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.     

Characterization of antigenic regions in the porcine reproductive and respiratory syndrome virus by the use of peptide-specific serum antibodies

The porcine reproductive and respiratory syndrome virus (PRRSV) is an RNA virus that causes reproductive failure in sows and boars, and respiratory disease in pigs of all ages. Antibodies against several viral envelope proteins are produced upon infection, and the glycoproteins GP4 and GP5 are known targets for virus neutralization. Still, substantial evidence points to the presence of more, yet unidentified neutralizing antibody targets in the PRRSV envelope proteins. The current study aimed to identify and characterize linear antigenic regions (ARs) within the entire set of envelope proteins of the European prototype PRRSV strain Lelystad virus (LV). Seventeen LV-specific antisera were tested in pepscan analysis on GP2, E, GP3, GP4, GP5 and M, resulting in the identification of twenty-one ARs that are capable of inducing antibodies upon infection in pigs. A considerable number of these ARs correspond to previously described epitopes in different European- and North-American-type PRRSV strains. Remarkably, the largest number of ARs was found in GP3, and two ARs in the GP3 ectodomain consistently induced antibodies in a majority of infected pigs. In contrast, all remaining ARs, except for a highly immunogenic epitope in GP4, were only recognized by one or a few infected animals. Sensitivity to antibody-mediated neutralization was tested for a selected number of ARs by in vitro virus-neutralization tests on alveolar macrophages with peptide-purified antibodies. In addition to the known neutralizing epitope in GP4, two ARs in GP2 and one in GP3 turned out to be targets for virus-neutralizing antibodies. No virus-neutralizing antibody targets were found in E, GP5 or M. Since the neutralizing AR in GP3 induced antibodies in a majority of infected pigs, the immunogenicity of this AR was studied more extensively, and it was demonstrated that the corresponding region in GP3 of virus strains other than LV also induces virus-neutralizing antibodies. This study provides new insights into PRRSV antigenicity, and contributes to the knowledge on protective immunity and immune evasion strategies of the virus.     

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.     

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.     

Live porcine reproductive and respiratory syndrome virus vaccines: Current status and future direction

Porcine reproductive and respiratory syndrome (PRRS) caused by PRRS virus (PRRSV) was reported in the late 1980s. PRRS still is a huge economic concern to the global pig industry with a current annual loss estimated at one billion US dollars in North America alone. It has been 20 years since the first modified live-attenuated PRRSV vaccine (PRRSV-MLV) became commercially available. PRRSV-MLVs provide homologous protection and help in reducing shedding of heterologous viruses, but they do not completely protect pigs against heterologous field strains. There have been many advances in understanding the biology and ecology of PRRSV; however, the complexities of virus-host interaction and PRRSV vaccinology are not yet completely understood leaving a significant gap for improving breadth of immunity against diverse PRRS isolates. This review provides insights on immunization efforts using infectious PRRSV-based vaccines since the 1990s, beginning with live PRRSV immunization, development and commercialization of PRRSV-MLV, and strategies to overcome the deficiencies of PRRSV-MLV through use of replicating viral vectors expressing multiple PRRSV membrane proteins. Finally, powerful reverse genetics systems (infectious cDNA clones) generated from more than 20 PRRSV isolates of both genotypes 1 and 2 viruses have provided a great resource for exploring many innovative strategies to improve the safety and cross-protective efficacy of live PRRSV vaccines. Examples include vaccines with diminished ability to down-regulate the immune system, positive and negative marker vaccines, multivalent vaccines incorporating antigens from other porcine pathogens, vaccines that carry their own cytokine adjuvants, and chimeric vaccine viruses with the potential for broad cross-protection against heterologous strains. To combat this devastating pig disease in the future, evaluation and commercialization of such improved live PRRSV vaccines is a shared goal among PRRSV researchers, pork producers and biologics companies.     

Development of a porcine reproductive and respiratory syndrome virus differentiable (DIVA) strain through deletion of specific immunodominant epitopes

The availability of a DIVA (differentiating infected from vaccinated animals) vaccine is very important for the control and eradication of endemic infectious diseases such as porcine reproductive and respiratory syndrome (PRRS). Previous studies in our laboratory identified several B-cell linear epitopes consistently recognized by convalescent sera obtained from pigs infected with a North American porcine reproductive and respiratory syndrome virus (PRRSV) strain. To ascertain if one or more of these immunodominant epitopes can be used as the basis of DIVA differentiation, we selected two epitope markers previously identified on the non-structural protein 2 (PRRSV NSP2, predictably the viral protein most likely to tolerate large deletions). The choice of these epitopes was primarily based on their immunodominance and their deletion were performed along the backbone of the wild-type cDNA infectious clone (FL12). We were able to successfully rescue a mutant that fulfilled the requirements for a DIVA marker strain, such as: efficient growth of the deletion mutant in vitro and in vivo and induction of specific seroconversion as measured by a commercial ELISA kit, with absence of a marker-specific peptide-ELISA response in 100% (n=15) of the inoculated animals. In summary, our results provide proof of concept that DIVA PRRSV vaccines can potentially be developed by deletion of individual "marker" immunodominant epitopes.     

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.     

Safety and protective efficacy of porcine reproductive and respiratory syndrome recombinant virus vaccines in young pigs

Three porcine reproductive and respiratory syndrome virus (PRRSV) recombinants, generated by mutagenesis of an infectious cDNA clone of the Lelystad virus (LV) isolate, were tested for their safety and protective efficacy as potential PRRSV vaccines in pigs. Recombinant vABV688 contains two amino acid substitutions in the minor structural protein GP(2) resulting in improved growth on cell line CL2621; in recombinant vABV707 the region encoding the ectodomain of the major unglycosylated membrane protein M has been replaced by that of the murine lactate dehydrogenase-elevating arterivirus; recombinant vABV746 lacks the six C-terminal amino acids of the nucleocapsid protein N. First, we determined the safety of these recombinant viruses by monitoring the stability of the introduced mutations in 8-week-old pigs. We showed that the introduced genomic mutations were maintained throughout the viraemic period. Second, the protective efficacy of immunization with the recombinant viruses against challenge with a homologous and a heterologous PRRSV strain was determined in two pigs and compared with the efficacy of vABV437, a virus derived from the parental LV cDNA. The viraemia in pigs immunized with the recombinant viruses was reduced compared to pigs immunized with vABV437. In addition, the length of viraemia was reduced in the sentinel pigs that were introduced into the groups immunized with vABV746, vABV688, and vABV707, however, all of the sentinel pigs became infected. Pigs immunized with vABV707 and vABV437 were protected against challenge with homologous virus LV-Ter Huurne and transmission of the latter virus. None of the immunized pigs were protected against heterologous challenge with the virulent US isolate SDSU#73, but the vABV707- and vABV746-immunized pigs were protected against transmission of this virus from challenged pigs. In conclusion, the obtained viral recombinants are interesting candidates to be further explored for their use as vaccines against PRRSV.     

Global distribution of rubella virus genotypes

Phylogenetic analysis of a collection of 103 E1 gene sequences from rubella viruses isolated from 17 countries from 1961 to 2000 confirmed the existence of at least two genotypes. Rubella genotype I (RGI) isolates, predominant in Europe, Japan, and the Western Hemisphere, segregated into discrete subgenotypes; international subgenotypes present in the 1960s and 1970s were replaced by geographically restricted subgenotypes after approximately 1980. Recently, active subgenotypes include one in the United States and Latin America, one in China, and a third that apparently originated in Asia and spread to Europe and North America, starting in 1997, indicating the recent emergence of an international subgenotype. A virus that potentially arose as a recombinant between two RGI subgenotypes was discovered. Rubella genotype II (RGII) showed greater genetic diversity than did RGI and may actually consist of multiple genotypes. RGII viruses were limited to Asia and Europe; RGI viruses were also present in most of the countries where RGII viruses were isolated.     

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.     

Recombination in Vaccine and Circulating Strains of Porcine Reproductive and Respiratory Syndrome Viruses

Em2007, a porcine reproductive and respiratory syndrome virus (PRRSV) variant with a unique 68 aa deletion in Nsp2, was recently isolated in China. Phylogenetic and molecular evolutionary analyses indicated that Em2007 is a natural recombinant between a vaccine strain of PRRSV and circulating virus. We also tested its pathogenicity in piglets.     

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.     

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.     

Genetic diversity of the Korean field strains of porcine reproductive and respiratory syndrome virus

Porcine reproductive and respiratory syndrome (PRRS) is one of the most economically significant diseases in the swine industry. The PRRS virus (PRRSV) has genetically diverse populations, like other RNA viruses, and various field strains continue to be reported worldwide. The molecular epidemiological study of PRRSV can provide important data for use in controlling the disease. In this study, 50 oral fluid samples from conventional farms in Korea were taken to analyze nucleotide sequences of the open reading frame 5 of PRRSV. The viruses present in more than 80% of oral fluid samples genetically originated from the type 2 PRRSV, which is North American (NA) lineage. In addition 8.9% of samples contained both of the type 1 PRRSV, which is European (EU) lineage and the type 2 PRRSV. About 60% of farms involved in this study had more than two strains of PRRSV. In phylogenetic analysis, the Korean field strains of PRRSV detected from the oral fluid samples were divided into several subgroups: four subgroups of Korean field strains clustered with the type 1 PRRSV, and other five subgroups of Korean field strains clustered with the type 2. These results suggest that the type 2 PRRSV is more prevalent than the type 1 in Korea and heterologous strains of PRRSV can simultaneously infect a single pig farm.     

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.     

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.     

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.     

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.     

Nipah virus infection of pigs in peninsular Malaysia

Between late 1998 and 1999, the spread of a new disease of pigs, characterized by a pronounced respiratory and neurological syndrome, sometimes accompanied by the sudden death of sows and boars, was recorded in pig farms in peninsular Malaysia. The disease appeared to have a close association with an epidemic of viral encephalitis among workers on pig farms. A previously unrecognised paramyxovirus was later identified from this outbreak; this virus was related to, but distinct from, the Hendra virus discovered in Australia in 1994. The new virus was named 'Nipah' and was confirmed by molecular characterization to be the agent responsible for the disease in both humans and pigs. The name proposed for the new pig disease was 'porcine respiratory and neurological syndrome' (also known as 'porcine respiratory and encephalitis syndrome'), or, in peninsular Malaysia, 'barking pig syndrome'. The authors describe the new disease and provide the epidemiological findings recorded among infected pigs. In addition, the control programmes which were instituted to contain the virus in the national swine herd are outlined.     

Antibody repertoire development in fetal and neonatal piglets. XV. Porcine circovirus type 2 infection differentially affects serum IgG levels and antibodies to ORF2 in piglets free from other environmental factors

Porcine circovirus type 2 (PCV2) is an important pathogen in the porcine respiratory disease complex (PRDC) and its persistence may be due to dysregulation of systemic immunity. We examined this contention using isolator piglets. We present data on Ig levels in serum and bronchio-alveolar lavage (BAL), on antibody response to PCV2 and to TNP conjugates used as model antigens in 48 PCV2-infected isolator piglets. We compared these to data from TNP-immunized isolator piglets colonized with a probiotic flora, those infected with swine influenza (S-FLU) and those infected with porcine respiratory and reproductive syndrome virus (PRRSV). We found that PCV2 infection does not cause generalized hypergammaglobulinemia that characterizes PRRSV infections, but causes an unexplained increase in serum IgA. All animals had serum IgG to the ORF2 gene product of PCR2, but neither IgA nor IgG anti-ORF2 responses in BAL. PCV2 infection is a poor adjuvant since only natural anti-TNP antibodies were found. Unexpectedly, immunization appeared to result in lower Ig levels and lower anti-ORF2 responses. There was extreme variation in serum Ig levels in response to infection that could in part be traced to genetic and gender differences. These data suggest that non-replicating vaccines are unlikely to result in a significant primary antibody response but may prime the system for a secondary antibody and cytotoxic response following actual infection. In any case, developers may have to contend with significant genetic differences in the response of piglets to PCV2.