Evaluation of Fluorescence Microsphere Immunoassay for Detection of Antibodies to Rift Valley Fever Virus Nucleocapsid Protein and Glycoproteins.

Rift Valley fever virus (RVFV) is a mosquito-borne, zoonotic virus that infects ruminants, including cattle, sheep, goats, camels, and buffalo. Multiplexing diagnostic assays that can simultaneously detect antibodies against multiple RVFV antigens offer a high-throughput test for disease surveillance and vaccine evaluations. We describe the improvement and evaluation of a previously developed fluorescence microsphere immunoassay (FMIA) for the detection of IgG and IgM antibodies against the RVFV glycoprotein (Gn) and the immunogenic nucleocapsid protein (Np). Well-characterized vaccinated and experimentally infected ruminant sera were used for the evaluation of the assay. Recombinant viral proteins were produced and then coupled to polystyrene magnetic beads for analysis using the Luminex MAGPIX system with xMAP technology. The FMIA was performed in parallel with virus neutralization tests. Our results revealed the highest median fluorescence intensity (MFI) values for the detection of IgG antibodies against RVFV Np, indicating that this antigen would be a good candidate for a screening assay. The Np and Gn targets could differentiate infected animals from animals vaccinated with a candidate subunit vaccine formulation based on the RVFV Gn and Gc proteins. The results presented in this report demonstrate that FMIA provides a rapid and robust serological diagnostic tool for the detection of antibodies against RVFV. The targets developed in this assay provide the basis for the development of a companion diagnostic test for an RVFV Gn/Gc subunit vaccine that is capable of differentiating infected from vaccinated animals (DIVA), as well as a multiplex serodiagnostic assay that can simultaneously screen for several ruminant diseases.

Comparison of Two US Sheep Scrapie Isolates Supports Identification as Separate Strains.

Scrapie is a naturally occurring transmissible spongiform encephalopathy of sheep and goats. There are different strains of sheep scrapie that are associated with unique molecular, transmission, and phenotype characteristics. However, in the United States, very little is known about the potential presence of scrapie strains. Scrapie strain and PRNP genotype could both affect susceptibility, potential for transmission, incubation period (IP), and control measures required for eliminating scrapie from a flock. The investigators evaluated 2 US scrapie isolates, No. 13-7 and x124, after intranasal inoculation to compare clinical signs, IPs, spongiform lesions, and patterns of PrPSc deposition in sheep with scrapie-susceptible PRNP genotypes (QQ171). After inoculation with x124, susceptibility and IP were associated with valine at codon 136 (V136) of the prion protein: VV136 sheep had short IPs (6.9 months), those in AV136 sheep were 11.9 months, and AA136 sheep did not develop scrapie. All No. 13-7 inoculated sheep developed scrapie, with IPs of 20.1 months for AA136 sheep, 22.8 months for AV136 sheep, and 26.7 months for VV136 sheep. Patterns of immunoreactivity in the brain were influenced by inoculum isolate and host genotype. Differences in PrPSc profiles versus isolate were most striking when examining brains from sheep with the VV136 genotype. Inoculation into C57BL/6 mice resulted in markedly different attack rates (90.5% for x124 and 5.9% for No. 13-7). Taken together, these data demonstrate that No. 13-7 and x124 represent 2 distinct strains of scrapie with different IPs, genotype susceptibilities, and PrPSc deposition profiles.

Phylogenetic analysis and victim contact tracing of rabies virus from humans and dogs in Bali, Indonesia.

SUMMARY: The emergence of human and animal rabies in Bali since November 2008 has attracted local, national and international interest. The potential origin and time of introduction of rabies virus to Bali is described. The nucleoprotein (N) gene of rabies virus from dog brain and human clinical specimens was sequenced using an automated DNA sequencer. Phylogenetic inference with Bayesian Markov Chain Monte Carlo (MCMC) analysis using the Bayesian Evolutionary Analysis by Sampling Trees (BEAST) v. 1.7.5 software confirmed that the outbreak of rabies in Bali was caused by an Indonesian lineage virus following a single introduction. The ancestor of Bali viruses was the descendant of a virus from Kalimantan. Contact tracing showed that the event most likely occurred in early 2008. The introduction of rabies into a large unvaccinated dog population in Bali clearly demonstrates the risk of disease transmission for government agencies and should lead to an increased preparedness and efforts for sustained risk reduction to prevent such events from occurring in future.

Diagnostic approaches for Rift Valley fever.

Disease outbreaks caused by arthropod-borne animal viruses (arboviruses) resulting in significant livestock and economic losses world-wide appear to be increasing. Rift Valley fever (RVF) virus is an important arbovirus that causes lethal disease in cattle, camels, sheep and goats in Sub-Saharan Africa. There is concern that this virus could spread because of global warming, increased animal trade or through bioterrorism. This paper discusses the current and developing approaches to diagnosis of RVF. Diagnostic assays are available for RVF, but availability can be limited and there is a need for global harmonization. Continued improvement of standard serological and viral genome amplification approaches, including new embedded/syndromic testing, biosensor, emerging virus detection and characterization technologies is needed.

Borna disease, a progressive meningoencephalomyelitis as a model for CD4+ T cell-mediated immunopathology in the brain.

A homogeneous T cell line NM1 with Borna disease (BD) virus reactivity could be established. The NM1 cells have been characterized as CD4+ T cells. Adoptive transfer revealed that this MHC class II-restricted immune cell is responsible for the immunopathological effect leading to BD, a progressive meningoencephalomyelitis.

Borna disease virus-specific T cells protect against or cause immunopathological Borna disease.

In this report we show that passive immunization of Lewis rats with viable CD4+, Borna disease virus (BDV)-specific T cells before infection with BDV resulted in protection against BD, whereas inoculation of these T cells after BDV infection induced clinical disease with more rapid onset than seen in BDV control animals. The protective as well as encephalitogenic effector functions of BDV-specific CD4+ T cells were mediated only by viable BDV-specific T cells. The protective situation was obtained by passive transfer of BDV-specific T cells into animals inoculated later with virus, whereas the immunopathological situation was observed when virus-specific T cells developed normally or after adoptive transfer, and appeared on the scene after considerable virus replication in the brain.

A borna virus cDNA encoding a protein recognized by antibodies in humans with behavioral diseases.

Borna disease virus (BDV) causes a rare neurological disease in horses and sheep. The virus has not been classified because neither an infectious particle nor a specific nucleic acid had been identified. To identify the genome of BDV, a subtractive complementary DNA expression library was constructed with polyadenylate-selected RNA from a BDV-infected MDCK cell line. A clone (B8) was isolated that specifically hybridized to RNA isolated from BDV-infected brain tissue and BDV-infected cell lines. This clone hybridized to four BDV-specific positive strand RNAs (10.5, 3.6, 2.1, and 0.85 kilobases) and one negative strand RNA (10.5 kilobases) in BDV-infected rat brain. Nucleotide sequence analysis of the clone suggested that it represented a full-length messenger RNA which contained several open reading frames. In vitro transcription and translation of the clone resulted in the synthesis of the 14- and 24-kilodalton BDV-specific proteins. The 24-kilodalton protein, when translated in vitro from the clone, was recognized by antibodies in the sera of patients (three of seven) with behavioral disorders. This BDV-specific clone will provide the means to isolate the other BDV-specific nucleic acids and to identify the virus responsible for Borna disease. In addition, the significance of BDV or a BDV-related virus as a human pathogen can now be more directly examined.

Retinal function and morphology are altered in cattle infected with the prion disease transmissible mink encephalopathy.

Transmissible spongiform encephalopathies (TSEs) are a group of diseases that result in progressive and invariably fatal neurologic disease in both animals and humans. TSEs are characterized by the accumulation of an abnormal protease-resistant form of the prion protein in the central nervous system. Transmission of infectious TSEs is believed to occur via ingestion of prion protein-contaminated material. This material is also involved in the transmission of bovine spongiform encephalopathy ("mad cow disease") to humans, which resulted in the variant form of Creutzfeldt-Jakob disease. Abnormal prion protein has been reported in the retina of TSE-affected cattle, but despite these observations, the specific effect of abnormal prion protein on retinal morphology and function has not been assessed. The objective of this study was to identify and characterize potential functional and morphologic abnormalities in the retinas of cattle infected with a bovine-adapted isolate of transmissible mink encephalopathy. We used electroretinography and immunohistochemistry to examine retinas from 10 noninoculated and 5 transmissible mink encephalopathy-inoculated adult Holstein steers. Here we show altered retinal function, as evidenced by prolonged implicit time of the electroretinogram b-wave, in transmissible mink encephalopathy-infected cattle before the onset of clinical illness. We also demonstrate disruption of rod bipolar cell synaptic terminals, indicated by decreased immunoreactivity for the alpha isoform of protein kinase C and vesicular glutamate transporter 1, and activation of Müller glia, as evidenced by increased glial fibrillary acidic protein and glutamine synthetase expression, in the retinas of these cattle at the time of euthanasia due to clinical deterioration. This is the first study to identify both functional and morphologic alterations in the retinas of TSE-infected cattle. Our results support future efforts to focus on the retina for the development of new strategies for the diagnosis of TSEs.

Characterization of a US sheep scrapie isolate with short incubation time.

Scrapie is a naturally occurring fatal neurodegenerative disease of sheep and goats. Susceptibility to the disease is partly dependent upon the genetic makeup of the host. In a previous study it was shown that sheep intracerebrally inoculated with US scrapie inoculum (No. 13-7) developed terminal disease within an average of 19 months. We have since produced an inoculum, No. x124 from pooled brains of US-origin sheep scrapie, that results in incubations nearly threefold shorter. The present study documents clinicopathologic findings and the distribution of abnormal prion proteins (PrP(Sc)) by immunohistochemical (IHC) and Western blot (WB) techniques, in tissues of sheep inoculated with No. x124. All inoculated sheep developed clinical disease and were euthanatized within an average of 7.7 months postinoculation (MPI). Sheep that had valine/valine or alamine/valine at codon 136 of prion protein (PRNP) gene developed the disease faster and were euthanatized at an average of 4.3 and 5.6 MPI, respectively. Also, the inoculum was able to induce disease in a short time (7 MPI) in a sheep that was relatively resistant (QR at codon 171) to scrapie. This indicates that inoculum No. x124 appears to induce scrapie in shorter time than inoculum No. 13-7, especially in sheep homozygous or heterozygous for valine at codon 136.

Genome Sequence of Classical Swine Fever Virus NIVEDI-165, Subtype 1.1, a Field Virus Strain Isolated from the Southern Part of India.

The whole-genome sequence of an Indian field isolate of classical swine fever virus, NIVEDI-165, was found to be subtype 1.1, and it showed 89 to 99% amino acid identity and 84 to 99% nucleotide identity with four and five Indian classical swine fever virus (CSFV) isolates, respectively. To the best of our knowledge, this is the first report on a full-genome sequence of CSFV from South India.

Phylogeny and evolution of infectious bursal disease virus circulating in Turkish broiler flocks.

The emergence of new infectious bursal disease virus (IBDV) variants can threaten poultry health and production all over the world causing significant economic losses. Therefore, this study was performed to determine IBDV molecular epidemilogy, VP2 gene variation, and corresponding pathological lesions in IBDV infected chickens in Turkey. For this, 1855 bursa of Fabricius samples were collected from 371 vaccinated broiler flocks. Atrophia and haemorrhages were seen in the bursa Fabricius of very virulent IBDV (vvIBDV) infected chickens. Partial VP2 gene was sequenced and phylogenetic, recombination, and evolutionary analyses were performed. 1548 (83.5%) out of 1855 of bursa of Fabricius samples were IBDV positive and 1525 of those could be sequenced. The recombination analysis did not detect occurrence of any recombination event among the Turkish strains. Among 1525 sequenced samples, 1380 of them were found to be classical strains. Among 1380 classical strains, 1317 were similar to IBDV 2512, 11 to Faragher 52/70, 40 to 228 E, and 12 to Lukert strain. Out of 1525 reverse transcriptase ploymerase chain reaction positive samples, 144 of them were found to be similar to vvIBDV-VP2 gene reported to GenBank previously. The phylogenetic tree performed on a broad sequence dataset demonstrated grouping of vvIBDV Turkish strains in three different clusters, including sequences collected also from Iraq and Kuwait (Cluster 1), Indian (Cluster 2), and a distinct Turkish-only cluster (Cluster 3). The evolutionary rate estimation on branches/clades including Turkish strain mirrored the expected one for RNA viruses and no significant differences were found among different considered branches. In conclusion, results of this study indicate that vvIBDV strains similar to those circulating in various countries in the Middle East are present and undergoing evolution in chickens from Turkish broiler flocks. This point needs to be taken into account in planning adequate control strategies.

Restricted expression of Borna disease virus glycoprotein in brains of experimentally infected Lewis rats.

AIM: Borna disease virus (BDV) induces a persistent infection in the central nervous system (CNS) accompanied by a non-purulent meningoencephalitis. BDV-infection of Lewis rats provides an important model to investigate basic principles of neurotropism, viral persistence and resulting dysfunctions. To date, the in vivo strategies of BDV to persist in the CNS are not fully understood. Viral glycoproteins are main targets of the antiviral defence implicating a controlled expression in case of persistent infections. Therefore, we analysed the expression profiles of the BDV-glycoprotein (BDV-GP) and corresponding BDV-intron II RNA in experimentally infected rat brains, focusing on their spatio-temporal occurrence, regional, cellular and intracellular locations. METHODS: This was carried out by immunohistochemistry and in situ hybridization. The expression pattern of the most abundantly expressed BDV-nucleoprotein (BDV-N) served as a reference. RESULTS: BDV-N mRNA was detected preferentially in the cytoplasm of neurones, whereas BDV-intron II mRNA was found predominantly in the nucleus of brain cells. The genomic RNA was restricted to the nucleus. Expression of BDV-GP was significantly lower than BDV-N expression and mainly limited to cerebral cortex, hippocampus, amygdala and thalamus. BDV-GP was restricted to larger neurones; BDV-N occurred also in astrocytes, oligodendrocytes and ependymal cells. CONCLUSIONS: The expression profiles of BDV-GP, BDV-N and their mRNAs are significantly different, indicating that BDV-GP expression is regulated in vivo. This might be achieved by restricted nuclear export and/or maturation of BDV-intron II mRNA or limited translation as a viral mechanism to escape from the immune response and enable persistence in the CNS.

Introduction: conceptualizing and partitioning the emergence process of zoonotic viruses from wildlife to humans.

This introduction provides a telegraphic overview of the processes of zoonotic viral emergence, the intricacies of host-virus interactions, and the distinct role of biological transitions and modifying factors. The process of emergence is conceptualized as two transition stages which are common and required for all disease emergence, (1) human contact with the infectious agent and (2) cross-species transmission of the agent, and two transition stages which are not required for emergence and appear unavailable to many zoonotic pathogens, (3) sustained human-to-human transmission and (4) genetic adaptation to the human host. The latter two transitions are presumably prerequisites for the pandemic emergence of a pathogen. The themes introduced herein are amplified and explored in detail by the contributors to this volume. Each author explores the mechanisms and unique circumstances by which evolution, biology, history, and current context have contrived to drive the emergence of different zoonotic agents by a series of related events; although recognizable similarities exist among the events leading to emergence the details and circumstances are never repetitive.

Influenza viruses in animal wildlife populations.

Influenza viruses belong to the family Orthomyxoviridae. Genus Influenza A viruses are true zoonotic agents with many animal reservoirs, whereas genus Influenza B viruses are generally considered to be a virus of humans. The genome of influenza A viruses consists of eight unique segments of single-stranded RNA of negative polarity; they are typed according to their surface proteins, hemagglutinin (HA) and neuraminidase (NA). HA and NA, the major antigenic determinants of influenza A viruses, are present in 16 and nine serologic subtypes, respectively. Annual epidemics and occasional pandemics of influenza in humans depend on the continued evolution of influenza viruses. Although they have numerous potential host populations, most of our genetic and biologic data are obtained from studies of domestic populations of species such as chickens, turkeys, swine, and horses. Concerning wildlife populations, including wild populations of these domesticated species, much less is known. The purpose of this review is to establish what role wildlife populations play in the continued evolution of influenza viruses. Future work needs to determine what chain of events makes it possible for an influenza virus to be successfully transmitted to, and more importantly within, an alternative host population. Even questions as fundamental as which hosts can transmit viruses to humans remain unanswered so far.

Preliminary observations on the experimental transmission of chronic wasting disease (CWD) from elk and white-tailed deer to fallow deer.

To determine the transmissibility of chronic wasting disease (CWD) to fallow deer (Dama dama) and to provide information about clinical course, lesions and suitability of currently used diagnostic procedures for detection of CWD in this species, 13 fawns were inoculated intracerebrally with CWD brain suspension from elk (n=6) or white-tailed deer (n=7). Three other fawns were kept as uninfected controls. Three CWD-inoculated deer were killed 7.6 months post-inoculation (mpi). None had abnormal prion protein (PrPd) in their tissues. One sick deer died at 24 mpi and one deer without clinical signs was killed at 26 mpi. Both animals had a small focal accumulation of PrPd in the midbrain. Between 29 and 37 mpi, three other deer became sick and were killed. All had shown gradual decrease in appetite and some loss of body weight. Microscopical lesions of spongiform encephalopathy were not observed, but PrPd was detected in tissues of the central nervous system (CNS) by immunohistochemistry, western blot and by two commercially available rapid diagnostic tests. This study demonstrates that intracerebrally inoculated fallow deer amplified CWD PrPd from white-tailed deer and elk in the absence of lesions of spongiform encephalopathy. Four years after CWD inoculation, the remaining five inoculated and two control deer are alive and apparently healthy.

Genetic evolution of classical swine fever virus under immune environments conditioned by genotype 1-based modified live virus vaccine.

Modified live vaccines (MLVs) based on genotype 1 strains, particularly C-strain, have been used to prevent and control classical swine fever virus (CSFV) worldwide. Nevertheless, a shift in the predominant CSFV strains circulating in the field from genotype 1 or 3 to genotype 2 is seen. Genotype 2 is genetically distant from the vaccine strains and was recently reported during outbreaks after vaccine failure; this has raised concerns that vaccination has influenced viral evolution. In Korea in 2016, there was an unexpected CSF outbreak in a MLV-vaccinated commercial pig herd. The causative CSFV strain was genetically distinct from previously isolated Korean strains but similar to recent Chinese strains exhibiting enhanced capacity to escape neutralization; this suggests the need for global cooperative research on the evolution of CSFV. We analysed global E2 sequences, using bioinformatics tools, revealing the evolutionary pathways of CSFV. Classical swine fever virus genotypes 1 and 2 experienced different degrees and patterns of evolutionary growth. Whereas genotype 1 stayed relatively conserved over time, the genetic diversity of genotype 2 has progressively expanded, with few fluctuations. It was determined that genotype 2 evolved under lower immune pressures and at a higher evolutionary rate than genotype 1. Further, several selected codons, under diversifying selection in genotype 1 but under purifying selection in genotype 2, correspond to antigenic determinants, which could lead to evasion of vaccine-induced immunity. Our findings provide evidence that evolutionary changes in CSFV are the result of the disproportionate usage of the CSF MLVs in endemic areas; this underscores the need to develop mitigation strategies to minimize the substantial risk associated with the emergence of vaccine-escaping mutants.

Experimental second passage of chronic wasting disease (CWD(mule deer)) agent to cattle.

To compare clinicopathological findings in first and second passage chronic wasting disease (CWD(mule deer)) in cattle, six calves were inoculated intracerebrally with brain tissue derived from a first-passage CWD-affected calf in an earlier experiment. Two uninoculated calves served as controls. The inoculated animals began to lose both appetite and weight 10-12 months later, and five subsequently developed clinical signs of central nervous system (CNS) abnormality. By 16.5 months, all cattle had been subjected to euthanasia because of poor prognosis. None of the animals showed microscopical lesions of spongiform encephalopathy (SE) but PrP(res) was detected in their CNS tissues by immunohistochemistry (IHC) and rapid Western blot (WB) techniques. Thus, intracerebrally inoculated cattle not only amplified CWD PrP(res) from mule deer but also developed clinical CNS signs in the absence of SE lesions. This situation has also been shown to occur in cattle inoculated with the scrapie agent. The study confirmed that the diagnostic techniques currently used for diagnosis of bovine spongiform encephalopathy (BSE) in the US would detect CWD in cattle, should it occur naturally. Furthermore, it raised the possibility of distinguishing CWD from BSE in cattle, due to the absence of neuropathological lesions and to a distinctive multifocal distribution of PrP(res), as demonstrated by IHC which, in this study, appeared to be more sensitive than the WB technique.

Distribution of Borna disease virus in the brain of rats infected with an obesity-inducing virus strain.

Experimental infection of Lewis rats with Borna disease virus (BDV), a nonsegmented, single-stranded RNA virus, usually causes an immune-mediated biphasic neurobehavioral disorder. Such animals develop a persistent infection of the CNS with viral antigen expression in all brain regions and a disseminated nonpurulent meningoencephalitis. Interestingly, intracerebral infection of Lewis rats with a BDV-variant (BDV-ob) causes a rapid increase of body weight with the development of an obesity syndrome without obvious neurological signs. The obese phenotype is correlated with a characteristic distribution of inflammatory lesions and BDV-antigen in the rat brain. Infiltration with mononuclear immune cells and viral antigen expression are restricted to the septum, hippocampus, amygdala and ventromedian tuberal hypothalamus. Therefore, infection with the obesity-inducing BDV-ob results most likely in neuroendocrine dysregulations leading to the development of an obesity syndrome. This might be due to the restriction of viral antigen expression and inflammatory lesions to brain areas which are involved in the regulation of body weight and food intake. The BDV-induced obesity syndrome represents a model for the study of immune-mediated neuroendocrine disorders caused by viral infections of the CNS.

Pathogenesis of Borna disease.

Borna disease represents a unique model of a virus-induced immunological disease of the brain. Naturally occurring in horses and sheep, the mechanisms of pathogenesis have been studied in experimental animals, namely in the rat. Many investigations have revealed that the infection of the natural hosts principally follows the same pathogenic pathways as observed in rats, leading to a severe encephalomyelitis. This affliction of the central nervous system results in severe neurological disorders that again, are fully comparable in laboratory animals to those in the natural and the different experimental hosts. In addition, alterations have been reported which are also based on the infection of the brain and do not result in the classical encephalitic clinical picture but rather in alterations of behavior. However, to all of our knowledge, the various clinical pictures of Borna disease are not caused by the infecting virus itself but rather by the hosts immune response towards it, i.e. by a virus-induced cell-mediated immunopathological reaction. The importance of virus-specific CD4+ T cells as exemplified by a cultured T cell line and of CD8+ T cells as shown by immunomodulatory substances and specific antibody treatment in vivo for the pathogenesis of acute Borna disease will be elucidated here. In addition, evidence will be provided that virus-specific CD8+ T cells are also responsible for the dramatic brain atrophy in the chronic phase of the disease in rats. Therefore, Borna disease not only lends itself exquisitely well to the study of the pathogenesis of an immunopathological disease of the brain but also represents one of the few models for immune-mediated tissue destruction that eventually leads to brain atrophy and clinically to dementia.

Borna disease virus: nature of the etiologic agent and significance of infection in man.

This review presents data on the characterization of Borna disease virus (BDV) and its potential as a possible causative agent in humans. The isolation of: (i) BDV-specific cDNA clones that encode various BDV-specific proteins and (ii) partially purified virus particles led to the conclusion that the viral genome consists of negative-sense, single-stranded RNA. The organization of the BDV-specific RNA species appears to be a nested set of overlapping subgenomic RNA transcripts. Furthermore, evidence is presented that BDV can infect humans and may cause certain psychiatric and neurological disorders. This concept is supported by: (i) the finding of virus-specific antibodies in sera of patients with neuropsychiatric diseases and (ii) results obtained during attempts to isolate BDV or a BDV-related agent from the cerebrospinal fluid of seropositive patients.