration in an animal model of AIDS. Such studies facilitate future examinations of the migratory profile of CNS-homing monocytes, the role of monocytes in virus import into the brain, and the disruption of blood-cerebrospinal fluid and blood-brain barrier functions in primates.     

Alternative migration routes of Ascaris suum in the pig

Experiments were conducted to investigate possible alternative routes of extraintestinal migration of Ascaris suum larvae in the pig. Pigs were infected with A. suum via injection of newly hatched larvae into cecal veins (i.v.), into cecal lymph nodes (LN), or intraperitoneally (i.p.), and control animals were inoculated orally with infective eggs (p.o.). Two pigs per inoculation route were necropsied on days 1, 4, and 13 postinoculation. The numbers of liver lesions and the percentage of larvae recovered was considerably greater in pigs inoculated i.v. or p.o. on each necropsy day. However, irrespective of inoculation route, at least a proportion of larvae passed through the livers and were able to complete migration to the small intestine by day 13. The results indicate that larval penetration of the intestinal wall is not necessary for liver-lung migration and that passage through the liver may be favorable for migrating A. suum larvae, although a delayed arrival in the small intestine cannot be ruled out for larvae following alternative routes.     

Adaptation of Enterovirus 71 to Adult Interferon Deficient Mice

Non-polio enteroviruses, including enterovirus 71 (EV71), have caused severe and fatal cases of hand, foot and mouth disease (HFMD) in the Asia-Pacific region. The development of a vaccine or antiviral against these pathogens has been hampered by the lack of a reliable small animal model. In this study, a mouse adapted EV71 strain was produced by conducting serial passages through A129 (α/β interferon (IFN) receptor deficient) and AG129 (α/β, γ IFN receptor deficient) mice. A B2 sub genotype of EV71 was inoculated intraperitoneally (i.p.) into neonatal AG129 mice and brain-harvested virus was subsequently passaged through 12 and 15 day-old A129 mice. When tested in 10 week-old AG129 mice, this adapted strain produced 100% lethality with clinical signs including limb paralysis, eye irritation, loss of balance, and death. This virus caused only 17% mortality in same age A129 mice, confirming that in the absence of a functional IFN response, adult AG129 mice are susceptible to infection by adapted EV71 isolates. Subsequent studies in adult AG129 and young A129 mice with the adapted EV71 virus examined the efficacy of an inactivated EV71 candidate vaccine and determined the role of humoral immunity in protection. Passive transfer of rabbit immune sera raised against the EV71 vaccine provided protection in a dose dependent manner in 15 day-old A129 mice. Intramuscular injections (i.m.) in five week-old AG129 mice with the alum adjuvanted vaccine also provided protection against the mouse adapted homologous strain. No clinical signs of disease or mortality were observed in vaccinated animals, which received a prime-and-boost, whereas 71% of control animals were euthanized after exhibiting systemic clinical signs (P<0.05). The development of this animal model will facilitate studies on EV71 pathogenesis, antiviral testing, the evaluation of immunogenicity and efficacy of vaccine candidates, and has the potential to establish correlates of protection studies.