Analysis of the swine tracheobronchial lymph node transcriptomic response to infection with a Chinese highly pathogenic strain of porcine reproductive and respiratory syndrome virus.

BACKGROUND: Porcine reproductive and respiratory syndrome virus (PRRSV) is a major pathogen of swine worldwide. Emergence in 2006 of a novel highly pathogenic PRRSV (HP-PRRSV) isolate in China necessitated a comparative investigation into the host transcriptome response in tracheobronchial lymph nodes (TBLN) 13 days post-infection with HP-PRRSV rJXwn06, PRRSV strain VR-2332 or sham inocula. RNA from each was prepared for next-generation sequencing. Amplified library constructs were directly sequenced and a list of sequence transcripts and counts was generated using an RNAseq analysis pipeline to determine differential gene expression. Transcripts were annotated and relative abundance was calculated based upon the number of times a given transcript was represented in the library. RESULTS: Major changes in transcript abundance occurred in response to infection with either PRRSV strain, each with over 630 differentially expressed transcripts. The largest increase in transcript level for either virus versus sham-inoculated controls were three serum amyloid A2 acute-phase isoforms. However, the degree of up or down-regulation of transcripts following infection with HP-PRRSV rJXwn06 was greater than transcript changes observed with US PRRSV VR-2332. Also, of 632 significantly altered transcripts within the HP-PRRSV rJXwn06 library 55 were up-regulated and 69 were down-regulated more than 3-fold, whilst in the US PRRSV VR-2332 library only 4 transcripts were up-regulated and 116 were down-regulated more than 3-fold. CONCLUSIONS: The magnitude of differentially expressed gene profiles detected in HP-PRRSV rJXwn06 infected pigs as compared to VR-2332 infected pigs was consistent with the increased pathogenicity of the HP-PRRSV in vivo.

Identification of the strain-specifically truncated nonstructural protein 10 of porcine reproductive and respiratory syndrome virus in infected cells.

The nonstructural protein 10 (nsp10) of porcine reproductive and respiratory syndrome virus (PRRSV) encodes for helicase which plays a vital role in viral replication. In the present study, a truncated form of nsp10, termed nsp10a, was found in PRRSV-infected cells and the production of nsp10a was strain-specific. Mass spectrometric analysis and deletion mutagenesis indicated that nsp10a may be short of about 70 amino acids in the N terminus of nsp10. Further studies by rescuing recombinant viruses showed that the Glu-69 in nsp10 was the key amino acid for nsp10a production. Finally, we demonstrated that nsp10a exerted little influence on the growth kinetics of PRRSV in vitro.

Efficacy evaluation of two commercial modified-live virus vaccines against a novel recombinant type 2 porcine reproductive and respiratory syndrome virus.

NADC30-like porcine reproductive and respiratory syndrome virus (PRRSV) causing clinical disease outbreaks has been recently reported in China. The recombination occurring among PRRSV strains could lead to the emergence of novel and more virulent viruses. In our previous study, a novel recombinant type 2 PRRSV (TJnh1501) between NADC30-like and modified-live virus (MLV)-like derived from the Chinese highly pathogenic PRRSV was shown to have higher pathogenicity than NADC30-like PRRSV. It remains unknown whether the emergence of the novel recombinant PRRSV strain can lead to variable protection efficacy of the MLV vaccines. In this paper, two typical commercial MLV vaccines were used to evaluate their efficacy to block TJnh1501 infection and onset of clinical symptoms. Our results showed that both MLV vaccines could shorten the period of fever and reduce viral loads in sera, but were not able to reduce the clinical signs and lung lesions indicating that the two commercial MLV vaccines provide limited cross-protection efficacy against the novel recombinant type 2 PRRSV infection. This study gives valuable suggestions for the use of MLV vaccines to control PRRSV infection in the field.

[Recovery of an infectious virus from the full-length cDNA of PRRSV BJ-4].

Six recombinant plasmids covering cDNA of porcine reproductive and respiratory syndrome virus BJ-4 were sequenced, respectively, and 23 point mutations were reverted with site-directed mutagenesis kit. The full-length cDNA clone pWSK-DCBA was assembled and re-sequenced. The capped viral genomic RNA was transcribed in vitro, mixed with liposome and transfected into MARC-145 cells, and an infectious virus (designated rV68) was rescued. The rescued virus was able to induce CPE typical of PRRSV on MARC-145 and stably propagated in vitro . Growth kinetics curve of the rV68 exhibited a delayed replication in MARC-145 cell, namely its peak titer time was 12h later than that of parental virus. However, there was no significant difference between the peak titers of the rescued and parental virus (P > 0.05). These results suggest that the full-length cDNA clone pWSK-DCBA of PRRSV BJ-4 is infectious, which provide a basis for further study on molecular pathogenicity and immunity, as well as developing novel vaccine of PRRSV.

Critical role of cytochrome c1 and its cleavage in porcine reproductive and respiratory syndrome virus nonstructural protein 4-induced cell apoptosis via interaction with nsp4.

Porcine reproductive and respiratory syndrome virus (PRRSV) actively induces cell apoptosis both in vitro and in vivo, which can contribute critically to viral pathogenesis. Previous studies have shown that the PRRSV nonstructural protein 4 (nsp4) is an important mediator of this process, but the underlying molecular details remain poorly understood. In this study, we found that the PRRSV nsp4 interacted with the mitochondrial inner membrane protein cytochrome c1 (cyto.c1) and induced its proteolytic cleavage. Interestingly, the cleaved N-terminal fragment of cyto.c1 was found to exert apoptotic activity, which could cause mitochondrial fragmentation, resulting in apoptotic cell death. And RNA interference (RNAi) silencing experiments further confirmed the crucial role which cyto.c1 played in nsp4- and PRRSV-induced cell apoptosis. Thus, our data provide an important piece of mechanistic clues for PRRSV-induced cell apoptosis and also elucidate a novel mechanism for the 3C-like proteases in this finding.

Enhancement of innate immunity with granulocyte colony-stimulating factor did not mitigate disease in pigs infected with a highly pathogenic Chinese PRRSV strain.

Porcine reproductive and respiratory syndrome virus (PRRSV) is responsible for one of the most economically important diseases in swine worldwide. It causes reproductive failure in sows and pneumonia in pigs that predisposes them to secondary bacterial infections. Methods to control PRRSV and/or limit secondary bacterial infections are desired to reduce the impact of this virus on animal health. Neutrophils play a major role in combatting infection; they can act as phagocytes as well as produce and release lytic enzymes that have potent antimicrobial effects leading to the destruction and clearance of bacterial pathogens. Granulocyte-colony stimulating factor (G-CSF) is a cytokine that controls the production, differentiation and function of granulocytes (including neutrophils) from the bone marrow. Recent work from our laboratory has shown that encoding porcine G-CSF in a replication-defective adenovirus (Ad5-G-CSF) and delivering a single dose to pigs induced a neutrophilia lasting more than two weeks. As secondary bacterial infection is a common occurrence following PRRSV infection, particularly following challenge with highly pathogenic (HP)-PRRSV, the aim of the current study was to evaluate the effectiveness of a single prophylactic dose of adenovirus-encoded G-CSF to mitigate secondary bacterial disease associated with HP-PRRSV infection. Administration of Ad5-G-CSF induced a significant neutrophilia as expected. However, between 1 and 2days following HP-PRRSV challenge the number of circulating neutrophils decreased dramatically in the HP-PRRSV infected group, but not the non-infected Ad5-G-CSF group. Ad5-G-CSF administration induced monocytosis as well, which was also reduced by HP-PRRSV challenge. There was no difference in the progression of disease between the Ad5-G-CSF and Ad5-empty groups following HP-PRRSV challenge, with pneumonia and systemic bacterial infection occurring in both treatment groups. Given the impact of HP-PRRSV infection on the neutrophilia induced by the Ad5-G-CSF administration, additional studies are warranted to evaluate the timing of Ad5-G-CSF induced neutrophilia and multiple G-CSF inoculations on protection against secondary bacterial infection following PRRSV infection. Nevertheless, this study may provide insight into the pathogenesis of HP-PRRSV.

Genetic characterization of porcine kobuvirus variants identified from healthy piglets in China.

In this study, two porcine kobuvirus strains, JS-01-CHN and JS-02a-CHN were detected from piglets with diarrhea and asymptomatic, respectively. The sequences of the two strains were analyzed using a bioinformatics software package. The full-length genome of JS-02a-CHN, was detected in healthy piglets was 8121 nucleotides (nt) long excluding the poly(A) tail. There was a 30 amino acid deletion in the 2B-coding region of JS-02a-CHN. We are the first to report a 30 amino acid deletion in porcine kobuvirus from asymptomatic piglets, indicating that porcine kobuvirus may have evolved differently based on geography and host differences. Fecal samples were obtained from pigs with diarrhea (n=91) and healthy (n=126) pigs and analyzed using RT-PCR. Of these, 64.8% (59/91) of diarrheic piglets and 19.8% (25/126) of healthy piglets were positive for PKV using VP1 specific primers. Twenty-eight (28) virus positive samples were randomly selected and the VP1 gene was analyzed. Phylogenetic analysis indicated that the 15 strains isolated from pigs with diarrhea clustered into different branches, while the VP1 sequences from clinically healthy pigs clustered into a single large group. These results indicate that the VP1 gene is diverse in pigs with diarrhea but conserved in healthy pigs in the Jiangsu Province.

Investigation on the co-infections of Toxoplasma gondii with PRRSV, CSFV or PCV-2 in swine in part of China.

The objective of the present investigation was to estimate the prevalence of Toxoplasma gondii infection and co-infection with porcine reproductive and respiratory syndrome virus (PRRSV), classical swine fever virus (CSFV) and porcine circovirus type 2 (PCV-2) in pigs in China. A total of 372 tissues or serum samples collected from pigs distributed in 9 provinces/municipalities of China during the period from February 2011 to November 2012 were assayed for T. gondii antigens and antibodies using enzyme linked immunosorbent assay (ELISA) technique, while the PCR was designed for the detection of the PRRSV, CSFV and PCV-2, respectively. The total positive rate of T. gondii, PRSSV, CSFV and PCV-2 was 9.14% (34/372), 50.00% (186/372), 37.10% (138/372) and 3.23% (12/372), respectively. Among the 34 T. gondii positive samples, 26 samples were simultaneously infected with T. gondii and viruses, while the remaining eight samples were infected with T. gondii alone. In addition, the co-infection rate of T. gondii with PRSSV, T. gondii with PRSSV and CSFV, T. gondii with PRSSV and PCV-2, T. gondii with CSFV and PCV-2, T. gondii with PRSSV, CSFV and PCV-2 was 1.61% (6/372), 4.03% (15/372), 0.27% (1/372), 0.27% (1/372) and 0.81% (3/372), respectively. The results of the present survey revealed that PRRSV and CSFV were the common pathogens co-existing with porcine toxoplasmosis in China, and both of them could increase the chances of T. gondii infection in pig. This is the first report of T. gondii co-infections with viruses in pigs. It is very important to understand the interactions of parasite and virus, and can be used as reference data for the control and prevention of co-infections of T. gondii and viruses in pigs.

[Purification and structural analysis of the nucleocapsid of PRRS virus expressed from Escherichia coli].

The DNA fragment encoding the nucleocapsid protein (N) of PRRSV BJ4 strain were cloned into the BamH I / EcoR I sites of pET28a vector to construct the expression plasmid pET28-N by designing special primers. The soluble protein (P28-N) were obtained by introducing the expression plasmid into E. coli BL21 (DE3) host cell, and the amount of recombinant protein reached to 28% of the total mass of bacterial protein. PET28-N were purified by nickel-affinity column of Proband resin. The circular dichroism (CD) analysis showed that the purified PET28-N shared a significant (26%) alpha-helical structure, beta-sheet (23.7%), beta-turn (19.8%), and random coil (30.3%), respectively. Finally,the secondary structure of N protein of PRRSV was deduced.

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.

Experimental infection of United States swine with a Chinese highly pathogenic strain of porcine reproductive and respiratory syndrome virus.

The pathogenesis of Type 2 highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) in 10-week old swine in the United States was investigated. rJXwn06, rescued from an infectious clone of Chinese HP-PRRSV, replicated in swine with at least 100-fold increased kinetics over U.S. strain VR-2332. rJXwn06 caused significant weight loss, exacerbated disease due to bacterial sepsis and more severe histopathological lung lesions in pigs exposed to HP-PRRSV than to those infected with VR-2332. Novel findings include identification of bacterial species present, the degree of thymic atrophy seen, and the inclusion of contact animals that highlighted the ability of HP-PRRSV to rapidly transmit between animals. Furthermore, comprehensive detailed cytokine analysis of serum, bronchoalveolar lavage fluid, and tracheobronchial lymph node tissue homogenate revealed a striking elevation in levels of cytokines associated with both innate and adaptive immunity in HP-PRRSV infected swine, and showed that contact swine differed in the degree of cytokine response.

Cytokine mRNA expression profiles in peripheral blood mononuclear cells from piglets experimentally co-infected with porcine reproductive and respiratory syndrome virus and porcine circovirus type 2.

Porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) significantly impact the swine industry worldwide. Co-infections with these viruses are common and several lines of evidence suggest that both PRRSV and PCV2 modify host immune responses that facilitate infection. This study examined cytokine mRNA expression profiles of peripheral blood mononuclear cells (PBMCs) from piglets experimentally co-infected with PRRSV and PCV2 to define the influence of co-infection on host immunity. PBMCs from infected and control piglets were stimulated with concanavalin A and the IL-2, IL-4, IL-6, IL-10, IL-12p40, IFN-gamma and TNF-alpha mRNA levels were determined by quantitative reverse transcription-polymerase chain reaction (RT-PCR). PBMCs from PRRSV/PCV2 co-infected piglets had significantly reduced IL-2, IL-4, IL-6, IL-12p40 and IFN-gamma and significantly increased TNF-alpha mRNA levels compared to those of the piglets infected with either PRRSV or PCV2 alone. The IL-10 mRNA levels in all virus-infected groups were significantly up-regulated early during infection. These results suggested that co-infection synergistically suppresses T helper 1 (Th1)-type and Th2-type cytokine production by PBMCs, indicating that co-infection likely compromises cell-mediated and humoral immune responses resulting in increased severity of the diseases in piglets.

A recombinant plasmid co-expressing swine ubiquitin and the GP5 encoding-gene of porcine reproductive and respiratory syndrome virus induces protective immunity in piglets.

We transfected a recombinant plasmid that co-expressed swine ubiquitin and a codon optimized GP5 encoding-gene of porcine reproductive and respiratory syndrome virus (PRRSV), designated pCA-U-optiGP5, as well as the plasmid pCA-optiGP5 encoding codon optimized GP5, and the plasmid pCA-GP5 expressing wild-type pGP5 into 293T cells. Expression of GP5 was measured by indirect immunofluorescence (IIF) assay and Western blot and found to be considerably higher in response to pCA-U-optiGP5 than the wild-type vector. GP5 protein was rapidly degraded in pCA-U-optiGP5-transfected 293T cells. The proteasome inhibitor, MG-132, however, successfully inhibited degradation. Immunogenicity of the three constructs was examined by measuring GP5-specific antibody production, lymphocyte proliferation, cytotoxic T lymphocyte (CTL) responses, and cytokine secretion in intramuscularly immunized pigs. Three weeks after the last inoculation, all animals were challenged intranasally with 2-ml 10(5)TCID(50)/ml PRRSV CH-1a. DNA immunization with pCA-optiGP5 produced a higher level of GP5-specific antibody than immunization with pCA-GP5, and the humoral response remained undetectable in the pCA-U-optiGP5 group. However, the fusion DNA had a significantly enhanced stimulation index (SI) and induced a stronger Th1 type cellular immune response than the single gene DNA, suggesting that ubiquitin conjugation improved the cellular but not the humoral immune response. Four of six pigs in the pCA-U-optiGP5 group, three of six in the pCA-optiGP5 group, and two of six in the pCA-GP5 group were devoid of visible pathological changes that were present in other vaccinated and control animals after challenge. Viral replication and distribution in the blood and tissues was lower in the pCA-U-optiGP5 vaccinated group than the other groups, suggesting that codon optimization, along with the development of rapidly processed antigen, represents a novel strategy to increase the immune efficacy of DNA vaccines against PRRSV.

Characterization of a highly pathogenic H5N1 influenza virus derived from bar-headed geese in China.

Influenza A viruses are usually non-pathogenic in wild aquatic birds, their natural reservoir. However, from May to July 2005, at Qinghai Lake in China, an unprecedented outbreak of highly pathogenic H5N1 avian influenza virus caused the death of thousands of wild migratory waterbirds. Herein, H5N1 influenza virus from bar-headed geese collected during the outbreak was characterized. Genomic analysis showed that A/Bar-headed Goose/Qinghai/0510/05 (Bh H5N1 virus) is a reassortant virus. Amino acid residue (lysine) at position 627 in the PB2 gene of the Bh H5N1 virus was the same as that of the human H5N1 virus (A/HK/483/97) and different from that of H5N1 avian influenza viruses deposited in GenBank. Antigenic analysis showed that significant antigenic variation has occurred in the Bh H5N1 virus. The Bh H5N1 virus induced systemic infections and caused 100 % mortality in chickens and mice, and 80 % mortality in ducks and geese. Bh H5N1 virus titres were higher in multiple organs of chickens, ducks and geese than in mice, and caused more severe histological lesions in chickens, ducks and mice than in geese. These results support the need to pay close attention and create control programmes to prevent the transmission of highly pathogenic avian influenza virus from wild migratory waterbirds into domestic chickens, ducks, geese and mammalian hosts.