Rapid Recovery of Classical Swine Fever Virus Directly from Cloned cDNA

The reverse genetics for classical swine fever virus （CSFV） is currently based on the transfection of in vitro transcribed RNA from a viral genomic cDNA clone, which is inefficient and time-consuming. This study was aimed to develop an improved method for rapid recovery of CSFV directly from cloned cDNA. Full-length genomic cDNA from the CSFV Shimen strain, which was flanked by a T7 promoter, the hepatitis delta virus ribozyme and T7 terminator sequences, was cloned into the low- copy vector pOK12, producing pOKShimen-RzTФ. Direct transfection of pOKShimen-RzTqb into PK/T7 cells, a PK-15- derived cell line stably expressing bacteriophage T7 RNA polymerase, allowed CSFV to be rescued rapidly and efficiently, i.e., at least 12 h faster and 31.6-fold greater viral titer when compared with the in vitro transcription-based rescue system. Furthermore, the progeny virus rescued from PK/T7 cells was indistinguishable, both in vitro and in vivo, from its parent virus and the virus rescued from classical reverse genetics. The reverse genetics based on intracellular transcription is efficient, convenient and cost-effective. The PK/T7 cell line can be used to rescue CSFV directly from cloned cDNA and it can also be used as an intracellular transcription and expression system for studying the structure and function of viral genes.

Generation and Immunogenicity of a Recombinant Adenovirus Co-Expressing the E2 Protein of Classical Swine Fever Virus and the GP5 Protein of Porcine Reproduction and Respiratory Syndrome Virus

Classical swine fever （CSF） and porcine reproduction and respiratory syndrome （PRRS） are both economically important, highly contagious diseases of swine worldwide. To develop an effective vaccine to control these two diseases, we constructed a recombinant adenovirus rAdV-GP52AE2, using a replication-defective human adenovirus serotype 5 as a delivery vector, to co-express the GP5 protein of highly pathogenic porcine reproduction and respiratory syndrome virus （PRRSV） and the E2 protein of classical swine fever virus （CSFV）. Foot-and-mouth disease virus （FMDV） 2A peptide was used as a linker between the GP5 and E2 proteins to allow automatic self-cleavage of the polyprotein. The GP5 and E2 genes were expressed as demonstrated by immunofluorescence assay and Western blotting. Immunization of mice resulted in a CSFV-neutralizing antibody titer of 1：128 and a PRRSV-neutralizing antibody titer of 1：16. The lymphoproliferative responses were detected by Cell Counting Kit-8 assay and the stimulation index of CFSV-specific and PRRSV-specific lymphocytes in the rAdV-GP52AE2 group was significantly higher than that in the negative control group. The results show that rAdV-GP52AE2 can induce both effective humoral and cell-mediated immune responses in mice. The protective efficacy of the recombinant virus against CSF was evaluated in immunized rabbits, which were protected from fever induced by challenge with C-strain. Our study provides supporting evidence for the use of FMDV 2A to develop a bivalent genetically-engineered vaccine.

Evaluation of the Pathogenicity of a Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus Variant in Piglets

Since May 2006,a highly pathogenic porcine reproductive and respiratory syndrome virus （HP-PRRSV） variant characterized by 30 amino acids deletion within its NSP2-coding region emerged and caused extensive economic losses to China＇s pig industry.To investigate the in vivo pathogenicity and immune responses of the newly emerging PRRSV,3 groups of 60-d-old conventional piglets were inoculated intranasally with a representative strain of the HP-PRRSV variant HuN4 with 3 different infection doses （3×103-3×105 TCID50）.The results revealed that the virus variant caused severe disease in piglets and the significant clinical characteristics consisted of persistently high fever （41.0-41.9oC） and high morbidity and mortality （60-100%）,the marked clinical signs of PRRS and severe histopathogenic damages in multiple organs.It induced rapid and intense humoral immune responses and seroconversion was detected in most infected pigs at 7 d post-infection （DPI）.The virus vigorously replicated in vivo and the highest virus average titer was 9.7 log copies mL-1 serum at 7 DPI.Elevated levels of IFN-g and IL-10 cytokine production in serum in this study were also observed.Taken together,our results demonstrated that the HP-PRRSV variant HuN4 strain is highly pathogenic for piglets and suitable to be a reference strain of highly virulent PRRSV for evaluating the efficacy of the new vaccines.

Enterogenous infection of Candida albicans in immunocompromised rats under severe acute pancreatitis

BACKGROUND:Opportunistic infection of Candida albicans(C.albicans) has become a serious problem in immunocompromised patients.The study aimed to explore the mechanism of enterogenous infection of C.albicans in immunocompromised rats under severe acute pancreatitis(SAP).METHODS:Sprague Dawley(SD) rats(n=100) were randomly assigned into 5 groups as the following:blank group,cyclophosphamide+ceftriaxone+SAP group,cyclophosphamide+ceftriaxone group,cyclophosphamide+SAP group,and cyclophosphamide group.The rats were sacrificed at 5and 10 days,and their jejunum,colon,mesenteric lymph nodes,pancreas,intestinal content,and blood were quickly collected to detect C.albicans.A region of the 25 S rRNA gene was chosen and amplified by polymerase chain reaction(PCR) to differentiate C.albicans genotypes.The amplified products were further sequenced and compared to judge their homology.RESULTS:Compared with the Cyclophosphamide group,the combination of immunosuppressants and broad-spectrum antibiotics significantly increased the colonization of C.albicans in intestine in 5 and 10 days.Pure SAP stress did not increase the opportunistic infection of C.albicans.The PCR products of C.albicans isolates all belonged to the genotype A family,and sequence alignment showed that the amplified fragments were homologous.CONCLUSION:The damage of immune system and broad-spectrum antimicrobial agents are important risk factors for opportunistic fungal infection.Intestinal tract is an important source for genotype-A C.albicans to translocate and invade into bloodstream.

Erythromycin Resistance of Streptococcus suis Isolates In Vivo

In order to study erythromycin resistance of Streptococcus suis under high or low concentration of selective drug pressure, Streptococcus suis strain LN was isolated from a diseased pig in 2005 and showed to be susceptible to erythromycin as determined by disc diffusion and tube dilution tests. In this study, clean level rabbits were divided into three groups of six rabbits each, including a prevention dosage group, a treatment dosage group, and a control group. After injection with S. suis strain LN, erythromycin （20 μg mL^-1） was taken orally in the prevention dosage group, erythromycin （5 mg kg^-1） was injected intramuscularly in the treatment dosage group, and no treatment was given in the control group. S. suis with intermediate resistance to erythromycin was isolated on the 5th day after infection from the prevention dosage group （5th PDG） and on the 7th day after infection from the treatment dosage group （7th TDG）. Both isolates were determined to be the constitutive macrolide-lincosamide-streptogramin B （cMLSB） resistance phenotype. The resistance gene ermB was detected in all of the isolates. The results suggested that both the 5th PDG and 7th TDG isolates had a mutation （A2372T） in the 23S rRNA gene. In addition, the 5th PDG isolates had a mutation in ribosomal protein L4 （detected as G268A） and a mutation in ribosomal protein L22 （A345C）; and the 7th TDG isolates had a C insertion at site 564. Each of these mutations is considered as a possible mechanism of erythromycin resistance in S. suis strain LN. This study demonstrated that erythromycin resistance was readily induced in S. suis at a low erythromycin dose creating selective pressure in vivo. Resistance appeared to be mediated by ribosome methylation, encoded by the ermB gene.

Biological Characteristics and Etiological Significance of Porcine Respiratory Coronavirus(PRCV)

Porcine respiratory coronavirus （PRCV）, a spike （S） gene natural deletion mutant of transmissible gastroenteritis virus （TGEV）, causes porcine respiratory disease complex. Research advances on porcine respiratory coronavirus were reviewed from four aspects of biological character, the model fimction for SARS-CoV research, contribution of the immunity to PRCV to protection against TGEV challenge exposure and other etiological significance

Unusual outcome of in utero infection and subsequent postnatal super-infection with different PCV2b strains

VC2002, isolated from postweaning multisystemic wasting syndrome(PMWS)-affected pig, is a mixture of two porcine circovirus genotype 2b(PCV2b) viruses, K2 and K39. Preliminary experiments disclosed short-term adverse effects of K39, but not K2, on porcine foetuses. These findings led to the hypothesis that infection of immuno-incompetent foetuses with K2 confers a status of immunotolerance, and postnatal super-infection with K39 triggers PMWS. To explore this hypothesis, nine 55-day-old foetuses were inoculated in utero(three with K2-104.3TCID50, three with K39-104.3TCID50 and three with medium), and foeto-pathogenicity examined. At 21 days post-inoculation(dpi), K2 did not induce pathology, whereas pathological effects of K39 were evident. Twenty-four 45-day-old foetuses were subsequently inoculated to examine the long-term effect of K2, including six with K2-high dose-104.3TCID50, six with K2-low dose-102.3TCID50 and 12 mock-inoculated controls. Both doses resulted in five mummified foetuses and one live-born piglet each(69dpi). K2 was recovered from all mummies. K2 and K2-specific antibodies were not detected in serum of the two live-born piglets at birth, indicating full control of K2 infection. The K2-low dose-infected piglet was immunostimulated at day 2, but not the K2-high dose-infected piglet. Both non-stimulated and stimulated K2-infected piglets were super-inoculated with K39 at day 6 or 8(taken as 0 days post super-inoculation). Low viral replication was observed in the non-stimulated K2-K39 piglet(up to 103.3 TCID50/g; identified as K39). In contrast, viral replication was extremely high in the stimulated K2-K39 piglet(up to 105.6TCID50/g) and identified as K2, indicating that K2 infection is controlled during foetal life, but emerges after birth upon immunostimulation. However, none of the piglets showed any signs of PMWS.

Effect of Candida albicans on Intestinal Ischemia-reperfusion Injury in Rats

Background： Inflammation is supposed to play a key role in the pathophysiological processes of intestinal ischemia-reperlhsion injury （IIRI）, and Candida albicans in human gut commonly elevates inflammatory cytokines in intestinal mucosa. This study aimed to explore the effect of C. albicans on IIRI. Methods： Fifty female Wistar rats were divided into five groups according to the status of C. a/bicans infection and IIRI operation： group blank and sham; group blank and IIRI; group cetbperazone plus IIR1; group C. albicans plus cetbperazone and IIRI （CCI）; and group C. albicans plus cefbperazone and sham. The levels of inflammatory factors tumor necrosis factor （TNF）-a, interleukin （IL）-6, IL- 1 β, and diamine oxidase （DAO） measured by enzyme-linked immunosorbent assay were used to evaluate the inflammation reactivity as well as the integrity of small intestine. Histological scores were used to assess the mucosal damage, and the C. albicans blood translocation was detected to judge the permeability of intestinal mucosal barrier. Results： The levels of inflammatory factors TNF-a, IL-6, and IL-1β in serum and intestine were higher in rats undergone both C. albicans infection and IIRI operation compared with rats in other groups. The levels of DAO （serum： 44.13 ± 4.30 pg/ml, intestine： 346.21 ± 37.03 pg/g） and Chiu scores （3.41 ± 1.09） which reflected intestinal mucosal disruption were highest in group CCI after the operation. The number ofC. albicans translocated into blood was most in group CCI （[33.80 ± 6.60] x 10-2 colony forming unit （CFU）/ml）. Conclusion： Intestinal C. albicans infection worsened the llRl-induced disruption of intestinal mucosal barrier and facilitated the subsequent C. alhicans translocation and dissemination.