Upregulation of HMGB1 secretion in lungs of pigs infected by highly pathogenic porcine reproductive and respiratory syndrome virus.

Porcine reproductive and respiratory syndrome (PRRS) remains a major driver for substantial economic losses to the swine industry across the world. Pulmonary inflammatory injury is a common manifestation in infected pigs. Previous studies reported that PRRS virus (PRRSV) induces secretion of high mobility group box 1 (HMGB1), a proinflammatory factor, in cultured cells. The objective of this study was to evaluate whether HMGB1 secretion is associated with PRRSV-induced pulmonary inflammatory responses in the early stage of infection in vivo. Three-week-old piglets were inoculated with either HuN4, a highly pathogenic PRRSV (HP-PRRSV) strain, or CH1R, an avirulent PRRSV vaccine strain. Necropsy was performed at 7 days post-infection. The results showed that HuN4 significantly induced the secretion of HMGB1 and inflammatory cytokines (IL-1ß, IL-6) into the bronchoalveolar lavage fluid (BALF). HuN4 infection induced severe interstitial pneumonia in the pigs. In contrast, pigs infected by CH1R had mild lung inflammation with minimal HMGB1 secretion. In addition, high viral load of HuN4 was detected in both pulmonary alveolar macrophages (PAMs) and lung tissue, whereas viral RNA of CH1R was confined to PAMs. In consistent with the pneumonia development, HuN4 induced inflammatory cytokines in both PAMs and lung tissue, while their expression in CH1R-infected pigs confined only to PAMs. These results indicate that the HuN4-induced HMGB1 secretion into BALF may enhance the pulmonary inflammatory response and exacerbate the lung injury. This finding provides insights to the inflammatory response and pathogenesis of the HP-PRRSV infection.

Porcine reproductive and respiratory syndrome virus induces HMGB1 secretion via activating PKC-delta to trigger inflammatory response.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes inflammatory injuries in infected pigs. PRRSV induces secretion of high mobility group box 1 (HMGB1) that enhances inflammatory response. However, the mechanism of PRRSV-induced HMGB1 secretion is unknown. Here, we discovered PRRSV induced HMGB1 secretion via activating protein kinase C-delta (PKCδ). HMGB1 secretion was positively correlated with PKCδ activation in PRRSV-infected cells in a dose and time-dependent manner. Suppression of PKCδ with inhibitor and siRNA significantly blocked PRRSV-induced HMGB1 translocation and secretion, which indicates PKCδ activation is essential for the PRRSV-mediated HMGB1 secretion. In addition, PKCδ knockdown in PRRSV-infected cells led to downregulation of inflammatory cytokines, including IL-1beta and IL-6. Moreover, PRRSV E and pORF5a proteins were found to activate PKCδ and consequent HMGB1 secretion. These results demonstrate PRRSV activates PKCδ to induce HMGB1 secretion via E and pORF5a. This finding provides insights on the inflammatory response and pathogenesis of PRRSV infection.

The middle half genome of interferon-inducing porcine reproductive and respiratory syndrome virus strain A2MC2 is essential for interferon induction.

Porcine reproductive and respiratory syndrome virus (PRRSV) is known to antagonize the innate immune response. An atypical PRRSV strain A2MC2 is capable of inducing synthesis of type I interferons (IFNs) in cultured cells. Here, we show that the middle half of the A2MC2 genome is needed for triggering the IFN synthesis. First, a cDNA infectious clone of this atypical strain was constructed as a DNA-launched version. Virus recovery was achieved from the infectious clone and the recovered virus, rA2MC2, was characterized. The rA2MC2 retained the feature of IFN induction in cultured cells. Infection of pigs with the rA2MC2 virus caused viremia similar to that of the wild-type virus. Chimeric infectious clones were constructed by swapping genomic fragments with a cDNA clone of a moderately virulent strain VR-2385 that antagonizes IFN induction. Analysis of the rescued chimeric viruses demonstrated that the middle two fragments, ranging from nt4545 to nt12709 of the A2MC2 genome, were needed for the IFN induction, whereas the chimeric viruses containing any one of the two A2MC2 fragments failed to do so. The results and the cDNA infectious clone of the IFN-inducing A2MC2 will facilitate further study of its biology, ultimately leading towards the development of an improved vaccine against PRRS.

Porcine Reproductive and Respiratory Syndrome Virus Antagonizes JAK/STAT3 Signaling via nsp5, Which Induces STAT3 Degradation.

Signal transducer and activator of transcription 3 (STAT3) is a pleiotropic signaling mediator of many cytokines, including interleukin-6 (IL-6) and IL-10. STAT3 is known to play critical roles in cell growth, proliferation, differentiation, immunity and inflammatory responses. The objective of this study was to determine the effect of porcine reproductive and respiratory syndrome virus (PRRSV) infection on the STAT3 signaling since PRRSV induces a weak protective immune response in host animals. We report here that PRRSV infection of MARC-145 cells and primary porcine pulmonary alveolar macrophages led to significant reduction of STAT3 protein level. Several strains of both PRRSV type 1 and type 2 led to a similar reduction of STAT3 protein level but had a minimal effect on its transcripts. The PRRSV-mediated STAT3 reduction was in a dose-dependent manner as the STAT3 level decreased, along with incremental amounts of PRRSV inocula. Further study showed that nonstructural protein 5 (nsp5) of PRRSV induced the STAT3 degradation by increasing its polyubiquitination level and shortening its half-life from 24 h to ∼3.5 h. The C-terminal domain of nsp5 was shown to be required for the STAT3 degradation. Moreover, the STAT3 signaling in the cells transfected with nsp5 plasmid was significantly inhibited. These results indicate that PRRSV antagonizes the STAT3 signaling by accelerating STAT3 degradation via the ubiquitin-proteasomal pathway. This study provides insight into the PRRSV interference with the JAK/STAT3 signaling, leading to perturbation of the host innate and adaptive immune responses. IMPORTANCE: The typical features of immune responses in PRRSV-infected pigs are delayed onset and low levels of virus neutralizing antibodies, as well as weak cell-mediated immunity. Lymphocyte development and differentiation rely on cytokines, many of which signal through the JAK/STAT signaling pathway to exert their biological effects. Here, we discovered that PRRSV antagonizes the JAK/STAT3 signaling by inducing degradation of STAT3, a master transcription activator involved in multiple cellular processes and the host immune responses. The nsp5 protein of PRRSV is responsible for the accelerated STAT3 degradation. The PRRSV-mediated antagonizing STAT3 could lead to suppression of a broad spectrum of cytokines and growth factors to allow virus replication and spread in host animals. This may be one of the reasons for the PRRSV interference with the innate immunity and its poor elicitation of protective immunity. This finding provides insight into PRRSV pathogenesis and its interference with the host immune responses.

Sustaining Interferon Induction by a High-Passage Atypical Porcine Reproductive and Respiratory Syndrome Virus Strain.

Porcine reproductive and respiratory syndrome virus (PRRSV) strain A2MC2 induces type I interferons in cultured cells. The objective of this study was to attenuate this strain by serial passaging in MARC-145 cells and assess its virulence and immunogenicity in pigs. The A2MC2 serially passaged 90 times (A2MC2-P90) retains the feature of interferon induction. The A2MC2-P90 replicates faster with a higher virus yield than wild type A2MC2 virus. Infection of primary pulmonary alveolar macrophages (PAMs) also induces interferons. Sequence analysis showed that the A2MC2-P90 has genomic nucleic acid identity of 99.8% to the wild type but has a deletion of 543 nucleotides in nsp2. The deletion occurred in passage 60. The A2MC2-P90 genome has a total of 35 nucleotide variations from the wild type, leading to 26 amino acid differences. Inoculation of three-week-old piglets showed that A2MC2-P90 is avirulent and elicits immune response. Compared with Ingelvac PRRS® MLV strain, A2MC2-P90 elicits higher virus neutralizing antibodies. The attenuated IFN-inducing A2MC2-P90 should be useful for development of an improved PRRSV vaccine.

Downregulation of protein kinase PKR activation by porcine reproductive and respiratory syndrome virus at its early stage infection.

The interferon-induced double-strand RNA activated protein kinase (PKR) plays an important role in antiviral response. The objective of this study was to assess the effect of porcine reproductive and respiratory syndrome virus (PRRSV) on PKR activation. Here we report that PRRSV inhibited PKR activation during its early stage infection of primary pulmonary alveolar macrophages (PAMs). PRRSV infection led to lower level of phosphorylated PKR in comparison with mock-infected cells. The PKR inhibition was sustained until 10h post infection in the presence of polyI:C, a synthetic analog of double-stranded RNA activating PKR. PKR-mediated phosphorylation of the eukaryotic translation initiation factor eIF2α was also lower in the PRRSV-infected PAMs during the early stage infection. Interestingly, inactivated PRRSV was capable to inhibit the PKR activation until 6h post infection. This suggests that structural components of PRRSV virions were responsible for the inhibition, although PRRSV replication was needed for longer inhibition. These results indicate that the downregulation of PKR activation during early infection stage should be essential for PRRSV to avoid the antiviral response to initiate replication. This finding contributes to our understanding on PRRSV interaction with host innate immune response and reveal a target for control of PRRSV infection.

Establishment and characterization of duck embryo epithelial (DEE) cell line and its use as a new approach toward DHAV-1 propagation and vaccine development.

The primary cell culture was derived from duck embryonic tissue, digested with collagenase type I. The existence of cell colonies with epithelial-like morphology, named duck embryo epithelial (DEE), were purified and optimally maintained at 37°C in M199 medium supplemented with 5% fetal bovine serum. The purified cells were identified as epithelial cell line by detecting Keratin-18 expression using immunofluorescence assay. Our findings demonstrated that DEE cell line can be propagated in culture with (i) a great capacity to adhere, (ii) a great proliferation activity, and (iii) a population doubling time of approximately 18h. Chromosomal features of the DEE cell line were remained constant after the 50th passage. Further characterizations of DEE cell line showed that cell line can normally be grown even after several passages and never converted to tumorigenic cells either in vitro or in vivo study. Susceptibility of DEE cell line was determined for transfection and duck hepatitis A type 1 virus (DHAV-1)-infection. Interestingly, the 50% egg lethal dose (ELD50) of the propagated virus in DEE cell line was higher than ELD50 of the propagated virus in embryonated eggs. Finally, DEE cell line was evaluated to be used as a candidate for DHAV-1 vaccine development. Our results showed that the propagated DHAV-1 vaccine strain SDE in DEE cell line was able to protect ducklings against DHAV-1 challenge. Taken together, our findings suggest that the DEE cell line can serve as a valuable tool for DHAV-1 propagation and vaccine production.

Generation and evaluation of a recombinant genotype VII Newcastle disease virus expressing VP3 protein of Goose parvovirus as a bivalent vaccine in goslings.

Newcastle disease virus (NDV) and Goose parvovirus (GPV) are considered to be two of the most important and widespread viruses infecting geese. In this study, we generated a recombinant rmNA-VP3, expressing GPV VP3 using a modified goose-origin NDV NA-1 by changing the multi-basic cleavage site motif RRQKR↓F of the F protein to the dibasic motif GRQGR↓L as that of the avirulent strain LaSota as a vaccine vector. Expression of the VP3 protein in rmNA-VP3 infected cells was detected by immunofluorescence and Western blot assay. The genetic stability was examined by serially passaging 10 times in 10-day-old embryonated SPF chicken eggs. Goslings were inoculated with rmNA-VP3 showed no apparent signs of disease and developed a strong GPV and NDV neutralizing antibodies response. This is the first study demonstrating that recombinant NDV has the potential to serve as bivalent live vaccine against Goose parvovirus and Newcastle disease virus infection in birds.

Enhancing neutralizing antibody production by an interferon-inducing porcine reproductive and respiratory syndrome virus strain.

Porcine reproductive and respiratory syndrome (PRRS) virus (PRRSV) continues to cause substantial economic losses to the global swine industry. PRRSV appears to inhibit synthesis of type I interferons (IFNs), such as IFN-α and -ß, which are critical for the innate immunity and play an important role in the modulation of adaptive immunity. An atypical PRRSV strain, A2MC2, is able to induce type I IFNs in vitro. In this study, A2MC2 induction of neutralizing antibodies in vivo was compared with the Ingelvac PRRS modified live virus (MLV) vaccine strain and VR-2385 (a moderate virulent strain). Three-week-old pigs were exposed to these PRRSV strains via intranasal or intramuscular routes to also account for a possible effect of inoculation routes. The interferon-inducing A2MC2 resulted in earlier onset and significantly higher levels of PRRSV neutralizing antibodies than the MLV. In addition, the A2MC2-induced neutralizing antibodies were capable of neutralizing VR-2385, a heterologous strain. The pigs exposed via intranasal route had higher titers of neutralizing antibodies than those injected via intramuscular route. Macroscopic and microscopic lung lesions 14 days post-exposure indicated that A2MC2 had similar virulence in vivo as VR-2385. Pulmonary alveolar macrophages (PAMs) collected during the necropsy 14 days post-exposure in the A2MC2 group had higher level expression of IFN-γ than the MLV group. These results indicate that A2MC2 can be further explored for development of an improved vaccine against PRRS.

Prevention of rabies virus infection in dogs by a recombinant canine adenovirus type-2 encoding the rabies virus glycoprotein.

Safe and effective vaccination is important for rabies prevention in animals. Although several genetically engineered rabies vaccines have been developed, few have been licensed for use, principally due to biosafety concerns or due to poor efficacy in animal models. In this paper, we describe the construction and characterization of a replication-competent recombinant canine adenovirus type-2 expressing the rabies virus glycoprotein (SRV9 strain) by a different strategy from that reported previously, i.e., the recombinant genome carrying the glycoprotein cDNA was generated by a series of strictly gene cloning steps, infectious recombinant virus was obtained by transfecting the recombinant genome into a canine kidney cell line, MDCK. This recombinant virus, CAV-E3delta-CGS, was subcutaneously injected into dogs. All vaccinated dogs produced effective neutralizing antibodies after one inoculation and a stronger anamnestic immune response was produced after booster injection. The immunized dogs could survive the challenge of 60,000 mouse LD50 CVS-24, which is lethal to all unimmunized dogs and is comparable to the conventional vaccines. The immunity lasts for months with a protective level of neutralizing antibody. This recombinant virus would be an alternative to the attenuated and the inactivated rabies vaccines and be prospective in immunizing dogs against rabies.

[Construction and immunogenicity of recombinant Canine adenovirus type 2 with the external part of glycoprotein of rabies virus].

The cases of rabies increase greatly in recent years in China and rabies continues to be a serious problem in developing countries due to the reservoirs of Rabies virus in dogs and wildlife vectors. The control of rabies depends on the development of safe, effective, economical vaccines that may be used for preexposure vaccination in animals. For this purpose, the external part of glycoprotein gene of Rabies virus strain SRV9 (RVG) was amplified by the RT-PCR and cloned into pEGFP-C1 with replacement of the GFP gene. The expression cassette pERVG2 is composed of CMV promoter, external glycoprotein gene and SV40 early mRNA polyadenylation signal. The expression cassette was released by Ase I /Mlu I double digestion and it was cloned rightwards and leftwards into Canine adenovirus type 2(CAV-2) E3 region plasmid pVAXE3 in which E3 region was deleted partly by Ssp I /Dra III digestion. After the replacement of CAV-2 E3 region in plasmid pCAV-2 which contains CAV-2 genome with the recombinant E3 region, recombinant CAV-2 genome plasmid was obtained. Recombinant CAV-2 genome was released from plasmid by Asc I /Cla I digestion, and then transfected into MDCK cells. Two replication-competent recombinant CAV2 expressing Rabies Virus external part of glycoprotein were produced. Vaccination experiment showed that the recombinant viruses can elicit an efficient antibody response in dogs.