Clinical Effect of Yinhuang Qingfei Capsules in Treatment of Asymptomatic and Mild/Common Severe Acute Respiratory Syndrome Coronavirus 2 Infection:An Analysis of 242 Cases

[Objectives]To investigate the clinical effect of Yinhuang Qingfei capsules in the treatment of asymptomatic and mild/common severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)infection.[Methods]A total of 362 patients with SARS-CoV-2 infection were divided into the treatment group with 242 patients and control group with 120 patients according to their treatment regimen.The patients in the control group were given standard treatment regimen and those in the treatment group were given Yinhuang Qingfei capsules in addition to the treatment in the control group.The two groups were observed in terms of average length of hospital stay,mean time for nucleic acid clearance,TCM syndrome score,and progression to severe/critical illness,and clinical outcome was compared between the two groups.[Results]There was a significant difference in the overall response rate between the treatment group and the control group[97.52%(236/242)vs 95.00%(114/120),P<0.05].Compared with the control group,the treatment group had significantly shorter length of hospital stay and time for nucleic acid clearance(P<0.05).After 7 days of treatment,both groups had a significant change in TCM syndrome score,and there was a significant difference in TCM syndrome score between the two groups(P<0.05);after 15 days of treatment,both groups had a TCM syndrome score of 0.Progression to severe/critical illness was not observed in either group.[Conclusions]Compared with the standard treatment regimen alone,standard treatment regimen combined with Yinhuang Qingfei capsules can effectively shorten the length of hospital stay and time for nucleic acid clearance and improve TCM symptoms in patients with asymptomatic and mild/common SARS-CoV-2 infection.

Huu S. TIEU’s Predicting Outcome of Severe Acute Respiratory Syndrome (SARS) and Preparing the Treatment for COVID-19 (Coronavirus) and Other Viral Pandemics

This project paper is to give a prediction for the future of other viral pandemics and to provide recommendations for preparing therapies that could help in the success of effective treatments and benefits for patients in life-threatening situations. The theory of prediction was proposed by Huu S. TIEU on March 25, 2019, and he hypothesized that any malfunctioning cell in the body could have a damaging effect. This paper discusses the prediction that Localized Oxygen Deprivation could be a contributing factor for a future epidemic or other viral pandemics that could affect body function. This paper is based on opinion and does not have sufficient evidence to support the claims made. Therefore, further in-depth study is needed to prove the findings. The author cites Hypoxia to support his idea, but he is not claiming that Hypoxia-Inducible Factor (HIF) has worked on his predictions. The author also tested a theory using cow blood curd for body function, but this test was not a structured test and the findings were not supported by other evidence. To further prove the idea or theory, further study into the subject should be conducted.

Severe acute respiratory syndrome coronavirus 2 may cause liver injury via Na^(+)/H^(+) exchanger

The liver has many significant functions,such as detoxification,the urea cycle,gluconeogenesis,and protein synthesis.Systemic diseases,hypoxia,infections,drugs,and toxins can easily affect the liver,which is extremely sensitive to injury.Systemic infection of severe acute respiratory syndrome coronavirus 2 can cause liver damage.The primary regulator of intracellular pH in the liver is the Na+/H+exchanger(NHE).Physiologically,NHE protects hepatocytes from apoptosis by making the intracellular pH alkaline.Severe acute respiratory syndrome coronavirus 2 increases local angiotensin II levels by binding to angiotensinconverting enzyme 2.In severe cases of coronavirus disease 2019,high angiotensin II levels may cause NHE overstimulation and lipid accumulation in the liver.NHE overstimulation can lead to hepatocyte death.NHE overstimulation may trigger a cytokine storm by increasing proinflammatory cytokines in the liver.Since the release of proinflammatory cytokines such as interleukin-6 increases with NHE activation,the virus may indirectly cause an increase in fibrinogen and D-dimer levels.NHE overstimulation may cause thrombotic events and systemic damage by increasing fibrinogen levels and cytokine release.Also,NHE overstimulation causes an increase in the urea cycle while inhibiting vitamin D synthesis and gluconeogenesis in the liver.Increasing NHE3 activity leads to Na+loading,which impairs the containment and fluidity of bile acid.NHE overstimulation can change the gut microbiota composition by disrupting the structure and fluidity of bile acid,thus triggering systemic damage.Unlike other tissues,tumor necrosis factor-alpha and angiotensin II decrease NHE3 activity in the intestine.Thus,increased luminal Na+leads to diarrhea and cytokine release.Severe acute respiratory syndrome coronavirus 2-induced local and systemic damage can be improved by preventing virus-induced NHE overstimulation in the liver.

Expression of Porcine Reproductive and Respiratory Syndrome Virus ORF7 Gene and Purification and Immunological Activity Analysis of the Recombinant Protein

[Objective] The aim of this study was to realize efficient expression of the porcine reproductive and respiratory syndrome virus （PRRSV） ORF7 gene in genetic engineering bacteria and analYze the immunological activity of the recombinant protein after purification. [ Method] The constructed recombinant expression vector pET-ORF7 was transformed into Escherichia co1BL21 （DE3） and induced by IPTG under the optimal condition. After analysis of SDS-PAGE and Western Blot, the expression products were purified by Ni-NTA His · Bind Resin chrom- atographic column under denaturing condition and renatured by gradient dialysis. Subsequently, the immunological activity of the renatured recombinant protein was detected by Westem Blot and indirect ELISA. [ Result] The recombinant plasmid pET-ORF7 expressed in E. coli successfully, and the fusion protein was in the form of inclusion body. By SDS-PAGE detection, the molecular weight of the expression protein was approximate 33 kD, according with the expectation. Analysis by Bandscan software showed that the expressed fusion protein was about 50% of total bacterial protein of BL21 （DE3）. Wastem Blot and indirect ELISA detection showed that the renatured protein could react with PRRSV positive serum specifically, indicating its good immunological activity. [ Conclusion] This study lays a foundation for the preparation of PRRSV monoclonal antibody and diagnostic kit.

Location of Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus in Tissues of Natural Cases

[ Objective] The aim of this study was to provide a theoretical basis for the prevention and treatment of highly pathogenic porcine reproductive and respiratory syndrome （HP-PRRS）. [Method] Antigen location and histopathological observation in natural cases infected by highly pathogenic porcine reproductive and respiratory syndrome virus （HP-PRRSV） were analyzed by immunohistochemistry and H. E. staining. [Result] The virus antigen mainly existed in epithelial calls, and also a few in mecrophages, lymphocytes and brain nerve cells. [ Conclusion] The cell and tissue tropism of HP-PRRSV strain in natural cases is different from that of previous strains.

Purification and Immunocompetence Analysis of GP5 Protein in Porcine Reproductive and Respiratory Syndrome

[Objective] The purification and immunocompetence of GPS protein in porcine reproductive and respiratory syndrome (PRRS) were analyzed in this study, which provided basis for establishing the corresponding serological method. [Method] The recombinant expression plasmid pGEX-6P-5 was transformed into BL21 and expressed after being induced with IPTG. The solubility analysis of expression products was carried out, and then the recombinant protein was purified for SDS-PAGE identification and Western-blot analysis. Finally, the recombinant antigen was used in the immune experiment of guinea pigs. [Result] The target protein content accounted for 30% of the total cells protein content according to the chromatography scanning, and the purity of target protein after purification reached 80%. The purified protein was analyzed by Western-blot and immune experiment of guinea pigs, and the results showed that the expressed protein had good reactionogenicity and immunogenicity. [Conclusion] This study provides materials for further studies on the function between PRRSV ORF5 gene and its editing protein, which also lays a foundation for porcine reproductive and respiratory syndrome virus genetic engineering products.

Analysis on Heredity and Variation of the ORF_5 Gene of Prevalence Strains Porcine Reproductive and Respiratory Syndrome Virus

[ Objective] The aim was to analyze the reason and epidemic trend of PRRSV, and provide theoretical basis for preventing and controlling PRRS. [Methed]According to the sequence of ATCC VR-2332 strain porcine reproductive and respiratory syndrome virus published by the GenBank, the primers were designed and synthesized. ORF5 gene sequences of seven prevalence strains were amplified by RT-PCR. The sequences of ORF5 genes were analyzed by DNAStar and compared with those of ATCC VR-2332, CH-1 a, B J-4, LV-M96262 and MLV vaccine strains, phylogenetic tree among isolates was analyzed. [Result] Analysis of nucleotide sequence showed that the homology was 88.1% - 98.8%, 89.9% -95.2%, 85.6% -98.7% between ORF5 genes of seven prevalence strains and VR-2332, CH-1a, BJ-4, and the homology was 54.7% -56.9% between ORF5 genes and LV. Analysis of amino acid sequence showed that the homology was 88.1% -96.8%, 88.1% - 94.5%, 86.1% -96.5% between ORF5 genes of seven prevalence strains and VR-2332, CH-1a, bBJ-4, the homology was 54.7% -56.2% between the ORF5 genes and LV.[ Conclusion] The variation of prevalence strains was great in the ORF5 gene region, the homology of ORF5 gene sequence was higher and genetic relationship was nearer during prevalence strains in the same region, or was far in different regions.

Fear can be more harmful than the severe acute respiratory syndrome coronavirus 2 in controlling the corona virus disease 2019 epidemic

The current corona virus disease 2019 outbreak caused by severe acute respiratory syndrome coronavirus 2 started in Wuhan,China in December 2019 and has put the world on alert.To safeguard Chinese citizens and to strengthen global health security,China has made great efforts to control the epidemic.Many in the global community have joined China to limit the epidemic.However,discrimination and prejudice driven by fear or misinformation have been flowing globally,superseding evidence and jeopardizing the anti-severe acute respiratory syndrome coronavirus 2 efforts.We analyze this phenomenon and its underlying causes and suggest practical solutions.

Genetic Variation of the ORF5 Gene of Porcine Reproductive and Respiratory Syndrome Virus in East China during 2008-2010

[Objective] A total of 260 swine samples of dead or sick pigs collected from 7 provinces （municipalities） Jiangsu, Anhui, Shanghai, Shandong, Zhejiang, Fujian and Jiangxi of China during 2008-2010 were detected for porcine reproductive and respiratory syndrome virus （PRRSV）. And the ORF5 genes of some isolates were amplified and sequenced for understanding the molecule epidemiology and the genetic evolution of PRRSV in East China. [Method] Using RT-PCR method, PRRSV was detected by RT-PCR from samples. The complete ORF5 genes of 36 PRRSV positive samples was amplified, sequenced and analyzed with other 15 strains available on GenBank. [Result] PRRSV was detected in 118/260 of the clinical samples, with a positive rate was 45.4%. Sequence analysis showed that the 36 isolates of this study belonged to the North American-type PRRSV strains and were closely related to the highly pathogenic PRRSV （HP-PRRSV） with 94.6%-100% amino acid sequence identities. The sequence analysis combined with the phylogenetic analysis indicated that all these North American-type PRRSV strains in East China were further divided into five subgenotypes, subgenotype Ⅲ showed closer identity with HPPRRSV; almost all subgenotypes were found to be variable in the primary neutralizing epitope; subgenotypes Ⅲ and IV had more glycosylation sites than others. Although these 36 isolates were collected from different provinces in East China, there were no obvious relations between the distribution of PRRSV and the region. [Conclusion] The PRRSV infection was widespread and HP-PRRSV was the popular strain in East China during 2008-2010. However some different genetic characteristics appeared in the genomes, the genetic evolution was relatively stable. There exists a cross-cutting phenomenon on the genetic relationship of PRRSV isolates obtained from different provinces. Subgenotypes IV and V only appeared in some provinces, but the distribution of PRRSV did not show apparent geographical characteristics.

Development of an Improved DNA-launched Porcine Reproductive and Respiratory Syndrome Virus Reverse Genetics System

[Objective] This study was to improve the virus replication efficiency of full length infectious cDNA clones by making use of the ribozyme＇s self incision property.[Method] By employing three-step PCR,HDV ribozyme（HdvRz）cDNA was isolated,and cloned into the downstream flanking the genome of the porcine reproductive and respiratory syndrome virus,and into which the bovine growth hormone polyadenylation sequence（BGH）was inserted via enzyme digestion and ligation,yielding pAPRRS-HB.The newly constructed pAPRRS-HB was used to transfect MARC-145 cells,in which the N protein and non-structural protein（nsp2）were determined by indirect immunofluorescence assay after 72 h of expression;meanwhile the virus titer of cell supernatant was tested using TCID50 assay.[Result] pAPRRS-HB containing complete infectious PRRSV cDNA has been successfully developed,and it performed about 10-fold higher virus rescue rate than pAPRRS without the engineered ribozyme element.[Conclusion] The results laid a foundation for revealing the structure and function of PRRSV gene.

Use of a Multiplex RT-PCR Assay for Simultaneous Detection of the North American Genotype Porcine Reproductive and Respiratory Syndrome Virus,Swine Influenza Virus and Japanese Encephalitis Virus

A multiplex reverse transcriptase-polymerase chain reaction(multiplex RT-PCR) assay was developed and subsequently evaluated for its efficacy in the detection of multiple viral infections simultaneously,in swine.Specific primers for each of the 3 RNA viruses,North American genotype porcine reproductive and respiratory syndrome virus,Japanese encephalitis virus,and swine influenza virus,were used in the testing procedure.The assay was shown to be highly sensitive because it could detect as little as 10-5 ng of each of the respective amplicons in a single sample containing a composite of all 3 viruses.The assay was also effective in detecting one or more of the same viruses in various combinations in specimens,including lymph nodes,lungs,spleens,and tonsils,collected from clinically ill pigs and in spleen specimens collected from aborted pig fetuses.The results from the multiplex RT-PCR were confirmed by virus isolation.The relative efficiency(compared to the efficiency of separate assays for each virus) and apparent sensitivity of the multiplex RT-PCR method show that this method has potential for application in routine molecular diagnostic procedures.

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.

Vaccination of Plasmid DNA Encoding Somatostatin Gene Fused with GP5 Gene of Porcine Reproductive and Respiratory Syndrome Virus Induces Anti-GP5 Antibodies and Promotes Growth Performance in Immunized Pigs

Somatostatin （SS） is a hormone that inhibits the secretion of growth hormone. Immunization against SS can promote the growth of animals. This paper described the effects of DNA immunization on the growth and antibody response in mice and pigs immunized with a plasmid DNA encoding SS fused with GP5 of porcine reproductive and respiratory syndrome virus （PRRSV）. A fragment of 180 bp encoding partial SS gene was amplified by PCR from the genomic DNA of peripheral blood mononuclear cells of pigs, and cloned as a fusion gene with PRRSV GP5 in plasmid pISGRTK3. Three times of immunization with the resulting plasmid pISG-SS/GP5 induced anti-GP5 antibodies in BALB/c mice and pigs, as demonstrated by GP5-specific ELISA and immunoblotting. Compared with pigs immunized with empty vector pISGRTK3, the growth performance of pigs immunized with pISG-SS/GP5 was increased by 11.1% on the 13th week after the last vaccination. The results indicated the plasmid DNA encoding SS and PRRSV GP5 fusion gene elicited anti-GP5 antibodies and improved the growth performance of immunized pigs.

Epidemiological Characteristics of An Outbreak of Severe Acute Respiratory Syndrome in Dongcheng District of Beijing From March to May 2003

Objective To describe epidemiologic features of an outbreak of severe acute respiratory syndrome (SARS) in Dongcheng District, Beijing occurred in a period between March and May 2003. Methods Data of SARS cases notified from Dongcheng District Center for Disease Control and Prevention (CDC )and supplemented by other channels were collected. Clinicians and officials of local hospitals were interviewed in groups and medical records of fatal cases of SARS were reviewed to verify the diagnosis. Stored serum specimens of the patients were detected for IgG antibody against SARS Co-V by enzyme-linked immunosorbent assay (ELISA). All the data were input into dataset files by Microsoft Excel-2000 software and analyzed with SPSS version 10.0 software. Results Outbreak of SARS in Dongcheng District started on March 14, 2003 with a peak in mid- and late April, and dropped in early May. A total of 572 reported cases were collected during this period in Dongcheng District, Beijing, and 99 of them were excluded from SARS, because of diagnosis of common cold, regular pneumonia, measles and rubella, etc. Actually, 473 SARS cases, which included 390 (82.5%) probable cases and 83 (17.5%) suspect cases, were analyzed. About 90% of the probable cases were positive for IgG antibody. Attack rate of SARS in permanent residents of Dongcheng District was 28.3 per 100 000. Forty-one of them died, with a case-fatality rate of 8.7%. Persons were all susceptible to infection of SARS Co-V, with the highest proportion at ages of 20-50 years, which accounted for 68.7% of the total cases. Average age of the patients at their onset was 40.7 years. No gender difference in SARS cases was found. Number of SARS cases in health-care workers (HCWs) accounted for 18.0% and that in retired workers accounted for 15.4% of the total cases. Cases occurred in all 10 sub-districts of Dongcheng, with the highest in Beixinqiao and Andingmen Sub-districts. Totally, 230 of the 572 notified cases (40.2%) were hospitalized at local hospitals under the jurisdiction of Dongcheng District. Eighteen of 85 cases of SARS occurred in HCWs of local hospitals, accounting for 4.5% of the total number of HCWs working at wards caring for SARS patients or fever clinics. There were 34.7% of SARS cases without any histories of contact before the onset of the disease. Familial aggregation phenomena were observed in 41.8% of the cases and 18.1% of households. And 7.4% (attack rate ) of those exposed to SARS cases suffered from the illness during the periods of quarantine. Conclusions SARS appeared to be infectious in origin and caused outbreak in Dongcheng District, Beijing introduced by an imported case traveling from Hong Kong in a period between March and May 2003. People are all susceptible to infection of SARS Co-V, which mainly threatens the young adults and the middle-aged, as well as HCWs and the retired workers. The main mode of transmission is direct exposure to SARS patients in a near distance at hospitals or families via droplets spread. Prevention and control of SARS should be focused on early isolation of patients and quarantine for close contacts. Current available measures to prevent and control SARS are proved to be effective.

Infectivity of Severe Acute Respiratory Syndrome during Its Incubation Period

Objective To evaluate the infectivity of severe acute respiratory syndrome （SARS） during its incubation period by investigating chains of transmission and individuals isolated for medical observation with a view to providing scientific evidence for updating protocols of medical isolation. Methods Individuals related with the two SARS chains of transmission in Beijing in 2003 and a group of individuals isolated for medical observation in Haidian district of Beijing during the SARS outbreak were selected as subjects of study. Contactors with SARS patients and those with symptom development following the contacts were investigated via questionnaire. Serum samples were collected from super transmitters and tested for SARS-CoV antibody by neutralization test and enzyme linked immunosorbent assay （ELISA）. Results A total of 1 112 contactors were investigated in three surveys. Of them, 669 had a history of close contact with symptomatic SARS patients, 101 developed symptoms with a rate of 15.1%, 363 had a history of close contact with patients in their incubation period, none of whom developed symptoms （0%）. Serum samples were collected fi＇om 32 highly-exposed individuals, of whom 13 developing SARS symptoms atter contact had serum samples positive for SARS-CoV antibody. Samples collected from the asymptomatic contactors were all negative for SARS-CoV antibody. Conclusion SARS cases are infectious only during their symptomatic period and are non-infectious during the incubation period. Isolation for medical observation should be placed for individuals who are in close contact with symptomatic SARS patients. The results of our study are of decisive significance for the Ministry of Health to the definition of SARS close contactor.

Elevated Plasma Tissue-type Plasminogen Activator (t-PA) and Soluble Throm-bomodulin in Patients Suffering From Severe Acute Respiratory Syndrome (SARS) as a Possible Index for Prognosis and Treatment Strategy

To detect the presence of endothelial injury in patients with severe acute respiratory syndrome （SARS） via enhanced levels of tissue-type plasminogen activator （t-PA） and soluble thrombomodulin （sTM）. Methods Case patients were from Xuanwu Hospital （Capital University of Medical Sciences, Beijing, China）, and all of them met clinical criteria for SARS. Healthy controls were some of the hospital employees. Endothelial injury bio-markers tPA and sTM were detected by commercial ELISA-methods. Results Classic plasma markers of endothelial injury, tPA and sTM significantly elevated in SARS patients in comparison to controls [t-PA： 1.48±0.16 nmol/L versus 0.25±0.03 nmol/L （P〈0.0001）, and sTM： 0.26±0.06 nmol/L versus 0.14±0.02 nmol/L （P〈0.05）]. The only patient who died had extremely high levels of these endothelial injury markers （t-PA： 2.77 nmol/L and sTM： 1.01 nmol/L）. The likelihood ratio analysis indicated the excellent discriminating power for SARS at the optimal cut-point of 0.49 nmol/L for tPA and 0.20 nmol/L for sTM, respectively. Significant numerical correlations were found among these endothelial injury markers in SARS patients. The numerical coefficient of correlation Pearson r between t-PA and sTM was 0.5867 （P〈0.05）. Conclusion Increased plasma concentrations of tPA and sTM in patients with SARS suggest the possibility of endothelial injury. SARS patients might need anticoagulant therapy or fibrinolytic therapy in order to reverse intraalveolar coagulation, microthrombi formation, alveolar and interstitial fibrin deposition. It may not only provide a useful treatment and prognostic index but also allow a further understanding of the pathological condition of the disease.

Effects of Integrative Chinese and Western Medicine on Arterial Oxygen Saturation in Patients with Severe Acute Respiratory Syndrome

Objective: To evaluate the effects of treatment of integrative Chinese and Western medicine (ICWM) on arterial oxygen saturation (SaO2) in patients with severe acute respiratory syndrome (SARS). Methods:The non-randomized, controlled trial was conducted on 447 SARS patients treated synchronously with western conventional treatment (WM group, n = 171) alone and ICWM (ICWM group, n = 276). The changes of the cases with normal level (≥95% ) or abnormal level (<95% ) SaO2 were observed dynamically. Results: In the 3rd-14th day of the therapeutic course, the percentage of patients with normal SaO2 in the ICWM group was higher than that in the WM group (OR = 0.5178, P = 0.0038), and this tendency was more evident in patients of the severe type (OR = 0. 18, P = 0. 0001). However, the statistical significance of difference was only shown in patients for whom the ICWM treatment started in the early period after the onset (≤7 days after it, OR = 0.3803, P = 0.006), but not shown in those who received ICWM treatment later in the mid-late period of SARS ( P>0. 05). Conclusion: ICWM treatment, particularly when it is used for intervention in the early stage, is beneficial for maintaining normal SaO2 in SARS patients.

Application of GP5 Protein to Develop Monoclonal Antibody against Porcine Reproductive and Respiratory Syndrome Virus

In this study, a panel of monoclonal antibodies （mAbs） against Porcine reproductive and respiratory syndrome virus（PRRSV）, named as 8C9 and4B4, were produced by fusing SP2/0 myeloma cells and spleen cells of BALB/c mice immunized with the PRRSV （TCID50=5.5）, screened by the indirect ELISA and subjected to several limiting dilutions, mAbs were then identified by biological characterization. Among the two fusion cell strains, 8C9 belonged to the IgG1 subclass and 4B4 belonged to the IgG2a subclass. The titers in cell culture supematant and abdomen liquor reached to 1：104and 1：105, respectively. The specificity test indicated that the two cells had specific reactions for the PRRSV and GP5 protein respectively, and no reaction with Classical swine fever virus （CSFV） or Swine vesicular disease virus （SVDV）. The molecular weights of the heavy chain and light chain were about 45.0 kDa and 25.0 kDa, respectively. In neutralization activity tests, the results showed that the prepared mAb 4B4 can protect 50% of cells with no CPE in dilution up to 1：512, but mAB 8C9 has no neutralization activities to PRRSV.

A direct real-time polymerase chain reaction assay for rapid high-throughput detection of highly pathogenic North American porcine reproductive and respiratory syndrome virus in China without RNA purification

Background： Porcine reproductive and respiratory syndrome virus （PRRSV）, and particularly its highly pathogenic genotype （HP-PRRSV）, have caused massive economic losses to the global swine industry. Results： To rapidly identify HP-PRRSV, we developed a direct reaL-time reverse transcription polymerase chain reaction method （dRT-PCR） that could detect the virus from serum specimen without the need of RNA purification Our dRT-PCR assay can be completed in 1.5 h from when a sample is received to obtaining a result. Additionally, the sensitivity of dRT-PCR matched that of conventional reverse transcription PCR （cRT-PCR） that used purified RNA The lowest detection limit of HP-PRRSV was 6.3 TCIDs0 using dRT-PCR. We applied dRT-PCR assay to 144 field samples and the results showed strong consistency with those obtained by cRT-PCR. Moreover, the dRT-PCR method was able to tolerate 5-20% （v/v） serum. Conclusions： Our dRT-PCR assay allows for easier, faster, more cost-effective and higher throughput detection of HP-PRRSV compared with cRT-PCR methods. To the best of our knowledge, this is the first report to describe a real-time RT-PCR assay capable of detecting PRRSV in crude serum samples without the requirement for purifying RNA. We believe our approach has a great potential for application to other RNA viruses.

Molecular Characterization of a Highly Pathogenetic Porcine Reproductive and Respiratory Syndrome Virus Variant in Hubei, China

Porcine reproductive and respiratory syndrome virus (PRRSV) has been recognized as one of the most important pathogens of pigs throughout the world. In 2006, more than 10 provinces of China have experienced an epizootic outbreak of pig diseases characterized by high fever, reddened skin and high morbidity and mortality. From June 2006 to April 2007, we have investigated some clinical samples in Hubei province by RT-PCR and cloned several major genes, N, GP5 and NSP2 gene, shown in this study. Phylogenetic analysis of these genes revealed that the highly pathogenic PRRSV variant, ZB, was responsible for 2006 emergent outbreak of pig disease in Hubei province similar with those variants isolated from other provinces in China in 2006, and belongs to the NA-type PRRSV. In the PRRSV variants, the N and GP5 shear about 90% identity with prototypic ATCC VR-2332 and some typical NA-type Chinese isolates, except the 2850bp NSP2 gene (only shares 65% identity with ATCC VR-2332). But they all shear more than and 97% identity with other highly pathogenetic Chinese PRRSV strains. Additionally, there are extensive amino acid (aa) mutations in the GP5 protein and 2 deletions in the Nsp2 protein when compared with the previous isolates. Most of the variants found in 2006 epizootic outbreak of pig diseases in China were the farthest variants from the typical NA-type PRRSV in phylogenetic distance, and these diversities may be responsible for the differences in the pathogenicity observed between these variants and original Chinese PRRSV strains.