A way forward of alternative methods in China: Implementation of skin corrosivity potential using in vitro reconstructed human epidermis.

The purpose of present study was to investigate the applicability of reconstructed human epidermis model to identify skin corrosive UN GHS Categories 1A, 1B/1C and non-corrosive chemicals in China. By using a commercialized reconstructed human epidermis model, China EpiSkin™ which had been proven to be applicable as a stand-alone test method to predict skin irritation in previous study, the predictive capacity of corrosion was assessed with 76 chemicals that included 30 reference chemicals recommended by OECD TG 431 in this study. The latter reference chemicals were tested in three runs, the within-laboratory reproducibility reached 100%, the accuracy was 90% for distinguishing corrosive and non-corrosive chemicals and 80% for sub-categorization (Cat. 1A vs Cat. 1B/1C vs non corrosive). Additional 46 chemicals were also tested, and the overall accuracy for sub-categorization of all 76 tested chemicals was 80.3% with 91.7% sensitivity for Category 1A, 82.1% sensitivity for category 1B/1C and 75% specificity which met all required predictive capacity by OECD. The present study results show that China EpiSkin™ model can be applied to predict sub-categorization 1A and 1B/1C of corrosive chemicals. The availability of skin corrosion in vitro test method provides the applicability of in vitro non-animal testing method for chemicals widely used in various industries, and will further support the implementation and promotion of alternative methods in China.

Short communication: Antiproliferative effect of 8 different Lactobacillus strains on K562 cells.

Some strains of Lactobacillus genus have antiproliferative activities against cancer cells. However, until now, the exact effector molecules of Lactobacillus strains with anticancer activity have not been identified. The aim of the present study was to explore which fraction of the Lactobacillus cells exerts the highest antiproliferative effect. For this purpose, the heat-killed bacterial cells, bacterial cell wall extract, and genomic DNA of 8 Lactobacillus strains were prepared to assess their antiproliferative activities against human myeloid leukemia cell lines K562. The heat-killed bacterial cells of the 8 lactobacilli strains exerted antiproliferative effect on K562 cells, and the inhibition rates exerted by the heat-killed bacterial cells of the strains G15AL, M5AL, SB31AL, SB5AL, and T3AL were significantly higher than those exerted by the cell walls and genomic DNA of the strains. The bacterial DNA of G15AL exerted higher antiproliferative effect on K562 cells. The exact effector molecules and the effect mechanism of the strains should be further explored for the application of these strains as probiotic strains or bioactive probiotic molecules.

Imported pigs may have introduced the first classical swine influenza viruses into Mainland China.

OBJECTIVES: The first classical swine influenza A H1N1 viruses were isolated in Mainland China in 1991. To aid surveillance of swine influenza viruses as part of pandemic preparedness, we sought to identify their origin. METHODS: We sequenced and phylogenically analyzed 19 swine influenza viruses isolated in 1991 and 1992 in China and compared them with viruses isolated from other regions during the same period. RESULTS: All 19 swine influenza viruses analyzed in our study shared the highest similarity with the classical swine influenza virus A/Swine/Maryland/23239/1991 (H1N1). Phylogenetic trees of eight segmented genes exhibited similar topology, with all segments in the cluster of classical swine influenza viruses. In addition, antigenic analysis also indicated that the tested isolated were related to classical swine influenza isolates. CONCLUSIONS: Classical swine H1N1 influenza viruses were predominant in Beijing pig herds during this period. Since both antibody and virus detections did not indicate the presence of CS H1N1 before 1991 in Mainland China, we combined with the data on pigs imported to and exported from China and concluded that these viruses might spread to China via pigs imported from North America and that they could affect the genetic evolution and transmission dynamics of swine influenza viruses in Hong Kong.

Mapping of H3N2 influenza antigenic evolution in China reveals a strategy for vaccine strain recommendation.

One of the primary efforts in influenza vaccine strain recommendation is to monitor through gene sequencing the viral surface protein haemagglutinin (HA) variants that lead to viral antigenic changes. Here we have developed a computational method, denoted as PREDAC, to predict antigenic clusters of influenza A (H3N2) viruses with high accuracy from viral HA sequences. Application of PREDAC to large-scale HA sequence data of H3N2 viruses isolated from diverse regions of Mainland China identified 17 antigenic clusters that have dominated for at least one season between 1968 and 2010. By tracking the dynamics of the dominant antigenic clusters, we not only find that dominant antigenic clusters change more frequently in China than in the United States/Europe, but also characterize the antigenic patterns of seasonal H3N2 viruses within China. Furthermore, we demonstrate that the coupling of large-scale HA sequencing with PREDAC can significantly improve vaccine strain recommendation for China.

Indications that live poultry markets are a major source of human H5N1 influenza virus infection in China.

Human infections of H5N1 highly pathogenic avian influenza virus have continued to occur in China without corresponding outbreaks in poultry, and there is little conclusive evidence of the source of these infections. Seeking to identify the source of the human infections, we sequenced 31 H5N1 viruses isolated from humans in China (2005 to 2010). We found a number of viral genotypes, not all of which have similar known avian virus counterparts. Guided by patient questionnaire data, we also obtained environmental samples from live poultry markets and dwellings frequented by six individuals prior to disease onset (2008 and 2009). H5N1 viruses were isolated from 4 of the 6 live poultry markets sampled. In each case, the genetic sequences of the environmental and corresponding human isolates were highly similar, demonstrating a link between human infection and live poultry markets. Therefore, infection control measures in live poultry markets are likely to reduce human H5N1 infection in China.

A comprehensive surveillance of adamantane resistance among human influenza A virus isolated from mainland China between 1956 and 2009.

BACKGROUND: Adamantane-derived drugs have been used for treatment and prophylaxis of influenza A virus infection for many years worldwide. Rapid surveillance of antiviral drug resistance is important for appropriate clinical guideline development. Here, we retrospectively assessed adamantane resistance among different influenza A subtypes (H1N1, H3N2 and H5N1) over 53 years (1956-2009) in mainland China. METHODS: A total of 1,451 viruses, including 773 H3N2 viruses, 647 H1N1 viruses and 31 human H5N1 viruses, were analysed by matrix gene sequencing and assayed for drug resistance. RESULTS: Our results show that the prevalence of adamantane-resistant H3N2 viruses was low between 1956 and 2002, but substantially increased in 2003 to the extent that since 2006 all H3N2 viruses have been drug resistant. The percentage of adamantane-resistant H1N1 viruses also increased from 50.0% in 2004 to 98.7% in 2007; however, this decreased to 46.7% in 2009. Only three adamantane-resistant H5N1 viruses have been detected since 2003, when the first case of human H5N1 virus infection was detected in mainland China. Phylogenetic analysis demonstrated that the increase of adamantane-resistant isolates was caused by point mutations or intrasubtype reassortment instead of intersubtype reassortment. CONCLUSIONS: Because of the high percentage of adamantane-resistant H3N2 and H1N1 viruses in mainland China, the use of amantadine and rimantadine drugs for prophylaxis and treatment of current seasonal influenza A infection is not recommended.

Development of a new candidate H5N1 avian influenza virus for pre-pandemic vaccine production.

BACKGROUND: Highly pathogenic H5N1 avian influenza viruses currently circulating in birds have caused hundreds of human infections, and pose a significant pandemic threat. Vaccines are a major component of the public health preparedness for this likely event. The rapid evolution of H5N1 viruses has resulted in the emergence of multiple clades with distinct antigenic characteristics that require clade-specific vaccines. A variant H5N1 virus termed clade 2.3.4 emerged in 2005 and has caused multiple fatal infections. Vaccine candidates that match the antigenic properties of variant viruses are necessary because inactivated influenza vaccines elicit strain-specific protection. OBJECTIVE: To address the need for a suitable seed for manufacturing a clade 2.3.4 vaccine, we developed a new H5N1 pre-pandemic candidate vaccine by reverse genetics and evaluated its safety and replication in vitro and in vivo. METHODS: A reassortant virus termed, Anhui/PR8, was produced by reverse genetics in compliance with WHO pandemic vaccine development guidelines and contains six genes from A/Puerto Rico/8/34 as well as the neuraminidase and hemagglutinin (HA) genomic segments from the A/Anhui/01/2005 virus. The multi-basic cleavage site of HA was removed to reduce virulence. RESULTS: The reassortant Anhui/PR8 grows well in eggs and is avirulent to chicken and ferrets but retains the antigenicity of the parental A/Anhui/01/2005 virus. CONCLUSION: These results indicate that the Anhui/PR8 reassortant lost a major virulent determinant and it is suitable for its use in vaccine manufacturing and as a reference vaccine virus against the H5N1 clade 2.3.4 viruses circulating in eastern China, Vietnam, Thailand, and Laos.

[Construction and characterization of reverse genetic system for H9N2 avian influenza].

OBJECTIVE: To provide a technology platform for vaccine development as well as the research on transmission and pathogenesis, the reverse genetic system for H9N2 avian influenza virus was established. METHODS: Eight full-length cDNAs of avian influenza virus A/Guangzhou/333/99 (H9N2) were amplified by RT-PCR and separately cloned into the transcription/expression vector, pCI-polI. The 8 plasmids DNA was cotransfected into 293T cell, the cell supernatant was collected and inoculated into embryonated eggs, the rescued virus from the allantoic fluid was identified by hemagglutinination assay. RESULTS: The avian influenza H9N2 virus was successfully rescued by 8 plasmids co-transfection in 293T cells. The hemagglutinination titer of the rescued virus is up to 2(9)/50 microl and its growth curve remained relatively as to the wild-type virus. CONCLUSION: The reverse genetic for avian influenza H9N2 subtype virus has been established successfully.

[Establishment of a method for rapid detection of the nucleic acid of the novel A (H1N1) influenza virus].

A new flu caused by a novel influenza A(H1N1) virus has spread over the United States, Mexico and more than 40 other countries. And because of the immediate global concern, WHO has announced that the current level of influenza pandemic alert is raised to phase 5, indicating approaching of an influenza pandemic. As patients suffering from the influenza A (H1N1) have the similar symptoms as patients with seasonal influenza, differential detection and identification of the influenza virus have to depend on specific laboratory tests. We have successfully developed a RT-PCR based method for detection of the influenza A (H1N1) virus, and had applied the method to detection of clinical samples.

[Laboratory confirmation of the first influenza A (H1N1) imported case in Mainland China].

The clinical throat swab specimen of an imported suspected case of influenza A (H1N1) was detec ted with real-time PCR, RT-PCR and subsequently confirmed by gene sequencing. The presence of influ enza A (H1N1) virus confirmed the first case with A (H1N1) infection in Mainland China.

[Study on the antigenicity and HA1 gene characteristics of influenza A viruses during 2004-2008 year in China].

OBJECTIVE: To under stand influenza A viruses epidemic, antigenicity and genetic characteristics variation between the vaccine and Circulation strains during 2004-2008 year in China. METHODS: The influenza A viruses (H1N1, H3N2) isolated from 2004-2008 year were under took antigenic and sequence analysis. Influenza A virus antigenicity and genetic characteristics were analyzed thought amino acid variation compassion of HA1 protein of influenza A virus isolates. RESULTS: The antigenicity of influenza H1N1 subtype viruses isolated from 2004 to 2007 is very similar with vaccine strain A/New Caledonia/20/1999 (HIN1)-like virus. The influenza H1N1 viruses circulated in 2008 year had similar antigenic characteristics with A/Brisben/59/2007 (H1N1) which is component of influenza vaccines for northern hemisphere 2008-2009 year. The influenza H3N2 subtype viruses of 2004-2005 year had antigenic variation comparatively with vaccine strain A/Fujian/411/12002 (H3N2), The antigenicity of 2006-2007 H3N2 viruses and 2008 year's is A/Wiscansin/67/2006(H3N2) and A/ Brisben/10/2006(H3N2) respectively. CONCLUSION: There is change of influenza A viruses (H1N1, H3N2) antigenic and genetic characteristics during 2004-2008 in China.