Purification of Semliki Forest virus RNA - dependent RNA polymerase

Replicase activity was found in the cytoplasm of the infected cell and, after removal of nuclei, the replication complex was characterized by isopycnic sucrose gradient centrifugation. A membranous band at 35% (w/w) sucrose contained replicase activity and was the site of RNA synthesis. At 1 3/4 hr post-infection the replicase was associated with input virion RNA but not input virion proteins. Replicase present in the post-nuclear supernatant was competent to synthesize all forms of virus-specific RNA found in the infected cell. However, on further purification there was a loss of the production of discrete single- stranded RNA in vitro. This loss was probably caused by endogenous ribonuclease activity and disruption of the replication complex. Recovery of the replicase rich band from isopycnic gradients allowed it to be analysed on linear sucrose gradients. The replication complex had an S-value of 250 which, on nonionic detergent treatment, was converted to a 25S structure. The replicase activity of this 25S complex was specifically bound to an oligo(dT)- cellulose column bearing attached 42S virion RNA. Analysis of the polypeptides eluted off in the fractions having replicase activity showed that there were only 3 labelled bands visible on polyacrylamide gels. Two of these comigrated with two non-structural virus polypeptides identified in infected cells. The third polypeptide of the purified replicase preparation (MW 40,000 daltons) is probably a host protein. Material from mock- infected cells purified exactly as the replicase contained only the 40,000 dalton protein. It is therefore concluded that SFV replicase contains 2 virus-specific polypeptides which have MWs of 90,000 and 63,000 daltons

Sequence Distances between env Genes of HIV-1 from Individuals Infected from the Same Source: Implications for the Investigation of Possible Transmission Events

AbstractPreviously described transmission studies have shown that HIV strains isolated from individuals infected from a common source are more homogeneous than HIV strains isolated from individuals with unrelated infections. This has been the basis, in at least four instances, for deciding whether apparently epidemiologically related cases represent actual transmissions. To date, HIV transmission studies have usually included sequence data from the most likely source of infection, and the probability of transmission from the donor to the recipient has been assessed by measuring sequence similarity against control data using likelihood analysis. We have recently studied a putative transmission involving a UK health care worker (CPHL1), a patient of CPHL1 (CPHL2), and CPHL3, a member of the same "sex circle" as CPHL2. We have used sequence distance and neighbour joining methods as well as likelihood analysis as means of determining genetic relatedness. Though no other source of infection was available our findings did not support the possibility that CPHL1 had infected CPHL2. Strain CPHL3 was closer to CPHL2 than to CPHL1. It is shown that control data from documented transmission events can be used to establish the source of infection in the absence of an index case. It is also shown that the C2-V3 region analysed in previous transmission studies is unreliable for accurate phylogenetic analysis. The results indicated that gp120 is a more informative region than C2-V3 for molecular transmission studies. Sequence distances between the env genes of related and unrelated infections have been derived in this work

Prevalence of disease related prion protein in anonymous tonsil specimens in Britain: cross sectional opportunistic survey

Objective To establish with improved accuracy the prevalence of disease related prion protein (PrPCJD) in the population of Britain and thereby guide a proportionate public health response to limit the threat of healthcare associated transmission of variant Creutzfeldt-Jakob disease (vCJD)

Molecular pathology of human prion disease

Human prion diseases are associated with a range of clinical presentations and are classified by both clinicopathological syndrome and aetiology with sub-classification according to molecular criteria. Considerable experimental evidence suggests that phenotypic diversity in human prion disease relates in significant part to the existence of distinct human prion strains encoded by abnormal PrP isoforms with differing physicochemical properties. To date, however, the conformational repertoire of pathological isoforms of wild-type human PrP and the various forms of mutant human PrP has not been fully defined. Efforts to produce a unified international classification of human prion disease are still ongoing. The ability of genetic background to influence prion strain selection together with knowledge of numerous other factors that may influence clinical and neuropathological presentation strongly emphasises the requirement to identify distinct human prion strains in appropriate transgenic models, where host genetic variability and other modifiers of phenotype are removed. Defining how many human prion strains exist allied with transgenic modelling of potentially zoonotic prion strains will inform on how many human infections may have an animal origin. Understanding these relationships will have direct translation to protecting public health

Characterization of a Novel Simian Immunodeficiency Virus (SIVmonNG1) Genome Sequence from a Mona Monkey (Cercopithecus mona)

A novel simian immunodeficiency virus (SIV) sequence has been recovered from RNA extracted from the serum of a mona monkey (Cercopithecus mona) wild born in Nigeria. The sequence was obtained by using novel generic (degenerate) PCR primers and spans from two-thirds into the gag gene to the 3′ poly(A) tail of the SIVmonNG1 RNA genome. Analysis of the open reading frames revealed that the SIVmonNG1 genome codes for a Vpu protein, in addition to Gag, Pol, Vif, Vpr, Tat, Rev, Env, and Nef proteins. Previously, only lentiviruses infecting humans (human immunodeficiency virus type 1 [HIV-1]) and chimpanzees (SIVcpz) were known to have a vpu gene; more recently, this has also been found in SIVgsn from Cercopithecus nictitans. Overall, SIVmonNG1 most closely resembles SIVgsn: the env gene sequence groups with HIV-1/SIVcpz env sequences, whereas the pol gene sequence clusters closely with the pol sequence of SIVsyk from Cercopithecus albogaris. By bootscanning and similarity plotting, the first half of pol resembles SIVsyk, whereas the latter part is closer to SIVcol from Colobus guereza. The similarities between the complex mosaic genomes of SIVmonNG1 and SIVgsn are consistent with a shared or common lineage. These data further highlight the intricate nature of the relationships between the SIVs from different primate species and will be helpful for unraveling these associations

HIV-1 pol gene variation is sufficient for reconstruction of transmissions in the era of antiretroviral therapy.

OBJECTIVES: We wished to assess the potential of using HIV-1 pol gene for the identification of transmissions events by phylogenetic means in the era of antiretroviral drug selective pressure. DESIGN: The relatedness of the viruses within a large database of pol sequences generated from HIV-1 infected individuals from the UK was reconstructed by phylogenetic analyses. METHODS: A total of 140 pol sequences were selected out of the 2500 database entries, on the basis of a pairwise genetic distance higher than 95%. Neighbour Joining and Maximum Likelihood trees were implemented. Trees were reconstructed after exclusion of codon positions associated with drug resistance from the original pol alignment. Trees based on the corresponding env and gag genes were implemented to confirm the linkages. RESULTS: Up to 23 transmission clusters were identified, supported by high bootstrap values (> 99), congruent epidemiological data and/or similar drug resistance motifs. The topology of the tree was consistent after exclusion of the drug resistance associated codons. Identical topologies were obtained in trees implemented from gag and env genes alignments. CONCLUSIONS: Despite its genetic conservation, the HIV-1 pol gene holds sufficient variability to permit the phylogenetic reconstruction of transmissions. Identical clusters were obtained whichever of the three principal genes is considered and no bias was induced by the presence of drug resistance mutations. These findings demonstrate the important epidemiological information inherent within routinely collected laboratory data, which can assist in estimating rates of recent HIV-1 transmission within a population