Transmission Of Pseudorabies Virus Research Articles

Pseudorabies Virus Associations in Wild Animals: Review of Potential Reservoirs for Cross-Host Transmission.

Pseudorabies virus (PRV) has received widespread attention for its potential health effects on humans, wildlife, domestic animals, and livestock. In this review, we focus on PRV dynamics in wildlife, given the importance of wild-origin PRV transmission to domestic and farm animals. Wild boars, pigs, and raccoons can serve as reservoirs of PRV, with viral transmission to domestic livestock occurring via several routes, such as wild herd exposure, contaminated meat consumption, and insect vector transmission. Many endangered feline and canine species can be infected with PRV, with acute disease and death within 48 h. The first confirmed human case of PRV infection in mainland China was reported in 2017. Thus, PRV exhibits potentially dangerous cross-host transmission, which is likely associated with inappropriate vaccination, poor awareness, and insufficient biosecurity. Currently, no vaccine provides full protection against PRV in all animals. Here, we summarize the epidemiology and pathogenesis of PRV infection in wild, domestic, and farmed animals, which may facilitate the design of novel therapeutics and strategies for controlling PRV infection and improving wildlife protection in China.

The Epidemiology and Variation in Pseudorabies Virus: A Continuing Challenge to Pigs and Humans.

Pseudorabies virus (PRV) can infect most mammals and is well known for causing substantial economic losses in the pig industry. In addition to pigs, PRV infection usually leads to severe itching, central nervous system dysfunction, and 100% mortality in its non-natural hosts. It should be noted that increasing human cases of PRV infection have been reported in China since 2017, and these patients have generally suffered from nervous system damage and even death. Here, we reviewed the current prevalence and variation in PRV worldwide as well as the PRV-caused infections in animals and humans, and briefly summarized the vaccines and diagnostic methods used for pseudorabies control. Most countries, including China, have control programs in place for pseudorabies in domestic pigs, and thus, the disease is on the decline; however, PRV is still globally epizootic and an important pathogen for pigs. In countries where pseudorabies in domestic pigs have already been eliminated, the risk of PRV transmission by infected wild animals should be estimated and prevented. As a member of the alphaherpesviruses, PRV showed protein-coding variation that was relatively higher than that of herpes simplex virus-1 (HSV-1) and varicella-zoster virus (VZV), and its evolution was mainly contributed to by the frequent recombination observed between different genotypes or within the clade. Recombination events have promoted the generation of new variants, such as the variant strains resulting in the outbreak of pseudorabies in pigs in China, 2011. There have been 25 cases of PRV infections in humans reported in China since 2017, and they were considered to be infected by PRV variant strains. Although PRV infections have been sporadically reported in humans, their causal association remains to be determined. This review provided the latest epidemiological information on PRV for the better understanding, prevention, and treatment of pseudorabies.

Porcine ZC3H11A Is Essential for the Proliferation of Pseudorabies Virus and Porcine Circovirus 2

Porcine epidemic viruses, such as pseudorabies virus (PRV) and porcine circovirus 2 (PCV2), are among the most economically damaging pathogens affecting the swine industry. Importantly, previous studies have shown that cases of human infection with PRV occur frequently, indicating the considerable risk of PRV transmission from pigs to humans. Zinc finger CCCH-type containing 11A (ZC3H11A) has been confirmed to play a crucial role in maintaining the nuclear export of mRNA under stress in humans, but its role in pigs remains unknown. In this study, we observed that ZC3H11A interacted with the transcription and export complex and played an important role in mRNA export. Specifically, we knocked out ZC3H11A in PK-15 cells with CRISPR/Cas9 and challenged them with PRV and PCV2. The results showed that the proliferation of the virus was significantly inhibited in ZC3H11A-/- cells, indicating that porcine ZC3H11A is indispensable for the proliferation of PRV and PCV2. Furthermore, our study demonstrated that the inactivation of ZC3H11A in host cells also inhibited the proliferation of PRV and PCV2. Taken together, the results of our study indicated that ZC3H11A is important for maintaining the export of mRNAs, which in turn facilitates the proliferation of PRV and PCV2, suggesting that it can be a potential target for producing antiviral pigs and drugs.

THE PRESENCE OF ANTIBODIES AGAINST PSEUDORABIES VIRUS IN WILD BOARS (SUS SCROFA) IN KOREA.

The control and elimination of pseudorabies (PR) is one of the most important goals in the pig industry. After the first PR outbreak in Korea in 1986, all pigs infected with PR virus (PRV) were removed, and a vaccination program for pigs was implemented. No PR has occurred in Korea since 2010, and vaccination was discontinued after 2013. Information on the seroprevalence of PRV in pigs, including wild boars (Sus scrofa), is important for evaluating the PR status in a country. In this study, 2.65% (28/1057) of the wild boars tested had antibodies against PRV in 2018, indicating that PRV has been circulating continuously in the wild boar population in Korea. Effective means should be implemented to prevent the transmission of PRV between wild and domestic pigs, because the wild boar is a potential reservoir host for PRV.

Evidence for interspecies transmission route of pseudorabies virus via virally contaminated fomites

Pseudorabies virus (PRV) is a zoonotic agent with a wide host range, causing significant economic losses in animal husbandry and potential public health risk globally. The causative agent has recently gained attention due to the inter-species transmission among different species of animals, even human beings. Although PRV's prevalence is found in many species of animals, regardless of whether the strain involved is a vaccine, classical or variant, few lines of evidence for the viral transmission route are available. Here, we reported that viral contamination is associated with the inter-species transmission of PRV. We found that PRV contamination was widely distributed in the environment of pig farms, that viral distribution in the environment is associated with the implementation of biosecurity measures, and that PRV could transmit from pigs to dogs through virally contaminated fomites. Collectively, our findings provide a basis for understanding the ecology and transmission route of PRV and underscore the importance of implementing biosecurity measures to control this disease.

Proteomics Analysis Identifies IRSp53 and Fascin as Critical for PRV Egress and Direct Cell-Cell Transmission.

Pseudorabies virus (PRV) has been widely used as a live trans-synaptic tracer for mapping neuronal circuits. Systematically identifying mature PRV virion proteomes and defining co-purified host proteins are necessary to fully understand the detailed mechanism underlying PRV transmission processes. Here, a PRV virion purification strategy based on sorting with flow cytometry is developed and the mature extracellular and intracellular PRV virion proteomes using LC coupled with MS/MS are characterized. In addition to viral proteins, a large number of host proteins are also identified, including proteins related to actin cytoskeletal dynamics and membrane protrusion. How many of these host proteins are true virion components are unknown and the majority of these may not be. Through functional analysis, it is found that IRSp53 and fascin are critical for the egress process and play a role in direct cell-cell transmission. Moreover, it is shown that CDC42 and Rac1 are also involved in the production of mature extracellular virions. The results suggest that the formation of the filopodia-like cytoskeleton and the rearrangement of the membrane, which are both associated with IRSp53 and fascin, may be important for the transmission of viruses used in neuronal tracing.

Gene Expression Profiling with Cre-Conditional Pseudorabies Virus Reveals a Subset of Midbrain Neurons That Participate in Reward Circuitry.

The mesolimbic dopamine pathway receives inputs from numerous regions of the brain as part of a neural system that detects rewarding stimuli and coordinates a behavioral response. The capacity to simultaneously map and molecularly define the components of this complex multisynaptic circuit would thus advance our understanding of the determinants of motivated behavior. To accomplish this, we have constructed pseudorabies virus (PRV) strains in which viral propagation and fluorophore expression are activated only after exposure to Cre recombinase. Once activated in Cre-expressing neurons, the virus serially labels chains of presynaptic neurons. Dual injection of GFP and mCherry tracing viruses simultaneously illuminates nigrostriatal and mesolimbic circuitry and shows no overlap, demonstrating that PRV transmission is confined to synaptically connected neurons. To molecularly profile mesolimbic dopamine neurons and their presynaptic inputs, we injected Cre-conditional GFP virus into the NAc of (anti-GFP) nanobody-L10 transgenic mice and immunoprecipitated translating ribosomes from neurons infected after retrograde tracing. Analysis of purified RNA revealed an enrichment of transcripts expressed in neurons of the dorsal raphe nuclei and lateral hypothalamus that project to the mesolimbic dopamine circuit. These studies identify important inputs to the mesolimbic dopamine pathway and further show that PRV circuit-directed translating ribosome affinity purification can be broadly applied to identify molecularly defined neurons comprising complex, multisynaptic circuits.SIGNIFICANCE STATEMENT The mesolimbic dopamine circuit integrates signals from key brain regions to detect and respond to rewarding stimuli. To further define this complex multisynaptic circuit, we constructed a panel of Cre recombinase-activated pseudorabies viruses (PRVs) that enabled retrograde tracing of neural inputs that terminate on Cre-expressing neurons. Using these viruses and Retro-TRAP (translating ribosome affinity purification), a previously reported molecular profiling method, we developed a novel technique that provides anatomic as well as molecular information about the neural components of polysynaptic circuits. We refer to this new method as PRV-Circuit-TRAP (PRV circuit-directed TRAP). Using it, we have identified major projections to the mesolimbic dopamine circuit from the lateral hypothalamus and dorsal raphe nucleus and defined a discrete subset of transcripts expressed in these projecting neurons, which will allow further characterization of this important pathway. Moreover, the method we report is general and can be applied to the study of other neural circuits.

Serologic, molecular, and pathologic survey of pseudorabies virus infection in hunted wild boars (Sus scrofa) in Italy.

To investigate pseudorabies-virus (PrV) -antibody and viral-DNA prevalence, we collected blood, nasal and genital swabs, and tonsillar and lymph-node tissue samples from 139 wild boars (Sus scrofa; 39 piglets, 30 juveniles, and 70 adults), during the hunting season of 2010-2011 in Tuscany, Central Italy. We performed immunohistochemistry with anti-PrV monoclonal antibodies on selected tissue samples. Forty-three of 139 (30.9%) boars were PrV-antibody positive and a 1,954-base-pair PrV-specific product was amplified from nine nasal (6.5%) and 26 genital (18.7%) swabs. Sequence analysis of PrV-positive PCR products revealed identity scores of 99-100% with Suid herpesvirus 1 strain Becker (JF797219) and confirmed the identification of PrV DNA in tested swabs. There was significantly higher antibody prevalence in adults than in juveniles and in piglets than in juveniles. The prevalence of viral DNA was significantly higher in genital swabs than in nasal specimens. The percentage of positive nasal swabs did not differ among age classes. Piglets had a higher percentage of PCR-positive genital swabs than juvenile and adult subjects (30.8% vs. 13.3% and 14.3%, respectively). Results confirmed that PrV infection is widespread in the wild boar population in the study area. The presence of anti-PrV antibodies and of the PrV virus in piglets could be related to vertical transmission of the virus. This hypothesis was also supported by a higher presence of viral genome in genital swabs than in nasal swabs. This field study supports the importance of vertical transmission of PrV, and the high prevalence of virus in genital swabs supports venereal transmission in adult feral boars.

The neutralization of pseudorabies virus by anti-α-galactocyl natural antibody in normal serum

Pseudorabies virus (PrV), a member of herpesviridae alphaherpes subfamily, can infect human cells in vitro. However, the transmission to Old World primates including humans is strongly restricted. In this study, we report the neutralizing activity of normal human serum against PrV grown in CPK cells derived from pig. PrV grown in all Old World primates-derived cells, which was tested in this study, were not neutralized by normal human serum. The virion of PrV grown in CPK cells harbored Galα1–3Galβ1–4GlcNAc-R (α-gal epitope) on its surface, while PrV grown in Vero cells did not. Depletion of antibodies reacting to surface antigens of CPK cells negated the neutralization activity of human serum. Blockade of anti-α-gal antibodies by adding soluble Galα1–3Gal to normal human serum also prevent the inactivation of PrV grown in CPK cells. Although normal swine serum did not neutralize PrV grown in CPK cells, swine serum supplemented with exogenous anti-α-gal antibodies did. These results indicate that anti-α-gal antibodies in normal human serum contribute to the neutralization of PrV. Anti-α-gal antibodies in normal human serum may prevent transmission of PrV into humans.

Transmission of pseudorabies virus from immune-masked blood monocytes to endothelial cells.

Pseudorabies virus (PRV) may cause abortion, even in the presence of vaccination-induced immunity. Blood monocytes are essential to transport the virus in these immune animals, including transport to the pregnant uterus. Infected monocytes express viral proteins on their cell surface. Specific antibodies recognize these proteins and should activate antibody-dependent cell lysis. Previous work showed that addition of PRV-specific polyclonal antibodies to PRV-infected monocytes induced internalization of viral cell surface proteins, protecting the cells from efficient antibody-dependent lysis in vitro (immune-masked monocytes). As a first step to reach the pregnant uterus, PRV has to cross the endothelial cell barrier of the maternal blood vessels. The current aim was to investigate in vitro whether immune-masked PRV-infected monocytes can transmit PRV in the presence of virus-neutralizing antibodies via adhesion and fusion of these monocytes with endothelial cells. Porcine blood monocytes, infected with a lacZ-carrying PRV strain, were incubated with PRV-specific antibodies to induce internalization. Then, cells were co-cultivated with endothelial cells for different periods of time. Only PRV-infected monocytes with internalized viral cell surface proteins adhered efficiently to endothelial cells. LacZ transmission to endothelial cells, as a measure for monocyte-endothelial cell fusion, could be detected after co-cultivation from 30 min onwards. Virus transmission was confirmed by the appearance of plaques. Adhesion of immune-masked PRV-infected monocytes to endothelial cells was mediated by cellular adhesion complex CD11b-CD18 and subsequent fusion was mediated by the virus. In conclusion, immune-masked PRV-infected monocytes can adhere and subsequently transmit virus to endothelial cells in the presence of PRV-neutralizing antibodies.

Pseudorabies virus is transmitted among vaccinated conventional pigs, but not among vaccinated SPF pigs

Whereas the reproduction ratio ( R) of pseudorabies virus (PRV) in vaccinated specific pathogen free (SPF) pigs without maternally derived antibodies under experimental conditions has repeatedly been shown to be significantly below 1, R in vaccinated conventional pigs in the field with maternally derived antibodies was significantly above 1. To exclude the difference in husbandry conditions as a cause for this discrepancy, we quantified and compared the transmission of PRV in both groups under identical experimental conditions. Whereas none of the SPF sentinel pigs became infected ( R=0, significantly<1), all conventional sentinel pigs did become infected ( R=2.5, significantly>1). Moreover, only one SPF pigs shed virus in saliva, the mean cumulative titre being almost a 100-fold less than in conventional pigs (17 pigs, P=0.003). In addition, the mean proliferation of peripheral blood lymphocytes in response to PRV antigens was significantly higher in SPF pigs than in conventional pigs at all points studied ( P<0.0001). Moreover, the virus-neutralising antibody titre after vaccination was significantly higher in SPF pigs than in conventional pigs. We conclude that the discrepancy in transmission between vaccinated SPF pigs and vaccinated conventional pigs cannot be attributed to the experimental conditions.

Venereal transmission of pseudorabies viruses indigenous to feral swine.

Between 1995 and 1998, we designed a series of studies in which we attempted to determine the main routes of transmission involved in the natural infection of pseudorabies virus (PRV) indigenous to free-ranging feral swine (Sus scrofa). Naturally infected feral sows transmitted the infection to uninfected feral boars, with which they had been commingled for a 6-wk period. Pseudorabies virus was isolated from boar preputial swabs, but not from nasal swabs. Three of the same PRV-infected feral sows did not transmit the infection to domestic boars during a 16 wk commingling period, despite the fact that they became pregnant. Feral boars, naturally infected with PRV transmitted the virus to domestic gilts while penned together during 6 wk. Pseudorabies virus was isolated from vaginal swabs, but not from nasal swabs of gilts, after 2 and 3 wk of commingling. When the same infected boars were commingled with either feral or domestic boars for 13 wk, PRV transmission did not occur. None of the exposed boars developed neutralizing antibodies or yielded virus from their preputial or nasal swabs. Our results indicate that PRV indigenous to feral swine is preferentially transmitted to feral or domestic swine of the opposite sex by the venereal route. This mode of transmission differs from that seen in the natural transmission of PRV prevalent in domestic swine, where contaminated secretions, excretions and aerosols are responsible for the spread of the virus. Based on these results, we feel that as long as feral swine do not come into direct contact with domestic swine, PRV-infected feral swine probably pose only a limited risk to the success of the National Pseudorabies Eradication Program. The fact that PRV is usually transmitted from feral to domestic swine at the time of mating would indicate that the isolation of domestic herds by the use of a "double fence," should be adequate protection against reinfection with PRV.

Genital infection and transmission of pseudorabies virus in feral swine in Florida, USA

Seventeen feral swine (FS) naturally infected with pseudorabies virus (PRV) and treated with dexamethasone (4 mg/kg body wt) on five consecutive days shed virus primarily from the genital tract and less frequently from the upper respiratory tract. The FS isolates were identified as PRV by virus neutralization with specific polyclonal antiserum and by direct immunofluorescence. Restriction endonuclease analysis with BamHI showed that representative samples from a total of 62 isolates were identical to each other, but differed in at least 5 DNA bands from the PRV Shope reference strain profile. DNA purified from FS isolates propagated in Vero cells or DNA extracted directly from genital swabs were amplified in the polymerase chain reaction using primers specific for the gpII (gB) gene of PRV. This amplification yielded a product of the expected size (200 bp), which specifically hybridized to a digoxigenin-labelled 30-mer probe complementary to an area within the region defined by the primers. In a transmission experiment, PRV was recovered from the vagina at 1 and 6 weeks after uninfected feral gilts were mixed with infected feral boars. PRV was not isolated from the upper respiratory tract of either gilts or boars. At eight weeks, 4 of the 5 gilts had developed low titer neutralizing antibodies to PRV. Our results indicate that PRV in FS is transmitted through sexual contact.

The influence of maternal immunity on the transmission of pseudorabies virus and on the effectiveness of vaccination

The purpose of this study was to investigate whether maternal immunity could prevent transmission of pseudorabies virus (PRV) among pigs, and whether it reduced the effectiveness of a single or double vaccination with regard to the transmission of PRV. In five experiments, the transmission of PRV, expressed as the reproduction ratio R, was compared in groups of pigs with maternal immunity and in groups of pigs without maternal immunity. Transmission of PRV among unvaccinated pigs with maternal immunity ( R=0.2) was significantly lower than among pigs without maternal immunity ( R=6.3). Furthermore, maternal immunity in young pigs prevented transmission of PRV, as R was significantly below one. In once-vaccinated groups, PRV spread extensively among pigs with maternal immunity ( R=23), but did not spread extensively among pigs without maternal immunity ( R=0.6). In twice-vaccinated groups, transmission of PRV among pigs with maternal immunity ( R=0.6) did not differ significantly from the transmission of PRV among pigs without maternal immunity ( R=0.3). Thus, a single vaccination of pigs with PRV strain 783 at 10 weeks of age, when they still possessed maternal immunity, seemed not sufficient to prevent transmission of PRV. Virus transmission could be reduced, however, if maternally immune pigs were vaccinated twice at 10 and 14 weeks of age.

Comparison of two pseudorabies virus vaccines, that differ in capacity to reduce virus excretion after a challenge infection, in their capacity of reducing transmission of pseudorabies virus

Pseudorabies virus (PRV) vaccines are often compared for their capacity to reduce virus excretion after a challenge infection. Vaccines, used for the eradication of PRV, however, should reduce transmission of PRV among pigs. The purpose of this study was to investigate whether the amount of virus excreted after a challenge infection is an accurate measure of the capacity of a vaccine to reduce transmission of PRV among pigs. Two experiments were carried out, each using two groups of 10 pigs. The pigs in group one were intramuscularly vaccinated once with the glycoprotein E (gE)-negative vaccine X, the pigs in group two with the gE-negative strain 783. Eight weeks later, 5 pigs in each group were inoculated with wild-type PRV. A gE-ELISA was used to detect PRV infection. The transmission of PRV was estimated from the number of contact infections and expressed as the reproduction ratio R. The inoculated pigs vaccinated with vaccine X shed significantly more virus than the inoculated pigs vaccinated with strain 783. However, despite the difference in virus excretion, the transmission of PRV between the two groups did not differ. We conclude that virus excretion is not an accurate measure for determining vaccine effectiveness. However, R of vaccine X ( R = 0.98) was not significantly below one, whereas R of vaccine 783 ( R = 0) was significantly below one. Consequently, we cannot exclude the possibility that major outbreaks of PRV occur among pigs vaccinated with vaccine X.

A retrospective study into characteristics associated with the seroprevalence of pseudorabies virus-infected breeding pigs in vaccinated herds in the southern Netherlands

Vaccination programs to eradicate pseudorabies virus (PRV) are being considered in several countries. Knowledge of factors that influence PRV transmission within vaccinated breeding herds may contribute to the success of these programs. A multivariate analysis of variance of the PRV-seroprevalence in sows in 209 herds (average seroprevalence 67.0% per herd) in the southern Netherlands revealed the following risk indicators: (1) presence of finishing pigs; (2) production type (producers of finishing piglets had a higher seroprevalence than producers of breeding stock); (3) vaccination of the sows during nursing (in comparison with vaccinating all sows simultaneously at 5 month intervals, or vaccination during the second half of gestation); (4) pig density in the municipality where the herd was located (seroprevalence increased with higher pig density); (5) herd size less than 100 sows; (6) average within-herd parity (seroprevalence increased with higher withinherd parity); (7) replacement pigs raised on the premises; (8) vaccine strain administered to the sows. Purchase policy (breeding pigs purchased between 10 weeks and 7 months of age, or use of home-bred gilts only) did not significantly contribute to the multivariate model.

Predicting the number of herds infected with pseudorabies virus in the United States

Summary Epidemiologic modeling of the likely herd-to-herd transmission of pseudorabies virus (prv) was developed to assess the progress and potential for the prv-eradication program in the United States. The herd-to-herd transmission of prv over a 20-year period (1993 to 2012) in the United States was simulated under various scenarios, which included variable program-funding levels and variable prevalences. A transition model (Markov process model) was used to predict yearly changes in herd prevalence of prv infection. Five mutually exclusive states of nature for herds were assumed: uninfected and not vaccinated; uninfected and vaccinated; known to be infected and not vaccinated; known to be infected and vaccinated; and infected, but not known to be infected. Three prevalences for states in the United States were assumed: higher prevalence, moderate prevalence, and lower prevalence. Three funding levels were assumed: no eradication program, continued funding at the current level, and increased funding of 25%. Estimates made by an expert panel for determining probabilities in the state-transition matrices were used. A model also was developed, and was considered to be the most optimistic scenario likely under increased funding of 25%. The most optimistic estimates of the probabilities that still lay within the range of estimates made by the expert panel were used for this model. Only the optimistic transmission matrices allowed for total eradication of prv. Using the optimistic matrices, all states in the United States of America had moved into the moderate- or low-level risk status by the year 2000. The longest time taken to achieve eradication was for the state of Iowa, where eradication was not achieved until 2012.

Prevalence and Transmission of Pseudorabies Virus in an Isolated Population of Feral Swine

Six hundred sixty-one feral swine (Sus scrofa) from Ossabaw Island, Georgia (USA) were captured, bled, and their sera tested for pseudorabies virus (PRV) antibody during a 6 yr period. Prevalence of seroconversion in females was somewhat higher than in males (10% versus 7%), but the difference was not statistically significant. Adults had a significantly higher prevalence than juveniles (29% versus 1%). An important finding in this study was that seroconversion occurred primarily in the adult feral swine.