Disinfection of Contaminated Heritage Surfaces from SARS-CoV-2 Virus

To increase the safety of adenovirus vector (AdV)-based therapy without reducing its efficacy, a single-cycle adenovirus vector (SC-AdV) with a deletion in the protease gene (PS) was developed in order to be used as a substitute for the replication-competent adenovirus (RC-AdV). Since no infectious viral particles are assembled, there is no risk of viral shedding. The complementary cell lines for this developed AdV proved to be suboptimal for the production of viral particles and require the presence of fetal bovine serum (FBS) to grow. In the current study, we produced both stable pools and clones using adherent and suspension cells expressing the PS gene. The best adherent cell pool can be used in the early stages for the generation of protease-deleted adenovirus, plaque purification, and titration. Using this, we produced over 3400 infectious viral particles per cell. Additionally, the best suspension subclone that was cultured in the absence of FBS yielded over 4000 infectious viral particles per cell. Harvesting time, culture media, and concentration of the inducer for the best suspension subclone were further characterized. With these two types of stable cells (pool and subclone), we successfully improved the titer of protease-deleted adenovirus in adherent and suspension cultures and eliminated the need for FBS during the scale-up production. Eight lots of SC-AdV were produced in the best suspension subclone at a scale of 2 to 8.2 L. The viral and infectious particle titers were influenced by the virus backbone and expressed transgene.

Syntaxin 17 is an autophagosomal SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) protein required for the fusion of autophagosomes with lysosomes to form autolysosomes and thereby to deliver the enclosed contents for degradation. Hepatitis C virus (HCV) induces autophagy. In light of the observation that the number of viral particles formed by HCV-infected cells is much greater than the number of infectious viral particles finally released by HCV-infected cells, the regulation of fusion between autophagosomes and lysosomes might fulfill a key function controlling the number of released virions. HCV-replicating cells possess a decreased amount of syntaxin 17 due to impaired expression and increased turnover of syntaxin 17. Overexpression of syntaxin 17 in HCV-replicating cells diminishes the number of released infectious viral particles and decreases the amount of intracellular retained viral particles by favoring the formation of autolysosomes, in which HCV particles are degraded. Inhibition of lysosomal acidification by bafilomycin rescues the decreased release of virions from syntaxin 17-overexpressing cells, while induction of autophagy by rapamycin enforces the impairment of release under these conditions. Vice versa, inhibition of syntaxin 17 expression by specific small interfering RNAs results in an elevated amount of intracellular retained viral particles and facilitates the release of HCV virions by impairment of autophagosome-lysosome fusion. HCV genome replication, however, is not affected by modulation of syntaxin 17 expression. These data identify syntaxin 17 to be a novel factor controlling the release of HCV. This is achieved by regulation of autophagosome-lysosome fusion, which affects the equilibrium between the release of infectious viral particles and lysosomal degradation of intracellular retained viral particles. Hepatitis C virus (HCV) induces autophagy. Syntaxin 17 is an autophagosomal SNARE protein required for the fusion of autophagosomes with lysosomes. In HCV-infected cells, a major fraction of the de novo-synthesized viral particles is not released but is intracellularly degraded. In this context, the effect of HCV on the amount and distribution of syntaxin 17 and the relevance of syntaxin 17 for the viral life cycle were investigated. This study demonstrates that the amount of syntaxin 17 decreased in HCV-replicating cells. In addition, syntaxin 17 is identified to be a novel factor controlling the release of HCV, and the relevance of autophagosome-lysosome fusion as a regulator of the amount of released viral particles is revealed. Taken together, these findings indicate that syntaxin 17 is involved in the regulation of autophagosome-lysosome fusion and thereby affects the equilibrium between the release of infectious viral particles and the lysosomal degradation of intracellularly retained viral particles.

The Japanese fulminant hepatitis-1 (JFH1)-based hepatitis C virus (HCV) infection system has permitted analysis of the complete viral replication cycle in vitro. However, lack of robust infection systems for primary, patient-derived isolates limits systematic functional studies of viral intrahost variation and vaccine development. Therefore, we aimed at developing cell culture models for incorporation of primary patient-derived glycoproteins into infectious HCV particles for in-depth mechanistic studies of envelope gene function. To this end, we first constructed a packaging cell line expressing core, p7, and NS2 based on the highly infectious Jc1 genotype (GT) 2a chimeric genome. We show that this packaging cell line can be transfected with HCV replicons encoding cognate Jc1-derived glycoprotein genes for production of single-round infectious particles by way of trans-complementation. Testing replicons expressing representative envelope protein genes from all major HCV genotypes, we observed that virus production occurred in a genotype- and isolate-dependent fashion. Importantly, primary GT 2 patient-derived glycoproteins were efficiently incorporated into infectious particles. Moreover, replacement of J6 (GT 2a) core, p7, and NS2 with GT 1a-derived H77 proteins allowed production of infectious HCV particles with GT 1 patient-derived glycoproteins. Notably, adaptive mutations known to enhance virus production from GT 1a-2a chimeric genomes further increased virus release. Finally, virus particles with primary patient-derived E1-E2 proteins possessed biophysical properties comparable to Jc1 HCVcc particles, used CD81 for cell entry, were associated with ApoE and could be neutralized by immune sera. This work describes cell culture systems for production of infectious HCV particles with primary envelope protein genes from GT 1 and GT 2-infected patients, thus opening up new opportunities to dissect envelope gene function in an individualized fashion.

Bovine vaccinia (BV) is a zoonosis caused by Vaccinia virus (VACV), which affects lactating cows and milkers. VACV DNA and infectious particles have been detected in milk of naturally infected cows. However, the period and pattern of VACV shedding in milk is unknown, as is whether the presence of VACV in milk is due to a localized or a systemic infection. To address those questions, eight lactating cows were inoculated with VACV in previously scarified teats. The experiment was divided in two phases. In Phase 1, milk samples were collected daily for 33 days, and in Phase 2, four animals from the first phase were immunosuppressed. In both phases, milk was collected with a sterile catheter on even days and by hand milking on odd days. All animals showed typical BV lesions in the inoculated teats. All milk samples were subjected to nested polymerase chain reaction (PCR) and real-time quantitative PCR to detect VACV DNA. PCR-positive samples were subjected to virus isolation. VACV DNA was intermittently detected in milk in both phases and infectious viral particles could be detected only in phase 2, on the 69th, 73rd, 74th, 77th, 79th, and 81st days postinfection. Despite the possibility of propagation of VACV through milk, it is known that milk continues to be drawn and marketed normally during outbreaks of the disease. The detection of both VACV DNA and infectious particles in milk samples draws attention to the potential public health risk associated with the consumption of milk from BV outbreaks. Detection of VACV in the milk from noninfected teats demonstrated that VACV shedding in milk might be related to a systemic infection. Moreover, it was shown that VACV DNA and viral infectious particles could be detected in milk even after healing of the lesions, demonstrating that VACV may cause a persistent infection in cattle.

IFIT1 Expression Patterns Induced by H9N2 Virus and Inactivated Viral Particle in Human Umbilical Vein Endothelial Cells and Bronchus Epithelial Cells.

Defective Interfering Particles of Negative-Strand RNA Viruses.

BackgroundViruses can fall prey to their defective interfering (DI) particles. When viruses are cultured by serial passage on susceptible host cells, the presence of virus-like DI particles can cause virus populations to rise and fall, reflecting predator-prey interactions between DI and virus particles. The levels of virus and DI particles in each population passage can be determined experimentally by plaque and yield-reduction assays, respectively.ResultsTo better understand DI and virus particle interactions we measured vesicular stomatitis virus and DI particle production during serial-passage culture on BHK cells. When the multiplicity of infection (MOI, or ratio of infectious virus particles to cells) was fixed, virus yields followed a pattern of progressive decline, with higher MOI driving earlier and faster drops in virus level. These patterns of virus decline were consistent with predictions from a mathematical model based on single-passage behavior of cells co-infected with virus and DI particles. By contrast, the production of virus during fixed-volume passages exhibited irregular fluctuations that could not be described by either the steady-state or regular oscillatory dynamics of the model. However, these irregularities were, to a significant degree, reproduced when measured host-cell levels were incorporated into the model, revealing a high sensitivity of virus and DI particle populations to fluctuations in available cell resources.ConclusionsThis study shows how the development of mathematical models, when guided by quantitative experiments, can provide new insight into the dynamic behavior of virus populations.

If tradition can be based on just two or three instances, I am following the custom of my two predecessors in the Frontispiece to this sesquicentennial issue of The Photogrammetric Record. In the 50th issue, published under the editorship of Keith Atkinson in October 1977, the Frontispiece contained two black-and-white aerial photographs of St Paul's Cathedral in the City of London, UK. They were taken in 1949 and 1975 (reproduced as (f) and (g) in the Frontispiece of the present issue) and were also included on the cover of that issue, the only occasion on which the Record has had other than a plain cover. Keith Atkinson also published two Ordnance Survey photographs of the cathedral and its surroundings from 1969 and 1989 on the occasion of the 75th issue in April 1990. Paul Newby continued this practice in the 100th issue, published in October 2002, with colour photographs of St Paul's taken in 2000 and 2002 ((h) and (j) in our Frontispiece) as well as copies of the 1949 and 1975 photographs. Mr Newby's decision to increase the number of issues from two to four per annum from 2003 means that this 150th issue is under 13 years from the 100th issue, rather than the previous 25-year spacing. My own Frontispiece selection of St Paul's Cathedral comprises: (a) an early Aerofilms oblique aerial photograph from 1921 (now in the archives held by Historic England – formerly English Heritage); (b) a Blom digital aerial photograph (4 cm ground sample distance (GSD)) from 2007; (c) a WorldView-2 satellite image (50 cm GSD) from 2013; (d) a Blom airborne lidar image (50 cm GSD) from 2007; and (e) a terrestrial laser-scanner image taken from the National Geographic Channel's series Time Scanners which was broadcast in 2014 and demonstrated the value of laser-scanning technology to a wider audience. I have invited Editors Emeriti Keith Atkinson and Paul Newby to write their own contributions later in this editorial to complement their thoughts published in the 50th and 100th issues. My own observations were amply aired on the occasion of the Record's Diamond Jubilee (Granshaw, 2013). Furthermore, my last two editorials have indicated the development of photogrammetry over the past century. My December 2014 contribution noted that aerial photography itself was a fledgling activity at the commencement of the First World War, and the first Frontispiece photograph (a), taken by Aerofilms (formed by war veterans Wills and Graham-White), is all the more remarkable for its quality only three years after the end of that terrible conflict. My March 2015 editorial demonstrated that current interaction with the cognate discipline of computer vision still has a long way to go, notwithstanding the modern trend to increasingly incorporate concepts, software and nomenclature from that subject into photogrammetric applications. The past 50 issues of the Record have demonstrated an inexorable trend compared with the previous 100 when aerial photography was dominant. In the 100th issue, Paul Newby used Fig. 1(a) to illustrate the growth of close range photogrammetry in the period since the 50th issue. This tendency has continued over the last 50 issues, alongside the increasing importance of satellite imagery together with both airborne and terrestrial laser-scanner data (illustrated by images (c), (d) and (e) in our Frontispiece, and also Fig. 1(b)). As far as publishing the Record is concerned, the past 50 issues have demonstrated, in true British fashion, continuity allied to gradual change. In many ways our journal is very conservative; for example, we still maintain an Index (one of only two Wiley journals to do so). The format of the journal has barely altered over the years though the balance has changed, with more emphasis on reviewed papers and less on reports. The topic of peer review leads me to record the excellent work of our International Editorial Board, steered magnificently by Jim Chandler from 2002 to 2010, and then by Simon Buckley since that date. Both Coordinators have demonstrated exceptionally hard work and innovation. Moreover, Jim Chandler and Paul Newby oversaw, in 2006, the digitisation of all issues of the Record back to issue 1 in 1953; Simon Buckley has transformed the review process with the introduction, this year, of the ScholarOne Manuscript electronic review system. Some aspects of the Record's content have grown; others have waned. For example, issue 105 (March 2004) saw Spanish abstracts join their French and German counterparts; these were further augmented by Chinese abstracts from issue 146 (June 2014). September 2008 (issue 123) saw the first special issue, devoted to the ISPRS Hannover Workshop 2007; further special issues constituted Nos. 124, 131, 136 and 137. Single-topic virtual issues were placed on the Wiley Online Library in 2012 (environmental applications) and 2014 (image matching). On the other hand, the advertising that had assisted the journal's finances eventually ended (other than for publishers' own publicity) in issue 127 (September 2009); Fig. 3(d) illustrates one of the last three photogrammetric advertisements, which were an integral part of a print era but sit less happily among the pop-up promotions of the Internet. The future holds many unknowns, both for our journal and the discipline of photogrammetry itself. The Photogrammetric Record remains the primary photogrammetric portal of the UK's Remote Sensing and Photogrammetry Society (RSPSoc). There will undoubtedly be changes, perhaps sooner rather than later, in online-only distribution (with print restricted to on-demand) and open-access developments. These changes need to be viewed in a positive light rather than with a Luddite mentality: both the current page and colour budgets (480 and 256 pages, respectively, per year), that are limiting financial features in a print issue, essentially disappear in a solely electronic issue. As Jim Chandler and Hans-Gerd Maas have noted in the Sesquicentennial Congratulations that follow this editorial, the Record is now the only international journal dedicated exclusively to photogrammetry, but its own financial health in the rapidly changing publishing industry needs to be carefully monitored. There are current fiscal issues which I am hopeful will be resolved, due in no small part to the supportive efforts of Pauline Miller, the chair of RSPSoc's Publications Committee, and Tina Thomson, the Society's treasurer. Photogrammetry itself has enjoyed a recent resurgence. Non-geomatics personnel in both academe and industry are now well aware of modern photogrammetry. They may be under the impression that our subject is a new discipline and believe that laser scanning (lidar) is waning. This is essentially due to the cost of data collection: it is much cheaper to acquire a camera than a laser scanner. Photogrammetric software has become extremely easy to use, handling large sets of images automatically. There is a danger here: with essentially no consideration of network geometry nor of the size of datasets, accuracy may be compromised and processing times can become excessive. The challenge for those with geomatic and photogrammetric backgrounds is to ensure that we remain relevant when it is so easy and inexpensive for non-specialists to achieve acceptable results. Aerial survey companies are also moving from lidar back to the traditional realm of photogrammetry. I am therefore hopeful that, despite these various challenges, a revival in photogrammetry means we will reach our 200th issue, and do so in good health. Furthermore, this will be achieved by embracing essential modifications yet respecting our tradition, embedded in every one of our 150 issues, of quality dissemination of photogrammetric research and practice. The annual RSPSoc Thompson Award is presented to the authors of the best paper in the Record (Fig. 2). I feel sure that E. H. Thompson would appreciate the hard work and dedication of the current editorial team and that we are maintaining the standards that he and his predecessors set, along with those of our two Editors Emeriti whose contributions now follow. January 2015 In the years since the first issue of The Photogrammetric Record was published in March 1953, six Editors have been responsible for its publication: P. G. Mott (1953–54); H. A. L. Shewell (1954–62); E. H. Thompson (1962–76); K. B. Atkinson (1976–99); P. R. T. Newby (1999–2011); and S. I. Granshaw (from 2011). All of them have adhered to the founding concepts of the journal so that the 150th issue of June 2015 still bears some similarities with the inaugural edition of 62 years earlier. Mott (1977) related the influence of The Polar Record (to which he was a regular contributor) in both name and presentation to the first volume of our own journal, praising its combination of clarity, boldness and artistic merit. The Geographical Journal was also reflected in The Photogrammetric Record's original style and content. The first issue of the Record contained three articles, all substantial contributions written by heavyweights of the photogrammetric firmament – G. C. Brock, J. A. Eden and E. H. Thompson. They set a standard of content which successive editors strove to maintain. By the time of the 50th issue in 1977, the Record had become well established. A typical issue would carry an editorial article (some of those by Thompson were classics of their kind and are still quoted from time to time); scientific and technical articles and shorter contributions; Photogrammetry Round the World (a collection of abstracts of current periodical literature); numerous book reviews; notes; correspondence; and obituaries. Foreign language abstracts of articles were only provided in French and German. The Photogrammetric Society's Technical Committee influenced the content of the Record over many years; its tone was set from the birth of the Society with E. H. Thompson as the very first chairman of the Committee. I found it vital to be a member of the Committee while also Editor. One volume of the Record comprised six issues published over three years and concluded with an Index of the content of that volume. The cost of publication was supported by the inclusion of paid advertisements, usually running to many pages in each issue (Fig. 3). For example, the 50th issue contained 27 pages of advertisements, including five pages from Wild Heerbrugg, four pages from Zeiss (Oberkochen) and three pages from Zeiss (Jena) (reminding us of the divided Germany after the Second World War). An article published in the same 50th issue sets out some of the details which fashioned the content and appearance of the Record (Atkinson, 1977). Although there are distinct similarities with the journal of today, it is the differences which are more significant. Fig. 1 in Atkinson (1977) illustrates in graph form the numbers of published articles originating from UK authors and from foreign authors. The former dominate. That situation changed many years ago and was in part responsible for the addition, in 1998, of the subtitle An International Journal of Photogrammetry, together with the establishment of an International Editorial Board. As was pointed out at the time, Volume 15 (published in 1995–97) contained 70 articles of which 30 originated in 15 different countries outside the UK. It has now become a rarity to read articles from British authors; there were two such papers in issue 148, none in issue 149 and just one co-author from the UK in the current issue. In 1977, the Record was then one of several periodicals which catered solely for articles on photogrammetry. Other contemporary titles included Bildmessung und Luftbildwesen, Bulletin de la Société Française de Photogrammétrie, Photogrammetria, Photogrammetric Engineering, Photogrammétrie and The South African Journal of Photogrammetry. Some titles have survived and changed with the inclusion of remote sensing or geomatics content while others have fallen by the wayside. Another marked contrast lies in author employment. The same Fig. 1 (Atkinson, 1977) shows that, in the 1970s, academic authors were outnumbered by others from commercial, industrial, government and military employment. For example, issue 46 (October 1975) contained nine articles, only three of which originated in universities whereas, in March 2015, issue 149 carried six articles which all originated in academic institutions. But what was the burning photogrammetric issue of the mid-1970s? There is no doubt that the birth of the analytical plotter was most significant with the International Congress of Photogrammetry at Helsinki in 1976 (Petrie, 1977) witnessing, for the first time, the introduction or announcement of new analytical plotters from eight instrument manufacturers. Their names constitute something of a trip down memory lane. The instruments actually on display at Helsinki were the Zeiss (Oberkochen) Planicomp C 100 (Fig. 3(b)), the Ottico Meccanica Italiana (OMI) AP/C-4 and the Officine Galileo Digital Stereocartograph, while there were prototype instruments from Matra (Traster 77), Instronics (Anaplot) and Zeiss (Jena) (Stereodicomat). In this context, it is also of interest to read a Note from The Photogrammetric Record, 9(49) (page 119). "A large number of members of The Photogrammetric Society gathered in the rooms of the Linnean Society on Tuesday, 14th December, 1976 to debate the motion that this house considers that the day of the analogue instrument is over." Following details of chairman and speakers, "serious argument and good humour were equally prevalent during the debate which resulted in the defeat of the motion by a substantial majority". February 2015 As the Editor who succeeded Keith Atkinson, I was very well aware of the legacy of excellence left for me in 1999 by Keith himself and by Veronica Brown, his loyal Assistant Editor, who stayed on to help me in my early years in the post. The first issue of The Photogrammetric Record which I received whilst a student at University College London (UCL) was No. 40 (October 1972). By happy chance this contained an article (Fagan, 1972) about photogrammetry at the Ordnance Survey to which I would be posted after my year at UCL, as well as an editorial which discussed the UK's relationship with the International Society for Photogrammetry (ISP) which, as ISPRS, became a prime focus of my attention many years later. By the time of the 50th issue in 1977 The Photogrammetric Record was clearly in business for the long term and, apart from the illustrated cover of that issue, seemed destined to remain unchanging in a changing world, despite the death of E. H. Thompson and the accession of K. B. Atkinson to the Editor's chair. The Photogrammetric Society's skilfully arranged winter programme of interesting lectures provided a steady stream of worthy contributions to the Record. Once every two years a distinguished overseas speaker was invited; although these would frequently be from Europe our network of contacts and therefore of invitees extended as far as North America, Australasia and, on one occasion, Japan. The members of the Technical Committee who designed the programme and invited the speakers, and of the Editorial team who cajoled them into becoming authors working to a publication deadline, put great voluntary effort into ensuring that the process worked smoothly and efficiently. The influence of the Technical Committee on the balance of the content of the Record has perhaps been underestimated, although both Keith Atkinson and I have commented over the years on the shift in our authorship from practitioners towards a preponderance of academics and researchers. The custom of recording and transcribing the lengthy discussions which followed the presentation of every paper demanded extra effort but was thoroughly worthwhile. This reasonably continuous flow of contributions was augmented every two years (or four issues) by a large proportion of the proceedings of each of the regular series of weekend Society Symposia, held then in Birmingham University under the aegis of R. G. Bird. In 1977 this became the Thompson Memorial Symposium. The subsequent series of Thompson Symposia on April weekends continued until soon after the merger with the Remote Sensing Society, when they were subsumed into the Annual Conferences of the Remote Sensing and Photogrammetry Society (RSPSoc). Another regular input has been the series of multi-authored papers reporting on ISP, then ISPRS, Congresses which take place every four years. Our efforts to present a truly independent analysis of these very large meetings have continued to be appreciated by readers around the world. Without extensive statistical analysis it is impossible to pin down the moment when the emphasis shifted from papers invited for presentation at Society meetings to unsolicited contributions from authors both in the UK and overseas who simply wished to publish the results of their work. Perhaps there was no specific moment, just an inexorable but gradual change. Keith Atkinson had long operated a peer review process, but this was purely informal and was based on taking advice from his private army of experts among his friends and colleagues. Along with the increasingly cutthroat world in which academics are obliged to publish in formally peer-reviewed journals in order to flourish, the shift to a global contribution base must have been one of the main factors which persuaded him to establish the International Editorial Board (IEB) in 1998, shortly before his retirement. The other momentous change in that same year was our first experiment in electronic publishing, a compact disc containing the whole of volume 15 (1995–97), instigated by Jim Chandler (who from No. 100 would also become Coordinator of the IEB and therefore of the peer review process); this CD was the true forerunner of the simultaneous online publication which we take for granted today. After the formation of RSPSoc in 2001, support for photogrammetric technical meetings was dwindling and the lecture programme became unsustainable. From early in the new century, the Record would have to rely entirely on a combination of external contributions and, a major new effort this, the collected proceedings of occasional technical symposia organised by members of our Society or friends of our journal. When I took over from Keith Atkinson immediately after the publication of his No. 94 in 1999, it was his boast, and his encouragement to me, that The Photogrammetric Record had never experienced a copy shortage and, despite the occasional vagaries of the British printing industry, had always appeared on schedule. Even in the global publishing and printing world which we now inhabit, that proud record has been maintained with very few exceptions; any occasional worries about a shortage of available copy have been replaced by the constant concern about the page budget set by our co-publishers, Wiley-Blackwell. That, however, is to anticipate the momentous change brought about in 2003: to secure the future of the Record it was decided, soon after the merger of the Photogrammetric Society with the Remote Sensing Society, to enter into a co-publishing agreement with a scientific publisher in the open market. After much investigation and negotiation, we selected Blackwell Publishing Ltd of Oxford. The hundredth issue (October 2002) became the last of the old, weighty numbers published half-yearly by the Society alone, and No. 101, March 2003, would bring in the new era of slim quarterly issues co-published by RSPSoc and Blackwell (eventually Wiley-Blackwell). Sadly No. 100 was also the last to include Veronica Brown's regular feature Photogrammetry Round the World in which, for over 25 years, she had faithfully provided abstracts from photogrammetric publications worldwide. Although in the days of easy access to the World Wide Web it could be argued that the need for this feature had become less pressing, it was a matter of regret that no volunteer came forward to continue her work. In all other respects we strove to maintain the style and content of the Record while taking advantage of the increased flexibility and immediacy offered by quarterly publication in both paper and online form. That was the future, forward from No. 101 to another milestone with the current issue, No. 150. March 2015

Replication-competent Vectors and Empty Virus-like Particles: New Retroviral Vector Designs for Cancer Gene Therapy or Vaccines

The mechanisms by which infectious hepatitis C virus (HCV) particles are assembled and released from infected cells remain poorly characterized. In this regard, many other enveloped viruses, notably human immunodeficiency virus type 1, have been shown to utilize the host vacuolar protein sorting machinery (also known as the endosomal sorting complex required for transport; ESCRT) to traffic through the cell and effect the membrane rearrangements required for the formation of enveloped particles. We postulated that this might also apply to HCV. To test this hypothesis, we established a method of conditional virus-like particle assembly involving trans-complementation of an envelope-deleted JFH-1 genome using plasmid transfection. This system reliably produced virus particles that were infectious and could be enumerated easily by focus-forming assay in Huh7 cells. Following co-transfection with plasmids expressing various dominant-negative forms of either components of the ESCRT-III complex or Vps4 (the AAA ATPase that recycles the ESCRT complexes), a reduction in particle production was seen. No significant effect was observed after co-transfection of dominant-negative ESCRT-I or Alix, an ESCRT associated protein. Dominant-negative Vps4 or ESCRT-III components had no effect on either virus genome replication or the accumulation of intracellular infectious particles. These data were confirmed using cell culture infectious HCV and we conclude that HCV requires late components of the ESCRT pathway for release of infectious virus particles.

Influenza A and B virus reproduction in the allantoic membrane of the intact chicken embryo was studied quantitatively with particle enumeration procedures. Virus particles were enumerated on the basis of two independent properties; capacity to infect and to cause hemagglutination. The infective property of influenza B virus (Lee) was even more unstable than that of influenza A virus (PR8). Inactivation occurred at a constant logarithmic rate which was independent of the concentration of particles and corresponded with first order reaction kinetics. In allantoic fluid at 35°C. either in vitro or in vivo, Lee virus had a half-life for infectivity of only 85 minutes. In contrast, the hemagglutinating property, like that of PR8, was relatively stable and was not appreciably affected by 12 hours at 35°C. On the basis that the number of non-infective particles is equal to the number of hemagglutinating particles minus the number of infective particles and that the number of cells lining the allnatoic membrane is 1.8 x 107, the effects of various particle-cell ratios on the reproductive process were analyzed. Adsorption of infective and non-infective Lee particles occurred at the same logarithmic rate, i.e. about 50 per cent in 72 minutes, and the rate was nearly independent of the particle-cell ratio up to a value of 55. The adsorption capacity of an allantoic cell was at least 44 Lee or 89 PR8 particles. The interval before new particles appeared in the allantoic fluid increased as the particle-cell ratio was decreased with both Lee and PR8. At ratios of 0.2 or less, the appearance time for infective particles was nearly identical to that for hemagglutinating particles with both viruses. At ratios of about 1.0, the "latent period" in the allantoic membrane per se was computed to be 150 to 160 minutes for both Lee and PR8. The number of particles, both infective and hemagglutinating, increased at a constant logarithmic rate for 6 hours or more after the adsorptive period. With Lee virus, at a particle-cell ratio of 5 or less, the doubling time was constant and had a value of 43 minutes. The dynamics of the logarithmic increase period suggest that reproduction corresponds to an autocatalytic reaction in which the rate is proportional to the amount of material produced. When the particle-cell ratio was increased to 10 or more, either with infective or non-infective (inactivated at 35°C. or 22°C.) particles, the doubling time increased to 65 minutes. Comparable effects from high ratios were found with PR8. Non-infective particles accumulated at a rapid rate after the interval of constant logarithmic increase regardless of the particle-cell ratio. This accumulation was even more striking with Lee than with PR8 as was expected because of the shorter half-life of the infective property. With both viruses at particle-cell ratios of 4 or more, a large proportion of the particles were non-infective within a few hours after new particles appeared. At particle-cell ratios of 0.2 or less, the maximal yield was relatively constant, i.e., about 900 to 1400 hemagglutinating particles per cell with Lee and 500 to 900 with PR8. However, even with very low ratios, i.e. 0.001 or less, it was not possible to obtain more than about 160 infective particles per cell with either virus regardless of the interval. As was expected, the lower the ratio, the longer was the interval before maximal yields were produced. At ratios of about 10, the maximal yield was reduced by 50 per cent or more with both viruses. Comparable reductions in yield were obtained whether the high particle-cell ratio was due to infective or non-infective (inactivated at 35°C. or 22°C.) particles. These findings indicate that there is a critical particle-cell ratio above which alterations appear in the dynamics of reproduction of influenza viruses. This ratio has a value of approximately 3. The observed alterations in the reproductive process are discussed in relation to the hypothesis that adsorption of 3 or more infective or non-infective particles per cell induces cell damage.

A decade since the epidemic of the acquired immunodeficiency syndrome (AIDS) was first recognized, a wealth of information has accumulated on the molecular biology of the causative agents, the human immunodeficiency viruses (HIV). Of particular interest is knowledge of the viral enzymes involved in the formation of new virus particles. Such enzymes constitute attractive targets for efforts aimed at selecting agents that interfere with virus multiplication and subsequent spread and pathogenesis. Already, several agents that inhibit the viral reverse transcriptase (e.g., nucleoside analogs such as Zidovudine) have proved to have a beneficial effect on the course off the disease, but their prolonged use has been associated with significant toxicity and the emergence of resistant mutants. A second enzyme that has recently attracted attention is the virus-coded protease. This enzyme is involved in the cleavage of viral precursor polyproteins into the final products that constitute the mature virus particle. Protease inhibitors interfere with the process of virus maturation which is required for the formation of infective virus particles. Several custom-made inhibitors with a high selective action against HIV protease have been produced recently. They are nonhydrolyzable peptide analogs that mimic the cleavage sequences of the natural substrate of the enzyme during the transition state of the cleavage reaction. It is hoped that a similar selectivity in vivo may make protease inhibitors a promising new category of AIDS therapeutics.

Photobleaching is a major obstacle in the real-time imaging of biological events, particularly at the single-molecule/particle level. Here, we report a strategy to delay photobleaching of a light-switch complex, [Ru(phen)2dppx]2+, by insertion of a six-cysteine peptide into virus particles. The six-cysteine peptide was inserted into viral protein R of HIV-1 and assembled into infectious HIV-1 viral particles, where it effectively delayed the photobleaching of the [Ru(phen)2dppx]2+ complex used to label viral genomic RNAs. This delay in photobleaching allowed for a monofluorescent assay to be constructed for the real-time monitoring of viral uncoating, a poorly understood process. This novel strategy to delay photobleaching in infectious viral particles provides a powerful method to analyze viral uncoating at the single-particle level in real time.

Mode of antiviral action of 5-iodouracil deoxyriboside

Chapter VIII - The Comparative Chemistry of Infective Virus Particles and of other Virus-Specific Products : Animal Viruses