Bilateral Necrotizing Retinitis following Encephalitis Caused by the Pseudorabies Virus Confirmed by Next-Generation Sequencing.

Purpose: The objective of this study was to report a case of bilateral necrotizing retinitis following viral encephalitis caused by the pseudorabies virus.Case report: A 49-year-old male had decreased bilateral visual acuity after the recovery of consciousness for one month. He had been in an unconsciousness status due to encephalitis for two months before the ocular symptoms developed. He was a pig slaughterer. Ocular ultrasound showed bilateral vitreous haze and retinal detachment. A vitrectomy and silicone oil tamponade were performed on the left eye. During surgery, massive periphery retinal necrosis appearing as a tattered fish net, and multiple retinal holes were observed. The pseudorabies virus was detected by next-generation sequencing in the vitreous specimen.Conclusion: The pseudorabies virus may cause bilateral necrotizing retinitis following viral encephalitis among those with close contact to pigs. Intraocular fluid provides a greater selection of samples and a longer time window for pathogenic detection.

Genetic evolution analysis of novel recombinant pseudorabies virus strain in Sichuan, China.

Pseudorabies is a disease that seriously endangers the pig industry in China. Recently, we successfully isolated a pseudorabies virus from the brain tissue of piglets at a farm in Sichuan, China, and named it the FJ62 strain. In order to understand the molecular biological characteristics of the strain, primers were designed for glycoproteins gB, gC, gD and gE, which were amplified by a polymerase chain reaction (PCR) and sequenced. After comparing the sequence with the GenBank 22 pseudorabies virus reference strains and establishing the genetic evolutionary tree, it was found that the gB gene of pseudorabies virus was highly homologous (up to 100%) with the MY-1 strain which is isolated from a wild boar in Japan (AP018925) but that homology with other strains in China was low. The gC gene was in the same branch as most of the representative strains in China, with 99.5% homology. The gD gene is in the same branch as the domestic strain LA in China (KU552118), and the homology was 99.9%. The gE gene was in the same branch as the domestic BJ/YT strain in China (KC981239), with 99.9% homology. The results showed that the FJ62 strain of the pseudorabies virus isolated here may be a variant strain of FJ62 isolated from a domestic pig after natural recombination of pseudorabies virus genotype I from wild boar and genotype II from pigs in China. There have been no similar reports in Sichuan. The discovery of the recombinant virus strain provides a reference basis for the prevention and control of pseudorabies and a design strategy for a vaccine in Sichuan, China, in the future.

Human encephalitis complicated with bilateral acute retinal necrosis associated with pseudorabies virus infection: A case report.

We report the case of a patient who presented with viral encephalitis and a pulmonary infection complicated with bilateral acute retinal necrosis after direct contact with diseased swine. Next-generation sequencing of the cerebrospinal fluid and vitreous humor detected pseudorabies virus (PRV) simultaneously. Intravenous acyclovir and dexamethasone treatment improved the symptoms of encephalitis, and vitrectomy surgery with silicone oil tamponade was used to treat the retinal detachment. This case implies that PRV can infect humans; thus, self-protection is imperative when there is contact with animals.

Characterization of a moderately pathogenic pseudorabies virus variant isolated in China, 2014.

In this study, we reported a moderately pathogenic pseudorabies virus (PRV) variant isolated from one Bartha-K61-vaccinated pig farm in Weifang, Shandong Province, China, 2014. The sick piglets in the farm were characterized by anorexia, weight loss and neurologic symptoms but did not die. Sequence alignment of the gE gene indicated that it belonged to a new mutated PRV strain and about 15% amino acid sites had mutations, deficiencies and insertions compared to the other PRV strains. The gD gene had two amino acid insertions and ten amino acid mutations in comparison with the Bartha-K61 vaccine strain. The TK and gM genes were the same as one highly pathogenic PRV TJ strain. Evidence from virus isolation, laboratory challenge, serological detection and histopathologic examination confirmed that the etiological agent of the disease is PRV SD1404, which is a moderately pathogenic strain and causes piglets to be sick but not to die. PRV SD1404 strain is different from other reports and should be paid more attention to avoid economic losses.

Epidemiological investigation of pseudorabies in Shandong Province from 2013 to 2016.

In late 2011, a variant pseudorabies virus (vPRV) emerged in Bartha-K61-vaccinated pig herds, resulting in high morbidity and mortality of piglets in China. Since 2013, the autopsy lesions, histological examinations, virus isolation, phylogenetic analysis and selection pressure analysis of the gE gene of vPRV were recorded for 395 clinical cases, and 5,033 pig serum samples were detected by PRV gE-coated enzyme-linked immunosorbent assay. The major clinical symptoms were abortion in pregnant sows, fatal neurological signs in piglets and respiratory disease in growing pigs. Necrotic splenitis, hepatitis and lymphadenitis, haemorrhagic nephritis and non-suppurative encephalitis were observed by histopathological examination. Typical eosinophilic inclusion bodies were found in the nuclei of liver cells. Using PCR, 110 samples among 395 clinical cases tested positive for the gE gene. Fifteen vPRV strains were isolated and confirmed by sequencing and phylogenetic analysis of the gE gene. The strains shared 97.1%-99.9% nucleotide (nt) and 96.6%-99.5% amino acid (aa) homology with PRV reference strains. Selection pressure analysis showed that one site in the codons of glycoprotein E was under positive selection. Of the 5,033 serum samples, 2,909 were positive by ELISA for a positive rate of 57.8%. These results showed that vPRV was still prevalent in Shandong Province, indicating severe PRV infectious pressure. The preparation of new vaccines against PRV is extremely urgent.

A sensitive and specific lateral flow assay for rapid detection of antibodies against glycoprotein B of Aujeszky's disease virus.

A direct double antibody lateral flow assay (DDA-gB-LFA) for the detection of antibodies against the glycoprotein B (gB) of Aujeszky's disease virus (ADV) in swine sera was developed. A native ADV gB was used for the preparation of a conjugate with colloidal gold particles and the immobilization on the strip membrane. The gB purified from ADV virions by immunoaffinity chromatography retained its native epitope structure after adsorption on the nitrocellulose membrane and the surface of colloidal gold particles. The diagnostic specificity and sensitivity of the DDA-gB-LFA were evaluated using 236 field swine sera. The diagnostic specificity and sensitivity of the DDA-gB-LFA compared to a commercially available gB-based ELISA were 98.0% and 98.6%, respectively, when determined with the use of the reader-detection mode, and 98.0% and 93.5%, respectively, when determined using visual detection. The DDA-gB-LFA provides a rapid, sensitive, and specific determination of ADV gB-directed antibodies in sera and can be used for the detection of ADV-exposed swine.

Comparison of Pathogenicity-Related Genes in the Current Pseudorabies Virus Outbreak in China.

There is currently a pandemic of pseudorabies virus (PRV) variant strains in China. Despite extensive research on PRV variant strains in the past two years, few studies have investigated PRV pathogenicity-related genes. To determine which gene(s) is/are linked to PRV virulence, ten putative virulence genes were knocked out using clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 technology. The pathogenicity of these mutants was evaluated in a mouse model. Our results demonstrated that of the ten tested genes, the thymidine kinase (TK) and glycoprotein M (gM) knockout mutants displayed significantly reduced virulence. However, mutants of other putative virulence genes, such as glycoprotein E (gE), glycoprotein I (gI), Us2, Us9, Us3, glycoprotein G (gG), glycoprotein N (gN) and early protein 0 (EP0), did not exhibit significantly reduced virulence compared to that of the wild-type PRV. To our knowledge, this study is the first to compare virulence genes from the current pandemic PRV variant strain. This study will provide a valuable reference for scientists to design effective live attenuated vaccines in the future.

Enhancing expression of the pseudorabies virus glycoprotein E in yeast and its application in an indirect sandwich ELISA.

AIMS: The purpose of this study was to produce a recombinant pseudorabies virus (PRV) glycoprotein E (gE) protein with the correct antigenicity for use as a low-cost diagnostic antigen. METHODS AND RESULTS: The gene fragment encoding the amino-terminal immunodominant region of PRV gE (codons 31-270) (gEN31-270) was codon optimized and expressed constitutively and secreted using a Pichia pastoris expression system. Yeast-expressed gEN31-270 (ygEN31-270) was harvested from the culture supernatant, and ygEN31-270 was shown to exhibit N-linked glycosylation. An indirect sandwich enzyme-linked immunosorbent assay (ELISA) was developed using ygEN31-270 as a coating antigen, and the results showed that the assay had high sensitivity and specificity, as well as almost perfect concordance with a commercial gE ELISA kit. CONCLUSIONS: The immunodominant region (amino acids 31-270) of gE was expressed successfully in P. pastoris using a codon optimization strategy. ygEN31-270 was secreted and N-glycosylated. The ygEN31-270-based indirect sandwich ELISA showed high sensitivity and specificity to detect gE-specific antibodies in swine serum samples. SIGNIFICANCE AND IMPACT OF THE STUDY: The ygEN31-270-based indirect sandwich ELISA may provide an alternative method for developing a diagnostic kit with easy manipulation and low cost.

Detección serológica de Brucella suis, virus de influenza y virus de la enfermedad de Aujeszky en criaderos porcinos familiares de menos de 100 madres en Argentina / Serological detection of Brucella suis, influenza virus and Aujeszky's disease virus in backyard and small swine holders in Argentina

Los criaderos porcinos de menos de 100 madres representan más del 99% de los de todo el país; sin embargo, existen escasos reportes sobre su situación sanitaria y productiva. Se recabó información productiva y se tomaron muestras para detectar anticuerpos contra Brucella suis (Bs), virus de la enfermedad de Aujeszky (VA) y virus de influenza (VI) en 68 establecimientos de menos de 100 madres ubicados en la región norte, centro y sur del país. El 80% de los establecimientos fueron positivos al VI H1 pandémico 2009, el 11% al H3 clúster 2, mientras que el 11,7% presentó anticuerpos contra el VA y el 6% contra Bs. Ninguno de los productores conocía los factores de riesgo para la transmisión de enfermedades del cerdo al humano. El 47% compra sus reproductores a pares o en ferias. En lo que respecta a normas de bioseguridad, solo el 16% de los establecimientos tenía cerco perimetral y el 37% de las granjas contaba con asesoramiento veterinario. Los resultados de este estudio demuestran que la caracterización productiva y el relevamiento sanitario son de suma importancia para mejorar la productividad y reducir el riesgo de transmisión de enfermedades. El conocimiento de la situación sanitaria y de los factores de riesgo es necesario para conseguir un mejor control y la erradicación de enfermedades en sistemas de baja tecnificación. Se deberían llevar a cabo estudios más representativos a nivel país para detectar los agentes circulantes y, sobre la base de esta información, implementar medidas de prevención y control.

Farmers raising less than 100 sows represent more than 99% of swine producers in Argentina, although little is known about their sanitary status and productive characteristics in the country. Sanitary and productive information was obtained. Furthermore, samples for serological studies were taken to detect antibodies against Brucella suis (Bs), Aujeszky's disease virus (AV) and influenza virus (IV) in 68 backyard and small producers with less than 100 sows located in the north, central and south regions of Argentina. Antibodies against H1 pandemic were detected in 80% of the farms while 11%, 11.7% and 6.0% of the producers were positive to influenza H3 cluster 2, AV and Bs, respectively. None of the producers was aware of the risk factors concerning the transmission of diseases from pigs to humans. A percentage of 47% of them buy pigs for breeding from other farmers and markets. With regard to biosecurity measures, only 16% of the farms had perimeter fences. The results of this study demonstrate that productive characterization and disease surveys are important to improve productivity and to reduce the risk of disease transmission among animals and humans. The study of sanitary status and risk factors is necessary for better control and eradication of diseases in backyard or small producers. More representative studies at country level should be carried out to detect the pathogensthat circulate and, with this knowledge, to implement prevention and control measures.

Development of a rapid, simple, and specific real-time PCR assay for detection of pseudorabies viral DNA in domestic swine herds.

Despite successful eradication of pseudorabies virus (PRV) from the commercial pig industry in the United States in 2004, large populations of feral swine in certain regions act as wildlife reservoirs for the virus. Given the threat of reintroduction of the virus into domestic herds, a rapid, reliable, easily implemented assay is needed for detection of PRV. Although a real-time PCR (rtPCR) assay exists, improvements in rtPCR technology and a greater understanding of the diversity of PRV strains worldwide require an assay that would be easier to implement, more cost effective, and more specific. We developed a single-tube, rapid rtPCR that is capable of detecting 10 copies of PRV glycoprotein B ( gB) DNA per 20-µL total volume reaction. The assay did not produce a false-positive in samples known to be negative for the virus. The assay was negative for genetically similar herpesviruses and other porcine viruses. Our assay is a highly specific and sensitive assay that is also highly repeatable and reproducible. The assay should be a useful tool for early detection of PRV in pigs in the case of a suspected introduction or outbreak situation.

Harmonizing methods for wildlife abundance estimation and pathogen detection in Europe-a questionnaire survey on three selected host-pathogen combinations.

BACKGROUND: The need for wildlife health surveillance as part of disease control in wildlife, domestic animals and humans on the global level is widely recognized. However, the objectives, methods and intensity of existing wildlife health surveillance programs vary greatly among European countries, resulting in a patchwork of data that are difficult to merge and compare. This survey aimed at evaluating the need and potential for data harmonization in wildlife health in Europe. The specific objective was to collect information on methods currently used to estimate host abundance and pathogen prevalence. Questionnaires were designed to gather detailed information for three host-pathogen combinations: (1) wild boar and Aujeszky's disease virus, (2) red fox and Echinococcus multilocularis, and (3) common vole and Francisella tularensis. RESULTS: We received a total of 70 responses from 19 European countries. Regarding host abundance, hunting bags are currently the most widely accessible data source for widely distributed mid-sized and larger mammals such as red fox and wild boar, but we observed large differences in hunting strategies among countries as well as among different regions within countries. For small rodents, trapping is the method of choice, but practical applications vary among study sites. Laboratory procedures are already largely harmonized but information on the sampled animals is not systematically collected. CONCLUSIONS: The answers revealed that a large amount of information is available for the selected host-pathogen pairs and that in theory methods are already largely harmonized. However, the comparability of the data remains strongly compromised by local differences in the way, the methods are applied in practice. While these issues may easily be overcome for prevalence estimation, there is an urgent need to develop tools for the routine collection of host abundance data in a harmonized way. Wildlife health experts are encouraged to apply the harmonized APHAEA protocols in epidemiological studies in wildlife and to increase cooperation.

Development of a competitive double antibody lateral flow assay for the detection of antibodies specific to glycoprotein B of Aujeszky's disease virus in swine sera.

Three lateral flow assays (LFAs) for the detection of antibodies against glycoprotein B (gB) of Aujeszky's disease virus (ADV) in swine sera: a competitive double antibody sandwich LFA without a preincubation step (CDAS-gB-LFA), a CDAS-gB-LFA with a preincubation step (pCDAS-gB-LFA), and a competitive direct gB-LFA have been developed and were compared with each other and with a gB-ELISA. The assays are based on monoclonal antibodies to immunodominant epitopes of ADV gB. The pCDAS-gB-LFA proved to be the most specific and sensitive assay to detect antibodies directed to ADV gB. The specificity and sensitivity of the pCDAS-gB-LFA with the use of an LFA reader for test line intensity measurements were 97.6 and 94.9%, respectively. The lower diagnostic sensitivity of the pCDAS-gB-LFA compared to a gB-ELISA reflects its reduced analytical sensitivity, which was shown in titration experiments with positive sera. The pCDAS-gB-LFA, using the reader-based and visual detection modes, showed good agreement in respect to specificity; however, the LFA reader detection provided a higher diagnostic and analytical sensitivity compared to visual detection. The developed pCDAS-gB-LFA is a rapid, sensitive, and specific method for the detection of antibodies to ADV gB and can be used for screening ADV-infected swine in unvaccinated herds.

Pseudorabies in farmed foxes fed pig offal in Shandong province, China.

Pseudorabies (PR, Aujeszky's disease) is an acute, highly contagious viral disease resulting in major economic losses to the swine industry. PR is endemic in wild and domestic animals, although its natural host is the pig. Here, we report an outbreak of PR in foxes on a fur-producing farm in Yuncheng county, Shandong, China, that were fed pig offal. The diagnosis of PR was based on nervous signs and standard PCR methods and by isolation of PRV from fox brain tissue in Vero cells. The diagnosis was confirmed by an indirect immunofluorescence assay and electron microscopy. Phylogenetic analysis of a partial (804 nt) viral glycoprotein gC gene sequence indicated that it was likely to be a field strain closely related to a cluster of PRV previously identified in China.

Pseudorabies virus variant in Bartha-K61-vaccinated pigs, China, 2012.

The widely used pseudorabies virus (PRV) Bartha-K61 vaccine has played a key role in the eradication of PRV. Since late 2011, however, a disease characterized by neurologic symptoms and a high number of deaths among newborn piglets has occurred among Bartha-K61-vaccinated pigs on many farms in China. Clinical samples from pigs on 15 farms in 6 provinces were examined. The PRV gE gene was detectable by PCR in all samples, and sequence analysis of the gE gene showed that all isolates belonged to a relatively independent cluster and contained 2 amino acid insertions. A PRV (named HeN1) was isolated and caused transitional fever in pigs. In protection assays, Bartha-K61 vaccine provided 100% protection against lethal challenge with SC (a classical PRV) but only 50% protection against 4 challenges with strain HeN1. The findings suggest that Bartha-K61 vaccine does not provide effective protection against PRV HeN1 infection.

A nanoparticle-assisted PCR assay to improve the sensitivity for rapid detection and differentiation of wild-type pseudorabies virus and gene-deleted vaccine strains.

Nanoparticle-assisted polymerase chain reaction (nanoPCR) is a novel method for the rapid amplification of DNA and has been adopted for the detection of virus because of its simplicity, rapidity, and specificity. A nanoPCR assay was developed to detect and differentiate wild-type and gene-deleted pseudorabies virus (PRV). Three pairs of primers for nanoPCR developed in this study were selected from conserved regions of PRV, producing specific amplicons of 431 bp (gB), 316 bp (gE), and 202 bp (gG). The sensitivity of this assay using purified plasmid constructs containing the specific gene fragments was 100-1000 fold higher than conventional PCR. The PRV nanoPCR assay did not amplify porcine parvovirus, porcine circovirus type 2, porcine reproductive and respiratory syndrome virus, porcine teschovirus, or African swine fever virus but produced three bands of expected size with PRV and two bands of expected size with the gene-deleted PRV-Bartha-K61. Of 110 clinical samples collected from seven provinces in China, 53% and 48% were positive for wild-type PRV according to the nanoPCR assay and virus isolation, respectively.

Expression and purification of Suid Herpesvirus-1 glycoprotein E in the baculovirus system and its use to diagnose Aujeszky's disease in infected pigs.

Suid Herpesvirus 1 (SHV-1) is the etiological agent of Aujeszky's disease (AD), which affects swine herds worldwide and causes substantial economic losses due to animal mortality and lost productivity. In order to eradicate SHV-1, vaccination programs using viruses lacking the gene encoding glycoprotein E (gE) are ongoing in several countries. These eradication programs have generated a currently unmet demand for affordable and sensitive tests that can detect SHV-1 infection, yet distinguish between infected and vaccinated pigs. To meet this demand, we used the baculovirus-insect cell system to produce immunologically authentic full-length recombinant gE protein for use in a serum ELISA assay. As previous efforts to clone the gE gene had failed due to its extremely high GC-content (75% average), we used betaine as a PCR enhancer to facilitate amplification of the entire gE gene from the Argentinian CL15 strain of SHV-1. The cloned gE gene was expressed at high levels in recombinant baculovirus-infected insect cells and reacted strongly with sera from SHV-1 infected pigs. We used the recombinant gE protein to develop a local indirect ELISA test with sensitivity and specificity comparable to currently available commercial tests. Thus, recombinant gE produced in baculovirus-infected insect cells is a viable source of antigen for the detection of SHV-1 in ELISA tests. We also provide evidence supporting a potential application of this recombinant form of gE as a SHV-1 subunit vaccine.

Evaluation of a real-time polymerase chain reaction assay for Pseudorabies virus surveillance purposes.

Pseudorabies virus (PRV) is the cause of Aujeszky's disease, a disease that is significant economically for the swine industry worldwide. A real-time polymerase chain reaction (PCR) assay based on the gB and gE genes was used to identify PRV nucleic acid in diagnostic samples. Using virus isolation (VI) as the gold standard, the PCR assay performed well in a variety of diagnostic matrices. Testing was conducted on 1,027 nasal swabs with the following findings: gB sensitivity: 94.6% (95% confidence interval [CI]: 92.3-96.4%), specificity: 71.0% (95% CI: 64.0-77.3%); gE sensitivity: 94.6% (95% CI: 92.3-96.4%), specificity: 79.3% (95% CI: 72.9-84.7%). Diagnostic performance of the real-time PCR assay developed as a testing method indicates that it is a rapid, accurate assay that can provide reliable results on clinical samples.

Atividade antiviral de extratos de plantas medicinais disponíveis comercialmente frente aos herpesvírus suíno e bovino / Antiviral activity of commercially available medicinal plants on suid and bovine herpesviruses

O presente trabalho teve como objetivo pesquisar a atividade antiviral in vitro de plantas medicinais disponíveis comercialmente sobre herpesvírus suíno (SuHV-1) e bovino (BoHV-1). As espécies adquiridas foram Mikania glomerata, Cymbopogon citratus, Equisetum arvense, Peumus boldus, Solanum paniculatum, Malva sylvestris, Piper umbellatun e Solidago microglossa. A citotoxicidade dos extratos foi avaliada na linhagem celular MDBK pelas alterações morfológicas das células e obtenção da concentração máxima não citotóxica (CMNC) de cada planta. A atividade antiviral foi realizada com os extratos em suas respectivas CMNC e avaliada com base na redução do título viral e expressos em porcentagem de inibição. Os extratos aquosos de Peumus boldus e Solanum paniculatum apresentaram atividade antiviral sobre o SuHV-1 com 98% de inibição viral enquanto o de Peumus boldus inibiu apenas o BoHV-1 em 99%.

This paper aims to find commercially available medicinal plants showing antiviral activity in vitro on suid and bovine herpesviruses. The following species were tested: Mikania glomerata, Cymbopogon citratus, Equisetum arvense, Peumus boldus, Solanum paniculatum, Malva sylvestris, Piper umbellatun and Solidago microglossa. The cytotoxicity was evaluated by morphological changes in cells determining the maximum not cytotoxic concentration (MNCC). The antiviral activity was evaluated by viral title reduction. The extracts from Peumus boldus and Solanum paniculatum showed antiviral activity against SuHV-1 with 98% of inhibition. The extract of Peumus boldus also showed activity against BoHV-1 with 99% of inhibition.

Phylogenetic analysis of Suid Herpesvirus 1 isolates from Argentina.

Argentinean Suid Herpesvirus 1 isolates were compared with reference strains and sequences available at GenBank and phylogenetically analyzed. A short fragment of the gE gene of the immunodominant epitopes was used for preliminary grouping of isolates by phylogenetic analysis. The analysis of the partial gC gene provided more precise genetic typing and segregation into the main genotypes I and II. Results confirmed that the Argentinean genotype I isolates predominate in our country. The topology of the partial gC gene was similar to that previously reported. The Argentinean type I isolates belonged to one cluster and grouped together with NIA-3 and American and Brazilian genotype I strains. However, the results obtained by the algorithms allow inferring that the Yamagata S-81 and Mer (genotype II) strains are grouped together.

PCR duplex para diferenciação de amostras vacinais e selvagens do vírus da doença de Aujeszky / A duplex PCR for differentiation of wild and vaccine virus of Aujeszkyïs disease

A duplex PCR was developed to differentiate the wild-type virus from the attenuated virus used in vaccinations. The PCR was able to amplify fragments of 493bp for glycoprotein E (gE) gene and 207bp for glycoprotein B (gB) gene. The analytical sensitivity was determined by addition of a virus field sample titled in the brain samples of pigs. The standard virus strain Shope, the vaccine strain Bartha, and ten other field isolates were subjected to PCR. The PCR was able to amplify fragments of gE and gB in all field samples and only fragments of gB were amplified in the attenuated virus, as expected. The technique was able to detect up to 100.5 TCID50/50mL virus in samples of brain. Duplex PCR proved to be an important tool for differentiation of naturally-infected animals and animals vaccinated with the virus deleted for gE.