(2,8,16,38), although they achieve this by distinct mechanisms (40). Thus, for example, we have shown that simian virus 5 (SV5) blocks IFN signaling by targeting STAT1, an essential component of IFN-␣/␤ and IFN-␥ signaling cascades, for proteasome-mediated degradation (3). However, the ability of SV5 to block IFN signaling is species dependent. Thus, wild-type (wt) SV5 blocks IFN signaling in human, monkey, and canine cells but fails to block IFN signaling in murine cells. Indeed, the ability to block IFN signaling in a particular species may be one factor that limits the host range of a given virus (2).SV5 is a prototype member of the Rubulavirus genus within the Paramyxoviridae family (19). Like other paramyxoviruses, SV5 has a nonsegmented, negative-sense, single-stranded RNA genome. Its genome is encapsidated within a helical nucleocapsid which is surrounded by a pleomorphic, lipid-containing envelope containing the HN and F virus glycoproteins. (For a review of the Paramyxoviridae, see reference 17.) Over the last few years, techniques have been developed to manipulate the genomes of many Paramyxoviridae, including SV5 (11,12,24,27). Thus, it is now possible to manipulate the genome of SV5 and to correlate genotypes with phenotypes. We report here that a single amino acid substitution in the V protein confers on SV5 the ability to block IFN signaling in murine cells, and we demonstrate protracted virus protein synthesis in murine cells infected with a recombinant virus which blocks IFN signaling in these cells compared to cells infected with wt virus. MATERIALS AND METHODSCells, viruses, and IFN. Murine BF cells (cloned from a primary cell culture of a BALB/c mouse embryo) and human 2fTGH cells (22) were grown as monolayers in 25-or 75-cm 2 tissue culture flasks in Dulbecco modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (growth medium). Monolayer cultures of A549, BHK-21, MDBK, and CV-1 cells were maintained in DMEM and 10% fetal calf serum as described previously (26). Chick embryo fibroblasts (CEFs) were grown as described previously (18). All cell lines were negative for mycoplasmas as screened by DAPI (4Ј,6Ј-diamidino-2-phenylindole) staining. Human and mouse cells were treated with recombinant human ␣A/D (rHuIFN-␣A/D) IFN (31) at 1,000 IU/ml in medium containing 2% bovine serum (maintenance medium). The wt (strain W3A) (1), rSV5-V/P N 100 D, and SV5 mci-2 strains of SV5 were grown and titrated under appropriate conditions in Vero cells using maintenance medium. High-multiplicity infections

Relationships and host range of human, canine, simian and porcine isolates of simian virus 5 (parainfluenza virus 5) Sequence comparison of the V/P and F genes of 13 human, canine, porcine and simian isolates of simian virus 5 (SV5) revealed a surprising lack of sequence variation at both the nucleotide and amino acid levels (0-3 %), even though the viruses were isolated over 30 years and originated from countries around the world. Furthermore, there were no clear distinguishing amino acid or nucleotide differences among the isolates that correlated completely with the species from which they were isolated. In addition, there was no evidence that the ability of the viruses to block interferon signalling by targeting STAT1 for degradation was confined to the species from which they were isolated. All isolates had an extended cytoplasmic tail in the F protein, compared with the original W3A and WR monkey isolates. Sequence analysis of viruses that were derived from human bone-marrow cells isolated in London in the 1980s revealed that, whilst they were related more closely to one another than to the other isolates, they all had identifying differences, suggesting that they were independent isolates. These results therefore support previous data suggesting that SV5 can infect humans persistently, although the relationship of SV5 to any human disease remains highly contentious. Given that SV5 has been isolated on multiple occasions from different species, it is proposed that the term simian virus 5 is inappropriate and suggested that the virus should be renamed parainfluenza virus 5. INTRODUCTIONSimian virus type 5 (SV5) is in the genus Rubulavirus of the subfamily Paramyxovirinae of the family Paramyxoviridae. SV5 was first isolated from rhesus and cynomolgus monkey kidney-cell cultures and two of the original monkey isolates are referred to as WR and W3A (Hull et al., 1956;Choppin, 1964). It was thus thought initially that monkeys were the natural host for SV5, but epidemiological studies in the 1960s showed that wild monkeys do not have antibodies against the virus. However, these animals seroconvert in captivity and, on this basis, it was suggested that infection of monkeys occurs either in transit or shortly after contact with humans (Tribe, 1966;Atoynatan & Hsiung, 1969;Hsiung, 1972). Indeed, Tribe (1966) suggested that monkeys that were brought into captivity should be immunized immediately against SV5 to prevent them being infected with the virus. There is also experimental evidence supporting the contention that SV5 naturally infects humans (Hsiung, 1972; Goswami et al., 1984). For example, SV5 has been isolated on numerous occasions from a variety of human tissues, including bone-marrow cells (Goswami et al., 1984). Despite this, infection of humans with SV5 has remained a subject of some debate and controversy, fuelled by the fact that SV5 can contaminate primary monkey kidney-cell cultures (and other cell lines), which are commonly used to isolate viruses from clinical samples (Chanock et al., 1961;Hsiung...

CPI(+) and CPI(-) are two canine isolates of simian virus 5 (SV5). CPI(+) was originally isolated from the cerebrospinal fluid of a dog with temporary posterior paralysis and CPI(-) was recovered at 12 days p.i. from the brain tissue of a dog experimentally infected with CPI(+). We have previously shown that the V protein of SV5 blocks interferon (IFN) signalling by targeting STAT1 for degradation. Here we report that whilst CPI(+) targets STAT1 for degradation, CPI(-) fails to and as a consequence, CPI(+) blocks IFN signalling but CPI(-) does not. Three amino acid differences in the P/V N-terminal common domain of the V protein are responsible for the observed difference in the abilities of CPI(+) and CPI(-) to block IFN signalling. In cells persistently infected with CPI(-) the virus may become repressed in response to IFN, under which circumstances virus glycoproteins are lost from the surface of infected cells and virus nucleocapsid proteins accumulate in cytoplasmic inclusion bodies. We suggest that in vivo cells infected with IFN-resistant viruses (in which there would be continuous virus protein synthesis) may be more susceptible to killing by cytotoxic T cells than cells infected with IFN-sensitive viruses (in which virus protein synthesis was repressed), and a model of virus persistence is put forward in which there is alternating selection of IFN-resistant and IFN-sensitive viruses depending upon the state of the adaptive immune response.

These findings suggest that sexual transmission constrains viral diversity at the population level, partially because of the preferential transmission of ancestral as opposed to contemporary strains circulating in the transmitting partner. Future successful vaccine strategies may need to target these transmitted ancestral strains.

The mechanism by which inflammation influences the adaptive response to vaccines is not fully understood. Here, we examine the role of lymph node macrophages (LNMs) in the induction of the cytokine storm triggered by inactivated influenza virus vaccine. Following vaccination, LNMs undergo inflammasome-independent necrosis-like death that is reliant on MyD88 and Toll-like receptor 7 (TLR7) expression and releases pre-stored interleukin-1α (IL-1α). Furthermore, activated medullary macrophages produce interferon-β (IFN-β) that induces the autocrine secretion of IL-1α. We also found that macrophage depletion promotes lymph node-resident dendritic cell (LNDC) relocation and affects the capacity of CD11b LNDCs to capture virus and express co-stimulatory molecules. Inhibition of the IL-1α-induced inflammatory cascade reduced B cell responses, while co-administration of recombinant IL-1α increased the humoral response. Stimulation of the IL-1α inflammatory pathway might therefore represent a strategy to enhance antigen presentation by LNDCs and improve the humoral response against influenza vaccines.

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The role of natural killer (NK) cells in the immune response against vaccines is not fully understood. Here, we examine the function of infiltrated NK cells in the initiation of the inflammatory response triggered by inactivated influenza virus vaccine in the draining lymph node (LN). We observed that, following vaccination, NK cells are recruited to the interfollicular and medullary areas of the LN and become activated by type I interferons (IFNs) produced by LN macrophages. The activation of NK cells leads to their early production of IFNg, which in turn regulates the recruitment of IL-6+ CD11b+ dendritic cells. Finally, we demonstrate that the interleukin-6 (IL-6)-mediated inflammation is important for the development of an effective humoral response against influenza virus in the draining LN.

As HIV-1 evolves over the course of infection, resistance against antiretrovirals may arise in the absence of drug pressure, especially against receptor and fusion blockers because of the extensive changes observed in the envelope glycoprotein. Here we show that viruses from the chronic phase of disease are significantly less sensitive to CCR5 receptor and fusion blockers compared to early infection variants. Differences in susceptibility to CCR5 antagonists were observed in spite of no demonstrable CXCR4 receptor utilization. No significant sensitivity differences were observed to another entry blocker, soluble CD4, or to reverse transcriptase, protease, or integrase inhibitors. Chronic as compared to early phase variants demonstrated greater replication when passaged in the presence of subinhibitory concentrations of fusion but not CCR5 receptor inhibitors. Fusion antagonist resistance, however, emerged from only one chronic phase virus culture. Because sensitivity to receptor and fusion antagonists is correlated with receptor affinity and fusion capacity, respectively, changes that occur in the envelope glycoprotein over the course of infection confer greater ability to use the CCR5 receptor and increased fusion ability. Our in vitro passage studies suggest that these evolving phenotypes increase the likelihood of resistance against fusion but not CCR5 receptor blockers.