Analysis of the recombination and evolution of the new type mutant pseudorabies virus XJ5 in China.

Pseudorabies have caused enormous economic losses in China's pig industry and have recurred on many large pig farms since late 2011. The disease is caused by highly pathogenic, antigenic variant pseudorabies virus (vPRV) strains. Our laboratory isolated a pseudorabies virus in 2015 and named it XJ5. The pathogenic ability of this mutant strain was much stronger than that of the original isolate. After we sequenced its whole genome (GenBank accession number: OP512542), we found that its overall structure was not greatly changed compared with that of the previous strain Ea (KX423960.1). The whole genome alignment showed that XJ5 had a strong genetic relationship with the strains isolated in China after 2012 reported in GenBank. Based on the isolation time of XJ5 and the mutation and recombination analysis of programs, we found that the whole genome homology of XJ5 and other strains with Chinese isolates was greater than 95%, while the homology with strains outside Asia was less than 94%, which indicated that there may be some recombination and mutation patterns. We found that virulent PRV isolates emerged successively in China in 2011 and formed two different evolutionary clades from foreign isolates. At the same time, this may be due to improper immunization and the presence of wild strains in the field, and recent reports have confirmed that Bartha vaccine strains recombine with wild strains to obtain new pathogenic strains. We performed genetic evolution analysis of XJ5 isolated and sequenced in our laboratory to trace its possible mutations and recombination. We found that XJ5 may be the result of natural mutation of a virus in a branch of mutant strains widely existing in China.

A case report of long-delayed diagnosis of pseudorabies virus encephalitis with endophthalmitis: lessons from metagenomic next generation sequencing.

BACKGROUND: Pseudorabies virus (PRV) was thought to only infect animals. Recent studies have shown that it can also infect human. CASE PRESENTATION: We report a case of pseudorabies virus encephalitis and endophthalmitis, diagnosed 89 days after onset, confirmed with intraocular fluid metagenomic next generation sequencing (mNGS) after the result of two cerebrospinal fluid (CSF) mNGS tests were negative. Although treatment with intravenous acyclovir, foscarnet sodium, and methylprednisolone improved the symptoms of encephalitis, significant diagnostic delay resulted in permanent visual loss. CONCLUSIONS: This case suggests that pseudorabies virus (PRV) DNA in the intraocular fluid may have a higher positivity than that in the CSF. PRV may persist in the intraocular fluid for an extended period and may thus require extended antiviral therapy. Patients with severe encephalitis and PRV should be examined with the focus on pupil reactivity and light reflex. A fundus examination should be performed in patients with a central nervous system infection, specifically, those in a comatose state, to help reduce eye disability.

A novel paper biosensor based on Fe3O4@SiO2-NH2and MWCNTs for rapid detection of pseudorabies virus.

In this study, a novel paper biosensor based on Fe3O4@SiO2-NH2magnetic polymer microspheres and multi walled carbon nanotubes (MWCNTs) for rapid detection of pseudorabies virus (PRV) was first developed. Fe3O4@SiO2-NH2were functionalized with PRV antibody and doped in cellulose nitrate paper to fabricate the magnetic paper biosensor with good magnetic response and biocompatibility. Using MWCNTs to build conductive network of sensors, PRV antigen binds specifically to the immunomagnetic microspheres on the sensor, and the resulting immune complex changes the magnetic domain structure of the sensor and the structural gap of MWCNTs, causing the magnetic property and impedance change. TEM and EDS characterization proved that the biosensor was successfully doped with Fe3O4@SiO2-NH2and effectively recognized PRV. Under optimized conditions, the impedance variation was found to be linearly related to the logarithm value of PRV concentrations in the range of 10-1 mg ml-1, with the detection limit of 10 ng ml-1. This paper biosensor demonstrated advantages of portability, high sensitivity and specificity, providing a valuable method for early control of PRV.

Multiplex and on-site PCR detection of swine diseases based on the microfluidic chip system.

BACKGROUND: At present, the process of inspection and quarantine starts with sampling at the customs port, continues with transporting the samples to the central laboratory for inspection experiments, and ends with the inspected results being fed back to the port. This process had the risks of degradation of biological samples and generation of pathogenic microorganisms and did not meet the rapid on-site detection demand because it took a rather long time. Therefore, it is urgently needed to develop a rapid and high-throughput detection assay of pathogenic microorganisms at the customs port. The aim of this study was to develop a microfluidic chip to rapidly detect swine pathogenic microorganisms with high-throughput and higher accuracy. Moreover, this chip will decrease the risk of spreading infection during transportation. RESULTS: A series of experiments were performed to establish a microfluidic chip. The resulting data showed that the positive nucleic acid of four swine viruses were detected by using a portable and rapid microfluidic PCR system, which could achieve a on-site real-time quantitative PCR detection. Furthermore, the detection results of eight clinical samples were obtained within an hour. The lowest concentration that amplified of this microfluidic PCR detection system was as low as 1 copies/µL. The results showed that the high specificity of this chip system in disease detection played an important role in customs inspection and quarantine during customs clearance. CONCLUSION: The microfluidic PCR detection system established in this study could meet the requirement for rapid detection of samples at the customs port. This chip could avoid the risky process of transporting the samples from the sampling site to the testing lab, and drastically reduce the inspection cycle. Moreover, it would enable parallel inspections on one chip, which greatly raised the efficiency of inspection.

Design of fusion protein for efficient preparation of cyanovirin-n and rapid enrichment of pseudorabies virus.

OBJECTIVE: Cyanovirin-N (CVN) is a cyanobacterial protein with potent neutralizing activity against enveloped virus. To achieve the economic and functional production of CVN, the CVN N-terminally fused with CL7(A mutant of the Colicin E7 Dnase) was utilized to improve the solubility and stability of CVN fusion protein (CL7-CVN). Additionally, to improve the detection limit of existing PRV diagnostic assays, CL7-CVN was used for Pseudorabies virus (PRV) enrichment from larger sample volumes. RESULTS: CVN fused with CL7 was efficiently expressed at a level of ~ 40% of the total soluble protein in E. coli by optimizing the induction conditions. Also, the stability of CVN fusion protein was enhanced, and 10 mg of CVN with a purity of ~ 99% were obtained from 1 g of cells by one-step affinity purification with the digestion of HRV 3C protease. Moreover, both purified CVN and CL7-CVN could effectively inhibit the infection of PRV to PK15 cells. Considering the bioactivity of CL7-CVN, we explored a strategy for PRV enrichment from larger samples. CONCLUSIONS: CL7 effectively promoted the soluble expression of CVN fusion protein and improved its stability, which was meaningful for its purification and application. The design of CVN fusion protein provides an efficient approach for the economical and functional production of CVN and a new strategy for PRV enrichment.

Single Virus Tracking with Quantum Dots Packaged into Enveloped Viruses Using CRISPR.

Labeling viruses with high-photoluminescence quantum dots (QDs) for single virus tracking provides a visual tool to aid our understanding of viral infection mechanisms. However, efficiently labeling internal viral components without modifying the viral envelope and capsid remains a challenge, and existing strategies are not applicable to most viruses. Here, we have devised a strategy using the clustered regularly interspaced short palindromic repeats (CRISPR) imaging system to label the nucleic acids of Pseudorabies virus (PRV) with QDs. In this strategy, QDs were conjugated to viral nucleic acids with the help of nuclease-deactivated Cas9/gRNA complexes in the nuclei of living cells and then packaged into PRV during virion assembly. The processes of PRV-QD adsorption, cytoplasmic transport along microtubules, and nuclear entry were monitored in real time in both Vero and HeLa cells, demonstrating the utility and efficiency of the strategy in the study of viral infection.

Development of a revised ICC-qPCR method used for Pseudorabies virus inactivation validation study of biologically sourced materials.

To develop a precise and convenient method to evaluate the virus transmission risk of biologically sourced materials, an integrated cell culture-qPCR (ICC-qPCR) method for Pseudorabies virus (PRV) was established and revised for applications to this new field. The optimized post-infection period was found at 12-hr to achieve a reasonable detection limit (-0.25 Log10TCID50/100 µL, Logs) and a quantitative range (0.75-3.75 Logs). The results of mimic samples suggested that three 10-fold dilutions at the time of virus inoculation combined with three washes after virus absorption, and the sets of non-amplified samples as controls could efficiently eliminate the false positive signals caused by high levels of noninfectious viruses. The virus inactivation validation studies of acellular porcine corneas suggested that the logs inactivation of PRV at 12 kGy irradiation dose obtained by general ICC-qPCR, revised ICC-qPCR and cell culture were 2.49, 4.85 and 5.08, respectively. At 25 kGy, those were 2.31, 4.85 and 5.08, respectively. The results obtained by the revised ICC-qPCR were consistent with cell culture and more precise than general ICC-qPCR. Therefore, the revised ICC-qPCR proposed in this study has an application prospect in the PRV inactivation validation studies of biologically sourced materials.

Development of a SYBR green I-based duplex real-time PCR assay for detection of pseudorabies virus and porcine circovirus 3.

In the present study, a specific and reliable duplex SYBR green I-based quantitative real-time polymerase chain reaction assay was established to detect pseudorabies virus (PRV) and porcine circovirus 3 (PCV3) simultaneously. Viral genomes of PRV and PCV3 in one specimen were identified by their different melting temperatures with melting peaks at 87 °C and 81 °C for PRV and PCV3 respectively, whilst other non-targeted swine pathogens exhibited no fluorescent signals. The assay displayed a high degree of linearity (R2 > 0.997), and the limits of detection were 37.8 copies/µL, 30.6 copies/µL and 60 copies/µL for PRV, PCV3 and the mixture of two recombinant plasmids, respectively. It had good repeatability and reproducibility, and the coefficients of variation in intra-batch and inter-batch assays were all less than 2.0%. In this research, the duplex assay was further evaluated using 117 clinical tissue specimens from diseased pigs in the field. The results revealed the infection rates of PRV and PCV3 were 23.08% (27/117) and 55.56% (65/117) respectively, and PRV and PCV3 co-infection rate was 14.53% (17/117). The assay could be utilized as a diagnostic tool with specificity, sensitivity, and reliability for molecular epidemiological surveillance of PRV and PCV3.

First report of a pseudorabies-virus-infected wolf (Canis lupus) in China.

We provide the first report of a wolf infected with pseudorabies virus (PRV) in China. We observed the clinical symptoms and also dissected tissue samples from the wolf. The samples were ground under sterile conditions and injected subcutaneously into the necks of rabbits, which subsequently developed intense pruritus symptoms and died. The PRV strain from the wolf was isolated in porcine kidney (PK)-15 cells and was specifically recognized by pig PRV antibody-positive serum, as shown by indirect immunofluorescence. Tissues from the dead wolf and rabbits were examined by polymerase chain reaction (PCR), and the PCR-amplified partial glycoprotein E gene was sequenced, which confirmed that the wolf had died as a result of PRV infection.

Isolation and molecular characterization of a variant of Chinese gC-genotype II pseudorabies virus from a hunting dog infected by biting a wild boar in Japan and its pathogenicity in a mouse model.

We isolated a variant of Chinese pseudorabies virus from a hunting dog with symptoms similar to Aujeszky's disease and designated the isolate MY-1 strain. The dog developed symptoms 6 days after hunting and biting a wild boar and died the day after onset. The Bam HI restriction profile of MY-1 DNA was different from those of the Japanese reference strain Yamagata-S81 and two vaccine strains, Bartha and Begonia, and resembled Bam HI-RFLP (restriction fragment length polymorphism) type IV. Complete nucleotide sequences were determined, and phylogenetic analyses revealed that MY-1 belonged to the same cluster of old Chinese strains and variant strains isolated recently in China, but most of the open reading frames of MY-1 were located on a different branch from those of these Chinese strains. Based on a gC phylogenetic analysis, MY-1 belonged to gC-genotype II composed of those Chinese strains. In mice, the 50% lethal dose (LD50) of MY-1 (103.0 TCID50) was almost the same as those of Yamagata-S81 and Bartha. The LD50 value of Begonia was 10≥4.5 TCID50. The mean survival periods of mice after infection with 104 TCID50 of MY-1, Yamagata-S81 and Bartha were 3.9 days, 2.3 days, and 8.0 days, respectively. The results suggested that the variant of Chinese PRV with slightly weaker pathogenicity than that of wild virulent viruses might be maintained in wild boars in Japan. Furthermore, we would like to propose that old Chinese strains, recent Chinese variant strains, and MY-1 should be grouped as an Asian type PRV.

Real-time RPA assay for rapid detection and differentiation of wild-type pseudorabies and gE-deleted vaccine viruses.

The objective of this study was to develop a dual real-time recombinase polymerase amplification (RPA) assay using exo probes for the detection and differentiation of pseudorabies virus (PRV). Specific RPA primers and probes were designed for gB and gE genes of PRV within the conserved region of viral genome. The reaction process can be completed in 20 min at 39 °C. The dual real-time RPA assay performed in the single tube was capable of specific detecting and differentiating of the wild-type PRV and gE-deleted vaccine strains, without cross-reactions with other non-targeted pig viruses. The analytical sensitivity of the assay was 102 copies for gB and gE genes. The dual real-time RPA demonstrated a 100% diagnostic agreement with the real-time PCR on 4 PRV strains and 37 clinical samples. Through the linear regression analysis, the R2 value of the real-time RPA and the real-time PCR for gB and gE was 0.983 and 0.992, respectively. The dual real-time RPA assay provides an alternative useful tool for rapid, simple, and reliable detection and differentiation of PRV, especially in remote and rural areas.

Vaccine resistant pseudorabies virus causes mink infection in China.

BACKGROUND: Pseudorabies, a highly contagious infectious disease of swine is caused by pseudorabies virus (PRV). PRV can cause fatal infection in other animal species. RESULTS: We report a deadly outbreak of pseudorabies that killed 87.2% (3522/4028) minks in a farm in 2014 in Shandong Province, China. PRV was isolated by using Vero cell culture and detected in mink samples by PCR from minks died during the outbreak. Epidemiological analysis indicated that 5.8% of minks (33/566) were PCR positive to PRV in Shandong Province. Phylogenetic analysis indicated that the PRV strains isolated from minks in this study were in the same clade with the Chinese porcine PRV isolates, which are resistant to the PRV vaccine. CONCLUSIONS: We demonstrated that pseudorabies virus caused an outbreak of minks in a farm in Shandong Province of China and the virus has a very high infection rate in minks in Shandong Province, which is a challenge for the fur industry in China.

Development of an isothermal recombinase polymerase amplification assay for rapid detection of pseudorabies virus.

Recombinase polymerase amplification assays using real-time fluorescent detection (real-time RPA assay) and lateral flow dipstick (RPA LFD assay) were developed targeting the gD gene of pseudorabies virus (PRV). Both assays were performed at 39 °C within 20 min. The sensitivity of the real-time RPA assay and the RPA LFD assay was 100 copies per reaction and 160 copies per reaction, respectively. Both assays did not detect DNAs from other virus or PRV negative samples. Therefore, the developed RPA assays provide a rapid, simple, sensitive and specific alternative tool for detection of PRV.

Outbreak of severe pseudorabies virus infection in pig-offal-fed farmed mink in Liaoning Province, China.

An outbreak of severe pseudorabies virus (PRV) infection in farmed mink occurred in northern China in late 2014, causing significant economic losses in the local fur industry. Here, we report the first case of a PRV outbreak in mink in northeastern China, caused by feeding farmed mink with raw pork or organs contaminated by PRV. Mink infected with virulent PRV exhibited diarrhea, neurologic signs, and higher mortality, which can be misdiagnosed as highly pathogenic mink enteritis virus (MEV), canine distemper virus (CDV), and food poisoning. However, these were excluded as causative agents by PCR or bacteria isolation. The duration of disease was 3-7 days, and the mortality rate was 80-90%. PRV was characterized using indirect immunofluorescence assays (IFA) and electron microscopy (EM). Phylogenetic analysis based on full-length genome sequences and those of individual genes of this novel virus strain showed that it clustered in an independent branch with several other PRV isolates from China.

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.

Pseudorabies virus infection (Aujeszky's disease) in an Iberian lynx (Lynx pardinus) in Spain: a case report.

BACKGROUND: The only natural hosts of Pseudorabies virus (PRV) are members of the family Suidae (Sus scrofa scrofa). In species other than suids infection is normally fatal. In these mammals, including carnivores, PRV typically causes serious neurologic disease. The endangered Iberian lynx (Lynx pardinus) is a wild feline endemic to south-western Europe (Iberian Peninsula). The Iberian lynx was found to be the world's most endangered felid species in 2002. In wild felines, PRV infection has only been previously reported once in a Florida panther in 1994. No seropositive lynxes have ever been found, nor has PRV been detected in dead Iberian lynxes to date. CASE PRESENTATION: We describe the first reported case of pseudorabies in an Iberian lynx (Lynx pardinus). Pseudorabies was diagnosed in a young wild Iberian lynx from Extremadura (SW Spain) by histopathological examination, immunohistochemistry, polymerase chain reaction (PCR) and sequence analysis. Gross lesions included alopecia of the ventral neck, bloody gastro-intestinal contents and congestion of the brain. Histopathological analysis showed a moderate nonsuppurative meningoencephalitis with diffuse areas of demyelination, necrotizing gastritis and enteritis of the small intestine. Pseudorabies virus (PRV) antigen was found in neuronal and non-neuronal cells of the brain, tonsils, and gastric glandular epithelial cells by immunohistochemical analysis. The presence of the virus in the brain was confirmed by nested PCR. The sequence analysis of the 146 bp fragment (from the viral glycoprotein B gene) showed that the amplified sequence matched (with 100% identity) the PRV genome. Furthermore, specific DNA from glycoprotein D and E encoding-genes was detected by conventional and real-time PCR, respectively, confirming the latter that this infection was produced by a wild-type PRV strain. CONCLUSIONS: This study supports the suspicion that PRV could infect the Iberian lynx. The detection of PRV in a dead Iberian lynx suggests that the virus may have a negative impact on the survival of endangered lynxes in the wild. However, because this is the first verified instance of lynx mortality resulting from pseudorabies, its true impact on the population is unknown.

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.

[Serological detection of Brucella suis, influenza virus and Aujeszky's disease virus in backyard and small swine holders in Argentina]. / 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.

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.

Pseudorabies virus isolates from domestic pigs and wild boars show no apparent in vitro differences in replication kinetics and sensitivity to interferon-induced antiviral status.

Pseudorabies virus is the causative agent of Aujeszky's disease. Domestic pigs and wild boars are its natural hosts, and strains circulating within both populations differ in their capacity to induce clinical disease. Cell biological and molecular explanations for the observed differences in virulence are, however, lacking. Different virulence determinants that can be assessed in vitro were determined for five domestic swine strains, four wild boar strains and the NIA3 reference strain. Replication kinetics and plaque formation capacity in continuous swine testicular cells and different primary porcine cell lines were highly similar for isolates from both populations. Treatment of these cell lines with IFNα, IFNγ or a combination of both provoked similar plaque-reducing effects for all strains. In conclusion, our results indicate that isolates from domestic swine and wild boar differ neither in intrinsic replication and dissemination capacity nor in sensitivity to antiviral effects of IFNs.

Age- and strain-dependent differences in the outcome of experimental infections of domestic pigs with wild boar pseudorabies virus isolates.

Although pseudorabies virus (PRV) has been eradicated in domestic swine in many countries, its presence in wild boars remains a threat for a reintroduction into the currently unprotected swine population. To assess the possible impact of such a reintroduction in a naive herd, an in vivo infection study using two genetically characterized wild boar PRV isolates (BEL24043 and BEL20075) representative for wild boar strains circulating in south-western and central Europe and the virulent NIA3 reference strain was performed in 2- and 15-week-old domestic pigs. Our study revealed an attenuated nature of both wild boar strains in 15-week-old pigs. In contrast, it showed the capacity of strain BEL24043 to induce severe clinical symptoms and mortality in young piglets, thereby confirming that the known age dependency of disease outcome after PRV infection also holds for wild boar isolates. Despite the absence of clinical disease in 15-week-old sows, both wild boar PRV strains were able to induce seroconversion, but to a different extent. Importantly, differences in infection and transmission capacity of both strains were observed in 15-week-old sows. Strain BEL24043 induced a more prolonged and disseminated infection than strain BEL20075 and was able to spread efficiently to contact animals, indicative of its capacity to induce a sustained infection. In conclusion, it was shown that a reintroduction of a wild boar isolate into the domestic swine population could have serious economic consequences due to the induction of clinical symptoms in piglets and by jeopardizing the PRV-negative status.