Jianer Wo

Human infections with the emerging avian influenza A H7N9 virus from wet market poultry: clinical analysis and characterisation of viral genome

Summary Background Human infection with avian influenza A H7N9 virus emerged in eastern China in February, 2013, and has been associated with exposure to poultry. We report the clinical and microbiological features of patients infected with influenza A H7N9 virus and compare genomic features of the human virus with those of the virus in market poultry in Zhejiang, China. Methods Between March 7 and April 8, 2013, we included hospital inpatients if they had new-onset respiratory symptoms, unexplained radiographic infiltrate, and laboratory-confirmed H7N9 virus infection. We recorded histories and results of haematological, biochemical, radiological, and microbiological investigations. We took throat and sputum samples, used RT-PCR to detect M, H7, and N9 genes, and cultured samples in Madin-Darby canine kidney cells. We tested for co-infections and monitored serum concentrations of six cytokines and chemokines. We collected cloacal swabs from 86 birds from epidemiologically linked wet markets and inoculated embryonated chicken eggs with the samples. We identified and subtyped isolates by RT-PCR sequencing. RNA extraction, complementary DNA synthesis, and PCR sequencing were done for one human and one chicken isolate. We characterised and phylogenetically analysed the eight gene segments of the viruses in the patients and the chickens isolates, and constructed phylogenetic trees of H, N, PB2, and NS genes. Findings We identified four patients (mean age 56 years), all of whom had contact with poultry 3–8 days before disease onset. They presented with fever and rapidly progressive pneumonia that did not respond to antibiotics. Patients were leucopenic and lymphopenic, and had impaired liver or renal function, substantially increased serum cytokine or chemokine concentrations, and disseminated intravascular coagulation with disease progression. Two patients died. Sputum specimens were more likely to test positive for the H7N9 virus than were samples from throat swabs. The viral isolate from the patient was closely similar to that from an epidemiologically linked market chicken. All viral gene segments were of avian origin. The H7 of the isolated viruses was closest to that of the H7N3 virus from domestic ducks in Zhejiang, whereas the N9 was closest to that of the wild bird H7N9 virus in South Korea. We noted Gln226Leu and Gly186Val substitutions in human virus H7 (associated with increased affinity for α-2,6-linked sialic acid receptors) and the PB2 Asp701Asn mutation (associated with mammalian adaptation). Ser31Asn mutation, which is associated with adamantane resistance, was noted in viral M2. Interpretation Cross species poultry-to-person transmission of this new reassortant H7N9 virus is associated with severe pneumonia and multiorgan dysfunction in human beings. Monitoring of the viral evolution and further study of disease pathogenesis will improve disease management, epidemic control, and pandemic preparedness. Funding Larry Chi-Kin Yung, National Key Program for Infectious Diseases of China.

SARS-coronavirus replicates in mononuclear cells of peripheral blood (PBMCs) from SARS patients

Abstract Background: The etiologic agent of severe acute respiratory syndrome (SARS) is a recently identified, positive single-stranded RNA (ssRNA) coronavirus (SARS-CoV). Little is known about the dynamic changes of the viral replicative form in SARS cases. Objectives: Evaluate whether SARS-CoV can infect and replicate in peripheral blood mononuclear cells (PBMCs) of infected persons and reveal any dynamic changes to the virus during the course of the disease. Study design: Peripheral blood mononuclear cells collected from SARS cases infected by the same infectious source were tested for both negative-stranded RNA (minus-RNA, “replicative intermediates”) and positive-stranded RNA (genomic RNA) of SARS-CoV during the course of hospitalization by reverse transcription-polymerase chain reaction (RT-PCR). Results: SARS-CoV minus-RNA was detected in PBMCs from SARS patients. The viral replicative forms in PBMCs were detectable during a period of 6 days post-onset of the disease, while the plus-RNA were detectable for a longer period (8–12 days post-onset). Conclusions: SARS-coronavirus can infect and replicate within PBMCs of SARS patients, but viral replication in PBMCs seems subject to self-limitation.

Construction and cellular immune response induction of HA-based alphavirus replicon vaccines against human-avian influenza (H5N1).

Several approaches are being taken worldwide to develop vaccines against H5N1 viruses; most of them, however, pose both practical and immunological challenges. One potential strategy for improving the immunogenicity of vaccines involves the use of alphavirus replicons and VP22, a herpes simplex type 1 (HSV-1) protein. In this study, we analysed the antigenic peptides and homogeneity of the HA sequences (human isolates of the H5N1 subtype, from 1997 to 2003) and explored a novel alphavirus replicon system of VP22 fused with HA, to assess whether the immunogenicity of an HA-based replicon vaccine could be induced and augmented via fusion with VP22. Further, replicon particles expressing VP22, and enhanced green fluorescent protein (EGFP) were individually used as controls. Cellular immune responses in mice immunised with replicons were evaluated by identifying specific intracellular cytokine production with flow cytometry (FCM). Animal-based experimentation indicated that both the IL-4 expression of CD4(+) T cells and the IFN-gamma expression of CD8(+) T cells were significantly increased in mice immunised with VPR-HA and VPR-VP22/HA. A dose titration effect vis-à-vis both IL-4 expression and IFN-gamma expression were observed in VPR-HA- and VPR-VP22/HA-vaccinated mice. Our results revealed that both VPR-VP22/HA and VPR-HA replicon particles presented a promising approach for developing vaccines against human-avian influenza, and VP22 could enhance the immunogenicity of the HA antigens to which it is fused.

Hepatitis C virus-specific DNA sequences in human DNA : Differentiation by means of restriction enzyme analysis at the DNA level in healthy, anti-HCV-negative individuals

Abstract This study aimed to look for further HCV-specific sequences at the DNA level of healthy, HCV-negative individuals. Here, the sequence section from nt 57 to nt 328 within the 5′-NCR was assayed. Different combinations of primers were used in the nested PCR without a preceding reverse transcriptase step, resulting in fragments of the expected molecular weight size and also shorter and longer ones. It shows that the major part of the 5′-non-coding region (5′-NCR) of the hepatitis C virus genome is present in the DNA fraction from peripheral blood mononuclear cells (PBMCs) of healthy, anti-HCV-negative individuals tested. Furthermore we designed experiments to prove the specificity of these findings, by using restriction enzymes for digest assays of the target DNA before PCR (pre-PCR digest) and of the products after PCR (post-PCR digest). In conclusion, our study indicates that the main part of the internal ribosome entry site (IRES) structure of HCV at least is contained in the DNA of the individuals tested.

Expression of H5N1 influenza virus hemagglutinin protein fused with protein transduction domain in an alphavirus replicon system.

Alphavirus replicons, in which structural protein genes are replaced by heterologous genes, express high levels of the heterologous proteins. On the basis of the potencies of replicons to self-replicate and express foreign proteins and the remarkable intercellular transport property of VP22, a novel alphavirus Semliki Forest virus (SFV) replicon system of VP22 fused with a model antigen, hemagglutinin (HA), of the human-avian H5N1 influenza virus, was explored in this study. Further, replicon particles expressing HA, VP22, and enhanced green fluorescent protein (EGFP) individually were used as controls. By flow cytometry based on the analysis of transfection efficiency, SFV-EGFP replicon particle titer was 1.13 x 10(7)transducing units (TU)/ml. The titers of SFV-HA, SFV-VP22 and SFV-VP22-HA replicon particles, which were titrated by using SFV-EGFP replicon particles, were 1.42 x 10(7), 3.23 x 10(7), and 1.01 x 10(7)TU/ml, respectively. HA and VP22-HA expression was observed in SFV-HA- and SFV-VP22-HA-transfected BHK-21 cells, respectively. Immunofluorescence staining revealed that the fluorescence intensity in the SFV-VP22-HA-transfected BHK-21 cells was more than that in the SFV-HA-transfected BHK-21 cells. Both SFV-VP22-HA and SFV-HA replicon particles presented a promising approach for developing vaccines against human-avian influenza. VP22-HA fusion protein with similar trafficking properties may also enhance vaccine potency.