Recent outbreaks of severe acute respiratory syndrome (SARS) have spurred intense research efforts around the world to deal with the serious threat to health posed by this novel coronavirus. A rapid, reliable diagnostic assay is needed for monitoring the spread of the disease. Here we report a method for eliminating falsenegative results and increasing test sensitivity, based on the hypothesis that the message encoded by the nucleocapsid (N) gene is the most abundant during viral infection. Nasopharyngeal aspirates and stool samples were obtained from suspected SARS patients with major clinical symptoms and a significant history of close contact with infected patients. Total RNAs were extracted in a 96-well format, together with pig kidney epithelial (PK-15) cells as an internal control for extraction efficiency. PCR inhibitors were removed by ethanol precipitation, and a PCR for the pig ␤-actin gene was used as a positive control for all clinical samples. Samples were analyzed by a reverse transcriptase PCR assay. Northern blot analysis was performed to demonstrate differences in subgenomic transcripts of the virus, and a real-time quantitative PCR was employed to compare the sensitivities of two loci (1b and N). The detection rate of the assay reached 44.4% on day 9 after the onset of the disease. The diagnostic PCR amplifying the N gene gave an average of a 26.0% (6.3 to 60.0%) stronger intensity signal than that for the 1b gene. In conclusion, the nucleocapsid gene represents an additional sensitive molecular marker for the diagnosis of the SARS coronavirus and can be further adapted for use in a high-throughput platform assay.

BackgroundUnderstanding the effects of pretreatment on anaerobic digestion of sludge waste from wastewater treatment plants is becoming increasingly important, as impetus moves towards the utilization of sludge for renewable energy production. Although the field of sludge pretreatment has progressed significantly over the past decade, critical questions concerning the underlying microbial interactions remain unanswered. In this study, a metagenomic approach was adopted to investigate the microbial composition and gene content contributing to enhanced biogas production from sludge subjected to a novel pretreatment method (maintaining pH at 10 for 8 days) compared to other documented methods (ultrasonic, thermal and thermal-alkaline).ResultsOur results showed that pretreated sludge attained a maximum methane yield approximately 4-fold higher than that of the blank un-pretreated sludge set-up at day 17. Both the microbial and metabolic consortium shifted extensively towards enhanced biodegradation subsequent to pretreatment, providing insight for the enhanced methane yield. The prevalence of Methanosaeta thermophila and Methanothermobacter thermautotrophicus, together with the functional affiliation of enzymes-encoding genes suggested an acetoclastic and hydrogenotrophic methanogenesis pathway. Additionally, an alternative enzymology in Methanosaeta was observed.ConclusionsThis study is the first to provide a microbiological understanding of improved biogas production subsequent to a novel waste sludge pretreatment method. The knowledge garnered will assist the design of more efficient pretreatment methods for biogas production in the future.

RNA-Seq was used to unveil the transcriptional profile of DF-1 cells at the early stage of caIBDV infection. Total RNAs were extracted from virus-infected cells at 0, 6 and 12 hpi. RNA-Seq datasets of respective samples mapped to 56.5–57.6% of isoforms in the reference genome Galgal4.73. At 6 hpi, 23 isoforms underwent an elevated expression, while 128 isoforms were up-regulated and 5 were down-regulated at 12 hpi in the virus-infected group. Besides, 10 isoforms were exclusively expressed in the virus-infected cells. Though no significant change was detected in cytokine and interferon expression levels at the first 12 hours of infection, modulations of the upstream regulators were observed. In addition to the reported regulatory factors including EIF2AK2, MX, OAS*A, GBP7 and IFIT, IBDV infection also triggered a IFIT5-IRF1/3-RSAD5 pathway in the DF-1 cells which potentially restricted the viral replication cycle in the early infection stage. Over-expression of LIPA and CH25H, together with the suppression of STARD4, LSS and AACS genes implied a modulation of membrane fluidity and lipid raft arrangement in the infected cells. Alternative splicing of the EFR3 homolog A gene was also through to be involved in the lipid membrane regulation, and these cumulative responses projected an inhibition of viral endocytosis. Recognition of viral RNA genomes and intermediates was presumably enhanced by the elevated levels of IFIH1, DHX58 and TRIM25 genes which possess properties on detecting viral dsRNA. On the other hand, the caIBDV arrested the host's apoptotic process by inducing the expression of apoptosis inhibitors including NFKBIA/Z, TNFAIP2/3 and ITA at the first 12 hours of infection. In conclusion, the differential expression landscape demonstrated with RNA-Seq provides a comprehensive picture on the molecular interactions between host cells and virus at the early stage of infection.

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