Herpes Simplex Virus 1 Induces Brain Inflammation and Multifocal Demyelination in the Cotton Rat Sigmodon hispidus.

Demyelinating central nervous system (CNS) disorders like multiple sclerosis (MS) and acute disseminated encephalomyelitis (ADEM) have been difficult to study and treat due to the lack of understanding of their etiology. Numerous cases point to the link between herpes simplex virus (HSV) infection and multifocal CNS demyelination in humans; however, convincing evidence from animal models has been missing. In this work, we found that HSV-1 infection of the cotton rat Sigmodon hispidus via a common route (lip abrasion) can cause multifocal CNS demyelination and inflammation. Remyelination occurred shortly after demyelination in HSV-1-infected cotton rats but could be incomplete, resulting in "scars," further supporting an association between HSV-1 infection and multifocal demyelinating disorders. Virus was detected sequentially in the lip, trigeminal ganglia, and brain of infected animals. Brain pathology developed primarily on the ipsilateral side of the brain stem, in the cerebellum, and contralateral side of the forebrain/midbrain, suggesting that the changes may ascend along the trigeminal lemniscus pathway. Neurologic defects occasionally detected in infected animals (e.g., defective whisker touch and blink responses and compromised balance) could be representative of the brain stem/cerebellum dysfunction. Immunization of cotton rats with a split HSV-1 vaccine protected animals against viral replication and brain pathology, suggesting that vaccination against HSV-1 may protect against demyelinating disorders.IMPORTANCE Our work demonstrates for the first time a direct association between infection with herpes simplex virus 1, a ubiquitous human pathogen generally associated with facial cold sores, and multifocal brain demyelination in an otherwise normal host, the cotton rat Sigmodon hispidus For a long time, demyelinating diseases were considered to be autoimmune in nature and were studied by indirect methods, such as immunizing animals with myelin components or feeding them toxic substances that induce demyelination. Treatment against demyelinating diseases has been elusive, partially because of their unknown etiology. This work provides the first experimental evidence for the role of HSV-1 as the etiologic agent of multifocal brain demyelination in a normal host and suggests that vaccination against HSV-1 can help to combat demyelinating disorders.

Neuropathogenicity of newly isolated avian leukosis viruses from chickens with osteopetrosis and mesenchymal neoplasms.

ABSTRACT The prototype fowl glioma-inducing virus (FGVp) causes fowl glioma and cerebellar hypoplasia in chickens. In this study, we investigated whether a strain of avian leukosis virus (ALV), associated with avian osteopetrosis and mesenchymal neoplasms, is able to induce fowl glioma. We encountered avian osteopetrosis and mesenchymal neoplasms, including myxosarcoma and rhabdomyosarcoma, in Japanese native chickens used for both egg-laying and meat production. These birds were also affected by non-suppurative encephalitis and glioma in their brains. Four ALV strains (GifN_001, GifN_002, GifN_004, GifN_005) were isolated, and a phylogenic analysis of envSU showed that these isolates were classified into different clusters from FGVp and the variants previously reported. Whereas the envSU shared a high identity (94.7%) with that of Rous sarcoma virus (strain Schmidt-Ruppin B) (RSV-SRB), the identity between envTM of GifN_001 and that of FGVp was high (94.5%), indicating that GifN_strains may emerge by recombination between FGVp and other exogenous ALVs. Specific-pathogen-free chickens inoculated in ovo with GifN_001 revealed fowl glioma and cerebellar hypoplasia. These results suggest that the newly isolated strains have acquired neuropathogenicity to chickens.

Cerebral patterns of neuropsychological disturbances in hepatitis C patients.

Neuropsychiatric symptoms and cognitive impairment have been consistently reported in patients with hepatitis C virus (HCV) infection. Since the mechanisms behind remain to be established, the present study attempted to assess whether neuropsychological impairments in HCV-infected patients are accompanied by structural alterations in the brain. Therefore, 19 anti-HCV-antibody-positive women with mild liver disease and 16 healthy controls underwent extensive neuropsychological testing and cranial magnetic resonance imaging (MRI) examination. Nine of the patients and five controls were followed up after 6-7 years. Voxel-based morphometry and magnetization transfer imaging were utilized to study HCV-associated structural gray and white matter changes. The HCV-infected patients had significantly worse fatigue and depression scores and significantly poorer performance on attention and memory tests than controls. The patients displayed gray matter (GM) atrophy in the bilateral insula and thalamus and a profound GM volume increases in the cerebellum. Microstructural GM changes in the insula were also evident by a reduced magnetization transfer ratio. Structural white matter changes were observed along several descending and crossing fiber tracts. Follow-up at 7 years revealed increased GM atrophy in the left amygdala and left parahippocampal regions over time. We conclude that our data provide evidence for structural alterations in the brains of patients with chronic HCV infection. Disturbances of cerebellothalamocortical regions and circuits, linking cerebellar projections to the prefrontal cortex through the thalamus, underpin the emotional and cognitive dysfunction characteristically observed in these patients.

Lessons Learned at the Epicenter of Brazil's Congenital Zika Epidemic: Evidence From 87 Confirmed Cases.

Congenital Zika virus infection has stimulated great international concern. A prospective case series of 87 infants with laboratory-confirmed congenital Zika syndrome (CZS) at the epicenter of the Brazilian Zika epidemic in Pernambuco state is presented. Mothers were interviewed for symptoms of possible Zika virus (ZIKV) infection during pregnancy, and fetal ultrasounds were obtained. Infant cerebrospinal fluid (CSF) samples were tested for ZIKV-specific antibodies, and sera were screened for other congenital infections. Neuroimaging and ophthalmologic evaluations were also performed. Sixty-six mothers (76%) reported symptoms of ZIKV infection during gestation. Fetal ultrasounds were available from 90% of the mothers, and all demonstrated brain structural abnormalities. All of the CSF samples tested positive for ZIKV immunoglobulin M. The majority of infants (89%) were term; the mean birth weight was 2577 ± 260 g, and the mean head circumference was 28.1 ± 1.8 cm. Severe microcephaly, defined as head circumference 3 SD below the mean for sex and gestational age, was found in 72 (82%) infants. All infants had an abnormal neurological exam, and 18 (20.7%) had arthrogryposis. The main abnormalities detected in computed tomography scans were calcifications (99%), followed by ventricular enlargement (94%), cortical hypogyration (81%), and less commonly, cerebellar hypoplasia (52%). Unilateral diaphragm paralysis was identified in 3 infants. Maternal young age, term infant, small for gestational age, and the presence of ophthalmologic abnormalities were significantly associated with a smaller head circumference Z score. Our findings, based on laboratory-confirmed ZIKV infection, add valuable evidence for the understanding of CZS.

Outbreaks of Neuroinvasive Astrovirus Associated with Encephalomyelitis, Weakness, and Paralysis among Weaned Pigs, Hungary.

A large, highly prolific swine farm in Hungary had a 2-year history of neurologic disease among newly weaned (25- to 35-day-old) pigs, with clinical signs of posterior paraplegia and a high mortality rate. Affected pigs that were necropsied had encephalomyelitis and neural necrosis. Porcine astrovirus type 3 was identified by reverse transcription PCR and in situ hybridization in brain and spinal cord samples in 6 animals from this farm. Among tissues tested by quantitative RT-PCR, the highest viral loads were detected in brain stem and spinal cord. Similar porcine astrovirus type 3 was also detected in archived brain and spinal cord samples from another 2 geographically distant farms. Viral RNA was predominantly restricted to neurons, particularly in the brain stem, cerebellum (Purkinje cells), and cervical spinal cord. Astrovirus was generally undetectable in feces but present in respiratory samples, indicating a possible respiratory infection. Astrovirus could cause common, neuroinvasive epidemic disease.

HIV Maintains an Evolving and Dispersed Population in Multiple Tissues during Suppressive Combined Antiretroviral Therapy in Individuals with Cancer.

UNLABELLED: While combined antiretroviral therapy (cART) can result in undetectable plasma viral loads, it does not eradicate HIV infection. Furthermore, HIV-infected individuals while on cART remain at an increased risk of developing serious comorbidities, such as cancer, neurological disease, and atherosclerosis, suggesting that during cART, tissue-based HIV may contribute to such pathologies. We obtained DNA and RNA env, nef, and pol sequences using single-genome sequencing from postmortem tissues of three HIV(+) cART-treated (cART(+)) individuals with undetectable viral load and metastatic cancer at death and performed time-scaled Bayesian evolutionary analyses. We used a sensitive in situ hybridization technique to visualize HIV gag-pol mRNA transcripts in cerebellum and lymph node tissues from one patient. Tissue-associated virus evolved at similar rates in cART(+) and cART-naive (cART(-)) patients. Phylogenetic trees were characterized by two distinct features: (i) branching patterns consistent with constant viral evolution and dispersal among tissues and (ii) very recently derived clades containing both DNA and RNA sequences from multiple tissues. Rapid expansion of virus near death corresponded to wide-spread metastasis. HIV RNA(+) cells clustered in cerebellum tissue but were dispersed in lymph node tissue, mirroring the evolutionary patterns observed for that patient. Activated, infiltrating macrophages were associated with HIV RNA. Our data provide evidence that tissues serve as a sanctuary for wild-type HIV during cART and suggest the importance of macrophages as an alternative reservoir and mechanism of virus spread. IMPORTANCE: Combined antiretroviral therapy (cART) reduces plasma HIV to undetectable levels; however, removal of cART results in plasma HIV rebound, thus highlighting its inability to entirely rid the body of infection. Additionally, HIV-infected individuals on cART remain at high risk of serious diseases, which suggests a contribution from residual HIV. In this study, we isolated and sequenced HIV from postmortem tissues from three HIV(+) cART(+) individuals who died with metastatic cancer and had no detectable plasma viral load. Using high-resolution evolutionary analyses, we found that tissue-based HIV continues to replicate, evolve, and migrate among tissues during cART. Furthermore, cancer onset and metastasis coincided with increased HIV expansion, suggesting a linked mechanism. HIV-expressing cells were associated with tissue macrophages, a target of HIV infection. Our results suggest the importance of tissues, and macrophages in particular, as a target for novel anti-HIV therapies.

Case report: microcephaly associated with Zika virus infection, Colombia.

BACKGROUND: Recently there has been a large outbreak of Zika virus infections in Colombia, South America. The epidemic began in September 2015 and continued to April 2017, for the total number of Zika cases reported of 107,870. For those confirmed Zika cases, there were nearly 20,000 (18.5%) suspected to be pregnant women, resulting in 157 confirmed cases of microcephaly in newborns reported by their health government agency. There is a clear under-estimation of the total number of cases and in addition no prior publications have been published to demonstrate the clinical aspects of the Zika infection in Colombia. We characterized one Zika presentation to be able to compare and contrast with other cases of Zika infection already reported in the literature. CASE PRESENTATION: In this case report, we demonstrate congenital microcephaly at week 19 of gestation in a 34-year-old mother who showed symptoms compatible with Zika virus infection from Sincelejo, State of Sucre, in the Colombian Caribbean. Zika virus RNA was detected in the placenta using real-time reverse transcriptase polymerase chain reaction (RT-PCR). At week 25, the fetus weigh estimate was 770 g, had a cephalic perimeter of 20.2 cm (5th percentile), ventriculomegaly on the right side and dilatation of the fourth ventricle. At week 32, the microcephaly was confirmed with a cephalic perimeter of 22 cm, dilatation of the posterior atrium to 13 mm, an abnormally small cerebellum (29 mm), and an augmented cisterna magna. At birth (39 weeks by cesarean section), the head circumference was 27.5 cm, and computerized axial tomography (Siemens Corp, 32-slides) confirmed microcephaly with calcifications. CONCLUSION: We report a first case of maternal Zika virus infection associated with fetal microcephaly in Colombia and confirmed similar presentation to those observed previous in Brazil, 2015-2016.

Generation of a Recombinant Akabane Virus Expressing Enhanced Green Fluorescent Protein.

UNLABELLED: We generated a recombinant Akabane virus (AKAV) expressing enhanced green fluorescence protein (eGFP-AKAV) by using reverse genetics. We artificially constructed an ambisense AKAV S genome encoding N/NSs on the negative-sense strand, and eGFP on the positive-sense strand with an intergenic region (IGR) derived from the Rift Valley fever virus (RVFV) S genome. The recombinant virus exhibited eGFP fluorescence and had a cytopathic effect in cell cultures, even after several passages. These results indicate that the gene encoding eGFP in the ambisense RNA could be stably maintained. Transcription of N/NSs and eGFP mRNAs of eGFP-AKAV was terminated within the IGR. The mechanism responsible for this appears to be different from that in RVFV, where the termination sites for N and NSs are determined by a defined signal sequence. We inoculated suckling mice intraperitoneally with eGFP-AKAV, which resulted in neurological signs and lethality equivalent to those seen for the parent AKAV. Fluorescence from eGFP in frozen brain slices from the eGFP-AKAV-infected mice was localized to the cerebellum, pons, and medulla oblongata. Our approach to producing a fluorescent virus, using an ambisense genome, helped obtain eGFP-AKAV, a fluorescent bunyavirus whose viral genes are intact and which can be easily visualized. IMPORTANCE: AKAV is the etiological agent of arthrogryposis-hydranencephaly syndrome in ruminants, which causes considerable economic loss to the livestock industry. We successfully generated a recombinant enhanced green fluorescent protein-tagged AKAV containing an artificial ambisense S genome. This virus could become a useful tool for analyzing AKAV pathogenesis in host animals. In addition, our approach of using an ambisense genome to generate an orthobunyavirus stably expressing a foreign gene could contribute to establishing alternative vaccine strategies, such as bivalent vaccine virus constructs, for veterinary use against infectious diseases.

Central nervous system infections caused by varicella-zoster virus.

We carried out a clinical and epidemiological study of adult patients with varicella-zoster virus central nervous system infection diagnosed by PCR in cerebrospinal fluid. Twenty-six patients were included. Twelve (46.2 %) patients were diagnosed with meningitis and fourteen (53.8 %) with meningoencephalitis. Twelve (46.2 %) had cranial nerves involvement (mainly the facial (VII) and vestibulocochlear (VIII) nerves), six (23.1 %) had cerebellar involvement, fourteen (53.8 %) had rash, and four (15.4 %) developed Ramsay Hunt syndrome. Three (11.5 %) patients had sequelae. Length of stay was significantly lower in patients diagnosed with meningitis and treatment with acyclovir was more frequent in patients diagnosed with meningoencephalitis. We believe routine detection of varicella-zoster virus, regardless of the presence of rash, is important because the patient may benefit from a different clinical management.

Glucocorticoid treatment of MCMV infected newborn mice attenuates CNS inflammation and limits deficits in cerebellar development.

Infection of the developing fetus with human cytomegalovirus (HCMV) is a major cause of central nervous system disease in infants and children; however, mechanism(s) of disease associated with this intrauterine infection remain poorly understood. Utilizing a mouse model of HCMV infection of the developing CNS, we have shown that peripheral inoculation of newborn mice with murine CMV (MCMV) results in CNS infection and developmental abnormalities that recapitulate key features of the human infection. In this model, animals exhibit decreased granule neuron precursor cell (GNPC) proliferation and altered morphogenesis of the cerebellar cortex. Deficits in cerebellar cortical development are symmetric and global even though infection of the CNS results in a non-necrotizing encephalitis characterized by widely scattered foci of virus-infected cells with mononuclear cell infiltrates. These findings suggested that inflammation induced by MCMV infection could underlie deficits in CNS development. We investigated the contribution of host inflammatory responses to abnormal cerebellar development by modulating inflammatory responses in infected mice with glucocorticoids. Treatment of infected animals with glucocorticoids decreased activation of CNS mononuclear cells and expression of inflammatory cytokines (TNF-α, IFN-ß and IFNγ) in the CNS while minimally impacting CNS virus replication. Glucocorticoid treatment also limited morphogenic abnormalities and normalized the expression of developmentally regulated genes within the cerebellum. Importantly, GNPC proliferation deficits were normalized in MCMV infected mice following glucocorticoid treatment. Our findings argue that host inflammatory responses to MCMV infection contribute to deficits in CNS development in MCMV infected mice and suggest that similar mechanisms of disease could be responsible for the abnormal CNS development in human infants infected in-utero with HCMV.

One-year follow-up of transgene expression by integrase-defective lentiviral vectors and their therapeutic potential in spinocerebellar ataxia model mice.

We examined integrase-defective lentiviral vectors (IDLVs) with a mutant (D64V) integrase in terms of their residual integration capability, the levels and duration of transgene expression and their therapeutic potential in comparison to wild-type lentiviral vectors (WTLVs) with a wild-type integrase gene. Compared with WTLVs, the IDLV-mediated proviral integration into host-cell chromosomes was approximately 1/3850 in HeLa cells and approximately 1/111 in mouse cerebellar neurons in vivo. At 2 months, transgene expression by IDLVs in the mouse cerebellum was comparable to that by WTLVs, but then significantly decreased. The mRNA levels at 6 and 12 months after injection in IDLV-infected cerebella were approximately 26% and 5%, respectively, of the mRNA levels in WTLV-injected cerebella. To examine the therapeutic potential, IDLVs or WTLVs expressing a molecule that enhances the ubiquitin-proteasome pathway were injected into the cerebella of spinocerebellar ataxia type 3 model mice (SCA3 mice). IDLV-injected SCA3 mice showed a significantly improved rotarod performance even at 1 year after-injection. Immunohistochemistry at 1 year after injection showed a drastic reduction of mutant aggregates in Purkinje cellsfrom IDLV-injected, as well as WTLV-injected, SCA3 mice. Our results suggest that because of the substantially reduced risk of insertional mutagenesis, IDLVs are safer and potentially effective as gene therapy vectors.

Eastern equine encephalitis cases among horses in Brazil between 2005 and 2009.

Eastern equine encephalitis is a viral zoonosis that exhibits complex distribution and epidemiology, and greater importance should be given to this disease by the public-health authorities. In Brazil, although eastern equine encephalitis virus (EEEV) has been identified in vectors and antibodies are sometimes detected in horses and humans, there have been no records of equine encephalitis in horses caused by this virus during the last 24 years. This study describes eighteen cases of eastern equine encephalomyelitis that occurred in six Brazilian states between 2005 and 2009. Viral RNA was identified using semi-nested RT-PCR to detect members of the genus Alphavirus, and by genetic sequencing. The gene encoding NSP1 was partially amplified, and after genetic sequencing, eighteen sequences were generated. All eighteen strains were classified as belonging to lineage III of American EEEV. These findings could be an indication of the importance of this virus in animal and human public health.

Cytomegalovirus infection lengthens the cell cycle of granule cell precursors during postnatal cerebellar development.

Human cytomegalovirus (HCMV) infection in infants infected in utero can lead to a variety of neurodevelopmental disorders. However, mechanisms underlying altered neurodevelopment in infected infants remain poorly understood. We have previously described a murine model of congenital HCMV infection in which murine CMV (MCMV) spreads hematogenously and establishes a focal infection in all regions of the brain of newborn mice, including the cerebellum. Infection resulted in disruption of cerebellar cortical development characterized by reduced cerebellar size and foliation. This disruption was associated with altered cell cycle progression of the granule cell precursors (GCPs), which are the progenitors that give rise to granule cells (GCs), the most abundant neurons in the cerebellum. In the current study, we have demonstrated that MCMV infection leads to prolonged GCP cell cycle, premature exit from the cell cycle, and reduced numbers of GCs resulting in cerebellar hypoplasia. Treatment with TNF-α neutralizing antibody partially normalized the cell cycle alterations of GCPs and altered cerebellar morphogenesis induced by MCMV infection. Collectively, our results argue that virus-induced inflammation altered the cell cycle of GCPs resulting in a reduced numbers of GCs and cerebellar cortical hypoplasia, thus providing a potential mechanism for altered neurodevelopment in fetuses infected with HCMV.

The basal ganglia communicate with the cerebellum.

The basal ganglia and cerebellum are major subcortical structures that influence not only movement, but putatively also cognition and affect. Both structures receive input from and send output to the cerebral cortex. Thus, the basal ganglia and cerebellum form multisynaptic loops with the cerebral cortex. Basal ganglia and cerebellar loops have been assumed to be anatomically separate and to perform distinct functional operations. We investigated whether there is any direct route for basal ganglia output to influence cerebellar function that is independent of the cerebral cortex. We injected rabies virus (RV) into selected regions of the cerebellar cortex in cebus monkeys and used retrograde transneuronal transport of the virus to determine the origin of multisynaptic inputs to the injection sites. We found that the subthalamic nucleus of the basal ganglia has a substantial disynaptic projection to the cerebellar cortex. This pathway provides a means for both normal and abnormal signals from the basal ganglia to influence cerebellar function. We previously showed that the dentate nucleus of the cerebellum has a disynaptic projection to an input stage of basal ganglia processing, the striatum. Taken together these results provide the anatomical substrate for substantial two-way communication between the basal ganglia and cerebellum. Thus, the two subcortical structures may be linked together to form an integrated functional network.

Emv2, the only endogenous ecotropic murine leukemia virus of C57BL/6J mice.

With the proliferation of sequence data, great challenges are posed in the correct annotation of endogenous retroviruses, which together comprise up to ten per cent of the genomes of many organisms. It is therefore essential that all sources of information are carefully considered before drawing conclusions concerning the phylogeny, distribution and biological properties of endogenous retroviruses. We suggest that such due diligence has not been applied in the description of an endogenous ecotropic retrovirus that recently appeared in Retrovirology.

Reply to Emv2, the only endogenous ecotropic murine leukemia virus of C57BL/6J mice.

This correspondence was written in response to the comments by Young et al. Following careful evaluation of the relevant dataset, each of the points brought up by Young et al. has been addressed in this response. We anticipate this will clarify our findings regarding ERVmch8, an ecotropic endogenous retrovirus that was shown to have cerebellum-specific and age-dependent expression patterns in C57BL/6J mice.

Viewpoint on Emv2, the onlhy endogenous ecotropic murine leukemia virus of C57BL/6 mice.

Here I comment on the articles by Lee and colleagues (Retrovirology 2011, 8:82) and Lee and Cho (Retrovirology 2012, 9:23) dealing with an endogenous ecotropic mouse leukemia virus found in C57BL mice.

Cerebellum-specific and age-dependent expression of an endogenous retrovirus with intact coding potential.

BACKGROUND: Endogenous retroviruses (ERVs), including murine leukemia virus (MuLV) type-ERVs (MuLV-ERVs), are presumed to occupy ~10% of the mouse genome. In this study, following the identification of a full-length MuLV-ERV by in silico survey of the C57BL/6J mouse genome, its distribution in different mouse strains and expression characteristics were investigated. RESULTS: Application of a set of ERV mining protocols identified a MuLV-ERV locus with full coding potential on chromosome 8 (named ERVmch8). It appears that ERVmch8 shares the same genomic locus with a replication-incompetent MuLV-ERV, called Emv2; however, it was not confirmed due to a lack of relevant annotation and Emv2 sequence information. The ERVmch8 sequence was more prevalent in laboratory strains compared to wild-derived strains. Among 16 different tissues of ~12 week-old female C57BL/6J mice, brain homogenate was the only tissue with evident expression of ERVmch8. Further ERVmch8 expression analysis in six different brain compartments and four peripheral neuronal tissues of C57BL/6J mice revealed no significant expression except for the cerebellum in which the ERVmch8 locus' low methylation status was unique compared to the other brain compartments. The ERVmch8 locus was found to be surrounded by genes associated with neuronal development and/or inflammation. Interestingly, cerebellum-specific ERVmch8 expression was age-dependent with almost no expression at 2 weeks and a plateau at 6 weeks. CONCLUSIONS: The ecotropic ERVmch8 locus on the C57BL/6J mouse genome was relatively undermethylated in the cerebellum, and its expression was cerebellum-specific and age-dependent.

Parvovirus-induced encephalitis in a juvenile raccoon.

A juvenile raccoon was euthanized because of severe neurologic signs. At postmortem examination, no significant gross lesions were present. Histologic evaluation demonstrated nonsuppurative encephalitis in thalamus, brainstem, and hippocampus, cerebellar Purkinje cell loss, as well as poliomyelitis and demyelination of the spinal cord. Parvovirus antigen-specific immunohistochemistry revealed immunopositive neurons in the brainstem, cerebral cortex, and hippocampus. A few Purkinje cells were also immunopositive. DNA extracted from formalin-fixed, paraffin-embedded brain tissue (thalamus, hippocampus, cerebral cortex) yielded a positive signal using PCR targeting both feline and canine parvovirus. Sequencing analyses from a fragment of the NS1 gene and a portion of the VP2 gene confirmed the presence of DNA of a recent canine parvovirus variant (CPV-2a-like virus) in the cerebellum. Our case provides evidence that a recent canine parvovirus (CPV) strain (Carnivore protoparvovirus 1) can infect cerebral and diencephalic neurons and cause encephalitis in an otherwise healthy raccoon. Parvovirus-induced encephalitis is a differential diagnosis of rabies and canine distemper in raccoons with neurologic signs.

Evolutionary changes affecting rapid identification of 2008 Newcastle disease viruses isolated from double-crested cormorants.

A morbidity-mortality event involving virulent Newcastle disease virus (NDV) in wild double-crested cormorants (Phalacrocorax auritus) occurred in North America in the summer of 2008. All 22 viruses isolated from cormorants were positively identified by the USDA-validated real-time reverse transcription-PCR assay targeting the matrix gene. However, the USDA-validated reverse transcription-PCR assay targeting the fusion gene that is specific for virulent isolates identified only 1 of these 22 isolates. Additionally, several of these isolates have been sequenced, and this information was used to identify genomic changes that caused the failure of the test and to revisit the evolution of NDV in cormorants. The forward primer and fusion probe were redesigned from the 2008 cormorant isolate sequence, and the revised fusion gene test successfully identified all 22 isolates. Phylogenetic analyses using both the full fusion sequence and the partial 374-nucleotide sequence identified these isolates as genotype V, with their nearest ancestor being an earlier isolate collected from Nevada in 2005. Histopathological analysis of this ancestral strain revealed morphological changes in the brain consistent with that of the traditional mesogenic pathotypes in cormorants. Intracerebral pathogenicity assays indicated that each of these isolates is virulent with values of >0.7 but not more virulent than earlier isolates reported from Canada.