Pseudorabies Virus Isolates Research Articles

Pathogenesis of natural and experimental Pseudorabies virus infections in dogs.

BackgroundSince late 2011, cases of suspected canine pseudorabies have increased in north China with the outbreak of swine pseudorabies in the same area, but the pathogenesis of canine Pseudorabies virus (PRV) infections in China is poorly understood. In this study, we investigated the pathogenesis of canine pseudorabies.MethodsThe pathological changes in 13 dogs that died of natural PRV infections (confirmed by pathogen detection) during 2011–2013 in Beijing were evaluated. An experimental study was also conducted in which healthy adult beagle dogs were administered PRV isolate BJ-YT by subcutaneous injection. The dog tissues were subjected to gross and microscopic examinations and immunohistochemical analysis and the dogs’ serum cardiac troponin-I (cTn-I) was measured.ResultsSystemic hemorrhage and/or congestion were the most marked pathological changes in both the naturally and experimentally PRV-infected dogs. Macroscopically, the major lesions consisted of petechiae and ecchymoses in both the endocardium and epicardium, thrombi in the mitral valves, hemorrhage in the lungs and thymus, and incomplete contraction of the spleen. Microscopically, the major histopathological findings were systemic hemorrhage and congestion, nonsuppurative ganglioneuritis (in the experimentally infected dogs, unexamined in the naturally PRV-infected dogs), brainstem encephalitis (in the naturally infected dogs), necrosis or exudation in the myocardium, and lymphoid depletion in many lymphoid organs and tissues. Viral antigens were only detected in the brainstems and peripheral ganglia of the infected dogs. Serum cTn-I was significantly higher in the experimentally PRV-infected dogs with myocardial lesions than in the dogs without myocardial lesions.ConclusionsBased on these results, we conclude that virally induced systemic hemorrhage, peripheral nervous system pathology, and/or cardiac injury can individually or collectively cause death in PRV-infected dogs. The respiratory signs of the disease are attributed to cardiogenic lesions.

Pathogenicity and genomic characterization of a pseudorabies virus variant isolated from Bartha-K61-vaccinated swine population in China

Pseudorabies (PR) or Aujeszky's disease (AD), caused by pseudorabies virus (PRV), is an economically important viral disease worldwide. Recently, PR outbreaks occurred in a large number of Bartha-K61-vaccinated swine herds in many regions of China. Here, we isolated a PRV variant, named TJ strain, from a Bartha-K61-vaccinated pig farm in China, evaluated the pathogenicity of the TJ strain in susceptible animals and analyzed its complete genomic sequence obtained by 454 pyrosequencing. Vaccination-challenge experiment in sheep showed that the classical Bartha-K61 vaccine could not provide complete protection against the challenge with the PRV TJ strain. In mice, the 50% lethal dose (LD50) of the TJ strain (102.3 TCID50) was lower than that of the classical PRV SC strain (103.0 TCID50). Furthermore, the TJ strain displayed higher mortality for pigs, as compared with the SC strain. The PRV TJ strain genome was determined to be 143,642bp in length, encoding 67 open reading frames. The TJ strain was clustered to an independent branch together with some recent PRV isolates in China in the phylogenetic tree, which was relatively distant from previous PRV isolates. The TJ strain showed unique variations in the viral proteins that play key roles in the viral replication cycle. Taken together, the TJ strain is a highly pathogenic PRV variant with unique molecular signatures. Further studies are needed to explore the relevance of the sequence differences to the virulence alteration of the PRV variant.

Molecular characterization of Belgian pseudorabies virus isolates from domestic swine and wild boar

Aujeszky's disease is an economically important disease in domestic swine caused by suid herpesvirus 1, also called pseudorabies virus (PRV). In several European countries, including Belgium, the virus has successfully been eradicated from the domestic swine population. The presence of PRV in the wild boar population however poses a risk for possible reintroduction of the virus into the domestic pig population. It is therefore important to assess the genetic relatedness between circulating strains and possible epidemiological links. In this study, nine historical Belgian domestic swine isolates that circulated before 1990 and five recent wild boar isolates obtained since 2006 from Belgium and the Grand Duchy of Luxembourg were genetically characterized by restriction fragment length polymorphism (RFLP) analysis and phylogenetic analysis. While all wild boar isolates were characterized as type I RFLP genotypes, the RFLP patterns of the domestic swine isolates suggest that a shift from genotype I to genotype II might have occurred in the 1980s in the domestic population. By phylogenetic analysis, Belgian wild boar isolates belonging to both clade A and B were observed, while all domestic swine isolates clustered within clade A. The joint phylogenetic analysis of both wild boar and domestic swine strains showed that some isolates with identical sequences were present within both populations, raising the question whether these strains represent an increased risk for reintroduction of the virus into the domestic population.

Molecular epidemiology of Brazilian pseudorabies viral isolates

Pseudorabies is a disease caused by pseudorabies virus (PRV) and is responsible for considerable economic losses in the swine industry. The objective of this work was to use molecular epidemiology as a tool to facilitate the study of PRV outbreaks in Brazil. The standard PRV strain Shope, the vaccine strain Bartha and isolates from the south and the southeast regions of Brazil, were amplified for gE and gC partial genes by PCR. Results indicated that Brazilian PRV isolates are grouped in two clusters, A and B, except for one isolate that grouped with Bartha and Shope. Most Brazilian PRV isolates belonged to cluster B and diverged from virus isolated from other countries.

Development of Real-Time Polymerase Chain Reaction Assays for Rapid Detection and Differentiation of Wild-Type Pseudorabies and Gene-Deleted Vaccine Viruses

The successful eradication of pseudorabies in U.S. domestic swine was accomplished through the use of glycoprotein E (gE) deleted modified live virus vaccines and an accompanying gE differential enzyme-linked immunosorbent assay (ELISA). Yet, pseudorabies virus (PRV) was established in feral swine in the United States, becoming a potential reservoir of PRV for infection of domestic swine and other native wildlife. A critical need for the current PRV surveillance program in the United States is the rapid detection of PRV infection. For this reason, a set of 2 real-time polymerase chain reaction (PCR) assays by using TaqMan chemistry was developed and evaluated for their capability in the detection and differentiation of field and vaccine strains of PRV. PCR primers and probes were designed for gB and gE genes of PRV, respectively. The newly developed PRV-specific real-time PCR assays could detect all wild-type PRV isolates from diagnostic submissions and differentiate them from vaccine strains. The analytical sensitivity of the assays was approximately 0.1 plaque-forming units per reaction. The assays were highly specific for PRV, because no positive results were obtained from testing other common swine viral pathogens and other animal herpesviruses. The results of testing samples from domestic and feral swine and from bovine showed that the real-time PCR assays are more sensitive than gel-based PCR. These results demonstrated the potential application of the developed real-time PCR assays as a differential test for rapid and specific detection of PRV in domestic and feral swine, as well as nonporcine species that can be infected with PRV and serve as carriers.

Potential sites of virus latency associated with indigenous pseudorabies viruses in feral swine.

Free-ranging feral swine (Sus scrofa) are known to be present in at least 32 states of the USA and are continuously expanding their range. Infection with pseudorabies virus (PRV) occurs in feral swine and the primary route of transmission in free-living conditions seems to be venereal. Between 1995 and 1999, naturally infected feral swine and experimentally infected hybrid progeny of feral and domestic swine, were kept in isolation and evaluated for occurrence of latent PRV indigenous to feral swine in sacral and trigeminal ganglia and tonsil. Sacral ganglia were shown, by polymerase chain reaction (PCR) amplification of the thymidine kinase (TK) gene of PRV, to be the most frequent sites of latency of PRV. Nine (56%) of 16 sacral ganglia, seven (44%) of 16 trigeminal ganglia, and five (39%) of 13 tonsils from naturally infected feral swine were positive for PCR amplification of TK sequences of PRV. These tissues were negative for PRV when viral isolation was attempted in Vero cells. DNA sequencing of cloned TK fragments from the sacral ganglia of two feral swine, showed only one nucleotide difference between the two fragments and extensive sequence homology to fragment sequences from various domestic swine PRV strains from China, Northern Ireland, and the USA. The hybrid feral domestic swine, experimentally inoculated with an indigenous feral swine PRV isolate by either the genital or respiratory route, acquired the infection but showed no clinical signs of pseudorabies. Virus inoculated into either the genital or respiratory tract could, at times, be isolated from both these sites. The most common latency sites were the sacral ganglia, regardless of the route and dose of infection in these experimentally infected hybrids. Nine of 10 sacral ganglia, six of 10 trigeminal ganglia, and three of 10 tonsils were positive for PCR amplification of TK sequences. No virus was isolated from these tissues in Vero cells. The demonstration of the sacral ganglia as the most common sites of latency of pseudorabies viruses indigenous to feral swine, supports the hypothesis that these viruses are primarily transmitted venereally, and not by the respiratory route as is common in domestic swine, in which the trigeminal ganglia are the predominant sites of virus latency.

Experimental infection of wild boar with pseudorabies virus isolates of different virulence

The wild boar is considered as a potential carrier for Pseudorabies virus (PrV) and could also function as a reservoir for the infection. So far, however, little is known about the susceptibility of the wild swine, especially the wild boar, for PrV. Although in recent years PrV has repeatedly been isolated from wild pigs in the USA, Italy and Germany, data on the virulence of these PrV isolates are sparse. Molecular characterisation of PrV isolates obtained from wild boars in Germany has revealed a unique DNA pattern which had not been observed before in domestic animals. In addition, studies on the virulence of these PrV isolates were required to evaluate the potential risk of PrV infections for the wild boar and also for the domestic pig population. Therefore, the objective of this study was to (1) determine the susceptibility of the European wild boar (Sus scrofa, L.) for PrV, and (2) to characterise the virulence of a PrV isolate from the wild boar for the wild boar itself and for domestic pigs. In separate trials for each PrV isolate, four juvenile wild boars were intranasally infected with 104 or 106 TCID50 and kept together with two control animals. Infections with PrV-Bartha and the wild boar isolate BFW1 showed no or very mild symptoms. By contrast, wild boars infected with PrV-Kaplan showed severe clinical disease which included anorexia, acute shortness of breath, foamy salivation, vomitus, pruritus, blindness, irritability, incoordination and paralysis. After two animals had died on day 7 p.i., this trial was ended. The virus was isolated from nasal and genital swabs for PrV-Bartha between day 1 and 5 p.i., between day 1 and 7 p.i. for PrV-Kaplan, and between day 5 and 12 p.i. for PrV-BFW1. Characteristic titres of excreted virus were found in each trial. Maximal titres ranged between 1.5 and 4.5 log10. While antibodies were first observed on day 7 p.i. with PrV-Bartha and Kaplan as measured by an indirect ELISA, a specific humoral immune response was detected only after 12 days p.i. after infection with PrV-BFW1. Neutralising antibody titres reached 1:22 $\pm 10$ in PrV-Bartha- and 1:6 $\pm 7$ in PrV-BFW1-infected animals. Virus isolation from tissues succeeded only after infection with PrV-Kaplan (tonsil, spleen, mediastinal lymph node, salivary gland, lung, brain, spinal cord, accessory genital glands). Following immunosupression, in animals infected with PrV-Bartha, viral DNA was detected in tonsils and salivary glands by PCR, while PrV-specific amplicons were obtained from the tonsils, lung, brain and spinal cord after infection with PrV-BFW1. Two groups of 6 or 8 domestic pigs each were infected with 104, 106 or 107 KID50 of PrV-BFW1. Similar results were obtained with the lower dose, as described for the wild boar. With the higher dose, however, all animals developed respiratory symptoms, while only some pigs showed convulsions for a short period, unphysiological movements and nausea. Antibodies were first detected on day 7 p.i. by ELISA while neutralising antibodies reached titres of 1:4 $\pm 17$. Virus or viral DNA were only found in the tonsils. Immunosuppression failed to reactivate latent PrV. It can be concluded that the European wild boar is susceptible to infection with PrV isolated from domestic pigs. Clinical symptoms depend on the virulence of the PrV isolate, the dose and route of infection. The wild boar PrV isolate, BFW1, seems to be well adapted to the wild boar whereas it leads to a differentiated clinical and antibody response in domestic pigs depending on the dose of infection.

Evaluation of nucleic acid amplification methods for the detection of hog cholera virus.

A blind panel was tested in a diagnostic evaluation of a reverse transcription (RT) polymerase chain reaction (PCR) method for detecting hog cholera virus (HCV) from pig tissues. The capability of the RT-PCR test to discriminate between HCV and related pestiviruses, bovine viral diarrhea virus (BVDV), and those viruses causing similar diseases in swine, including African swine fever virus (ASFV) and pseudorabies virus (PRV), was also considered. Nucleic acid extraction involved either kit-based or conventional phenol:chloroform:isoamyl alcohol methods. A single-round PCR assay, using primers that hybridize to the conserved p120 nonstructural gene region, was 82.5% sensitive (n = 17) and 100% specific (n = 18) in the detection of the presence of HCV RNA. However, the sensitivity was increased to 100% following a second PCR test. In all, 4 HCV, 7 BVDV, 2 ASFV, and 1 PRV isolates were studied. Novel nucleic acid sequences were generated for 9 HCV strains. Analysis of a portion of the p120 region using these methods was suitable for HCV isolate characterization.

Sequence variation of the gC gene among pseudorabies virus strains

We have determined the nucleotide sequences of the major glycoprotein C (gC) gene of 3 pseudorabies virus (PRV) strains isolated in Japan, the USA, and Northern Ireland after gene amplification mediated by a polymerase chain reaction (PCR). The nucleotide and deduced amino acid sequence homologies among the strains were >98% and >97%, respectively. The restriction patterns of the amplified DNA fragments generated by restriction endonucleases SalI and SmaI revealed three genomic variations among the 15 PRV strains. The Japanese PRV isolates have identical restriction fragment patterns and differ from those of the non-Japanese isolates examined. Among 3 PRV strains with distinctive genotype each other, there is no significant difference in pathogenicity for the ddY mouse.

Structural organization of the termini of the L and S components of the genome of pseudorabies virus

The sequences of several hundred nucleotides around the junctions between the L and S components in concatemeric DNA and in mature virion DNA were ascertained. The two ends of the mature genome (which are joined in concatemeric DNA) show no sequence homology. Several directly repeated elements are present near both ends of the genome. Furthermore, the last 82 nucleotides at the left end of the L component (and of the genome) are repeated in inverted form (inverted repeat within the L component [IRL]) approximately 350 to 600 nucleotides downstream (depending on the virus isolate) bracketing the UL2 component. A comparison between the sequences at the right and left ends of the L component of the genome showed patchy homology, probably representing a vestigial inverted repeat bracketing the L component (IRL). Furthermore, less than 5% of the genomes have an L component that is in the orientation opposite to that of most of the viral genomes, indicating that the vestigial IRL that brackets the UL sequence may be sufficient to mediate inversion of the L component in some of the genomes. On the other hand, the UL2 component, which is bracketed by a perfect IRL, does not invert to a greater extent than does the L component (if it inverts at all). Analysis of the nucleotide sequence at the concatemeric junction of three different pseudorabies virus isolates showed almost complete sequence conservation. The sequence and organization of the repeated elements in the different isolates were almost identical, despite their different histories and origins. The high degree of conservation of these repeated elements implies that they may fulfill an essential function in the life cycle of the virus.

Update and comparison of the immediate-early gene DNA sequences of two pseudorabies virus isolates

Differences between the immediate-early gene DNA sequences of two pseudorabies virus isolates (Indiana-Funkhauser and Ka) were resolved and confirmed. The deduced amino acid sequences showed that regions 2 and 4 have fewer changes than the rest of the molecules. These two conserved regions may be functionally important.

Analysis of pseudorabies virus glycoprotein gIII localization and modification by using novel infectious viral mutants carrying unique EcoRI sites.

We have constructed two pseudorabies virus (PRV) mutants, each with a unique EcoRI restriction site in the nonessential gIII envelope glycoprotein gene. Since no natural PRV isolate has been reported to contain EcoRI sites, the isolation and single-step growth curve analysis of these mutants established that PRV can carry such a site with little ill effect in tissue culture. Virus carrying these defined mutations produced novel gIII proteins that enabled us to begin functional assignment of protein localization information within the gIII gene. Specifically, one viral mutant contained an in-frame synthetic EcoRI linker sequence that was flanked on one side by the first one-third of the gIII gene and on the other side by the last one-third of the gene. The resulting protein lacked the middle one-third of the parental species, including five of eight putative N-linked glycosylation signals, but was still glycosylated and found in enveloped virions; it was not secreted into the medium. A second viral mutant contained an in-frame synthetic EcoRI linker sequence that additionally specified a nonsense codon at position 158, producing a gIII protein that was glycosylated and secreted into the medium; the fragment was not found in enveloped virions. By endoglycosidase and pulse-chase analyses, we established a precursor-product relationship between the various forms of gIII expressed in the parental and mutant strains, and perhaps determined certain features of the gIII protein that are required for its efficient export within the cell.