Production and immunogenicity of chimeric virus-like particles containing porcine reproductive and respiratory syndrome virus GP5 protein

Porcine reproductive and respiratory syndrome virus (PRRSV) poses a severe threat in swine industry and causes heavy economic losses worldwide. Currently, the available vaccines are the inactivated and attenuated virus vaccines, but the use of PRRSV in their production raises the issue of safety. We developed a chimeric virus-like particles (VLPs) vaccine candidate for PRRSV protection. The chimeric VLPs was composed of M1 protein from H1N1 influenza virus and a fusion protein, denoted as NA/GP5, containing the cytoplasmic and transmembrane domains of H1N1 virus NA protein and PRRSV GP5 protein. Vaccination of BALB/c mice with 10 μg of chimeirc VLPs by intramuscular immunization stimulated antibody responses to GP5 protein, and induced cellular immune response. The data suggested that the chimeric VLP vaccine candidate may provide a new strategy for further development of vaccines against PRRSV infection.     

Enhancing heterologous protection in pigs vaccinated with chimeric porcine reproductive and respiratory syndrome virus containing the full-length sequences of shuffled structural genes of multiple heterologous strains

Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of arguably the most economically important global swine disease. The extensive genetic variation of PRRSV strains is a major obstacle for heterologous protection of current vaccines. Previously, we constructed a panel of chimeric viruses containing only the ectodomain sequences of DNA-shuffled structural genes of different PRRSV strains in the backbone of a commercial vaccine, and found that one chimeric virus had an improved cross-protection efficacy. In this present study, to further enhance the cross-protective efficacy against heterologous strains, we constructed a novel chimeric virus VR2385-S3456 containing the full-length sequences of shuffled structural genes (ORFs 3-6) from 6 heterologous PRRSV strains in the backbone of PRRSV strain VR2385. We showed that the chimeric virus VR2385-S3456 induced a high level of neutralizing antibodies in pigs against two heterologous strains. A subsequent vaccination and challenge study in 48 pigs revealed that the chimeric virus VR2385-S3456 conferred an enhanced cross-protection when challenged with heterologous virus strain NADC20 or a contemporary heterologous strain RFLP 1-7-4. The results suggest that the chimera VR2385-S3456 may be a good PRRSV vaccine candidate for further development to confer heterologous protection.     

Negative impact of porcine reproductive and respiratory syndrome virus infection on the efficacy of classical swine fever vaccine

Recent findings suggest that porcine reproductive and respiratory syndrome virus (PRRSV) possesses immunomodulatory properties. To investigate the effect of PRRSV infection on classical swine fever (CSF) vaccine efficacy, 17-day-old pigs were divided into five groups. The experimental group was infected with a Thai PRRSV (US genotype) a week before CSF vaccination and challenged with a virulent CSF virus (CSFV) 3 weeks following vaccination. The control groups received no PRRSV infection, no CSF vaccination, no CSF challenge, or in combination were included. The results demonstrated that PRRSV infection significantly inhibited host immune response that resulted in vaccination failure in the subsequent CSFV exposure. Following CSF challenge, the PRRSV-infected, vaccinated pigs exhibited clinical, virological and pathological features resembled to those of the non-vaccinated groups. The findings indicated that CSF immunization during an acute phase of PRRSV infection could result in vaccination failure.     

Development of a broadly protective modified-live virus vaccine candidate against porcine reproductive and respiratory syndrome virus

Modified-live virus (MLV) vaccines are widely used to protect pigs against porcine reproductive and respiratory syndrome virus (PRRSV). However, current MLV vaccines do not confer adequate levels of heterologous protection, presumably due to the substantial genetic diversity of PRRSV isolates circulating in the field. To overcome this genetic variation challenge, we recently generated a synthetic PRRSV strain containing a consensus genomic sequence of PRRSV-2. We demonstrated that our synthetic PRRSV strain confers unprecedented levels of heterologous protection. However, the synthetic PRRSV strain at passage 1 (hereafter designated CON-P1) is highly virulent and therefore, is not suitable to be used as a vaccine in pigs. In the present study, we attenuated CON-P1 by continuously passaging the virus in MARC-145 cells, a non-natural host cell line. Using a young pig model, we demonstrated that the synthetic virus at passages 90 and 122 (designated as CON-P90 and CON-P122, respectively) were fully attenuated, as evidenced by the significantly reduced viral loads in serum and tissues and the absence of lung lesion in the infected pigs. Most importantly, CON-P90 confers similar levels of heterologous protection as its parental strain CON-P1. Taken together, the results indicate that CON-P90 is an excellent candidate for the formulation of next generation of PRRSV MLV vaccines with improved levels of heterologous protection.     

Inhibition of porcine reproductive and respiratory syndrome virus by Cecropin D in vitro

Porcine reproductive and respiratory syndrome virus (PRRSV) continues to cause substantial economic losses to the pig industry worldwide. Although vaccines are commercially available for the control of PRRSV infection, no vaccination regimen has been proved sustained success in terms of generating a protective immune response. Therefore, the development of novel antivirals is urgently needed. Antimicrobial peptides display broad-spectrum antimicrobial activities against bacteria, fungi, and viruses and play an important role in host innate immune response. Here, we tested whether Cecropin D (CD) could inhibit PRRSV infection and replication in vitro. The inhibitory effect of CD occurred during viral attachment and the early period of viral entry into Marc-145 cells. CD also attenuated virus-induced apoptosis during the late phase of PRRSV infection and suppressed virus release in Marc-145 cells, which might contribute to the inhibition of PRRSV infection. Similar inhibitory effects on PRRSV infection were also found with CD treatment in porcine alveolar macrophages, the major target cell type of PRRSV infection in pigs in vivo. These findings suggest that CD has the potential to develop a new therapeutic agent against PRRSV infection.     

Inactivated and subunit vaccines against porcine reproductive and respiratory syndrome: Current status and future direction

Within a few years of its emergence in the late 1980s, the PRRS virus had spread globally to become the foremost infectious disease concern for the pork industry. Since 1994, modified live-attenuated vaccines against porcine reproductive and respiratory syndrome virus (PRRSV-MLV) have been widely used, but have failed to provide complete protection against emerging and heterologous field strains of the virus. Moreover, like many other MLVs, PRRSV-MLVs have safety concerns including vertical and horizontal transmission of the vaccine virus and several documented incidences of reversion to virulence. Thus, the development of efficacious inactivated vaccines is warranted for the control and eradication of PRRS. Since the early 1990s, researchers have been attempting to develop inactivated PRRSV vaccines, but most of the candidates have failed to elicit protective immunity even against homologous virus challenge. Recent research findings relating to both inactivated and subunit candidate PRRSV vaccines have shown promise, but they need to be pursued further to improve their heterologous efficacy and cost-effectiveness before considering commercialization. In this comprehensive review, we provide information on attempts to develop PRRSV inactivated and subunit vaccines. These includes various virus inactivation strategies, adjuvants, nanoparticle-based vaccine delivery systems, DNA vaccines, and recombinant subunit vaccines produced using baculovirus, plant, and replication-deficient viruses as vector vaccines. Finally, future directions for the development of innovative non-infectious PRRSV vaccines are suggested. Undoubtedly there remains a need for novel PRRSV vaccine strategies targeted to deliver cross-protective, non-infectious vaccines for the control and eradication of PRRS.     

Porcine reproductive and respiratory syndrome in hybrid wild boars, china

We conducted a serologic investigation of porcine reproductive and respiratory syndrome virus (PRRSV) in hybrid wild boar herds in China during 2008-2009. PRRSV isolates with novel genetic markers were recovered. Experimental infection of pigs indicated that hybrid wild boars are involved in the epidemiology of PRRSV.     

Immunogenicity and protective efficacy of recombinant pseudorabies virus expressing the two major membrane-associated proteins of porcine reproductive and respiratory syndrome virus

Porcine reproductive and respiratory syndrome virus (PRRSV) infection still remains today as the most significant health threat to swine and poses a challenge to current vaccination strategies. To develop a new generation of vaccine against PRRSV, a live attenuated pseudorabies virus (PRV) was used as vaccine vector to express the two major membrane-associated proteins (GP5 or M) of PRRSV in various forms. Four PRV recombinants, rPRV-GP5 (expressing native GP5), rPRV-GP5m (expressing GP5m, a modified GP5), rPRV-GP5-M (co-expressing GP5 and M proteins), rPRV-GP5m-M (co-expressing GP5m and M proteins) were generated. Mouse immunized with all these recombinants developed comparable PRV-specific humoral immune responses and provided complete protection against a lethal PRV challenge. However, the highest level of PRRSV-specific neutralizing antibodies and lymphocyte proliferative responses was observed in mice immunized with rPRV-GP5m-M. The immunogenicity and protective efficiency of rPRV-GP5m-M were further evaluated in the piglets. Compared to commercial PRRSV killed vaccine, detectable PRRSV-specific neutralizing antibody and higher lymphocyte proliferative responses could be developed in piglets immunized with rPRV-GP5m-M before virus challenge. Furthermore, more efficient protection against a PRRSV challenge was obtained in piglets immunized with rPRV-GP5m-M, as showed by the balanced body-temperature fluctuation, shorter-term viremia, lower proportion of virus load in nasal and oropharyngeal scrapings and tissues, and milder lung lesions. These data indicate that the recombinant rPRV-GP5m-M is a promising candidate bivalent vaccine against both PRV and PRRSV infection.     

miRNAs and genes expression in MARC-145 cell in response to PRRSV infection

The regulation of viral replication is under control of miRNAs and their target genes. Several articles report the cross-talk between host and virus. The drastic effects of Porcine reproductive and respiratory syndrome virus (PRRSV) pressed us to investigate the expression profiling of miRNAs and immunity related genes during PRRSV infection. This was performed by qPCR in MARC145 cells during PRRSV infection. It was observed that miRNAs and genes show different expression patterns at different time points during PRRSV infection. The early infected stage was accompanied with increased expression of some miRNAs including miR-204, miR-21, miR-181a, miR-29 while a decrease was observed for the same in late infection stage. The opposite condition also existed in parallel. An interesting observation was seen when miR-145 was strongly induced by PRRSV infection, whereas miR-127 expression was significantly reduced in all infection points. Taken together, our studies have revealed that the expressions of miRNAs and immune-related genes were regulated in PRRSV infected MARC-145 cells and had important roles in the immune response, providing a basis for further investigations.     

A novel baculovirus vector shows efficient gene delivery of modified porcine reproductive and respiratory syndrome virus antigens and elicits specific immune response

Porcine reproductive and respiratory syndrome (PRRS) is an economically devastating epizootic of porcine species. Current vaccines are inadequate to control the disease burden and outbreaks in the field. We report a novel baculovirus vaccine vector with White spot syndrome virus immediate early 1 shuttle promoter, with strong activity in both insect cells and mammalian cells, for immunization against PRRSV. The insect cell cultured baculovirus vector produces PRRSV envelope glycoproteins ORF2a, ORF3, ORF4 and ORF5, which are similar to the antigens in the infectious PRRS virion, and these antigens are stably incorporated on the surface of the baculovirus. Further, the baculovirus vector efficiently transduces these antigens in cells of porcine origin, thereby simulating a live infection. The baculovirus vectored PRRSV antigens, upon inoculation in mice, elicits robust neutralizing antibodies against the infective PRRS virus. Further, the experiments indicate that hitherto under emphasized ORF2a and ORF4 are important target antigens for neutralizing PRRSV infectivity.     

Whole genome characterization of a novel porcine reproductive and respiratory syndrome virus 1 isolate: Genetic evidence for recombination between Amervac vaccine and circulating strains in mainland China

Genotype 1 porcine reproductive and respiratory syndrome virus (PRRSV 1) have been continuously isolated in China in recent years. Complete genome sequences of these isolates are important to investigate the prevalence and evolution of Chinese PRRSV 1. Herein, we describe the isolation of a novel PRRSV 1 isolate, denominated HLJB1, in the Heilongjiang province of China. Complete genome sequencing of HLJB1 showed that it shares 90.66% and 58.21% nucleotide identities with PRRSV 1 and 2 prototypic strains Lelystad virus and ATCC VR-2332, respectively. HLJB1 has a unique 5-amino-acid insertion in nsp2, which has never been described in other PRRSV 1 isolates. Whole genome-based phylogenetic analysis revealed that all Chinese PRRSV 1 isolates are clustered in pan-European subtype 1 and can be divided into four subgroups. HLJB1 resides in the subgroup of BJEU06-1-like isolates but is also closely related to the Amervac-like isolates. Additionally, recombination analyses suggested that HLJB1 is a recombinant from the Amervac vaccine and the BJEU06-1 isolate. To our best knowledge, our results provide the first genetic evidence for recombination between Amervac vaccine and circulating strains. These findings are also beneficial for studying the origin and evolution of PRRSV 1 in China.     

Interplay of autophagy and apoptosis during PRRSV infection of Marc145 cell

Autophagy and apoptosis play essential roles 'in virus infection. Our study was performed to investigate the interplay between autophagy and apoptosis in PRRSV replication. In our present study, autophagy and apoptosis were induced by PRRSV infection. Viral replication was dampened/attenuated by autophagy deficient and potentiated by apoptosis inhibition. Furthermore, PRRSV replication was restored by apoptosis inhibition in autophagy deficient cells. Taken together, our findings unveil the functional relationship between autophagy and apoptosis during PRRSV replication.     

Immunogenicity study of plant-made oral subunit vaccine against porcine reproductive and respiratory syndrome virus (PRRSV)

Currently, killed-virus and modified-live PRRSV vaccines are used to control porcine reproductive and respiratory syndrome disease (PRRS). However, very limited efficacy of killed-virus vaccines and serious safety concerns for modified-live virus vaccines demand the development of novel PRRSV vaccines. In this report, we investigated the possibility of using transgenic plants as a cost-effective and scalable system for production and delivery of a viral protein as an oral subunit vaccine against PRRSV. Corn calli were genetically engineered to produce PRRSV viral envelope-associated M protein. Both serum and intestine mucosal antigen-specific antibodies were induced by oral administration of the transgenic plant tissues to mice. In addition, serum and mucosal antibodies showed virus neutralization activity. The neutralization antibody titers after the final boost reached 6.7 in serum and 3.7 in fecal extracts, respectively. A PRRSV-specific IFN-γ response was also detected in splenocytes of vaccinated animals. These results demonstrate that transgenic corn plants are an efficient subunit vaccine production and oral delivery system for generation of both systemic and mucosal immune responses against PRRSV.     

Protective humoral immune response induced by an inactivated porcine reproductive and respiratory syndrome virus expressing the hypo-glycosylated glycoprotein 5

Porcine reproductive and respiratory syndrome (PRRS) causes significant economic losses to the swine industry worldwide. Although inactivated and live vaccines are commercially available for the control of PRRS, both types of vaccine have not always proven successful in terms of generating a protective immune response, particularly in the case of inactivated vaccines. In this study, we tested whether an inactivated vaccine could induce a humoral immune response to PRRS during a homologous challenge. Amino acid substitutions were introduced into glycoprotein (GP) 5 of the FL12 strain of the PRRS virus (PRRSV) using site-directed mutagenesis with a pFL12 infectious clone. The substitutions led to double deglycosylation in the putative glycosylation moieties on GP5. The mutant virus was subsequently inactivated with binary ethylenimine. The efficacy of the inactivated mutant virus was compared with that of the inactivated wild-type PRRSV. Only the inactivated mutant PRRSV induced serum neutralizing antibodies at six weeks post-vaccination. The group that was administered the inactivated mutant virus twice exhibited a significantly increased neutralizing antibody titer after a challenge with the virulent homologous strain and exhibited more rapid clearing of viremia compared to other groups, including the groups that were administered either the inactivated mutant or wild-type virus only once and the group that was administered the inactivated wild-type virus twice. Histopathological examination of lung tissue sections revealed that the group that was administered the inactivated mutant virus twice exhibited significantly thinner alveolar septa, whereas the thickness of the alveolar septa of the other groups were markedly increased due to lymphocyte infiltration. These results indicated that the deglycosylation of GP5 enhanced the immunogenicity of the inactivated mutant PRRSV and that twice administrations of the inactivated mutant virus conferred better protection against the homologous challenge. These findings suggest that the inactivated PRRSV that expresses a hypo-glycosylated GP5 is a potential inactivated vaccine candidate and a valuable tool for controlling PRRS for the swine industry.     

Immune responses of pigs inoculated with a recombinant fowlpox virus coexpressing GP5/GP3 of porcine reproductive and respiratory syndrome virus and swine IL-18

Two recombinant fowlpox viruses (rFPV-ORF5-ORF3 and rFPV-IL-18-ORF5-ORF3) containing the ORF5/ORF3 cDNAs of PRRSV (strain Chang Chun) and IL-18 of swine were constructed and evaluated for theirs abilities to induce humoral and cellular responses in piglets. In addition, their abilities to protect piglets against homologous virus challenge were examined. All piglets were given booster vaccinations at 21 days after the initial inoculation, and all piglets were challenged at 60 after the initial inoculation. Control groups were inoculated with wild-type fowlpox virus (wtFPV). All animals vaccinated with rFPV-ORF5-ORF3 and rFPV-IL-18-ORF5-ORF3 developed specific anti-PRRSV ELISA antibody and neutralizing antibody, as well as T-lymphocyte proliferation response. To evaluate the cellular immune function, IFN-gamma production in pigs serum and T-lymphocytes (CD4 and CD8 T cells) in peripheral blood were examined. Following challenge with a pathogenic strain of PRRSV (strain Chang Chun), piglets inoculated with recombinant fowlpox virus (rFPV) showed lower (P<0.05) temperature, viremia and virus load in bronchial lymph nodes than control animals, suggesting the establishment of partial protection against PRRSV infection. The results demonstrated the potential use of a fowlpox virus-based recombinant vaccine in the control and prevention of PRRSV infections.     

Effect of vaccination on the potentiation of porcine reproductive and respiratory syndrome virus (PRRSV)-induced pneumonia by Mycoplasma hyopneumoniae

Porcine reproductive and respiratory syndrome virus (PRRSV) and Mycoplasma hyopneumoniae are frequently isolated pathogens from pigs with respiratory disease. A previous study conducted in our laboratory found that infection with M. hyopneumoniae increased the duration and severity of respiratory disease induced by PRRSV. The purpose of this experiment was to determine whether vaccination against M. hyopneumoniae and/or PRRSV decreased the enhancement of PRRSV-induced pneumonia. Both M. hyopneumoniae bacterin and PRRSV vaccine decreased the severity of clinical respiratory disease. Infection or vaccination with PRRSV appeared to decrease the efficacy of the M. hyopneumoniae bacterin. Vaccination with M. hyopneumoniae bacterin decreased the potentiation of PRRSV-induced pneumonia observed in the dual infected pigs. However, PRRSV vaccination in combination with M. hyopneumoniae bacterin eliminated this benefit and the amount of pneumonia induced by PRRSV increased. PRRSV vaccine alone did not decrease the potentiation of PRRSV pneumonia by M. hyopneumoniae.     

Infectious clone-derived viruses from virulent and vaccine strains of porcine reproductive and respiratory syndrome virus mimic biological properties of their parental viruses in a pregnant sow model

Understanding of the molecular basis of virulence and attenuation of porcine reproductive and respiratory syndrome virus (PRRSV) is important for the development of a safe and efficacious vaccine. Prime Pac (PP) is an attenuated vaccine strain of PRRSV which is being used in our laboratories as a source of gene(s) for the generation of chimeric constructs in the background of a highly virulent PRRSV derived from an infectious clone (FL12) to examine the molecular determinants of virulence and attenuation. To facilitate these studies, we generated a full-length cDNA clone of the PP vaccine strain by serially replacing the genomic fragments of the FL12 with the corresponding regions from the PP strain. The virus rescued from this newly assembled cDNA clone (PP18) exhibited in vitro growth properties and in vivo apathogenic characteristics of the parental PP virus. Using pregnant sows as the experimental model of reproductive pathogenesis, we have been able to unequivocally demonstrate the clearly contrasting phenotypes of the virulent and the attenuated viruses derived from the infectious clones (FL12 and PP18). The development of an infectious clone derived from a bona fide attenuated PRRSV vaccine strain should significantly facilitate ongoing studies to determine the molecular basis of virulence and attenuation.     

Efficacy of Type 2 PRRSV vaccine against Chinese and Vietnamese HP-PRRSV challenge in pigs

Porcine reproductive and respiratory syndrome virus (PRRSV) causes significant reproductive losses in the sow herd and respiratory disease in growing pigs. The virus belongs to the family Arteriviridae and there are two major genotypes. Type 1 is represented by Lelystad virus, the European prototype virus, and Type 2 is represented by the North American prototype virus, VR-2332. Depending on husbandry, immune status of the herd, and virulence of the isolate, the severity of disease and magnitude of economic loss can be variable. Vaccine use is not always successful indicating a lack of cross-protection between vaccine strains and circulating wild-type viruses. To date, there is no clear method to demonstrate if a vaccine confers protection against a specific isolate except for empirical animal studies. In 2006, a new lineage of Type 2 PRRSV emerged in Chinese swine herds that were suffering dramatic losses resulting in those viruses being described as “Highly Pathogenic PRRSV” (HP-PRRSV). Experimental reproduction of severe disease with HP-PRRSV isolates and virus derived from HP-PRRSV clones demonstrated the causal role of this virus. Recently, partial heterologous protection has been reported for Type 1 and Type 2 attenuated PRRSV vaccines against challenge by different Chinese HP-PRRSV isolates providing some hope for reducing economic loss. This paper reports the efficacy of a commercially available Type 2 attenuated vaccine in young pigs against heterologous challenge with a Chinese and Vietnamese HP-PRRSV isolate. When compared to unvaccinated pigs, vaccination decreased the length of viremia and viral titer, diminished the time of high fever and reduced macroscopic lung scores following homologous and heterologous PRRSV challenge. These results demonstrate the potential use of vaccine as an aid in the control of HP-PRRSV outbreaks.     

Commercial vaccines provide limited protection to NADC30-like PRRSV infection

NADC30-like PRRSV has been recently reported and became endemic in vaccinated pig herds in China. The outbreaks of disease in vaccinated pigs indicated the inefficacy of commercial PRRSV vaccines. In this study, five commercial PRRSV vaccines that have been widely used in China were used to evaluate the efficacy to a NADC30-like PRRSV infection. The vaccinated pigs were challenged with HNjz15, a NADC30-like PRRSV at 28 days post vaccination. Compared to unvaccinated pigs, the vaccinated pigs clinically shortened the period of fever with less pig numbers of clinical manifestations and had improved body weight gain at the end of the study. However, the vaccinated pigs developed viremia with similar kinetics and suffered pathological lesions in lung and lymphoid tissues as the unvaccinated pigs. The virus load in tonsil, lung and lymph nodes detected by immunohistochemistry staining in vaccinated pigs was also similar to that in unvaccinated pigs which indicated the inability of vaccination to eradicate the virus from tissues of vaccinated pigs. Therefore, the above results suggested current commercial PRRSV vaccines could not provide complete protection to the NADC30-like PRRSV infection.     

Chlorine dioxide inhibits the replication of porcine reproductive and respiratory syndrome virus by blocking viral attachment

Porcine reproductive and respiratory syndrome virus (PRRSV) causes a great economic loss to the swine industry globally. Current prevention and treatment measures are not effective to control the outbreak and spread of porcine reproductive and respiratory syndrome (PRRS). In other words, new antiviral strategies are urgently needed. Chlorine dioxide (ClO₂) is regarded as a broad-spectrum disinfectant with strong inhibitory effects on microbes and parasites. The purpose of this study was to evaluate the inhibitory effects and underlying molecular mechanisms of ClO₂ against PRRSV infection in vitro. Here, we identified ClO₂ (the purity is 99%) could inhibit the infection and replication of PRRSV in both Marc-145 cells and porcine alveolar macrophages (PAMs). ClO₂ could block PRRSV binding to cells rather than internalization and release, suggesting that ClO₂ blocks the first stage of the virus life cycle. We also demonstrated that the inhibition exerted by ClO₂ was attributed to the degradation of PRRSV genome and proteins. Moreover, we confirmed that ClO₂ could decrease the expression of inflammatory cytokines induced by PRRSV. In summary, ClO₂ is an efficient agent and potently suppressed PRRSV infection in vitro.