Pseudorabies virus membrane proteins gI and gE facilitate anterograde spread of infection in projection-specific neurons in the rat

The membrane proteins gI and gE of Pseudorabies virus (PRV) are required for viral invasion and spread through some neural pathways of the rodent central nervous system. Following infection of the rat retina with wild-type PRV, virus replicates in retinal ganglion neurons and anterogradely spreads to infect all visual centers in the brain. By contrast, gI and gE null mutants do not infect a specific subset of the visual centers, e.g., the superior colliculus and the dorsal lateral geniculate nucleus. In previous experiments, we suggested that the defect was not due to inability to infect projection-specific retinal ganglion cells, because mixed infection of a gE deletion mutant and a gI deletion mutant restored the wild-type phenotype (i.e., genetic complementation occurred). In the present study, we provide direct evidence that gE and gI function to promote the spread of infection after entry into primary neurons. We used stereotaxic central nervous system injection of a fluorescent retrograde tracer into the superior colliculus and subsequent inoculation of a PRV gI-gE double null mutant into the eye of the same animal to demonstrate that viral antigen and fluorescent tracer colocalize in retinal ganglion cells. Furthermore, we demonstrate that direct injection of a PRV gI-gE double null mutant into the superior colliculus resulted in robust infection followed by retrograde transport to the eye and replication in retinal ganglion neuron cell bodies. These experiments provide additional proof that the retinal ganglion cells projecting to the superior colliculus are susceptible and permissive to gE and gI mutant viruses. Our studies confirm that gI and gE specifically facilitate anterograde spread of infection by affecting intracellular processes in the primary infected neuron such as anterograde transport in axons or egress from axon terminals.     

Lesion of the Olfactory Epithelium Accelerates Prion Neuroinvasion and Disease Onset when Prion Replication Is Restricted to Neurons

Natural prion diseases of ruminants are moderately contagious and while the gastrointestinal tract is the primary site of prion agent entry, other mucosae may be entry sites in a subset of infections. In the current study we examined prion neuroinvasion and disease induction following disruption of the olfactory epithelium in the nasal mucosa since this site contains environmentally exposed olfactory sensory neurons that project directly into the central nervous system. Here we provide evidence for accelerated prion neuroinvasion and clinical onset from the olfactory mucosa after disruption and regeneration of the olfactory epithelium and when prion replication is restricted to neurons. In transgenic mice with neuron restricted replication of prions, there was a reduction in survival when the olfactory epithelium was disrupted prior to intranasal inoculation and there was >25% decrease in the prion incubation period. In a second model, the neurotropic DY strain of transmissible mink encephalopathy was not pathogenic in hamsters by the nasal route, but 50% of animals exhibited brain infection and/or disease when the olfactory epithelium was disrupted prior to intranasal inoculation. A time course analysis of prion deposition in the brain following loss of the olfactory epithelium in models of neuron-restricted prion replication suggests that neuroinvasion from the olfactory mucosa is via the olfactory nerve or brain stem associated cranial nerves. We propose that induction of neurogenesis after damage to the olfactory epithelium can lead to prion infection of immature olfactory sensory neurons and accelerate prion spread to the brain.     

Murine retrovirus-induced spongiform encephalomyelopathy: host and viral factors which determine the length of the incubation period.

A molecular clone of wild mouse ecotropic retrovirus CasBrE (clone 15-1) causes a spongiform neurodegenerative disease with a long incubation period, greater than or equal to 6 months. This virus infects the central nervous system (CNS) at low levels. In contrast, a chimeric virus, FrCasE, containing env and 3' pol sequences of 15-1 in a Friend murine leukemia virus background, infects the CNS at high levels and causes a rapid neurodegenerative disease with an incubation period of only 16 days. With both viruses, the induction of neurologic disease is dependent on inoculation during the perinatal period. Since the length of the incubation period of this disease appears to be a function of the relative level of CNS infection, we have attempted to identify the viral and host factors which determine the relative level of virus infection of the CNS. It was previously shown that the CNS is susceptible to infection only during the perinatal period (M. Czub, S. Czub, F. J. McAtee, and J. L. Portis, J. Virol. 65:2539-2544, 1991). Here we have found that the susceptibility of the CNS wanes progressively or gradually as a function of the age of the host, this age-dependent resistance being complete by 12 to 14 days of age. Utilizing a group of chimeric viruses, we found that the relative level of CNS infection achieved after inoculation of mice at 1 day of age was a function of the kinetics of virus replication and spread in peripheral organs. Viruses which reached peak viremia titers early (5 to 7 days of age) infected the CNS at high levels, and viruses which reached peak titers later infected the CNS at lower levels. Among the group of viruses examined in the current study, the kinetics of peripheral virus replication and spread appeared to be influenced primarily by sequences within the R-U5-5' leader region of the viral genome. These results suggested that the relative level of CNS infection was determined very early in life and appeared to be a function of a dynamic balance between the kinetics of virus replication in the periphery and a progressively developing restriction of virus replication in the CNS.     

Prion interference is due to a reduction in strain-specific PrPSc levels

When two prion strains infect a single host, one strain can interfere with the ability of the other to cause disease but it is not known whether prion replication of the second strain is also diminished. To further investigate strain interference, we infected hamsters in the sciatic nerve with the long-incubation-period transmissible mink encephalopathy (TME) agent DY TME prior to superinfection of hamsters with the short-incubation-period HY TME agent. Increases in the interval between TME agent inoculations resulted in an extension of the incubation period of HY TME or a complete block of the ability of the HY TME agent to cause disease. The sciatic nerve route of inoculation gave the two TME strains access to the same population of neurons, allowing for the potential of prion interference in the lumbar spinal cord. The ability of the DY TME agent to extend the incubation period of HY TME corresponds with detection of DY TME PrP(Sc), the abnormal isoform of the prion protein, in the lumbar spinal cord. The increased incubation period of HY TME or the inability of the HY TME agent to cause disease in the coinfected animals corresponds with a reduction in the abundance of HY TME PrP(Sc) in the lumbar spinal cord. When the two strains were not directed to the same populations of neurons within the lumbar spinal cord, interference between HY TME and DY TME did not occur. This suggests that DY TME agent replication interferes with HY TME agent replication when the two strains infect a common population of neurons.     

Prion interference with multiple prion isolates

Co-inoculation of prion strains into the same host can result in interference, where replication of one strain hinders the ability of another strain to cause disease. The drowsy (DY) strain of hamster-adapted transmissible mink encephalopathy (TME) extends the incubation period or completely blocks the hyper (HY) strain of TME following intracerebral, intraperitoneal or sciatic nerve routes of inoculation. However, it is not known if the interfering effect of the DY TME agent is exclusive to the HY TME agent by these experimental routes of infection. To address this issue, we show that the DY TME agent can block hamster-adapted chronic wasting disease (HaCWD) and the 263K scrapie agent from causing disease following sciatic nerve inoculation. Additionally, per os inoculation of DY TME agent slightly extends the incubation period of per os superinfected HY TME agent. These studies suggest that prion strain interference can occur by a natural route of infection and may be a more generalized phenomenon of prion strains.     

Characterization of nerve growth factor-dependent herpes simplex virus latency in neurons in vitro.

Primary sympathetic neuronal cultures were maintained for up to 5 weeks after inoculation with herpes simplex virus (HSV) without evidence of viral infection. Treatment with acyclovir for the first 7 days after viral inoculation prevented lytic infections in 100% of the cultures and resulted in viral latency in 100% of the cultures; reactivation occurred as the result of nerve growth factor (NGF) deprivation. Treatment of the cultures with several different inhibitors of viral DNA polymerase (acyclovir, aphidicolin, and phosphonoacetic acid) for 7 days after viral inoculation did not prevent the establishment of latency, which suggests that viral DNA replication was not required. During the latent phase of the infection, viral antigens were not detected with HSV-specific immunohistochemistry. However, 24 h after NGF deprivation, viral antigens were detected in essentially all of the neurons, indicating that the majority of neurons harbored latent HSV. The establishment of latency was not strain or type specific since latency was established with HSV type 2 and four strains of HSV type 1 and reactivation occurred in response to NGF deprivation.     

Adenovirus pneumonia in guineapigs: an experimental reproduction of the disease

The recently described virus-induced pneumonia in guineapigs (Naumann et al., 1981) was experimentally reproducible in newborn animals, though not in preadult animals. Baby hamsters and newborn rats were also not susceptible to infection. 10 of 11 infected newborn guineapigs developed pathological changes identical with those found in spontaneous cases. The incubation period was from 5 to 10 days. The agent could not be cultivated in vitro, and therefore no applicable serological tests could be established. The morphology of the virus, its intranuclear location, the course of the disease and the histopathological and ultrastructural changes strongly suggest that the virus is an adenovirus specific for guineapigs. The virus did not cross-react with human or fowl adenoviruses. It was ether resistant and non-oncogenic in baby rats and hamsters. During a 5-year period we registered a total of 51 spontaneous death cases diagnosed as adenovirus pneumonia in our experimental guineapigs, 4 from own breeding colony.     

The strain-encoded relationship between PrP replication, stability and processing in neurons is predictive of the incubation period of disease

Prion strains are characterized by differences in the outcome of disease, most notably incubation period and neuropathological features. While it is established that the disease specific isoform of the prion protein, PrP(Sc), is an essential component of the infectious agent, the strain-specific relationship between PrP(Sc) properties and the biological features of the resulting disease is not clear. To investigate this relationship, we examined the amplification efficiency and conformational stability of PrP(Sc) from eight hamster-adapted prion strains and compared it to the resulting incubation period of disease and processing of PrP(Sc) in neurons and glia. We found that short incubation period strains were characterized by more efficient PrP(Sc) amplification and higher PrP(Sc) conformational stabilities compared to long incubation period strains. In the CNS, the short incubation period strains were characterized by the accumulation of N-terminally truncated PrP(Sc) in the soma of neurons, astrocytes and microglia in contrast to long incubation period strains where PrP(Sc) did not accumulate to detectable levels in the soma of neurons but was detected in glia similar to short incubation period strains. These results are inconsistent with the hypothesis that a decrease in conformational stability results in a corresponding increase in replication efficiency and suggest that glia mediated neurodegeneration results in longer survival times compared to direct replication of PrP(Sc) in neurons.     

Temporal replication of the Pullman strain of Aleutian disease virus in royal pastel mink.

Information was sought on the temporal replication of Aleutian disease virus in 27 royal pastel mink. Groups of three were examined 8 to 126 days after they were inoculated subcutaneously with 10(3) 50% lethal doses of the Pullman strain. Much individual variation was noted in the onset of infection, occurrence of viremia, and extent of virus replication in the tissues. Thus, virus was detected in lymph nodes regional to the site of inoculation in only some mink during the first 14 days after inoculation. During this period, virus was often present as well in the mesenteric lymph node and spleen. First detected on day 10, viremia was present in all mink examined on day 28 but occurred irregularly thereafter, even when virus was widespread in the tissues. Except in five mink succumbing to the disease, the tissue distribution of virus after day 28 tended to be more limited, and the titers were generally lower than they had been earlier. Even though present in the lymph nodes and spleen, virus was often absent from the kidney, liver, and intestine after day 28. Specific antibody was detected on day 28 and was present in all mink thereafter, ostensibly without any adverse effect on virus replication. In most mink, the infection was considered subclinical, for it was usually not accompanied by a rise in serum gamma globulin or by morphologic evidence of the disease. The virologic findings in this study have a bearing on the relationship of subclinical infections to both horizontal and vertical transmission of the virus.     

The R-U5-5'' leader sequence of neurovirulent wild mouse retrovirus contains an element controlling the incubation period of neurodegenerative disease.

The wild mouse ecotropic retrovirus CasBrE causes a spongiform neurodegenerative disease after neonatal inoculation, with an incubation period ranging from 2 to 12 months. We previously showed that introduction of long terminal repeat (LTR) and gag-pol sequences from a strain of Friend murine leukemia virus (FB29) resulted in a dramatic acceleration of the onset of the disease. The chimeric virus FrCasE, which consisted of the FB29 genome containing 3' pol and env sequences from the wild mouse virus, induced a highly predictable, lethal neurodegenerative disease with an incubation period of only 16 days. Here we report that the sequences which are primary determinants of the length of the incubation period are located in the 5' end of the viral genome between a KpnI site in the R region of the LTR and a PstI site immediately 5' of the start codon for pr65gag (R-U5-5' leader). This region contains the tRNA primer binding site, splice donor site for the subgenomic env mRNA, and the packaging sequence. Computer-assisted sequence analysis failed to find evidence of a consensus sequence for a DNA enhancer in this region. In addition, sequences within a region of the genome between a ClaI site at the 3' end of env to the KpnI site in the R region of the LTR (inclusive of U3) also influenced the incubation period of the disease, but the effect was distinctly weaker than that of the R-U5-5' leader sequence. This U3 effect, however, appeared to be independent of the number of direct repeats, since deletion of one of two duplicated 42-base repeats containing consensus sequences of nuclear-factor binding domains had no effect on the incubation period of the disease. On the basis of Southern blot analysis of total viral DNA in the tissues, the effect of these sequences on the incubation period appeared to be related to the level of virus replication in the central nervous system. All of the chimeric viruses analyzed, irrespective of neurovirulence, replicated to comparable levels in the spleen and induced comparable levels of viremia.     

Slow development of measles virus (Edmonston strain) infection in the brain of nude mice

The Edmonston strain of measles virus caused neurologic disease in athymic nude mice by intracerebral inoculation. The incubation periods of the disease, however, were extremely long, ranging from 59 to 140 days when the mice were inoculated with 10(4) plaque forming units (PFU) of the virus. The Edmonston strain was highly infectious in the nude mouse brain since virus infection was established even with 1 PFU of the virus. Virus titers in the brains of infected mice increased with the time of incubation. These results indicate that the extremely long incubation period of the disease is ascribed to very slow development of virus infection in the mouse brain. On the other hand, the incubation periods of the Biken strain of SSPE virus were very short (generally within 2 weeks) even with inoculations of 1 PFU of the virus. However, the extent of the dissemination of infection in brains was not significantly different between the two viruses as examined by immunofluorescent staining.     

Identification of a sequence in the unique 5' open reading frame of the gene encoding glycosylated Gag which influences the incubation period of neurodegenerative disease induced by a murine retrovirus.

Neonatal inoculation of the wild-mouse ecotropic retrovirus CasBrE (clone 15-1) causes a noninflammatory spongiform neurodegenerative disease with an incubation period of > or = 6 months. Introduction of sequences from Friend murine leukemia virus (clone FB29) into the genome of CasBrE results in a marked shortening of the incubation period. The FB29 sequences which influence the incubation period were previously localized to the 5' leader sequence of the viral genome (M. Czub, F. J. McAtee, and J. L. Portis, J. Virol. 66:3298-3305, 1992). In the current study, we constructed a series of chimeric viruses consisting of the genome of CasBrE containing various segments of the leader sequence from FB29. A 41-nucleotide element (positions 481 through 521) near the 3' end of the leader was found to have a strong influence on the incubation period. This element influenced the kinetics of virus replication and/or spread in nonneuronal tissues, a property which was shown previously to determine the extent of central nervous system infection (M. Czub, F. J. McAtee, and J. L. Portis, J. Virol. 66:3298-3305, 1992). Curiously, this sequence had no demonstrable effect on virus replication in vitro in a fibroblastic cell line from Mus dunni. This segment encodes 14 of the unique 88-amino-acid N terminus of pr75gag, the precursor of a glycosylated form of the gag polyprotein which is expressed at the cell surface. Previous in vitro studies of mutants of Moloney murine leukemia virus lacking expression of glycosylated Gag failed to reveal a function for this protein in virus replication. We mutated the Kozak consensus sequence around the initiation codon for this protein in the chimeric virus CasFrKP, a virus which induces neurologic disease with a short (18- to 23-day) incubation period. M. dunni cells infected with the mutants lacked detectable cell surface Gag, but, compared with CasFrKP, no effect on replication kinetics in vitro was observed. In contrast, there was a marked slowing of the replication kinetics in vivo and a dramatic attenuation of neurovirulence. These studies indicate that glycosylated Gag has an important function in virus replication and/or spread in the mouse and further suggest that the sequence of its N terminus is a critical, though likely indirect, determinant of neurovirulence.     

Transgenic mice expressing the nucleoprotein of Borna disease virus in either neurons or astrocytes: decreased susceptibility to homotypic infection and disease

The nucleoprotein (N) of Borna disease virus (BDV) is the major target of the disease-inducing antiviral CD8 T-cell response in the central nervous system of mice. We established two transgenic mouse lines which express BDV-N in either neurons (Neuro-N) or astrocytes (Astro-N). Despite strong transgene expression, neurological disease or gross behavioral abnormalities were not observed in these animals. When Neuro-N mice were infected as adults, replication of BDV was severely impaired and was restricted to brain areas with a low density of transgene-expressing cells. Notably, the virus failed to replicate in the transgene-expressing granular and pyramidal neurons of the hippocampus (which are usually the preferred host cells of BDV). When Neuro-N mice were infected within the first 5 days of life, replication of BDV was not suppressed in most neurons, presumably because the onset of transgene expression in the brain occurred after these cells became infected with BDV. Astro-N mice remained susceptible to BDV infection, but they were resistant to BDV-induced neurological disorder. Unlike their nontransgenic littermates, Neuro-N mice with persistent BDV infection did not develop neurological disease after immunization with a vaccinia virus vector expressing BDV-N. In contrast to the situation in wild-type mice, this treatment also failed to induce N-specific CD8 T cells in the spleens of both transgenic mouse lines. Thus, while resistance to BDV infection in N-expressing neurons appeared to result from untimely expression of a viral nucleocapsid component, the resistance to BDV-induced neuropathology probably resulted from immunological tolerance.     

Borna disease virus glycoprotein is required for viral dissemination in neurons

Borna disease virus (BDV) is a nonsegmented negative-strand RNA virus with a tropism for neurons. Infection with BDV causes neurological diseases in a wide variety of animal species. Although it is known that the virus spreads from neuron to neuron, assembled viral particles have never been visualized in the brains of infected animals. This has led to the hypothesis that BDV spreads as nonenveloped ribonucleoproteins (RNP) rather than as enveloped viral particles. We assessed whether the viral envelope glycoprotein (GP) is required for neuronal dissemination of BDV by using primary cultures of rat hippocampal neurons. We show that upon in vitro infection, BDV replicated and spread efficiently in this system. Despite rapid virus dissemination, very few infectious viral particles were detectable in the culture. However, neutralizing antibodies directed against BDV-GP inhibited BDV spread. In addition, interference with BDV-GP processing by inhibiting furin-mediated cleavage of the glycoprotein blocked virus spread. Finally, antisense treatment with peptide nucleic acids directed against BDV-GP mRNA inhibited BDV dissemination, marking BDV-GP as an attractive target for antiviral therapy against BDV. Together, our results demonstrate that the expression and correct processing of BDV-GP are necessary for BDV dissemination in primary cultures of rat hippocampal neurons, arguing against the hypothesis that the virus spreads from neuron to neuron in the form of nonenveloped RNP.     

Age-dependent poliomyelitis of mice: expression of endogenous retrovirus correlates with cytocidal replication of lactate dehydrogenase-elevating virus in motor neurons.

The widespread presence of endogenous retroviruses in the genomes of animals and humans has suggested that these viruses may be involved in both normal and abnormal developmental processes. Previous studies have indicated the involvement of endogenous ecotropic murine leukemia virus (MuLV) in the development of age-dependent poliomyelitis caused by infection of old C58 or AKR mice by lactate dehydrogenase-elevating virus (LDV). The only genetic components which segregate with susceptibility to LDV-induced paralytic disease are multiple proviral copies of ecotropic MuLV and the permissive allele, at the Fv-1 locus, for N-tropic, ecotropic virus replication (Fv-1n/n). Using in situ hybridization and Northern (RNA) blot hybridization, we have correlated the expression of the endogenous MuLV, both temporally and spatially, with LDV infection of anterior horn motor neurons and the development of paralysis. Our data indicate that treatment of 6- to 7-month-old C58/M mice with cyclophosphamide, which renders these mice susceptible to LDV-induced paralytic disease, results in transient increases in ecotropic MuLV RNA levels in motor neurons throughout the spinal cord. Peripheral inoculation of C58/M mice with LDV, at the time of elevated MuLV RNA levels, results in a rapid spread of LDV to some spinal cord motor neurons. LDV infections then spread slowly but progressively throughout the spinal cord, involving an increasing number of motor neurons. LDV replication is cytocidal and results in neuron destruction and paralysis of the infected animals 2 to 3 weeks postinfection. The slow replication of LDV in the spinal cord contrasts sharply with the rapid replication of LDV in macrophages, the normal host cells for LDV, during the acute phase of infection. The data indicate that the interaction between the endogenous MuLV with the generally nonpathogenic murine togavirus LDV occurs at the level of the motor neuron. We discuss potential mechanisms for the novel dual-virus etiology of age-dependent poliomyelitis of mice.     

Extraneural prion neuroinvasion without lymphoreticular system infection

While prion infection of the lymphoreticular system (LRS) is necessary for neuroinvasion in many prion diseases, in bovine spongiform encephalopathy and atypical cases of sheep scrapie there is evidence to challenge that LRS infection is required for neuroinvasion. Here we investigated the role of prion infection of LRS tissues in neuroinvasion following extraneural inoculation with the HY and DY strains of the transmissible mink encephalopathy (TME) agent. DY TME agent infectivity was not detected in spleen or lymph nodes following intraperitoneal inoculation and clinical disease was not observed following inoculation into the peritoneum or lymph nodes, or after oral ingestion. In contrast, inoculation of the HY TME agent by each of these peripheral routes resulted in replication in the spleen and lymph nodes and induced clinical disease. To clarify the role of the LRS in neuroinvasion, the HY and DY TME agents were also inoculated into the tongue because it is densely innervated and lesions on the tongue, which are common in ruminants, increase the susceptibility of hamsters to experimental prion disease. Following intratongue inoculation, the DY TME agent caused prion disease and was detected in both the tongue and brainstem nuclei that innervate the tongue, but the prion protein PrP(Sc) was not detected in the spleen or lymph nodes. These findings indicate that the DY TME agent can spread from the tongue to the brain along cranial nerves and neuroinvasion does not require agent replication in the LRS. These studies provide support for prion neuroinvasion from highly innervated peripheral tissues in the absence of LRS infection in natural prion diseases of livestock.     

Recombinant type 5 adenoviruses expressing bovine parainfluenza virus type 3 glycoproteins protect Sigmodon hispidus cotton rats from bovine parainfluenza virus type 3 infection.

Cotton rats were used to study the replication and pathogenesis of bovine parainfluenza virus type 3 (bPIV3) and to test the efficacy of the F and HN glycoproteins in modulating infection. In vitro cultures of cotton rat lung cells supported the growth of bPIV3 as shown by virus recovery, immunofluorescence, immunoprecipitation, and syncytium induction. Intranasal (i.n.) inoculation of cotton rats with 10(7) PFU resulted in peak recovery of virus after 2 days (8 x 10(4) PFU/g of lung tissue) and significant bronchiolitis with lymphocyte infiltration 5 to 7 days postinfection. Immunohistochemical staining of lungs and trachea demonstrated that virus antigen-positive cells increased in frequency over the course of infection to a maximum on day 5. Serum antibody responses were evaluated by enzyme-linked immunosorbent assays (ELISA), hemagglutination inhibition (HAI), and serum neutralization (SN). Following a single i.n. inoculation, serum antibody levels were 1/40,960, 1/32, and 1/80, as detected by ELISA, HAI, and SN, respectively. When an intramuscular inoculation of 10(7) PFU was administered 10 days prior to the i.n. inoculation, a secondary response which resulted in an ELISA titer of 1/163,000, an HAI titer of 1/640, and an SN titer of 1/512 was induced. IN inoculation of recombinant adenoviruses type 5 containing the bPIV3 F or HN protein or a combination of the two viruses protected cotton rats from bPIV3 challenge. Protection was evaluated serologically by ELISA, HAI, and SN titers, histopathology, immunohistochemistry, and virus recovery.     

Early infection of the central nervous system by the GDVII and DA strains of Theiler's virus.

The DA strain of Theiler's virus, a murine picornavirus, causes a persistent infection of glial cells of the white matter of the spinal cord, associated with chronic inflammation and primary demyelination. The GDVII strain causes an acute fatal grey matter encephalomyelitis. We characterized the target cells of GDVII and DA viruses 4 days following intracerebral inoculation, and we compared the levels of viral RNA within these cells. GDVII virus infected approximately 10 times more cells than DA virus. Whereas GDVII virus infected neurons exclusively, DA virus infected also astrocytes and possible macrophage-microglial cells. The levels of viral RNA in neurons infected with GDVII and DA viruses were of the same order. These results show that DA virus infects glial cells already at the beginning of the disease and that the more efficient spread of GDVII virus is probably not due to a higher level of RNA replication per cell.     

The role of the blood-brain barrier during Venezuelan equine encephalitis virus infection

Venezuelan equine encephalitis (VEE) virus is a mosquito-borne alphavirus associated with sporadic outbreaks in human and equid populations in the Western Hemisphere. After the bite of an infected mosquito, the virus initiates a biphasic disease: a peripheral phase with viral replication in lymphoid and myeloid tissues, followed by a neurotropic phase with infection of central nervous system (CNS) neurons, causing neuropathology and in some cases fatal encephalitis. The mechanisms allowing VEE virus to enter the CNS are currently poorly understood. Previous data have shown that the virus gains access to the CNS by infecting olfactory sensory neurons in the nasal mucosa of mice. However, at day 5 after inoculation, the infection of the brain is multifocal, indicating that virus particles are able to cross the blood-brain barrier (BBB). To better understand the role of the BBB during VEE virus infection, we used a well-characterized mouse model system. Using VEE virus replicon particles (VRP), we modeled the early events of neuroinvasion, showing that the replication of VRP in the nasal mucosa induced the opening of the BBB, allowing peripherally administered VRP to invade the brain. Peripheral VEE virus infection was characterized by a biphasic opening of the BBB. Further, inhibition of BBB opening resulted in a delayed viral neuroinvasion and pathogenesis. Overall, these results suggest that VEE virus initially enters the CNS through the olfactory pathways and initiates viral replication in the brain, which induces the opening of the BBB, allowing a second wave of invading virus from the periphery to enter the brain.     

Simian immunodeficiency virus disease course is predicted by the extent of virus replication during primary infection

To elucidate the relationship between early viral infection events and immunodeficiency virus disease progression, quantitative-competitive and branched-DNA methods of simian immunodeficiency virus (SIV) RNA quantitation were cross-validated and used to measure viremia following infection of rhesus macaques with the pathogenic SIVmac251 virus isolate. Excellent correlation between the methods suggests that both accurately approximate SIV copy number. Plasma viremia was evident 4 days postinfection, and rapid viral expansion led to peak viremia levels of 10(7) to 10(9) SIV RNA copies/ml by days 8 to 17. Limited resolution of primary viremia was accompanied by relatively short, though variable, times to the development of AIDS (81 to 630 days). The persistent high-level viremia observed following intravenous inoculation of SIVmac251 explains the aggressive disease course in this model. Survival analyses demonstrated that the disease course is established 8 to 17 days postinfection, when peak viremia is observed. The most significant predictor of disease progression was the extent of viral decline following peak viremia; larger decrements in viremia were associated with both lower steady-state viremia (P = 0.0005) and a reduced hazard of AIDS (P = 0.004). The data also unexpectedly suggested that following SIVmac251 infection, animals with the highest peak viremia were better able to control virus replication rather than more rapidly developing disease. Analysis of early viral replication dynamics should help define host responses that protect from disease progression and should provide quantitative measures to assess the extent to which protective responses may be induced by prophylactic vaccination.