The antiviral activity of kaempferol against pseudorabies virus in mice.

BACKGROUND: Pseudorabies virus (PRV), a member of the Alphaherpesviruses, is one of the most important pathogens that harm the global pig industry. Accumulated evidence indicated that PRV could infect humans under certain circumstances, inducing severe clinical symptoms such as acute human encephalitis. Currently, there are no antiviral drugs to treat PRV infections, and vaccines available only for swine could not provide full protection. Thus, new control measures are urgently needed. RESULTS: In the present study, kaempferol exhibited anti-PRV activity in mice through improving survival rate by 22.22 %, which was higher than acyclovir (Positive control) with the survival rate of 16.67 % at 6 days post infection (dpi); meanwhile, the survival rate was 0 % at 6 dpi in the infected-untreated group. Kaempferol could inhibit the virus replication in the brain, lung, kidney, heart and spleen, especially the viral gene copies were reduced by over 700-fold in the brain, which was further confirmed by immunohistochemical examination. The pathogenic changes induced by PRV infection in these organs were also alleviated. The transcription of the only immediate-early gene IE180 in the brain was significantly inhibited by kaempferol, leading to the decreased transcriptional levels of the early genes (EPO and TK). The expression of latency-associated transcript (LAT) was also inhibited in the brain, which suggested that kaempferol could inhibit PRV latency. Kaempferol-treatment could induce higher levels of IL-1ß, IL-4, IL-6, TNF-α and IFN-γ in the serum at 3 dpi which were then declined to normal levels at 5 dpi. CONCLUSIONS: These results suggested that kaempferol was expected to be a new alternative control measure for PRV infection.

Fusion of pseudorabies virus glycoproteins to IgG Fc enhances protective immunity against pseudorabies virus.

Molecular adjuvants are vaccine delivery vehicle to increase specific antigens effectiveness. Herein, we concentrated on IgG Fc, an effective molecular adjuvant, to develop novel pseudorabies virus (PRV) subunit vaccines. Two major protective antigen genes of PRV were constructed and linked into the mouse IgG Fc fragment. The gD, gD-IgG2aFc, gB and gB-IgG2aFc proteins were expressed using a baculovirus system. Mice intranasally immunized with gD-IgG2aFc or gB-IgG2aFc subunit vaccine exhibited significantly higher PRV-specific antibodies, neutralizing antibodies and intracellular cytokines than the mice intranasally immunized with gD or gB subunit vaccine. Moreover, no histopathological lesions were observed in mice immunized with gB-IgG2aFc subunit vaccine via histopathology examination. Further, the gB-IgG2aFc subunit vaccine was efficient for PRV infection compared with live attenuated vaccine. Overall, these results suggest that IgG2a Fc fragment, as a potential molecular adjuvant, fused with PRV antigen might be a promising and efficient PRV vaccine candidate.

Virulent Pseudorabies Virus Infection Induces a Specific and Lethal Systemic Inflammatory Response in Mice.

Pseudorabies virus (PRV) is an alphaherpesvirus that infects the peripheral nervous system (PNS). The natural host of PRV is the swine, but it can infect most mammals, including cattle, rodents, and dogs. In these nonnatural hosts, PRV always causes a severe acute and lethal neuropathy called the "mad itch," which is uncommon in swine. Thus far, the pathophysiological and immunological processes leading to the development of the neuropathic itch and the death of the animal are unclear. Using a footpad inoculation model, we established that mice inoculated with PRV-Becker (virulent strain) develop a severe pruritus in the foot and become moribund at 82 h postinoculation (hpi). We found necrosis and inflammation with a massive neutrophil infiltration only in the footpad and dorsal root ganglia (DRGs) by hematoxylin and eosin staining. PRV load was detected in the foot, PNS, and central nervous system tissues by quantitative reverse transcription-PCR. Infected mice had elevated plasma levels of proinflammatory cytokines (interleukin-6 [IL-6] and granulocyte colony-stimulating factor [G-CSF]) and chemokines (Gro-1 and monocyte chemoattractant protein 1). Significant IL-6 and G-CSF levels were detected in several tissues at 82 hpi. High plasma levels of C-reactive protein confirmed the acute inflammatory response to PRV-Becker infection. Moreover, mice inoculated with PRV-Bartha (attenuated, live vaccine strain) did not develop pruritus at 82 hpi. PRV-Bartha also replicated in the PNS, and the infection spread further in the brain than PRV-Becker. PRV-Bartha infection did not induce the specific and lethal systemic inflammatory response seen with PRV-Becker. Overall, we demonstrated the importance of inflammation in the clinical outcome of PRV infection in mice and provide new insights into the process of PRV-induced neuroinflammation.IMPORTANCE Pseudorabies virus (PRV) is an alphaherpesvirus related to human pathogens such as herpes simplex virus 1 and varicella-zoster virus (VZV). The natural host of PRV is the swine, but it can infect most mammals. In susceptible animals other than pigs, PRV infection always causes a characteristic lethal pruritus known as the "mad itch." The role of the immune response in the clinical outcome of PRV infection is still poorly understood. Here, we show that a systemic host inflammatory response is responsible for the severe pruritus and acute death of mice infected with virulent PRV-Becker but not mice infected with attenuated strain PRV-Bartha. In addition, we identified IL-6 and G-CSF as two main cytokines that play crucial roles in the regulation of this process. Our findings give new insights into neuroinflammatory diseases and strengthen further the similarities between VZV and PRV infections at the level of innate immunity.

Antiviral Effect of Resveratrol in Piglets Infected with Virulent Pseudorabies Virus.

Pseudorabies virus (PRV) is one of the most important pathogens of swine, resulting in devastating disease and economic losses worldwide. Nevertheless, there are currently no antiviral drugs available for PRV infection. Resveratrol (Res) was identified to exert its antiviral activity by inhibiting the PRV replication in preliminary investigations. In our previous study, we found that Res has anti-PRV activity in vitro. Here, we show that Res can effectively reduce the mortality and increase the growth performance of PRV-infected piglets. After Res treatment, the viral loads significantly (p < 0.001) decreased. Pathological symptoms, particularly inflammation in the brain caused by PRV infection, were significantly (p < 0.001) relieved by the effects of Res. In Res-treated groups, higher levels of cytokines in serum, including interferon gama, interleukin 12, tumor necrosis factor-alpha and interferon alpha were observed at 7 days post infection. These results indicated that Res possesses potent inhibitory activity against PRV-infection through inhibiting viral reproduction, alleviating PRV-induced inflammation and enhancing animal immunity, suggesting that Res is expected to be a new alternative control measure for PRV infection.

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.

Variations in glycoprotein B contribute to immunogenic difference between PRV variant JS-2012 and Bartha-K61.

A newly emerged pseudorabies virus (PRV) variant has been identified in many Bartha-K61-vaccinated pig farms. This variant has caused great economic losses to the swine industry in China since 2011. Sequence analysis demonstrated that the gB gene of the emerging PRV variant JS-2012 had multiple variations compared with the vaccine strain Bartha-K61. In the study, a specific CRISPR/Cas9 system combined with homologous recombination was used to construct two recombinant viruses, BJB (Bartha-K61+JS-2012gB) and JBJ (JS-2012-ΔgE/gI+Bartha-K61gB), by interchanging the full-length gB genes between Bartha-K61 and JS-2012-ΔgE/gI. The two recombinant viruses showed similar characteristics in growth kinetics in vitro and similar pathogenicity in mice, as compared to their parental strains. Immunization of mice with inactivated BJB or JBJ followed by challenge of JS-2012 showed that BJB could increase protective efficacy to 80%, compared to only 40% protection by the parental Bartha-K61 strain. JBJ had a decreased protective efficacy of 65%, as compared to 90% protection by its parental JS-2012-ΔgE/gI strain. Exchange of the gB gene markedly altered the immunogenicity of the recombinant PRV. These data suggest that variations in gB might play an important role in the virulence of the reemergent PRV variant in China. Our results demonstrate the importance of gB in protective immunity and suggest that the recombinant virus BJB could be a promising vaccine candidate for eradication of the PRV variant.

Bartha-k61 vaccine protects growing pigs against challenge with an emerging variant pseudorabies virus.

Since late 2011, pseudorabies (PR) has resurfaced in many large pig farms, causing great economic loss for the swine industry in China. The PRV variant strain with high virulence and antigenic variation has been considered to be the main cause, and much attention has been focused on how to prevent and control the reoccurrence of this disease in China. In this study, two kinds of vaccination strategy were employed to evaluate the protective effects of Bartha-k61vaccine against both variant PRV (XJ5) and classical PRV (Ra) strain challenge. Humoral immunity response, clinical signs, survival rate, body weight, virus shedding and pathology were assessed in commercial pigs. The results showed that Bartha-k61vaccine, administered either once or twice, was effective against the PRV variant (XJ5) challenge, while no significant differences were observed between single and prime-boost vaccinated pigs. However, pigs vaccinated twice had better body weight gains than those vaccinated once, following challenge with the classical PRV strain (Ra) (p<0.01). Therefore, the Bartha-k61 vaccine appears to be an effective vaccine to control the spread of PRV variants in China in the absence of new powerful candidate vaccines specific to these PRV strains.

Emergence of virulent pseudorabies virus infection in northern China.

Our investigation was conducted in order to verify a recent severe epidemic at several swine farms in northern China that indicated a newly emerging disease. Evidence confirmed that the epidemic was caused by a virulent Pseudorabies virus infection in swine herds.

Oral immunization of wild boar and domestic pigs with attenuated live vaccine protects against Pseudorabies virus infection.

In domestic pigs strict control measures and the use of gene-deleted marker vaccines resulted in the elimination of pseudorabies virus (PrV) infections in many parts of Europe and North America. In free-roaming feral pigs and wild boar populations, however, serological surveys and monitoring in The Americas, Europe and North Africa provided serological and virological evidence that PrV is more widely distributed than previously assumed. Thus, there is a constant risk of spillover of PrV infection from wild pig populations to domestic animals which could require intervention to limit the infection in wild pigs. To investigate whether oral immunization of wild boar by live-attenuated PrV could be an option, wild boar and domestic pigs were orally immunized with 2×10(6) TCID(50) of the attenuated live PrV vaccine strain Bartha supplied either with a syringe or within a blister, and subsequently intranasally challenged with 10(6) TCID(50) of the highly virulent PrV strain NIA-3. Oral immunization with live-attenuated PrV was able to confer protection against clinical signs in wild boar and against transmission of challenge virus to naïve contact animals. Only two vaccinated domestic pigs developed neurological signs after challenge infection. Our results demonstrate that oral immunization against PrV infection in wild boar is possible. In case increasing PrV infection rates in wild boar may enhance the risk for spillover into domestic pig populations, oral immunization of wild boar against PrV in endemic areas might be a feasible control strategy.

Antiviral effects of a transgenic RNA-dependent RNA polymerase.

Transgenic expression of the RNA-dependent RNA polymerase 3D(pol) inhibited infection of Theiler's murine encephalitis virus (TMEV), a picornavirus from which it was derived. Here, we infected 3D(pol) transgenic mice with another picornavirus, as well as an alphaherpesvirus and a rhabdovirus. 3D(pol) transgenic FVB mice had significantly lower viral loads and survived longer after infection with all three types of viruses than nontransgenic FVB mice. Viral inhibition among three different types of virus by transgenic 3D(pol) suggests that the mechanism of action is not the direct interference with picornaviral 3D(pol) but instead may be the changing of host cells to an antiviral state before or after viral infection occurs, as basal interferon levels were higher in 3D(pol) transgenic mice before infection. Further study of this mechanism may open new possibilities for future antiviral therapy.

Effects of vitamin E on reproductive protection in pregnant mice infected with pseudorabies virus (PRV) via regulating expression of Toll-like receptors (TLRs) and cytokine balance.

Vitamin E supplement and pseudorabies virus (PRV) infection have a reciprocal role in influencing the maternal immune response, a key determinant of the success or failure of pregnancy. However, it remains unknown whether vitamin E supplement provides protection against PRV-induced failure of pregnancy. This study was therefore conducted to investigate the effect of dietary vitamin E level (0, 75, 375, 750 and 1,500 mg/kg) on the reproduction performance, immunity and expression of Toll-like receptors (TLRs) of PRV-challenged mice. The mortality and abortion rate of PRV-challenged mice decreased with the increase in vitamin E consumption. Overall, PBS-injected mice had a higher live embryo number and live litter size than PRV-challenged mice. Both live embryo number and live litter size of PRV-challenged mice increased with increasing vitamin E levels. Vitamin E supplement resulted in decreased concentration of serum IL-2 and IFN-γ, but increased concentration of serum IL-10. The concentration of serum IgG, IgA and IgM increased with increasing vitamin E levels. In the uterine and embryo mRNA abundance of TLR3, TLR7 and TLR9 was higher in PRV-challenged mice than that in PBS-injected mice fed on the same dosage of vitamin E. The mRNA abundance of embryonic TLR3, TLR7 and TLR9 in PRV-challenged mice decreased with increasing vitamin E levels. Collectively, vitamin E supplement may improve reproductive performance of PRV-challenged mice by attenuating PRV-induced negative effects on the cytokine profile, immunoglobulin synthesis and TLR expression.

Identification of functional domains within the essential large tegument protein pUL36 of pseudorabies virus.

Proteins of the capsid proximal tegument are involved in the transport of incoming capsids to the nucleus and secondary envelopment after nuclear egress. Homologs of the essential large capsid proximal tegument protein pUL36 are conserved within the Herpesviridae. They interact with another tegument component, pUL37, and contain a deubiquitinating activity in their N termini which, however, is not essential for virus replication. Whereas an internal deletion of 709 amino acids (aa) within the C-terminal half of the alphaherpesvirus pseudorabies virus (PrV) pUL36 does not impair its function (S. Böttcher, B. G. Klupp, H. Granzow, W. Fuchs, K. Michael, and T. C. Mettenleiter, J. Virol. 80:9910-9915, 2006), deletion of the very C terminus does (J. Lee, G. Luxton, and G. A. Smith, J. Virol. 80:12086-12094, 2006). For further characterization we deleted several predicted functional and structural motifs within PrV pUL36 and analyzed the resulting phenotypes in cell culture and a mouse infection model. Extension of the internal deletion to encompass aa 2087 to 2981 exerted only minor effects on virus replication but resulted in prolonged mean survival times of infected mice. Any additional extension did not yield viable virus. Deletion of an N-terminal region containing the deubiquitinating activity (aa 22 to 248) only slightly impaired viral replication in cell culture but slowed neuroinvasion in our mouse model, whereas a strong impairment of viral replication was observed after simultaneous removal of both nonessential domains. Absence of a region containing two predicted leucine zipper motifs (aa 748 to 991) also strongly impaired virus replication and spread. Thus, we identify several domains within the PrV UL36 protein, which, though not essential, are nevertheless important for virus replication.

Efficacy of intracerebral immunization against pseudorabies virus in mice.

To evaluate the efficacy of intracerebral (IC) immunization, mice were immunized with formalin-inactivated pseudorabies virus (PRV) by either subcutaneous (SC) or IC injection, and then 10(6) plaque-forming units of PRV were introduced into the hindleg of the immunized or non-immunized mice by intramuscular injection. The antibody titer in serum was elevated and boosted by additional immunization via both the SC and IC routes, but was higher after IC immunization. Intracerebrally immunized mice were completely protected from mortality and neurological signs, whereas all the non-immunized and 80% of the subcutaneously immunized mice died after developing neurological signs. In mouse models, IC immunization is more effective at inducing a protective immune response against the transneural spread of PRV than SC immunization.

Epidemiological survey of viral diseases of pigs in the Mekong delta of Vietnam between 1999 and 2003.

In the Mekong delta, backyard pig rearing plays an integral role in recycling nutrients in farming systems and generating valuable cash income. However, development has been hampered by fatal epizootics of piglets and reproductive failure of sows. Diseases are named by symptoms and blindly treated with antibiotics. As antibiotics are often ineffectual, involvement of viral diseases are suspected. To identify the causative agent, we first sero-surveyed porcine reproductive and respiratory syndrome (PRRS) and pseudorabies with 478 sera from non-vaccinated pigs collected from backyard farms, state farms and slaughterhouses in Can Tho province between 1999 and 2002. Antibodies for PRRS were first detected in 2002 in backyard farms and at high prevalence in state farms with increased piglet mortality. A few backyard breeder pigs had antibodies for pseudorabies in 2000 and 2002. With compulsory classical swine fever (CSF) vaccination, we examined the relationship between vaccination and antibodies in 70 serum samples. Seventy-nine percent of vaccinated breeders had CSF antibodies-higher than expected with irregular vaccination. Since circulation of CSF virus was suspected, isolation was attempted at 10 farms with fatal epizootics between 2002 and 2003. The viruses were detected at all farms and clustered within genogroup 2, despite vaccines corresponding to genogroup 1. This study demonstrated virologically/serologically the existence of PRRS, pseudorabies and CSF viruses in the Mekong delta of Vietnam. We also identified CSF as a cause of piglet mortality that disastrously affected backyard farming. Vaccine standardization and proper instructions are needed to simplify diagnosis and complement established simultaneous vaccination of sows with piglets.

Influence of tegument proteins of pseudorabies virus on neuroinvasion and transneuronal spread in the nervous system of adult mice after intranasal inoculation.

Pseudorabies virus (PrV) is a neurotropic alphaherpesvirus that, after intranasal infection of adult mice, enters peripheral neurons and propagates to the central nervous system. In recent years we have analyzed the contribution of virus-encoded glycoproteins to neuroinvasion and transneuronal spread (reviewed in T. C. Mettenleiter, Virus Res. 92:197-206, 2003). We now extend our studies to analyze the role of tegument proteins in these processes. To this end, PrV mutants unable to express the UL11, UL37, UL46, UL47, and UL48 tegument proteins, as well as the corresponding rescued viruses, were intranasally instilled into 6- to 8-week-old CD1 strain mice. First, mean survival times were determined which showed that mice infected with the UL46 deletion mutant succumbed to the disease as early as wild-type PrV-infected animals. Survival times increased in the order: PrV-DeltaUL47-, PrV-DeltaUL11-, and PrV-DeltaUL48-infected animals, a finding which parallels the growth phenotype of these viruses in cell culture. In contrast, none of the PrV-DeltaUL37-infected animals died. Upon closer histological examination, all viruses except PrV-DeltaUL37 were able to infect the nasal cavity and propagate to first- and second-order neurons as shown by two-color immunofluorescence. However, neuroinvasion was delayed in PrV-DeltaUL47, PrV-DeltaUL11, and PrV-DeltaUL48, a finding that correlated with the extended survival times. Surprisingly, whereas PrV-DeltaUL48 and PrV-DeltaUL37 replicated to similar titers in cell culture which were approximately 500-fold lower than those of wild-type virus, after intranasal infection of mice PrV-DeltaUL48 was able to infect areas of the brain like wild-type PrV, although only after a considerably longer time period. In contrast, PrV-DeltaUL37 was not able to enter neurons and was restricted to the infection of single cells in the nasal respiratory epithelium. Thus, our data demonstrate the importance of herpesviral tegument proteins in neuronal infection and show a different contribution of tegument proteins to the neuroinvasion phenotype of a neurotropic alphaherpesvirus.

A study of the progress of the Aujeszky's disease control programme in Italy using survival analysis.

A 3-year study (1997-2000) was performed on 294 swine herds from Italy, where a National Programme of Control of Aujeszky's Disease (AD) based on compulsory vaccination has been operative since 1997. Aim of the study was to evaluate the progress of this control programme using a survival approach applied to gE-seropositive herds at the beginning of the programme. The cumulative proportion of herds still gE-seropositive at the end of the study was 0.57. No significant difference in the probability of becoming gE-seronegative during the study period was found between herds of different type (breeding versus farrow-to-finish) whereas significant differences were seen between herds from different areas. The Cox's proportional hazards regression, performed on data from 79 herds, showed that the only risk factor significantly associated with a higher probability of becoming gE-seronegative is again the geographical location. Other risk factors considered in the analysis were: type of enterprise, type of replacement of animals, herd size, pig and pig herds densities around the farm, distance from the nearest pig herd and year of beginning of the vaccination with a gE-deleted vaccine.

Natural Aujeszky's disease in a Spanish wild boar population.

We describe an outbreak of Aujeszky's disease (AD) in a wild boar (Sus scrofa) population from central Spain. Mortality was estimated to be at least 14% (14/100) in juveniles and 7.5% (3/40) in adults. Most of the affected animals (12/17) were between 4 and 8 months of age. Gross lesions mainly consisted of enlarged and congestive tonsils and lymph nodes, petechial hemorrhages on the small intestine, and engorged blood vessels in the brain and meninges. Histopathology revealed mild nonsuppurative meningoencephalitis. Positivity to the fluorescent antibody test was found in tissues from the affected animals. Seroprevalence of antibodies to AD virus (ADV) was 56% (9/16). To our knowledge, this is the first description of clinical cases in a wild suid population.

Vaccination of pigs with a recombinant porcine adenovirus expressing the gD gene from pseudorabies virus.

Five week old, commercially available large white pigs were vaccinated with either a single dose or two doses of a recombinant porcine adenovirus expressing the glycoprotein D gene from pseudorabies virus (PRV). Pigs were monitored for the development of serum neutralizing antibodies to PRV and challenged 3 weeks after final vaccination. Prior to challenge, pigs given 2 doses of the vaccine demonstrated boosted levels of antibody compared with those given a single dose, and all surviving pigs had increased neutralization titres over pre-challenge levels. Following challenge, pigs were monitored for clinical signs of disease, with blood and nasal swabs collected for virus isolation. All control animals became sick with elevated temperatures for 6 days post challenge, whereas; vaccinated animals displayed an increase in body temperature for only 2-3 days. Control pigs and those given a single dose all lost condition, but the group given 2 doses remained healthy. At postmortem, gross lesions of pneumonia only occurred in control animals and those given a single dose of vaccine. Histology carried out on the brains of all animals demonstrated a difference in severity of infection and frequency of immunohistochemical antigen detection between test animals, with control and single dose groups being most severely affected and pigs given 2 doses the least. Virus isolation studies demonstrated that no viraemia could be detected, but virus was found in nasal swabs from some animals in both groups of vaccinates following challenge.