Full-length cDNA cloning, molecular characterization and differential expression analysis of peroxiredoxin 6 from Ovis aries.

Peroxiredoxin 6 (Prdx6), an important antioxidant enzyme that can eliminate reactive oxygen species (ROS) to maintain homeostasis, is a bifunctional protein that possesses the activities of both glutathione peroxidase and phospholipase A2. In this study, a novel full-length Prdx6 cDNA (OaPrdx6) was cloned from Sheep (Ovis aries) using rapid amplification of cDNA ends (RACE). The full-length cDNA of OaPrdx6 was 1753bp containing a 5'-untranslated region (UTR) of 93bp, a 3'-UTR of 985bp with a poly(A) tail, and an open reading frame (ORF) of 675bp encoding a protein of 224 amino acid residues with a predicted molecular weight of 25.07kDa. The recombinant protein OaPrdx6 was expressed and purified, and its DNA protection activity was identified. In order to analyze the Prdx6 protein expression in tissues from O. aries, monoclonal antibodies against OaPrdx6 were prepared. Western blotting results indicated that OaPrdx6 protein could be detected in heart, liver, spleen, lung, kidney, stomach, intestine, muscle, lymph node and white blood cells, and the highest expression was found in lung while the lowest expression in muscle. Compared to the normal sheep group, the mRNA transcription level of Prdx6 in buffy coat was up-regulated in the group infected with a virulent field strain of Brucella melitensis, and down-regulated in the group inoculated with a vaccine strain S2 of brucellosis. The results indicated that Prdx6 was likely to be involved in the host immune responses against Brucella infection, and probably regarded as a molecular biomarker for distinguishing between animals infected with virulent Brucella infection and those inoculated with vaccine against brucellosis.

Fever with thrombocytopenia associated with a novel bunyavirus in China.

BACKGROUND: Heightened surveillance of acute febrile illness in China since 2009 has led to the identification of a severe fever with thrombocytopenia syndrome (SFTS) with an unknown cause. Infection with Anaplasma phagocytophilum has been suggested as a cause, but the pathogen has not been detected in most patients on laboratory testing. METHODS: We obtained blood samples from patients with the case definition of SFTS in six provinces in China. The blood samples were used to isolate the causal pathogen by inoculation of cell culture and for detection of viral RNA on polymerase-chain-reaction assay. The pathogen was characterized on electron microscopy and nucleic acid sequencing. We used enzyme-linked immunosorbent assay, indirect immunofluorescence assay, and neutralization testing to analyze the level of virus-specific antibody in patients' serum samples. RESULTS: We isolated a novel virus, designated SFTS bunyavirus, from patients who presented with fever, thrombocytopenia, leukocytopenia, and multiorgan dysfunction. RNA sequence analysis revealed that the virus was a newly identified member of the genus phlebovirus in the Bunyaviridae family. Electron-microscopical examination revealed virions with the morphologic characteristics of a bunyavirus. The presence of the virus was confirmed in 171 patients with SFTS from six provinces by detection of viral RNA, specific antibodies to the virus in blood, or both. Serologic assays showed a virus-specific immune response in all 35 pairs of serum samples collected from patients during the acute and convalescent phases of the illness. CONCLUSIONS: A novel phlebovirus was identified in patients with a life-threatening illness associated with fever and thrombocytopenia in China. (Funded by the China Mega-Project for Infectious Diseases and others.).

[Development of a protocol on pulsed field gel electrophoresis analysis for Streptococcus suis].

OBJECTIVE: To develop a PFGE protocol for Streptococcus suis. METHODS: We developed and optimized a PFGE protocol for S. suis, in terms of plug preparation, choice of restriction endonucleases and optimized electrophoresis parameters. By analyzing the genome sequences of S. suis P1/7 with Mapdraw of DNAStar, we found three restriction enzymes, Swa I, Sma I and Apa I, were more suitable than others. RESULTS: Analysis of 100 isolates of S. suis including 34 of 35 serotypes identified, 59, 53 and 43 patterns were obtained from Swa I, Sma I and Apa I restriction, respectively. The enzyme Swa I had the greatest power for discrimination ability. CONCLUSION: By optimization of the protocol at various conditions, a rapid, reproducible, economic and practical PFGE method for S. suis was developed.

[An outbreak of human Streptococcus suis serotype 2 infections presenting with toxic shock syndrome in Sichuan, China].

OBJECTIVE: In mid-July 2005, five patients presented with septic shock to a hospital in Ziyang city in Sichuan, China, to identify the etiology of the unknown reason disease, an epidemiological, clinical, and laboratory study were conducted. METHODS: An enhanced surveillance program were established in Sichuan, the following activities were introduced: active case finding in Sichuan of (a) laboratory diagnosed Streptococcus suis infection and (b) clinically diagnosed probable cases with exposure history; supplemented by (c) monitoring reports on meningococcal meningitis. Streptococcus suis serotype 2 infection was confirmed by culture and biochemical reactions, followed by sequencing for specific genes for serotype and virulence factors. RESULTS: From June 10 to August 21, 2005, 68 laboratory confirmed cases of human Streptococcus suis infections were reported. All were villagers who gave a history of direct exposure to deceased or sick pigs in their backyards where slaughtering was performed. Twenty six (38%) presented with toxic shock syndrome of which 15 (58%) died. Other presentations were septicaemia or meningitis. All isolates were tested positive for genes for tuf, species-specific 16S rRNA, cps2J, mrp, ef and sly. There were 136 clinically diagnosed probable cases with similar exposure history but incomplete laboratory investigations. CONCLUSION: An outbreak of human Streptococcus suis serotype 2 infections occurred in villagers after direct exposure to deceased or sick pigs in Sichuan. Prohibition of slaughtering in backyards brought the outbreak to a halt. A virulent strain of the bacteria is speculated to be in circulation, and is responsible for the unusual presentation of toxic shock syndrome with high case fatality.

[Surveillance on severe acute respiratory syndrome associated coronavirus in animals at a live animal market of Guangzhou in 2004].

OBJECTIVE: To study the prevalence of severe acute respiratory syndrome coronavirus (SARS-CoV) like virus in animals at a live animal market of Guanzhou in 2004 before and after culling of wild animal action taken by the local authority, in order to predict the re-emerging of SARS from animal originals in this region. METHODS: Animals at live animal market were sampled for rectal and throat swabs in triplicate. A single step realtime reverse transcription-polymerase chain reaction (RT-PCR) diagnostic kit was performed for screening SARS-CoV like virus, the manual nested RT- PCR and DNA sequencing were performed for confirmation. Only specimens which tested positive for both of the N and P genes by nested RT-PCR were scored as positive. RESULTS: In 31 animals sampled in January 5 2004 before culling of wild animals at Guangdong Province, including 20 cats (Felis catus), 5 red fox (Vulpes vulpes) and 6 Lesser rice field rats (Rattus losea), 8 (25.8%) animals were tested positive for SARS-CoV like virus by RT-PCR methods, of which 4 cats, 3 red fox and one Lesser rice field rats were included. However, two weeks after culling of animals and disinfection of the market were implemented, in 119 animals sampled in January 20 2004, including 6 rabbits (Oryctolagus cuniculus), 13 cats, 46 red jungle fowl (Gallus gallus), 13 spotbill duck (Anas platyrhynchos), 10 greylag goose (Anser anser), 31 Chinese francolin (Franclinus pintadeanus), only rectal swab from one greylag goose was tested positive for SARS-CoV like virus. Furthermore, in 102 animals that including 14 greylag gooses, 3 cats, 5 rabbits, 9 spotbill duck (Anaspoecilorhyncha), 2 Chinese francolin (Franclinus pintadeanus), 8 common pheasant (Phasianus colchicus), 6 pigeons, 9 Chinese muntjac (Muntiacus reevesi), 19 wild boar (Sus scrofa), 16 Lesser rice field rats, 5 dogs, 1 mink (Mustela vison), 3 goats, 2 green peafowl (Pavo muticus) sampled in April, May, June, July, August and November, only rectal swab from one pig was tested positive. However, of 12 and 10 palm civets sampled in November and December including five of which had been at the live animals market for 2 days, none of them was tested positive. CONCLUSION: This findings revealed that animals being sampled in April, May, June, July, August and November of 2004, only one rectal swab from a pig was tested positive as SARS-CoV like virus, much lower than the results from the previous year, suggesting that the possibility of re-emerging of human infection from animal origins is low for the winter of 2004-2005.