Progress in research on the molecular biological detection techniques of avian encephalomyelitis.

Avian encephalomyelitis (AE) is a highly infectious disease caused by the avian encephalomyelitis virus (AEV), which primarily affects the central nervous system of 1- to 4-week-old chicks and causes significant economic losses in the worldwide poultry industry. Despite heavy dependency on vaccine immunization, AEV has persisted on farms for extended periods, which increases its virulence and makes quick and accurate detection crucial to preventing and controlling the disease. Classical diagnostic methods have been unable to meet the current requirements for rapid diagnosis of AE cases. To address this issue, this paper reviews the etiological and molecular biological detection techniques of AE, and it seeks to provide a reference for future research and to establish differential diagnostic techniques for AE epidemiological investigation, identification of epidemic strains, and early diagnosis of clinical cases. Through improving our understanding of AE, we can better combat the disease and protect the global poultry industry.

Advances in viral encephalitis: Viral transmission, host immunity, and experimental animal models.

Viral infections have led to many public health crises and pandemics in the last few centuries. Neurotropic virus infection-induced viral encephalitis (VE), especially the symptomatic inflammation of the meninges and brain parenchyma, has attracted growing attention due to its high mortality and disability rates. Understanding the infectious routes of neurotropic viruses and the mechanism underlying the host immune response is critical to reduce viral spread and improve antiviral therapy outcomes. In this review, we summarize the common categories of neurotropic viruses, viral transmission routes in the body, host immune responses, and experimental animal models used for VE study to gain a deeper understanding of recent progress in the pathogenic and immunological mechanisms under neurotropic viral infection. This review should provide valuable resources and perspectives on how to cope with pandemic infections.

Curcumin-dependent phenotypic transformation of microglia mediates resistance to pseudorabies-induced encephalitis.

Pseudorabies virus (PRV) causes viral encephalitis, a devastating disease with high mortality worldwide. Curcumin (CUR) can reduce inflammatory damage by altering the phenotype of microglia; however, whether and how these changes mediate resistance to PRV-induced encephalitis is still unclear. In this study, BV2 cells were infected with/without PRV for 24 h and further treated with/without CUR for 24 h. The results indicated that CUR promoted the polarization of PRV-infected BV2 cells from the M1 phenotype to the M2 phenotype and reversed PRV-induced mitochondrial dysfunction. Furthermore, M1 BV2 cell secretions induced signalling pathways leading to apoptosis in PC-12 neuronal cells, and this effect was abrogated by the secretions of M2 BV2 cells. RNA sequencing and bioinformatics analysis predicted that this phenotypic shift may be due to changes in energy metabolism. Furthermore, Western blot analysis showed that CUR inhibited the increase in AMP-activated protein kinase (AMPK) phosphorylation, glycolysis, and triacylglycerol synthesis and the reduction in oxidative phosphorylation induced by PRV infection. Moreover, the ATP levels in M2 BV2 cells were higher than those in M1 cells. Furthermore, CUR prevented the increase in mortality, elevated body temperature, slowed growth, nervous system excitation, brain tissue congestion, vascular cuffing, and other symptoms of PRV-induced encephalitis in vivo. Thus, this study demonstrated that CUR protected against PRV-induced viral encephalitis by switching the phenotype of BV2 cells, thereby protecting neurons from inflammatory injury, and this effect was mediated by improving mitochondrial function and the AMPK/NF-κB p65-energy metabolism-related pathway.

Seroprevalence and risk factors associated with seropositivity for equine encephalomyelitis virus in horses in Rio Grande do Norte, Brazil

The present objective was to investigate the presence of anti-equine viral encephalomyelitis (EVE) antibodies and the possible risk factors for its dissemination in horses raised in the East and West Potiguar mesoregions of the state of Rio Grande do Norte, Brazil. Serological diagnosis for neutralizing antibodies against Eastern (EEEV), Western (WEEV) and Venezuelan (VEEV). Equine viral encephalomyelitis was performed using a seroneutralization technique on 811 blood samples from horses from ninety properties and sixteen municipalities between July 2018 and February 2019. Factors associated with EVE were evaluated using an investigative epidemiological questionnaire, and the data were statistically analyzed using the Epi Info 3.5.2 software with a confidence level of 95%. The seroprevalence of anti-EVE antibodies was 14.2% (115), with 10.36% (84) for EEEV, 6.9% (56) for WEEV, and null for EVE. When analyzing risk factors, it can be concluded that horses raised in properties that do not clean installations and/or rent out their pasture are more likely to have anti-EVE antibodies. These results show evidence that horses raised in the East and West Potiguar mesoregions were exposed to EEEV and WEEV, thus reinforcing the importance of vaccination and serological survey of nonvaccinated horses as a means of monitoring the disease.

Astrovirus Infection in Cattle with Nonsuppurative Meningoencephalitis in South Korea.

Neurological diseases in cattle can be caused by several infectious agents. Astroviruses are increasingly recognized as the causative agent of encephalitis in various animals, including humans. In this study, a neuroinvasive astrovirus (BoAstV 20B05) was discovered in the brain tissues of an 81-month-old Korean native cattle with neurological symptoms. Lymphocyte infiltration and multifocal perivascular cuffing were observed in the cerebrum and brain stem, and viral antigens were also detected in the meninges. In particular, the concentration of the astroviral genome was high in the brain tissues. Korean BoAstV 20B05 was classified into the CH13/NeuroS1 clade and was closely related to the Neuro-Uy and KagoshimaSR28-462 strains. Our evolutionary analysis showed that Korean BoAstV 20B05 belongs to the sub-lineage NeuroS1 and evolved independently of BoAstV KagoshimaSR28-462. These results suggest that neuroinvasive astroviruses were first introduced in Korea. However, analysis is limited by the lack of reference astrovirus sequences reported in various countries within Asia, and further analysis should be performed using more strains. In this study, we identified a neuroinvasive astrovirus infection with neurological symptoms for the first time in South Korea and confirmed that BoAstV 20B05 may have been introduced in South Korea a long time ago.

Comparative Analysis of Novel Strains of Porcine Astrovirus Type 3 in the USA.

Porcine astrovirus type 3 (PoAstV3) has been previously identified as a cause of polioencephalomyelitis in swine and continues to cause disease in the US swine industry. Herein, we describe the characterization of both untranslated regions, frameshifting signal, putative genome-linked virus protein (VPg) and conserved antigenic epitopes of several novel PoAstV3 genomes. Twenty complete coding sequences (CDS) were obtained from 32 diagnostic cases originating from 11 individual farms/systems sharing a nucleotide (amino acid) percent identity of 89.74-100% (94.79-100%), 91.9-100% (96.3-100%) and 90.71-100% (93.51-100%) for ORF1a, ORF1ab and ORF2, respectively. Our results indicate that the 5'UTR of PoAstV3 is highly conserved highlighting the importance of this region in translation initiation while their 3'UTR is moderately conserved among strains, presenting alternative configurations including multiple putative protein binding sites and pseudoknots. Moreover, two predicted conserved antigenic epitopes were identified matching the 3' termini of VP27 of PoAstV3 USA strains. These epitopes may aid in the design and development of vaccine components and diagnostic assays useful to control outbreaks of PoAstV3-associated CNS disease. In conclusion, this is the first analysis predicting the structure of important regulatory motifs of neurotropic mamastroviruses, which differ from those previously described in human astroviruses.

Neurotropic Astroviruses in Animals.

Astrovirus infections are among the main causes of diarrhea in children, but their significance for animal health has remained underestimated and largely unknown. This is changing due to the increasing amount of newly identified neurotropic astroviruses in cases of nonsuppurative encephalitis and neurological disease in humans, pigs, ruminant species and minks. Neurological cases in ruminants and humans usually occur sporadically and as isolated cases. This contrasts with the situation in pigs and minks, in which diseases associated with neurotropic astroviruses are endemic and occur on the herd level. Affected animals show neurological signs such as mild ataxia to tetraplegia, loss of orientation or trembling, and the outcome is often fatal. Non-suppurative inflammation with perivascular cuffing, gliosis and neuronal necrosis are typical histological lesions of astrovirus encephalitis. Since astroviruses primarily target the gastrointestinal tract, it is assumed that they infect the brain through the circulatory system or retrograde following the nerves. The phylogenetic analysis of neurotropic astroviruses has revealed that they are genetically closely related, suggesting the presence of viral determinants for tissue tropism and neuroinvasion. In this review, we summarize the current knowledge on neurotropic astrovirus infections in animals and propose future research activities.

Isolation and genetic characteristics of a neurotropic teschovirus variant belonging to genotype 1 in northeast China.

Porcine teschovirus (PTV) is a causative agent of reproductive disorders, encephalomyelitis, respiratory diseases, and diarrhea in swine, with a worldwide distribution. In this work, we identified PTV-associated nonsuppurative encephalitis as a potential cause of posterior paralysis in neonatal pigs in northeast China. Using indirect immunofluorescence assay, western blot, electron microscopy, and genome sequencing, we identified a neurotropic PTV strain, named CHN-NP1-2016, in the supernatants of pooled cerebrum and cerebellum samples from an affected piglet. Nucleotide sequence alignment revealed that the whole genome of CHN-NP1-2016 shared the highest sequence similarity (86.76% identity) with PTV 1 strain Talfan. A combination of phylogenetic and genetic divergence analysis was applied based on the deduced amino acid sequence of the P1 gene with a cutoff value of the genetic distance (0.102 ± 0.008) for defining PTV genotypes, and this showed that CHN-NP1-2016 is a variant of genotype 1. In total, 16 unique mutations and five mutant clusters were detected in the capsid proteins VP1 and VP2 of CHN-NP1-2016 when compared to other PTV1 isolates. Importantly, we detected three mutant clusters located in the exposed surface loops of the capsid protein, potentially indicating significant differences in major neutralization epitopes. Moreover, a potential recombination event in the P1 region of PTV CHN-NP1-2016 was detected. These findings provide valuable insights into the role of recombination in the evolution of teschoviruses. To our knowledge, this is the first case report of PTV-1-associated encephalitis in northeast China. Future investigations will narrow on the serology and pathogenicity of this novel isolate.

Recombinant bovine IL17A acts as an adjuvant for bovine herpesvirus vaccine.

The Bovine herpes virus type 5 glycoprotein D (gD) is essential for viral penetration into host permissive cells. The Herpes virus gD glycoprotein has been used for bovine immunization, being efficient in reduction of viral replication, shedding and clinical signs, however sterilizing immunity is still not achieved. Recombinant subunit vaccines are, in general, poorly immunogenic requiring additional adjuvant components. Interleukin 17A (IL17A) is a pro-inflammatory cytokine produced by T helper 17 cells that mediate mucosal immunity. IL17 production during vaccine-induced immunity is a requirement for mucosal protection to several agents. In this study, we investigated the potential of a recombinant IL17A to act as an adjuvant for a recombinant BoHV-5 glycoprotein D vaccine in cattle. Three cattle groups were divided as: group 1) rgD5 + alumen + rIL-17A; 2) rgD5 + alumen; and 3) PBS + alumen. The cattle (3 per group) received two doses of their respective vaccines at an interval of 21 days. The group that received rIL17 in its vaccine formulation at the 7th day after the prime immunization had significant higher levels of specific rgD-IgG than the alumen group. Addition of rIL17 also led to a significant fold increase in specific anti-rgD IgG and neutralizing antibodies to the virus, respectively, when compared with the alumen group. Cells stimulated with rIL17A responded with IL17 transcription, as well IL2, IL4, IL10, IL15, Bcl6 and CXCR5. Our findings suggest that the rIL17A has adjuvant potential for use in vaccines against BoHV-5 as well as potentially other pathogens of cattle.

The S2 Subunit of QX-type Infectious Bronchitis Coronavirus Spike Protein Is an Essential Determinant of Neurotropism.

Some coronaviruses (CoVs) have an extra furin cleavage site (RRKR/S, furin-S2' site) upstream of the fusion peptide in the spike protein, which plays roles in virion adsorption and fusion. Mutation of the S2' site of QX genotype (QX-type) infectious bronchitis virus (IBV) spike protein (S) in a recombinant virus background results in higher pathogenicity, pronounced neural symptoms and neurotropism when compared with conditions in wild-type IBV (WT-IBV) infected chickens. In this study, we present evidence suggesting that recombinant IBV with a mutant S2' site (furin-S2' site) leads to higher mortality. Infection with mutant IBV induces severe encephalitis and breaks the blood-brain barrier. The results of a neutralization test and immunoprotection experiment show that an original serum and vaccine can still provide effective protection in vivo and in vitro. This is the first demonstration of IBV-induced neural symptoms in chickens with encephalitis and the furin-S2' site as a determinant of neurotropism.

Differential expression of cyclins mRNA in neural tissues of BoHV-1- and BoHV-5- infected cattle.

Bovine alphaherpesvirus types 1 (BoHV-1) and 5 (BoHV-5) are closely related alphaherpesviruses. BoHV-5 causes non-suppurative meningoencephalitis in calves. BoHV-1 is associated with several syndromes and, occasionally, can cause encephalitis. Although both viruses are neurotropic and they share similar biological properties, it is unknown why these alphaherpesviruses differ in their ability to cause neurological disease. Neural tissue samples were collected from BoHV-1- and BoHV-5-intranasally inoculated calves during acute infection, latency and reactivation and the levels of cyclins mRNA expression were analyzed by qRT-PCR. Striking differences in the levels of cyclins mRNA were particularly detected in trigeminal ganglion (TG). The expression levels of cyclins in TG during BoHV-5 latency suggest that these viruses utilize different strategies to persist in the host. It is apparent that a relationship between virus loads and cyclin mRNA levels can be established only during acute infection and other factors might be involved in the regulation of cell cycle components during BoHV latency and reactivation. Bovine alphaherpesviruses neuropathogenicity might be influenced by the differential control of cell cycle components by these herpesviruses. This is the first report on BoHV-5 modulation of cyclins expression in neural tissues from its natural host.

Biotic concerns in generating molecular diagnosis matrixes for 4 avian viruses with emphasis on Marek's disease virus.

The great advance in the field of diagnosis of avian viruses is reflecting the highly sophisticated molecular assays of the human and general virology in providing highly sensitive and fast methods of diagnosis. The present review will discuss the biotic factors and the complexities that became evident with the evolution of the novel molecular diagnostic assays with emphasis on 4 avian viruses, chicken anemia, infectious laryngotracheitis, turkey meningoencephalitis, but mainly on Marek's disease virus. To create a biologically meaningful diagnosis, attention should be dedicated to various biotic factors and not only of the diagnostic assay. Included among the important factors are, (a) the sample examined and the sampling strategy, (b) the outcomes of the pathogen amplification ex vivo, (c) the sampling time and its reflection on the disease diagnosis, (d) the impact of simultaneous multiple virus-infections regarding the ability to demonstrate all pathogens and inter- and intra-interactions between the pathogens. A concerted consideration of the relevant factors and the use of advanced molecular diagnostic assay would yield biologically significant diagnosis in real-time that would beneficiate the poultry industry.

Porencephaly and Periventricular Encephalitis in a 4-month-old Puppy: Detection of Canine Parvovirus Type 2 and Potential Role in Brain Lesions.

A 4-month-old puppy died after showing intracranial signs a few days after a suspected viral enteritis. Grossly, the right cerebral hemisphere had a large irregular cavity external to the internal capsule. Histopathological examination revealed a cystic lesion in the right hemisphere and non-suppurative inflammation of the diencephalon and periaqueductal nervous tissue. Porencephaly associated with periventricular non-suppurative encephalitis was diagnosed. A nested polymerase chain reaction (PCR) identified the presence of parvovirus DNA in the brain and real-time PCR typed this as canine parvovirus (CPV) type 2a. Immunohistochemistry revealed the presence of CPV antigen in the cytoplasm of scattered cells in the subependymal layers and choroid plexus epithelium. The porencephaly was not associated with inflammatory lesions or CPV antigen and was considered to have preceded the neurological signs. In contrast, the detection of CPV antigen in the subependymal layers and choroid plexus epithelium supported the association of this virus with the periventricular encephalitis.

Neuron-specific enolase as biomarker for possible neuronal damage in dogs with distemper vírus / Enolase neuronal específica como um possível biomarcador para lesão neuronal em cães com cinomose

Neuron-specific enolase (NSE) is a biomarker of neuronal cell lysis, which demonstrates stability in extracellular fluids such as blood and cerebrospinal fluid. To the authors knowledge there is no research information comparing the use of NSE in dogs with and without encephalitis, putting in evidence the importance of that biomarker to detect neuronal damage in dogs. The objective was to compare the serum NSE levels in dogs with and without encephalitis, and to determine the serum NSE levels in normal dogs. Thirty eight dogs were evaluated, 19 dogs with encephalitis (EG Group) and 19 dogs without encephalitis (CG Group). The criteria for inclusion in the EG Group were presence of neurological signs in more than one part of the CNS (multifocal syndrome) and positive molecular diagnosis for canine distemper virus; for the CG Group were an age between 1 to 7 years and be clinically normal; NSE were measured in serum using an ELISA assay, and the results were compared. In the EG Group the NSE values were higher with significant difference (P=0.0053) when compared with the CG Group. NSE is a biomarker that can be measured in serum samples of dogs to monitor neuronal lesions in encephalitis.(AU)

Enolase neuronal específica (NSE) é um biomarcador de lise de neurônios, que demonstra estabilidade em fluidos extracelulares como sangue e líquido cerebrospinal. Para o conhecimento dos autores, não há informações de pesquisa que comparem o uso de NSE em cães com e sem encefalite, evidenciando a importância desse biomarcador para detectar danos neuronais em cães. O objetivo foi comparar os níveis séricos de NSE em cães com e sem encefalites, e determinar os níveis séricos de NSE em cães saudáveis. Trinta e oito cães foram avaliados, 19 cães com encefalites (Grupo EG) e 19 cães sem encefalite (Grupo CG). O critério para inclusão no Grupo EG foi presença de sinais neurológicos em mais de uma estrutura do SNC (síndrome multifocal) e positividade no diagnóstico molecular para o vírus da cinomose canina; para o Grupo CG foi idade entre 1 e 7 anos e ser clinicamente normal; NSE foram mensuradas em amostras séricas usando o método de ELISA, e os resultados comparados. No Grupo EG os valores de NSE foram altos com diferença significativa (P=0.0053) quando comparado com o Grupo CG. NSE é um biomarcador que pode ser mensurado em amostras séricas de cães para monitorar lesões neuronais em encefalites.(AU)

Accurate and precise real-time RT-PCR assays for the identification of astrovirus associated encephalitis in cattle.

A novel bovine astrovirus genotype species (BoAstV-CH13/NeuroS1) was recently identified in brain tissues of cattle as a plausible cause of encephalitis. The purpose of the present study was to develop and validate real time RT-PCR assays for the detection of BoAstV-CH13/NeuroS1 in brain tissues of cattle. Three different primer-probe combinations were designed based on BoAstV-CH13/NeuroS1 full-genome sequences of 11 different strains identified in cattle, and established in three distinct one-step real time RT-PCR protocols. These protocols were compared regarding their diagnostic performance using brain tissues of cattle with and without astrovirus associated encephalitis. The limit of detection (LOD) of all three assays was between 1.34 × 101 and 1.34 × 102 RNA copies, leading to an analytical sensitivity two orders of magnitude superior compared to a conventional pan-astrovirus RT-PCR protocol (LOD 1.31 × 104 RNA copies). Amplification efficiency was in the range of 97.3% to 107.5% with linearity (R2) > 0.99. The diagnostic sensitivity and specificity of the assays was determined as 100%, and all three revealed good intra- and inter-test repeatability. In conclusion, the newly developed RT-qPCRs are sensitive, specific, and reliable test formats that will facilitate BoAstV-CH13/NeuroS1 detection in routine diagnostics as well as in research settings.

Encephalitis induced by a newly discovered ruminant rhadinovirus in a free-living Formosan sambar deer (Rusa unicolor swinhoei).

We documented a case of a free-living Formosan sambar deer (Rusa unicolor swinhoei) infected with a newly discovered ruminant Rhadinovirus (RuRv). Non-purulent encephalitis was the primary histological lesion of the sambar deer. We conducted nested PCR to screen for herpesvirus using generic primers targeting the DNA polymerase gene. In addition, we found that DNA polymerase gene of the sambar deer RuRv was present in the macrophage distributed in the Virchow Robin space with histopathologic lesions by chromogenic in-situ hybridization (CISH). The phylogenetic analysis indicated a high similarity between the viral sequence isolated from fallow deer and our case. This result suggests the possibility of cross-species transmission from other exotic Cervidae reservoir to the Formosan sambar deer.

West Nile and St. Louis encephalitis viral genetic determinants of avian host competence.

West Nile virus (WNV) and St. Louis encephalitis (SLEV) virus are enzootically maintained in North America in cycles involving the same mosquito vectors and similar avian hosts. However, these viruses exhibit dissimilar viremia and virulence phenotypes in birds: WNV is associated with high magnitude viremias that can result in mortality in certain species such as American crows (AMCRs, Corvus brachyrhynchos) whereas SLEV infection yields lower viremias that have not been associated with avian mortality. Cross-neutralization of these viruses in avian sera has been proposed to explain the reduced circulation of SLEV since the introduction of WNV in North America; however, in 2015, both viruses were the etiologic agents of concurrent human encephalitis outbreaks in Arizona, indicating the need to re-evaluate host factors and cross-neutralization responses as factors potentially affecting viral co-circulation. Reciprocal chimeric WNV and SLEV viruses were constructed by interchanging the pre-membrane (prM)-envelope (E) genes, and viruses subsequently generated were utilized herein for the inoculation of three different avian species: house sparrows (HOSPs; Passer domesticus), house finches (Haemorhous mexicanus) and AMCRs. Cross-protective immunity between parental and chimeric viruses were also assessed in HOSPs. Results indicated that the prM-E genes did not modulate avian replication or virulence differences between WNV and SLEV in any of the three avian species. However, WNV-prME proteins did dictate cross-protective immunity between these antigenically heterologous viruses. Our data provides further evidence of the important role that the WNV / SLEV viral non-structural genetic elements play in viral replication, avian host competence and virulence.

Porcine Astrovirus Type 3 in Central Nervous System of Swine with Polioencephalomyelitis.

Using next-generation sequencing, we identified and genetically characterized a porcine astrovirus type 3 strain found in tissues from the central nervous system of 1 piglet and 3 sows with neurologic signs and nonsuppurative polioencephalomyelitis. Further studies are needed to understand the potential for cross-species transmission and clinical impact.

Indication of Cross-Species Transmission of Astrovirus Associated with Encephalitis in Sheep and Cattle.

We report the identification of a neurotropic astrovirus associated with encephalitis in a sheep. This virus is genetically almost identical to an astrovirus recently described in neurologically diseased cattle. The similarity indicates that astroviruses of the same genotype may cause encephalitis in different species.

Infections of horses and shrews with Bornaviruses in Upper Austria: a novel endemic area of Borna disease.

Borna disease, a lethal infection with Borna disease virus-1 (BoDV-1), was diagnosed in four horses from Upper Austria in 2015 and 2016. All cases occurred in winter (two cases in February 2015 and two cases in December 2016), and the maximal distance of the affected stables was 17 km. To demonstrate whether the causative agent was also harbored by its reservoir host, the bicolored white-toothed shrew (Crocidura leucodon), 28 shrews from this geographic area were collected in 2015 and investigated for the presence of BoDV-1. The shrew species were identified according to taxonomic clues and molecular barcodes. Affected horses and all shrews were investigated using histology, immunohistochemistry (IHC) and reverse transcription PCR. The horses exhibited severe nonpurulent encephalitis. Large amounts of BoDV-1 antigen were identified in their CNS. Among the 28 shrews, nine were identified as C. leucodon and 13 as Sorex araneus (Common shrew; Eurasian shrew). Six C. leucodon (66.7%) and one S. araneus (7.7%) had BoDV-1 infections. In accordance with previous findings, the IHC of C. leucodon exhibited a high amount of viral antigen in many neural and extraneural tissues. By contrast, the single positive S. araneus had an exclusively neural staining pattern. Of all positive samples, whole-genome BoDV-1 sequences were generated. The acquired sequences of the affected shrews were not identical to each other and clustered around the sequences of the diseased horses belonging, surprisingly, to the German 'strain V' cluster.