The cellular receptor for measles virus--elusive no more

The identity of the measles virus receptor has been controversial. Several years ago CD46 was identified as a cellular receptor for the Edmonston strain of measles virus, but most clinical isolates of measles virus, which are most efficiently isolated in the marmoset B cell line B95a, cannot grow in many CD46+ cell lines. Although some researchers attributed it to post-entry block in viral replication, others believed that there is a receptor other than CD46 for wild-type measles viruses. A new study showed that human signalling lymphocytic activation molecule (SLAM; also known as CDw150) is a cellular receptor for measles virus, including the Edmonston strain. SLAM is expressed on lymphocytes and dendritic cells, and plays an important role in lymphocyte activation. The identification of SLAM as a measles virus receptor nicely explains the pathogenesis of measles virus infection.     

Monkey CV1 cell line expressing the sheep-goat SLAM protein: a highly sensitive cell line for the isolation of peste des petits ruminants virus from pathological specimens

Peste des petits ruminants (PPR) is an important economically transboundary disease of sheep and goats caused by a virus which belongs to the genus Morbillivirus. This genus, in the family Paramyxoviridae, also includes the measles virus (MV), canine distemper virus (CDV), rinderpest virus (RPV), and marine mammal viruses. One of the main features of these viruses is the severe transient lymphopaenia and immunosuppression they induce in their respective hosts, thereby favouring secondary bacterial and parasitic infections. This lymphopaenia is probably accounted for by the fact that lymphoid cells are the main targets of the morbilliviruses. In early 2000, it was demonstrated that a transmembrane glycoprotein of the immunoglobulin superfamily which is present on the surface of lymphoid cells, the signalling lymphocyte activation molecule (SLAM), is used as cellular receptor by MV, CDV and RPV. Wild-type strains of these viruses can be isolated and propagated efficiently in non-lymphoid cells expressing this protein. The present study has demonstrated that monkey CV1 cells expressing goat SLAM are also highly efficient for isolating PPRV from pathological samples. This finding suggests that SLAM, as is in the case for MV, CDV and RPV, is also a receptor for PPRV.     

2B4 utilizes ITAM-containing receptor complexes to initiate intracellular signaling and cytolysis

2B4 is a member of the SLAM receptor family capable of activating NK cell cytotoxicity in the context of EBV infection. SAP (SLAM Associated Protein) deficiency causes defective signaling downstream of SLAM family receptors and high susceptibility to EBV. 2B4 costimulates natural cytotoxicity receptor (NCR) and TCR initiated signals to induce cellular cytotoxicity and cytokine release. The 2B4-SAP signal transduction pathway is not predicted to overlap with the TCR-ITAM pathway, although SAP is required for some TCR-induced signals. We therefore examined the functional relationship between SLAM family receptor 2B4 and ITAM-containing adaptor complexes. Removal of Fc?RIγ or CD3ζ-containing complexes, using genetically manipulated cell lines or siRNA specific suppression, significantly reduces 2B4-initiated functions in NK and T cells, respectively. Consistent with this relationship, Syk and ZAP-70 are capable of transducing 2B4 signals for calcium mobilization and cytolysis. Furthermore, ITAM-containing molecules constitutively associate with SAP. These results suggest a potential physical association between 2B4 and the ITAM receptor complexes that is required for 2B4-initiated signaling and cell-mediated killing.     

Infection of HeLa cells by avian infectious bronchitis virus is dependent on cell status.

To investigate the adaptation of avian infectious bronchitis virus (IBV) in a human cell line may be beneficial to understanding the potential mechanisms of coronavirus interspecies infection. The current study addressed the poor replication of IBV in the HeLa human cell line demonstrated in previous reports. We showed that IBV strains M41, H52, H120 and Gray could be propagated in HeLa cells with distinct cytopathic effect. The virus titre in freshly dispersed HeLa cells was 1000-fold higher than in cell monolayers. Trypsin was not the determinant for the viral replication, suggesting that the restriction of IBV replication in HeLa cells is the result of intracellular events rather than the binding to or fusion with host cells. These IBV strains replicated to an average titre of 10(3.4+/-0.2)/0.1 ml median tissue culture infectious doses in freshly dispersed HeLa cells and maintained this titre for the first 12 passages. Then an approximately 10-fold increase (10(4.20+/-0.19)/0.1 ml) occurred in passage 13, which was maintained to passage 16, after which there was another, bigger rise to 10(6.6+/-0.3)/0.1 ml in passage 17. This titre was maintained until passage 24 when the experiment was terminated. The IBV M41 S1 gene was amplified and sequenced for passages 0, 5 and 21. There was only one amino acid replacement in the S1 protein, in passage 21. The presence of sialic acid on HeLa cells contributed to efficient virus replication, while human aminopeptidase N was not involved in the infection. Haemagglutinin activity gradually reduced with increased passages. These results indicated that the virus adaptation would probably be determined by host cell modification such as receptor glycosylation and different receptor utilization instead of viral gene mutation.     

RNAi抑制XJ-160病毒复制的研究

目的通过建立RNA干扰(RNA interference，RNAi)抑制XJ-160病毒复制的模型，在序列特异性、位置效应和量效关系等方面，探讨RNAi对XJ-160病毒复制的影响，为进一步研究RNAi的抗病毒作用和机制奠定基础。方法按照siRNA(short interferencing RNA)的设计要求合成XJ-160病毒特异siRNA，脂质体法转染BHK-21细胞后感染XJ-160病毒，通过测定病毒滴度、蛋白表达量及XJ-160病毒：RNA合成研究siRNA对XJ-160病毒复制的抑制。结果成功建立了RNAi抑制XJ-160病毒复制的模型，探讨了RNAi抑制该病毒复制作用的特点。结论外源导入的siRNA能够抑制XJ-160病毒的复制，并且这种抑制作用具有明显的序列特异性、位置效应和存在量效关系等特点；siRNA是通过降解病毒RNA实现抑制病毒复制作用的。     

Cross-inhibition to heterologous foot-and-mouth disease virus infection induced by RNA interference targeting the conserved regions of viral genome

RNA interference (RNAi) is the process by which double-stranded RNA (dsRNA) directs sequence-specific degradation of messenger RNA in animal and plant cells. In mammalian cells, RNAi can be triggered by 21-23 nucleotide duplexes of small interfering RNA (siRNA). Strategies to inhibit RNA virus multiplication based on the use of siRNAs have to consider the high genetic polymorphism exhibited by this group of virus. Here we described a significant cross-inhibition of foot-and-mouth disease (FMD) virus (FMDV) replication in BHK-21 cells by siRNAs targeted to various conserved regions (5'NCR, VP4, VPg, POL, and 3'NCR) of the viral genome. The results showed that siRNAs generated in vitro by human recombinant dicer enzyme gave an inhibition of 10- to 1000-fold in virus yield of both homologous (HKN/2002) and heterologous (CHA/99) isolates of FMDV serotype O at 48 h post-infection (hpi). The inhibition extended to at least 6 days post-infection. For serotype Asia1, the virus yield in YNBS/58-infected cells examined at 12, 24, and 48 hpi decreased by approximately 10-fold in cells pretreated with HKN/2002-specific siRNAs, but there was no significant decrease at 60 hpi. The inhibition was specific to FMDV replication, as no reduction was observed in virus yield of pseudorabies virus, an unrelated virus. Moreover, we also demonstrated an enhanced viral suppression could be achieved in BHK-21 cells with siRNA transfection after an infection had been established. These results suggested that siRNAs directed to several conserved regions of the FMDV genome could inhibit FMDV replication in a cross-resistance manner, providing a strategy candidate to treat high genetic variability of FMDV.     

A critical role for CD63 in HIV replication and infection of macrophages and cell lines

HIV infection typically involves interaction of Env with CD4 and a chemokine coreceptor, either CCR5 or CXCR4. Other cellular factors supporting HIV replication have also been characterized. We previously demonstrated a role for CD63 in early HIV infection events in macrophages via inhibition by anti-CD63 antibody pretreatment. To confirm the requirement for CD63 in HIV replication, we decreased CD63 expression using CD63-specific short interfering RNAs (siRNA), and showed inhibition of HIV replication in macrophages. Surprisingly, pretreatment with CD63 siRNA not only silenced CD63 expression by 90%, but also inhibited HIV-1 replication in a cultured cell line (U373-MAGI) which had been previously shown to be insensitive to CD63 monoclonal antibody inhibition. Although the anti-CD63 antibody was previously shown to inhibit early HIV infection events only in macrophages, we now show a potential role for CD63 in later HIV replication events in macrophages and cell lines. Further delineation of the role of CD63 in HIV replication may lead to development of novel therapeutic compounds.     

Potiskum virus: enhancement of replication in a macrophage-like cell line

Replication of Potiskum virus was studied in P388D1 macrophage-like cell line in the presence and absence of subneutralizing concentrations of specific antiviral antibody. The cultures were infected at multiplicities of infection (MOI) ranging from 0.4 to 0.0004. The virus replicated to high titres at all MOI tested, but there was an enhancement of virus replication in cultures supplemented with the antibody. Enhancement of replication was MOI dependent, the highest ratios being obtained in cultures infected at lowest MOI. In enhancement assays using various dilutions of immune mouse ascitic fluid (IMAF), the highest enhancement ratio was observed at dilution 1 : 500; the enhancing antibody titre was 5,000.     

siRNA-mediated inhibition of respiratory syncytial virus replication

The effect of siRNA against respiratory syncytial virus (RSV) was investigated in the cultured cells. MA104 cells were inoculated by RSV and transfected by different variants of siRNA against RSV phosphoprotein (P) mRNA or non-specific siRNA as a control. The inhibition of RSV was assessed by microscopically studying the cells, titrating the virus, and estimating viral RNA quantity in the culture supernatants by real-time polymerase chain reaction (PCR). The most potent variants of siRNA caused an up to 8-day delay of microscopically detectable syncytium generation to 8 days, an up to 280-fold decrease in viral titers, and an up to 40-fold reduction in viral RNA quantity in the supernatants, as compared to the controls (p < 0.001). RSV mRNA is a suitable target for siRNA-mediated RSV replication inhibition, promising an advance in the treatment of RSV infection.     

Host range and receptor utilization of canine distemper virus analyzed by recombinant viruses: Involvement of heparin-like molecule in CDV infection

We constructed recombinant viruses expressing enhanced green fluorescent protein (EGFP) or firefly luciferase from cDNA clones of the canine distemper virus (CDV) (a Japanese field isolate, Yanaka strain). Using these viruses, we examined susceptibilities of different cell lines to CDV infection. The results revealed that the recombinant CDVs can infect a broad range of cell lines. Infectivity inhibition assay using a monoclonal antibody specific to the human SLAM molecule indicated that the infection of B95a cells with these recombinant CDVs is mainly mediated by SLAM but the infection of 293 cell lines with CDV is not, implying the presence of one or more alternative receptors for CDV in non-lymphoid tissue. Infection of 293 cells with the recombinant CDV was inhibited by soluble heparin, and the recombinant virus bound to immobilized heparin. Both F and H proteins of CDV could bind to immobilized heparin. These results suggest that heparin-like molecules are involved in CDV infection.     

短双链RNA对柯萨奇B组3型病毒体外感染的抑制作用研究

目的利用RNA干扰技术，在细胞、蛋白和基因水平上观察短双链RNA对柯萨奇病毒体外感染Hela细胞的增殖抑制作用、对病毒RNA复制和蛋白合成的影响。方法应用病毒致细胞病变作用保护实验、空斑形成减少实验、Western Blot、RT-PCR等方法，在体外检测其抗CVB3病毒作用。结果设计、合成的8条SiRNA中，针对基因组2B、VP4、2A、3C区的SiRNA-2、SiRNA-3、SiRNA-6、SiRNA-7具有不同程度的抑制病毒作用。其中，病毒基因组2B的SiRN．2作用效果最好，对Hela细胞CVB3感染72h后致细胞病变和空斑形成的抑制率为95％，抑制病毒蛋白的合成，病毒基因的复制水平也显著降低，在病毒0．1、0．01、0．001、0．0001 MOI感染水平上对CVB3致细胞病变作用的抑制率分别为30％、70％、88％和99％（48h）。结论针对CVB3基因组2B的SiRNA-2具有较强的抑制柯萨奇B3型病毒感染的作用。     

A monoclonal antibody specific to the HA2 glycoprotein of influenza A virus hemagglutinin that inhibits its fusion activity reduces replication of the virus

Monoclonal antibody (MAb) CF2, which binds to the fusion peptide of influenza A virus hemagglutinin (HA) (amino acids (aa) 1-35 of the N-terminus of the light chain of HA), inhibited the fusion activity of HA. This MAb preferentially bound to pH 5-treated virus (with conformationally altered HA) and bound only weakly to the native wild type (wt) virus. However, a significant binding of MAb CF2 to the amantadine resistant virus mutant Ab4 (with a mutation at aa 17 of HA1 leading to a destabilization of HA trimer) was obtained without pH 5 treatment. Exploiting the fusion-inhibition activity of MAb CF2 the effect of this antibody on the virus replication in vitro was followed using both the wt virus and the amantadine resistant mutant Ab4. No reduction of replication of wt virus and a low reduction of replication of Ab4 mutant (by about 20%) was detected by radioimmunoassay after preincubation of the virus with a high concentration of MAb CF2 at room temperature. An increased reduction of replication of Ab4 mutant (by about 40%) was observed in cell radioimmunoassay (RIA) and in plaque assay when the virus was preincubated with MAb at 37 degrees C. Under these conditions a reduction of the wt virus replication also occurred by about 40%. This is the first report on the capacity of a MAb specific to HA2 gp, the light chain of influenza A virus HA, to reduce replication of the virus. This capacity in relation to (i) the affinity of the antibody to the virus, and (ii) the accessibility of corresponding epitopes on the virus surface as well as the proposed mechanism of inhibition of replication of the virus are discussed.