The authors discuss the implications of a new study that presents compelling data to show that norovirus evolution is driven by immune selection pressure. First called Norwalk virus  in reference to the Ohio town where specimens from a school outbreak enabled the seminal work that first characterised the virus, it was later dubbed the Small Round Structured Virus following visualisation by electron microscopy. The International Committee on Taxonomy of Viruses later settled on the present name of “norovirus”, classifying it as a member of the Caliciviridae family based on both morphology and phylogeny. A new study published in this issue of PLoS Medicine suggests that the colloquial “gastric flu” may have been the most apt term of all [Linguistically speaking, the predominant viral cause of gastroenteritis has been evolving. Once evocatively called m of all . When mem of all . There aIn the new study, Ralph Baric and colleagues present compelling data to demonstrate that norovirus evolution is driven by immune selection pressure. The domain of the exposed viral capsid protein that binds carbohydrates in the human gut evolves in the face of herd immunity. Histoblood group antigens (HBGAs), a heterogeneous group of related carbohydrates on mucosal surfaces, provide a “docking station” for noroviruses, and there is a large variety of such HBGAs. These sites are similar, but distinct, so as the capsid mutates and subtly changes its shape, it can still find new binding sites on the mucosal surface of the gut. The virus survives despite the build-up of immunity in the population because there is room in antigenic space for the virus to evolve and remain infectious. As with influenza, the predominant circulating norovirus may also change its antigenicity frequently, giving it the potential to cause pandemics and necessitate regular reformulation of vaccine.PLoS Medicine:This Perspective discusses the following new study published in 10.1371/journal.pmed.0050031Lindesmith LC, Donaldson EF, LoBue AD, Cannon JL, Zheng DP, et al. (2008) Mechanisms of GII.4 norovirus persistence in human populations. PLoS Med 5(2): e31. doi:Through phylogenetic analysis of norovirus isolates, Ralph Baric and colleagues show that new epidemic strains arise as the variety of available cellular receptors permits antigenic drift in the viral capsid.Noroviruses are the most commonly detected pathogen both in sporadic cases and outbreaks of gastroenteritis. They are particularly problematic in environments where groups congregate and infection can be rapidly transmitted through both faecal and vomitus routes. Outbreaks affect health care facilities worldwide, and may cause massive disruption to providing care, substantial economic loss, and, according to some reports, mortality in vulnerable patient populations . As is tSome individuals, upon being challenged with norovirus infection, develop gastroenteritis, while others develop asymptomatic infection and some show no signs of infection at all. This points to a role of acquired immunity as well as innate resistance to infection. Indeed, individuals who genetically encode the enzyme FUT2 a-fucosyltransferase and are secretor-positive  are susceptible to Norwalk virus (a GI.1 virus) infection. Distinct binding patterns have been described for a range of other GI and GII strains, including GII.4. Acquired immunity is not thought to last until a subsequent norovirus season, though a few individuals may acquire longer-lasting immunity. With these factors combined, one might think that immune selection pressure would be rather transient—only heavy at the end of a season—and that an evolutionarily stable strategy for norovirus might be to wait out the summer low season and attack again when population immunity has waned. This is not what Baric and colleagues have found.Instead, their findings suggest (1) that noroviruses are under heavy selective pressure and (2) that the norovirus capsid, which contains both antigenic sites and carbohydrate binding ligands, seems to have been finely tuned to evolve. The P2 region of the capsid is attached to the virus shell by a flexible hinge, so—with minor genetic tweaking—the virus can nuzzle up to a range of HBGA sites. This domain is evolving at a faster rate than regions not coding for surface residues.But perhaps Baric's most compelling evidence that the virus evolves in the face of human immunity comes in the form of serology . Pre-epidemic anti-serum binds poorly to post-epidemic virus-like particles; this observation was most extreme for the 2002–2003 epidemic, which by many accounts ,7 was thThere are, however, important differences between influenza and norovirus evolution and epidemiology. Although GII.4 viruses predominate, they do so within a large population of co-circulating genotypes. In contrast, a new influenza subtype generally replaces existing types. Following antigenic shift, replaced influenza types tend to go extinct, while usurped norovirus variants continue to circulate at low levels. Such differences are probably driven by the short-lived non-sterilising immunity to norovirus compared with the longer-term protection to influenza antigens. Indeed, the other norovirus genotypes may well be operating under different selective pressure than GII.4.Baric and colleagues' landmark paper sets forth a wide research agenda for norovirus vaccinology, virology, and epidemiology. The authors are bullish that their findings take us towards the development of norovirus vaccines. This is undoubtedly true—though it is unlikely that a major pharmaceutical company will invest the massive sums required to bring to market a product for such an antigenically diverse virus that confers short-lived immunity and is perceived to cause a relatively low burden of disease. We think this lack of investment in developing a vaccine would be unfortunate; vaccination targeted at vulnerable populations and health care workers could mitigate the most severe health and economic consequences caused by noroviruses. Formulation of a norovirus vaccine would be challenging, but tools developed for understanding influenza evolution could prove useful. For example, data generated from Baric and colleagues' serological experiments lend themselves to fascinating antigenic mapping methods used to quantify and visualise antigenic differences between circulating influenza strains , and mayWhat is really needed—but has been lacking—is a good in vitro cell culture system. Such a system would facilitate investigation of the relationship between antigen and receptor and how antibody interferes with binding. The finding that secretor-negative individuals are still susceptible to infection suggests that the HBGAs are only one link in a binding hierarchy.An area absent from Baric and colleagues' paper is a discussion of the seasonality of norovirus. Like influenza, noroviruses exhibit a strong wintertime seasonality (see


H9N2 avian influenza viruses continue to circulate worldwide; in Asia, H9N2 viruses have caused disease outbreaks and established lineages in land-based poultry. Some H9N2 strains are considered potentially pandemic because they have infected humans causing mild respiratory disease. In addition, some of these H9N2 strains replicate efficiently in mice without prior adaptation suggesting that H9N2 strains are expanding their host range. In order to understand the molecular basis of the interspecies transmission of H9N2 viruses, we adapted in the laboratory a wildtype duck H9N2 virus, influenza A/duck/Hong Kong/702/79 (WT702) virus, in quail and chickens through serial lung passages. We carried out comparative analysis of the replication and transmission in quail and chickens of WT702 and the viruses obtained after 23 serial passages in quail (QA23) followed by 10 serial passages in chickens (QA23CkA10). Although the WT702 virus can replicate and transmit in quail, it replicates poorly and does not transmit in chickens. In contrast, the QA23CkA10 virus was very efficient at replicating and transmitting in quail and chickens. Nucleotide sequence analysis of the QA23 and QA23CkA10 viruses compared to the WT702 virus indicated several nucleotide substitutions resulting in amino acid changes within the surface and internal proteins. In addition, a 21-amino acid deletion was found in the stalk of the NA protein of the QA23 virus and was maintained without further modification in the QA23CkA10 adapted virus. More importantly, both the QA23 and the QA23CkA10 viruses, unlike the WT702 virus, were able to readily infect mice, produce a large-plaque phenotype, showed faster replication kinetics in tissue culture, and resulted in the quick selection of the K627 amino acid mammalian-associated signature in PB2. These results are in agreement with the notion that adaptation of H9 viruses to land-based birds can lead to strains with expanded host range. The first isolation of a H9N2 subtype avian influenza virus was in 1966 in the U.S. To date, the mechanism of adaptation and interspecies transmission of H9N2 viruses is poorly understood. Accumulating data suggests that among the land-based poultry, quail play a crucial role for the genesis of influenza viruses. We have previously shown that quail provide an environment where most influenza subtypes are able to replicate, primarily in the respiratory tract In order to understand the molecular basis of adaptation and transmission of H9N2 viruses in different animal species in nature, we developed a laboratory-based model to adapt an H9N2 virus to land-based poultry. Our results suggest that laboratory adaptation of a duck H9N2 virus through serial lung passages in quail and chickens generated a variant virus that is capable of efficiently replicating and transmitting in chickens. More importantly, the adapted virus gained the ability to replicate efficiently in mice when inoculated intranasally. The results from this laboratory-based adaptation study mimic the ongoing activities of H9N2 viruses in nature and highlight the potential role of land-based poultry in the genesis of influenza viruses with expanded host range.Initial studies in our laboratory confirmed previous findings and showed that the WT702 virus replicates and transmits in Japanese quail but replicates poorly and does not transmit in White leghorn chickens In order to make the WT702 virus more transmissible in chickens we followed a strategy that we have previously employed for adaptation of a mallard H2N2 virus in quail and chickens 50) assays and analyzed tracheal and lung virus titers with the three viruses, WT702, QA23, and QA23CkA10. As shown in 50) for the WT702, QA23 and QA23CkA10 viruses were similar, between 1×102 and 5×101 EID50. In contrast, the chicken infectious dose 50 (CID50) required 1,000-fold more WT702 virus (5×105 EID50) than for the QA23 and QA23CkA10 viruses . We then analyzed the virus titers in the lungs of quail and chickens infected with different doses of the WT702, QA23 and QA23CkA10 viruses . More importantly, replication of the QA23 and QA23CkA10 viruses in mice was clearly evident. All mice infected with the QA23 or QA23CkA10 viruses at doses of 105 EID50 or 107 EID50 had positive lung virus titers  a group of 4 mice. Body weight changes and clinical signs of disease were monitored every day for 14 days  and used to infect intranasally , the adapted virus gained transmission properties in quail and replication/transmission properties in chickens Several amino acid substitutions were identified scattered throughout the genome of the QA23 and QA23CkA10 viruses compared to the WT702 virus. It was also evident the loss of 21 amino acids spanning the stalk region of the NA protein in the QA23 and QA23CkA10 viruses. Once again, our studies show that quail is one of potentially many other land-based birds from which NA genes of various lengths could be produced. To our knowledge this is the longest amino acid deletion among the NA of H9N2 viruses to date based on the available public influenza sequence database. H9N2 viruses isolated from land based poultry often contain deletions in the stalk region of the NA; however these deletions are restricted to just a single or few amino acids Alignment of the deduced amino acid sequence of the adapted viruses with available published H9N2 sequences provided important clues regarding mutations in the HA protein: For example, most duck H9N2 viruses contain theronine at position 29 (40 in the H3 HA) in the HA1 region, whereas those from land-based poultry contain alanine. Likewise, most duck H9N2 viruses encode for glutamic acid at position 43 in the HA2 portion but most land-based poultry derived viruses encode for lysine rd quail lung passage. The chicken-adapted (QA23CkA10) virus was isolated from the 10th chicken lung passage of the QA23 virus. Stocks of QA23 and QA23CkA10 viruses were prepared by the inoculation of lung homogenates into the allantoic cavity of 10-day-old embryonated chicken eggs. Subsequently, the QA23 and QA23CkA10 viruses were plaque-purified using confluent Madin-Darby canine kidney (MDCK) cells. MDCK cells were regularly maintained in Modified Eagle's medium (MEM)  containing 5% fetal bovine serum (FBS) (Sigma-Aldrich). Four days following the plaque assay, a single plaque from each virus was isolated and used for a second round of plaque purification in MDCK cells. Following 2-plaque purifications, 2 plaques from each virus were grown in embryonated eggs to produce a working stock of virus. Alternatively, viruses were grown in chicken embryo kidney (CEK) cells where specified. CEK cells were prepared from 17-days-old chicken embryo according to the method described previously The A/Duck/Hong Kong/702/79 (H9N2) virus (WT702) was kindly provided by Dr. Robert G. Webster at St. Jude Children's Research Hospital, Memphis, TN. The quail-adapted (QA23) virus was isolated from the 2350 in CEK cells according to the method described previously Growth kinetics of WT702 and plaque purified QA23 and QA23CkA10 viruses were monitored in MDCK cells and CEK cells. Confluent monolayers of MDCK or CEK cells were infected with the viruses at a multiplicity of infection (m.o.i) of 0.001. Following 1 h adsorption at 37°C, cells were washed three times with HBSS (Sigma-Aldrich) containing antibiotic/antimycotic. Cells were then maintained in Opti-MEM  or Medium 199 (Sigma-Aldrich) supplemented with TPCK-trypsin  at a concentration of 1 µg/ml. Supernatants were collected every 12 h for 72 hpi and stored at −80°C following clarification by centrifugation. Virus titers in the supernatant were measured by TCIDCoturnix coturnix, University of Maryland, College Park, Central Research Facility) and 3 to 4-weeks old specific-pathogen free White Leghorn chickens  were used throughout the study. Groups of three birds were inoculated intraocularly, intranasally, intratracheally and orally with different doses of virus diluted in 0.6 ml (quail) or 1.0 ml (chicken) in PBS. Tracheal and cloacal swabs were collected at 1, 3, 5, 7, 9, 11, and 13 dpi and were stored in 1 ml freezing medium at −70°C (50% glycerol in PBS containing 1% antibiotics). Virus titers in the swabs were determined by EID50 as previously described. Birds were observed and scored daily for clinical signs of infection and general well being. They were evaluated on appetite, activity, fecal output and signs of distress including cyanosis of the tongue or legs, ruffled feathers and respiratory distress. Experiments were carried out under BSL-2 conditions with investigators wearing appropriate protective equipment and compliant with all Institutional Animal Care and Use Committee (IACUC) of University of Maryland approved protocols and under the Animal Welfare (AWA) regulations.4- to 6-weeks old Japanese quail (6 EID50) by the ocular, nasal, oral and tracheal routes. Three quail were used for each passage. At 3 dpi, quail were sacrificed and lung homogenates were prepared using a mortar and sterile sea sand. The homogenates were resuspended in 3 ml sterile brain heart infusion (BHI) medium containing antibiotics as described 8 EID50. At 3 dpi, lung homogenates were prepared as described above. Pooled lung homogenates (1 ml) prepared from 3 separate chickens were used to inoculate the next group of chickens by nasal, oral, tracheal and cloacal routes. After 10 sequential lung passages in chickens (QA23CkA10), a stock virus was prepared in 10-days-old embryonated chickens eggs.For quail lung passages, quail were inoculated with the WT702 virus  and chickens (lung and intestine) a group of 3 birds were sacrificed at 3 dpi and tissue homogenates were prepared in BHI medium and stored at −80°C. Virus shedding through trachea and cloaca were monitored by the inoculation of swabs into 10-days-old embryonated chicken eggs. The virus titers in lung homogenates were measured also by the inoculation into 10-days-old embryonated chicken eggs.For replication and transmission studies, groups of 3 quail or chickens were inoculated with 5×105-week-old female BALB/c mice, 4 per group, were infected intranasally with different doses of the WT702, QA23, QA23-PP, QA23CkA10, QA23CkA10-PP, and QA23ML-3 Egg viruses in 50 µL PBS under light anesthesia using isoflurane . Body-weight and signs of disease were monitored everyday until they were sacrificed (4 or 14 dpi as indicated). Lung homogenates and virus titers were determined as described above.5′-AGCAAAAGCAAAGG-3′) and AMV reverse transcriptase . PCR amplification was performed using the universal primers described by Hoffman et al. Total RNA was extracted from stock virus in allantoic fluid or from the mouse lung homogenate using the Rneasy kit  according to manufacturer's instructions. Reverse transcription was carried out with the uni-12 primer (


The phylogenetics of the genus Alphavirus have historically been characterized usingpartial gene, single gene or partial proteomic data. We have mined cDNA andamino acid sequences from GenBank for all fully sequenced and some partiallysequenced alphaviruses and generated phylogenomic analyses of the genus Alphavirusgenus, employing capsid encoding structural regions, non-structural coding regionsand complete viral genomes. Our studies support the presence of the previouslyreported recombination event that produced the Western Equine Encephalitis clade,and confirm many of the patterns of geographic radiation and divergence of themultiple species. Our data suggest that the Salmon Pancreatic Disease Virus andSleeping Disease Virus are sufficiently divergent to form a separate clade fromthe other alphaviruses. Also, unlike previously reported studies employing limitedsequence data for correlation of phylogeny, our results indicate that the BarmahForest Virus and Middelburg Virus appear to be members of the Semliki Forestclade. Additionally, our analysis indicates that the Southern Elephant Seal Virusis part of the Semliki Forest clade, although still phylogenetically distant from allknown members of the genus Alphavirus. Finally, we demonstrate that the wholeRubella viral genome provides an ideal outgroup for phylogenomic studies of thegenus Alphavirus.


Plasmodium falciparum-hosting RBC (Pf-RBC) were retained in a parasite developmental stage-dependent way, the retention rates of a subset of ring-RBC being similar in micro-beads and in isolated-perfused human spleens. We found that this retention might be linked principally to the reduced surface-area-to-volume ratio of Pf-RBC. Interestingly, other rigid RBC, such as heat-treated RBC, and RBC from hereditary spherocytosis patients were also retained in micro-beads without any hemolysis. Micro-beads allow (i) depletion of heterogeneous RBC population from its rigid-RBC subpopulation ii) characteriziation of distinct molecular signatures of rigid versus deformable RBC subpopulations. This simple method portends wide medical applications, such as improving the quality of stored RBC concentrates prior to transfusion.Experimental tools to identify human red blood cells (RBC) prone to mechanical retention upstream from the spleen venous sinus inter-endothelial slits are currently suboptimal. We designed a micro-bead device mimicking the geometry of the human narrow and short inter-endothelial slits. Upon filtration through a mixture of 5-25 μm diameter micro-beads,


These "new viruses" are now commonly reported in the virological literature and are too numerous to cite here, two studies are given as typical examples [The wide availability of PCR-based methods for the amplification of nucleic acids homologous to known viral genomes plus automated DNA sequencing have led to explosive growth of a new field, 'proxy' virus isolation. Typically, genomic fragments are amplified and subjected to phylogenetic analysis showing that they are related to, but distinct from, known 'examples ,2. Althobona fide viruses we propose the term 'frag-virus'.To distinguish presumptive viruses known primarily through genomic sequence fragments from bona fide virus status. At the same time the frag-virus status will be a 'career end-point' for sequences belonging to non-infectious 'pseudo-viruses' or those having their origins in artifact.The 'frag-virus status' may be intermediary between 'full' recognition as a new virus, as occurred, for example, with hepatitis C virus and Kaposi sarcoma herpesvirus -6. Frag-Whatever the 'fate' of frag-viruses, this simple term is highly informative and may prove useful in preventing misconceptions about new viruses.


Noroviruses are shed in feces up to 8 weeks after infection. 9  genomic copies/g feces as measured by quantitative RT-PCR. Virus shedding was first detected by antigen ELISA ≈33 hours (median 42 hours) after inoculation and lasted 10 days (median 7 days) after inoculation. Understanding of the relevance of prolonged fecal norovirus excretion must await the development of sensitive methods to measure virus infectivity.Noroviruses are the most common cause of viral gastroenteritis in the United States. To determine the magnitude and duration of virus shedding in feces, we evaluated persons who had been experimentally infected with Norwalk virus. Of 16 persons, clinical gastroenteritis (watery diarrhea and/or vomiting) developed in 11; symptomatic illness lasted 1–2 days. Virus shedding was first detected by reverse transcription–PCR (RT-PCR) 18 hours after participant inoculation and lasted a median of 28 days after inoculation (range 13–56 days). The median peak amount of virus shedding was 95 × 10 Norovirus, and many of the human experimental infection studies have used this strain (Noroviruses are the most common cause of epidemic gastroenteritis in the United States (7 reverse transcription–PCR [RT-PCR] U/mL). The participants were recontacted and screened for health . The new challenge inoculum (lot 42399) was prepared from liquid feces of 1 participant by clarification, centrifugation, and serial filtration through filters with progressively smaller pore size to a final 0.45-μm filter size. The inoculum, which contained no other enteric viruses or adventitious agents, was packaged and stored at –80°C.Liquid feces from persons who participated in a previous Norwalk virus challenge study . The samples were delivered to the laboratory within a day of collection and were processed and stored as described above. Participants were educated about the importance of hand washing and hand hygiene at the beginning of the study, and these concepts were reinforced at each study visit. The clinical protocol was reviewed and approved by the Institutional Review Board at Baylor College of Medicine, and an Investigational New Drug application describing the clinical protocol and study inoculum was reviewed by the US Food and Drug Administration.Challenge studies were conducted from September 2004 through October 2006. Healthy adults (18–50 years of age) provided informed consent and successfully completed a test of understanding. In addition, eligible persons were secretor positive , myalgia, or headache.Infection was defined as seroresponse  or fecal virus excretion as detected by RT-PCR or presence of antigen. Viral gastroenteritis was defined as illness with moderate diarrhea  for any continuous 24-hour period (moderate diarrhea is >200 g of watery feces that immediately take the shape of the collection container) or 1 vomiting episode plus 1 of the following: abdominal cramps or pain, nausea, bloating, loose feces (if not fulfilling the definition of diarrhea), fever , anorexia (n = 8), headache (n = 7), myalgia (n = 4), temperature >37.6oC (n = 4), and chills (n = 3). The 5 participants who did not fulfill the criteria for gastroenteritis had nausea (n = 5), anorexia (n = 5), malaise (n = 4), abdominal cramps (n = 3), myalgia (n = 3), headache (n = 3), temperature >37.6oC (n = 2), chills (n = 2), and watery diarrhea <200 g (n = 2). Although the number of infected participants in each dosage group was relatively small, no differences in signs and symptoms based on inoculum dosage were apparent. The median duration of signs and symptoms was 23 (range 10–61) hours and was similar for both groups of participants.A total of 16 persons inoculated with Norwalk virus met the criteria for having Norwalk virus infection. Of these, 11 (69%) met the predefined definition for viral gastroenteritis. The 5 who did not meet this predefined definition had >4-fold rise in serum antibody level, and all but 2 also shed virus as measured by antigen ELISA  genomic copies/g feces, and 5 participants shed >100 × 106 copies/g until at least day 14 , and the average total number of viral genomic copies measured in the feces over the first 2 weeks after inoculation also was higher in the clinical gastroenteritis group . The virus concentrations in feces collected later after inoculation were low . Correlation between virus titer in feces and optical density results obtained in the antigen ELISA was strong  were higher than would be expected from electron microscopic studies  was much higher than the 107–108 median viral loads reported in the prior studies (>4 days) associated with infection caused by GII.4 norovirus. Amar et al. (Only a few studies have used quantitative RT-PCRs to examine fecal viral load, and these studies have been primarily of GII norovirus strains. Chan et al. (>3 weeks in ≈25% of infected persons, and Murata et al. (The development of more sensitive methods to detect noroviruses has been associated with a corresponding increase in the duration of recognized virus shedding (


Miniopterus bats collected in Kenya ranged from 17% to 26%. Seropositive bats were detected in 4 of 5 locations sampled across the country. These findings provide evidence that WCBV, originally isolated in Europe, may emerge in other continents.The prevalence of neutralizing antibody against West Caucasian bat virus (WCBV) in  Lyssavirus : Rabies virus (RABV), Lagos bat virus (LBV), Mokola virus (MOKV), Duvenhage virus (DUVV), European bat lyssavirus, types 1 and 2, and Australian bat lyssavirus. Another 4 lyssaviruses, all isolated from bats, have been assigned to the genus as putative species: Aravan virus, Khujand virus, Irkut virus, and West Caucasian bat virus (WCBV) .During 2006–2007, bats of at least 30 species were collected from 25 locations in Kenya A; . The samoC.The brain and other organs of bats were collected into sterile plastic tubes. Oral swabs were collected and placed in tubes containing Minimum Essential Medium  for virus isolation or in TRIzol  for RNA extraction. Serum was separated from the blood clot by centrifugation. All samples were transported on dry ice and stored at –80The brains  were subjected to the direct fluorescent antibody test for lyssavirus antigen  and subjected to nested reverse transcription–PCR, as described previously  Teflon-coated glass slides  as described elsewhere , this difference was consistent across locations 1, 13, and 20. Only females were available from location 8. At all locations, Miniopterus bats shared caves with other species of insectivorous and fruit bats. However, no serologic activity against WCBV was detected in these other species. Of note, serum from fruit bats R. aegyptiacus that shared caves with Miniopterus bats neutralized LBV but not WCBV. Conversely, serum from Miniopterus bats neutralized WCBV but not LBV. This finding suggests that bats of different species, even those roosting in the same caves, do not readily expose each other to lyssaviruses.Circulation of LBV was detected in fruit bats ous bats  from 4 oous bats . Among 7ous bats , panel BWe found WCBV-neutralizing antibodies in bats in Africa. Because limited serologic cross-reactivity between lyssaviruses and other rhabdoviruses has been demonstrated , even when seroprevalence is as high as 40%–70% (Miniopterus schreibersii (Miniopterus bats is intriguing. Perhaps WCBV and similar viruses are specifically associated with Miniopterus bats and distributed quite broadly. Miniopterus bats are common in the tropics and subtropics of the Old World (Miniopterus colonies consisted of thousands of bats. Many of these caves are regularly visited by local residents and by tourists. Although no data exist for WCBV pathogenicity in humans, the absence of reliable vaccine protection against this virus and the ability of WCBV to cause fatal encephalitis in animal models (WCBV was first isolated in 2002 in southeastern Europe from Lyssavirus and WCBV-neutralizing antibody detection in bat tissues, Kenya, 2006-2007*


Phlebovirus that causes periodic outbreaks of animal and human disease in Africa and the Arabian Peninsula. On the basis of its many competent vectors, its potential for aerosol transmission, and its progressive spread from East Africa to neighboring regions between 1950 and 2000, RVFV is ranked as a high-priority, emerging health threat for humans, livestock, and wildlife in all parts of the world.Rift Valley fever virus (RVFV) is a mosquito-borne Aedes species. Most often, local virus propagation is reactivated as these mosquitoes emerge from temporary ponds (dambos) formed by heavy rainfall in enzootic/endemic areas.Culex. Because livestock miscarriage and mortality rates are high, humans can also become occupationally exposed to RVFV by handling infected animal tissues or by aerosolization of body fluids.Rift Valley fever virus is typically maintained by vertical transmission among floodwater


West Nile virus (WNV) epidemic in the United States. We necropsied the dead birds and examined them by various methods; pathologic and immunohistologic investigations suggested a WNV infection. Subsequently, the virus was isolated, identified, partially sequenced, and subjected to phylogenetic analysis. The isolates exhibited 97% identity to Usutu virus (USUV), a mosquito-borne Flavivirus of the Japanese encephalitis virus group; USUV has never previously been observed outside Africa nor associated with fatal disease in animals or humans. If established in central Europe, this virus may have considerable effects on avian populations; whether USUV has the potential to cause severe human disease is unknown.During late summer 2001 in Austria, a series of deaths in several species of birds occurred, similar to the beginning of the  Usutu virus (USUV) is a relatively unknown member of the mosquito-borne cluster within the Flavivirus genus, closely related to important human pathogens such as Japanese encephalitis virus (JEV), Murray Valley encephalitis virus (MVEV), Dengue virus (DENV), Yellow fever virus (YFV), Saint Louis encephalitis virus (SLEV), and West Nile virus (WNV) (Praomys sp. (African soft-furred rats) and one from a man with fever and rash us (WNV) –3. Isolaus (WNV) –7. Only Turdus merula) was observed in and around Vienna, Austria. Some observers reported obviously sick blackbirds, which showed signs of apathy and ruffled plumage. Within 5 days in mid-August, five Great Gray Owls (Strix nebulosa) died in the Tiergarten Schönbrunn Vienna Zoo. In addition, many dead Barn Swallows (Hirundo rustica) were observed in the Austrian federal state of Upper Austria, 200 km west of Vienna. Investigating this episode of avian deaths in Austria, we determined USUV as the causative agent.From the beginning of August through mid-September 2001, a considerable die-off of Eurasian Blackbirds , tissue samples were fixed in 7% buffered formalin. After embedding in paraffin wax, 4-µm sections were stained with hematoxylin and eosin.Tick-borne encephalitis virus  using the Avidin-Biotin Complex technique Paraffin-embedded tissue samples were immunostained with a polyclonal mouse antibody to WNV  and a polyclonal rabbit antibody to 2 concentration 2.5 mM), 0.4 mM deoxynucleoside triphosphate (dNTP), 10 U recombinant RNasin Ribonuclease Inhibitor , 40 pmol forward and reverse primers, 1 µL enzyme mix, and 2.5 µL template RNA. Reverse transcription was performed for 30 min at 50°C. Following an initial denaturation for 15 min at 95°C, the reaction mixture was subjected to 45 cycles of heat denaturation at 94°C for 30 s, primer annealing at 60°C for 30 s, and DNA extension at 72°C for 1 min, completed by a final extension of 10 min at 72°C. Following RT-PCR, we performed electrophoresis on 20 µL of the amplicons in a 1.2% Tris acetate-EDTA-agarose gel. The gel was stained with ethidium bromide, and the bands were observed under UV light.RNA was extracted from 140-µL organ homogenates or cell culture suspensions by using the QIAamp Viral RNA Mini Kit . After aligning available nucleotide (nt) sequences of various mosquito-borne flaviviruses and determining highly conserved genomic regions, we designed three pairs of oligonucleotide primers (to amplify a wide range of mosquito-borne flaviviruses) and used them in the reverse transcription-polymerase chain reaction (RT-PCR) assays: 5´-TACAACATGATGGGVAARAGAGAGA-3´ (nt position 9031–9055 of WNV GenBank accession no. NC 001563) and 5´-AGCATGTCTTCYGTBGTCATCCAYT-3´ (nt position 10115–10091), resulting in a 1,084-bp amplification product; 5´-GARTGGATGACVACRGAAGACATGCT-3´ (nt position 10090–10115) and 5´-GGGGTCTCCTCTAACCTCTAGTCCTT-3´ (nt position 10832–10807), amplifying a 743-bp PCR product; and 5´-GCCACCGGAAGTTGAGTAGA-3´ (nt position 10460–10479 of WNV no. NC 001563) and 5´-GCTGGTTGTGCAGAGCAGAA-3´ (nt position 10908–10889), resulting in a 449-bp amplicon. Reverse transcription and amplifications were performed in a continuous RT-PCR method by using the QIAGEN OneStep RT-PCR Kit (Qiagen GmbH). Each 25-µL reaction mixture contained 5 μL 5X buffer ; M12294, WNV; AF013367, Cacipacore virus; U15763, JEV; AF013360, Alfuy virus; AF013389, MVEV; AF013412, USUV (South Africa); AF452643, USUV (Austria); AF013416, SLEV; M93130, DENV (type 3); and AF013417, YFV. The phylogenetic analysis was carried out by using Phylogeny Interference Program Package (PHYLIP), version 3.57c (available from: URL: http://evolution.genetics.washington.edu/phylip.html). We generated bootstrap resampling analysis of 100 replicates with the SEQBOOT program. Distance matrices were generated with the DNADIST/Neighbor-Joining program; a translation/transversion ratio of 2.0. AF013417 (YFV) was used as outgroup.We excised the fragments from gel, extracted DNA, and performed sequencing PCR. The PCR products were sequenced in both directions by using the ABI Prism 310 genetic analyzer automated sequencing system . The nucleotide sequences were compiled and aligned with the corresponding sequences deposited in the GenBank database. Finally, we constructed a phylogenetic tree based on a 1,035-nt fragment in the NS5 genomic region. The following sequences have been included in the phylogenetic analysis: AF013384, Paraffin-embedded tissue samples were processed as described Necropsy showed grossly swollen livers and spleens, as well as seromucous enteritis in all blackbirds and owls; histology showed various degrees of multifocal acute necrosis in liver and spleen. Although the blackbirds did not show obvious histologic brain lesions, the owls had encephalitis, predominantly shown as multifocal areas of neuronophagia and microgliosis , A and DImmunohistochemistry (IHC) with polyclonal antibodies to WNV was positive in 10 of 11 brains, showing reaction products in neurons and their processes and in the cytoplasm of microglial cells in glial nodules , B and EOrgan homogenates of blackbirds and owls were added to Vero cell cultures. After 24–48 hours, a cytopathic effect of cell rounding could be observed; 1 to 2 days later, the affected cells detached and floated in the medium.RT-PCRs with universal flavivirus primers resulted in clear PCR amplification products of the expected lengths. The primers were designed to amplify overlapping PCR products in the NS5 genomic region of mosquito-borne flaviviruses. RT-PCRs were performed both on the original organ homogenates and on cell culture suspensions, with identical results. Despite a poor state of preservation, organ homogenates of the swallow also proved positive by RT-PCR.The PCR products  were directly sequenced in both directions; the compiled nucleotide sequences  were aligned and compared with other sequences by using the Basic Local Alignment Search Tool (BLAST) search . The sequence obtained from the Austrian dead birds was 97% identical with a 1,035-nt fragment of USUV . A partial nucleotide sequence of this USUV proved to be 100% identical to the sequences of the 2001 USUV isolates. Although no severe bird die-offs were observed in 2000, we think that USUV may already be established and be overwintering in Austria (rather than being newly introduced in 2 consecutive years). Comparable with the introduction of WNV to North America in 1999, where the virus propagated in local mosquito species and then rapidly spread from New York to >20 states in the United States and Canada This study shows for the first time that USUV is highly pathogenic for several different species of birds. Because full sequence data of USUV isolates are not available , we cannot fully compare the strain isolated here with the African isolates. We cannot provide information on amino acid changes that might contribute to altered pathogenic properties. For the closely related WNV, the pathogenicity for birds seems to depend on the virus strain and whether the virus affects a previously exposed or unexposed population. The WNV strain that appeared in North American birds in 1999 is closely related to a strain isolated in Israel; this strain is associated with avian deaths in both countries –14. Cert The emergence of WNV in the United States in 1999 and USUV in central Europe in 2000–2001 is an indication of future virus activity. The next mosquito-borne flavivirus, which might be introduced to regions far from its original habitats, may be highly pathogenic for humans, farm animals, or pets, as many strains of JEV group are. We could speculate whether global warming or other environmental factors may have contributed to the introduction and maintenance of USUV, formerly restricted to tropical and subtropical areas, in a much colder climate. As a consequence of the introduction of USUV to Central Europe, surveillance programs for mosquito-borne flaviviruses in general  should be established in Europe, like those initiated in the United States after the first occurrence of WNV (


Avian influenza viruses of the H7 subtype have caused multiple outbreaks in domestic poultry and represent a significant threat to public health due to their propensity to occasionally transmit directly from birds to humans. In order to better understand the cross species transmission potential of H7 viruses in nature, we performed biological and molecular characterizations of an H7N3 virus isolated from mallards in Canada in 2001.Sequence analysis that the HA gene of the mallard H7N3 virus shares 97% identity with the highly pathogenic avian influenza (HPAI) H7N3 virus isolated from a human case in British Columbia, Canada in 2004. The mallard H7N3 virus was able to replicate in quail and chickens, and transmitted efficiently in quail but not in chickens. Interestingly, although this virus showed preferential binding to analogs of avian-like receptors with sialic acid (SA) linked to galactose in an α2–3 linkage , it replicated to high titers in cultures of primary human airway epithelial (HAE) cells, comparable to an avian H9N2 influenza virus with human-like α2–6 linkage receptors . In addition, the virus replicated in mice and ferrets without prior adaptation and was able to transmit partially among ferrets.in vitro and in vivo analysis of avian influenza viruses isolated from the natural reservoir in order to define their zoonotic potential.Our findings highlight the importance and need for systematic  Influenza A viruses are classified based on the antigenic properties of the surface proteins hemagglutinin (HA) and neuraminidase (NA). To date, 16 HA and 9 NA subtypes have been described. Wild aquatic birds (Orders Anseriformes and Charadriiformes) are considered the major reservoir of influenza A viruses in nature -5. On rain vitro and in vivo in order to better assess their zoonotic potential.Avian influenza viruses with the H7 HA (herein H7 viruses) have been associated with numerous outbreaks in poultry worldwide with devastating effects on the industry due to mass mortality, depopulation and trade restrictions . Prior tRecent studies show that some HPAI and LPAI H7 subtypes isolated from domestic poultry can infect mice and/or cause disease in ferrets without previous adaptation -17. Howein vitro and in vivo characterizations of a A/mallard/Albetera/24/01 (H7N3) virus isolated in Alberta, Canada, 2001 . We found that the Mal/01 virus possesses characteristics of a typical avian influenza virus in vitro, i.e. it binds strongly to α2–3 avian-like receptors, and infects almost exclusively ciliated cells in air-liquid interface cultures of human airway epithelial cells. The Mal/01 virus transmitted to direct contact ferrets, although no airborne respiratory droplet transmission was observed. Our studies suggest that changes in receptor specificity, might not be necessarily required for some H7 viruses to infect and/or transmit in mammals. These studies highlight the importance of carefully scrutinizing the characteristics of viruses from the wild bird reservoir on a systematic basis in order to ascertain their zoonotic and pandemic potential.In this study, we performed The molecular features conducive to interspecies transmission of influenza viruses are largely unknown. We were interested in determining the biological and molecular characteristics as well as the host range of a typical mallard H7N3 virus that was circulating in wild ducks in Canada in 2001. This virus circulated in ducks in Canada prior to the H7 outbreak in poultry that transmitted to humans in 2004. Genome sequence analysis revealed that Mal/01 virus shares between 97.5% to 100% amino acid identity with the HPAI A/Canada/rv504/2004 (H7N3) virus with the exception of the NS gene , whereas a prototypic human H3N2 virus (Pan/99) and an avian H9N2 virus (RGWF10) with human-like receptor specificity bound exclusively to SAα2–6Gal-resialylated CRBCs . In thisin vitro model of human respiratory tract epithelium. The apical surface of HAE cells cultured at the air-liquid interface contains both ciliated cells and mucus-secreting non-ciliated cells. These cultures express SAα2–3Gal receptors predominantly on ciliated cells, while SAα2–6Gal receptors are presented mainly on non-ciliated cells  and titrated in 10-day embryonated chicken eggs by the method of Reed and Muench [Four to six-week old Japanese quail  were anesthetized and infected intranasally (i.n.) with the RGMal/01 virus at a dose of 50, 500, 5.0 × 105TCID50 of RGMal/01 in PBS, 250 μl per nostril. Twenty-four hours later, two naïve ferrets were introduced. One (direct contact) was introduced directly into the cage with the infected ferret while the other (aerosol contact) was placed in a cage separated from the infected ferrets by a wire mesh (1 mm openings). The wire mesh prevented physical contact between the aerosol and the infected and direct contacts, allowing only air to be shared between the ferrets. All materials inside the cage of inoculated ferrets were removed and replaced before introducing the direct contact to preclude fomite transmission. Temperatures and body weight readings were taken daily. To monitor viral shedding, nasal washes were collected daily for up to 14 days. Briefly, ferrets were anesthetized as described above, and 1 ml of PBS was used to induce sneezing. Nasal washes were collected into petri dishes and brought to a total volume of 1 ml with PBS. Nasal washes were immediately tested for virus using the Flu DETECT™ Antigen Capture Test Strip  and additional aliquots were stored at -80°C before performing TCID50 assays. Two weeks after infection, blood was collected and seroconversion was monitored by determining HI antibody titers.Female Fitch ferrets, 3 to 6 months old , were used to test virus replication and transmission. Prior to infection, ferrets were monitored for 3–5 days to measure body weight and establish baseline body temperatures. Temperature readings were recorded daily through a transponder  implanted subcutaneously in each ferret. Blood was collected at 3 days before infection, and serum was tested for antibodies against RGMal/01 virus using the hemagglutination inhibition (HI) assay. Ferrets with HI titers at or lower than 10 were considered "influenza A-free" and were used in the study. Ferrets were housed in wire cages placed inside Hepa-filter isolators. Each ferret group housed in a single isolator unit consisted of three ferrets: one infected, one direct contact and one aerosol contact, as described previously ,31. Thre10 numbers of TCID50 per ml of 10% (w/v) tissue homogenate.To study the kinetics of virus replication and tissue tropism in ferrets, 3 ferrets were inoculated i.n. with 5 × 105 TCID50 of RGMal/01 as described above. At 8, 24, and 68 hours post-infection (hpi), each ferret was euthanized. Nasal turbinates, lungs, kidneys, olfactory bulbs (OB), brains, livers, spleens and intestines were harvested, and samples were both fixed with buffered neutral formalin for histological evaluation and stored at -80°C for virus titration. For histopathology, paraffin-embedded sections of 5 μm thicknesses were cut and stained with hematoxylin and eosin (H&E) . Representative microscopic photos were taken with SPOT ADVANCED software . The virus titer for each organ was determined by the method described by Reed and Muench and was expressed as logAnimal experiments were carried out under BSL-2 (birds and mice study) or BSL-2+ (ferret study) conditions and conducted under guidelines approved by the IACUC of the University of Maryland.The authors declare that they have no competing interests.HS generated the reverse genetics version of the Mal/01 virus, performed in vitro and in vivo studies, and wrote the manuscript. HW performed in vitro studies and wrote the manuscript. YA performed in vivo studies. DRP developed the conceptual aspects of the work and wrote and edited the manuscript. All authors read and approved the final manuscript.


This paper reviews the evidence on external costs of risky behaviors in the U.S. and provides a framework for estimating them. External costs arise when a person does not bear all the costs of his or her behavior. They provide one of the strongest rationales for government interventions. Although the earlier estimates of external costs no longer have policy relevance, they demonstrated that the existence of external costs was an empirical question. We recommend that the estimates of external costs be updated as insurance structures, environments, and knowledge about these behaviors change. The general aspects of external costs may apply to countries other than the U.S. after taking into account differences in institutional, policy and epidemiological characteristics. The costs that a person bears as a consequence of his or her actions are called internal (private) costs. External costs are not considered in a person’s decision making because they are borne by others. Possible external costs of any health-related behavior primarily derive from public or private group health insurance and from events directly causing harm to others . External costs are relevant on the grounds of fairness and efficiency. They become an issue of fairness when someone pays unwillingly or unknowingly for the actions of others. Grounds of economic efficiency apply when paying the full costs of their own action leads to an overall higher level of efficiency by a change in personal behavior, This is the first comprehensive study to examine the existing evidence on the external costs of the leading actual causes of death—smoking, poor diet/physical inactivity, and excessive alcohol consumption. Despite of the large number of studies on total costs, which do not differentiate between internal and external costs –9, relat2.Because risky health behaviors contribute to acute and chronic diseases which lead to high medical expenses, it seems straightforward that these behaviors also lead to an overall financial burden on those with healthier behaviors due to group health insurance programs. This, however, is more complex and requires three considerations: An analysis of cost differential between the group with risky behavior and the group without it should be conducted within a lifecycle framework. The attribution of differentials in costs to each behavior should be determined while controlling for other risky behaviors and characteristics that could contribute to this cost difference. And, finally, individual contributions to health care financing and other aspects of possible financial programs through Social Security, life insurance, or sickness benefits should be included in estimation of external costs.If a risky behavior leads to premature death, there are competing financial influences. On the one hand, higher health care costs may be imposed in the short run, but on the other hand, the society may spend less additional resources for two possible reasons: Health care expenditures increase dramatically with age and those expenses are more heavily subsidized for older adults than for younger persons. And persons with risky health behaviors may collect Social Security and pension benefits for a shorter time than those with healthier behaviors; however, the dependents of the deceased may receive public financial support. Therefore, the net financial effect of premature death is an empirical question.There are disputable issues related to external costs. Some may argue that because Medicare and Medicaid are entitlement programs which the society has chosen to fund, they should not be considered in the estimation of external costs. However, the willingness to fund health care for elderly and poor may differ from the willingness to pay for modifiable behaviors, such as smoking or excessive drinking.Another disputable issue is the costs born by family members. Economists generally view the family as the decision-making unit, therefore costs to family are usually considered private. However, society may cover costs associated with developmental problems of infants and children that are attributable to maternal smoking and drinking. In addition, costs imposed on children could be viewed external. When deciding to get married an adult presumably accepts the consequences of the spouse’s risky health behaviors, but children do not choose their parents.3.We searched PubMed and EconLit databases for the period 1982–2008, using both indexed (keyword) and free-text terms. Boolean logic was used to establish relationships between the concepts risky behavior and cost as well as fetal alcohol syndrome and cost. We also hand-searched the working papers of the National Bureau of Economic Research and checked references of related articles. We then screened them to identify those with empirical estimation of external costs and distinguished studies which had considered the important components of external cost estimation from those which had not.per se. The same construct should be applied to other habits. (2) Cost of illness and human capital approach, which estimates costs of medical treatment and productivity losses; and (3) Willingness to pay (WTP) and value of statistical life (VSL) approaches. WTP assesses what people are willing to pay (or accept) for relatively small changes in the risk of death. Based on WTP estimates, the economists developed VSL. To understand this concept, it helps to think in terms of risk exposure. When the risk of fatal accident is 1:100,000, statistically there is 1 death per 100,000 people. Changes in the risk level imply changes in the number of statistical lives saved. The economic value is essentially the marginal rate of substitution of wealth for risk of death.Limitations with settings, methods and assumptions of the available studies did not allow synthesis of the findings. Therefore, the findings are presented as they appeared in the literature, followed by the amount adjusted for inflation in 2009 dollars. The main methods used in estimating external costs were: (1) non-smoking smoker construct, which is based on epidemiological observation that smokers differ systematically from non-smokers . Therefo4.4.1.The earlier studies on external costs of smoking were primarily motivated by the question whether the society subsidized smoking. It was based on the traditional view of tobacco policy, where tobacco was treated as any other product and tobacco taxes were to be set equal to external costs it imposed on society. These studies indicated that the current external costs were lower than the taxes –14.The estimated lifetime external costs were $1,000 per smoker or a net external cost of 15 cents per pack of cigarettes at a 5% discount rate in 1986 U.S. dollars, not including costs imposed on family. This adjusts to $1,958 per smoker or a net external cost of 29 cents per pack of cigarettes in 2009 U.S. dollars. If the costs of maternal smoking and smoking-related fires were included, this estimate would range from 31 to 52 cents per pack in 1986 U.S. dollars ($0.61 to $1.02 in 2009 U.S. dollars) ,12. The et al. estimated the costs of maternal smoking to be 42 to 72 cents per pack in 1994 dollars ($0.61 to $1.04 in 2009 dollars) [Viscusi  updated dollars) , which wdollars) .Our understanding of the epidemiology, economics and overall effects of smoking has changed since these studies were conducted, and the above estimates do not have direct policy relevance today. Moreover, a new approach in behavioral economics views tobacco consumption as different from other products, suggesting that internal costs could also play a role in taxation policy. Taxation becomes a self-control device for smokers who may want to protect their future self from their current self. In this framework, the appropriate tax levels exceed the external costs .4.2.When the above studies were conducted, the health and associated economic effects of ETS were a major point of empirical contention in the literature . A 2006 A study by the U.S. Environmental Protection Agency  concludeA summary of several economic studies on ETS found that the lowest grand total estimated costs of ETS were $14.7 billion, the average were $75.4 billion and the highest were $167 billion in 2000 U.S. dollars . This ad4.3.et al. were the only study to provide a systematic accounting of how the costs of smoking were distributed among smokers and nonsmokers [Sloan nsmokers . The comnsmokers .i.e., $1.44 net of taxes . In the year 2000, the average retail price of a pack of cigarettes was $3.12 ($3.89 in 2009 dollars).In 2000 the estimated present value of private cost of smoking to a 24-year old smoker was $141,181 over the lifecycle and $32.78 per pack in 2000 dollars . The costs to spouses and for infant deaths  were an estimated $23,407 over the lifecycle or $5.44 per pack . The external costs were much smaller—$6,201 per 24-year-old smoker, net of federal and state cigarette excise taxes paid by smokers . On a per-pack basis, the external cost was $2.20 in contrast to $0.76 in excise taxes paid per pack, In later studies the estimates of private mortality costs ranged from about $25 per pack (15 percent or higher discount rates) to an estimated $222 per pack for men and $94 per pack for women (3 percent discount rate) in 2006 dollars . DiffereOverall, estimates of external costs of smoking depended greatly on the choice of discount rate, on inclusion of costs of ETS-related deaths and maternal smoking, and on the value of statistical life used.5.An early study related to externalities of physical inactivity  estimateBhattacharya and Sood  estimatei.e., the non-obese may benefit from the obesity epidemic due to induced innovation in biomedical research and medicine [A new aspect in this area is the issue of positive externalities of obesity, medicine . First, medicine .Another new area of research is the immediate and long-term impact of maternal obesity on life and health course of the child , which c6.Several studies provided strong evidence of high external costs associated with alcohol abuse ,12,33–42Another source of external costs is drinking during pregnancy which can lead to fetal alcohol spectrum disorders (FASD). The lifetime total costs per individual with FAS were estimated to be $2 and $2.9 million in 2002 dollars –42. If dCarpenter and Dobkin  found th7.i.e., paying the full costs of their actions leads to reduction in risky behaviors. For smoking and alcohol consumption it is well documented that increasing costs through excise taxes reduces consumption [The estimates of external costs must be updated as the health insurance structures, medical practices and technologies, environments and our knowledge about the effects of these risky behaviors change. Although many of the early estimates of externalities are no longer relevant for today’s policy, they demonstrate that the existence and magnitude of external costs are empirical questions, and that the assumptions of the models may lead to qualitatively different results. External costs are most relevant for policy when people change their behaviors in response to costs, sumption ,2. If thIt is useful discussing how these potential external effects can be addressed most directly and why they have not been resolved yet. Aligning the private costs of risky behaviors with the social costs appear to be straight-forward to implement. However, there have been significant differences in the tax policies of smoking and alcohol. Despite of the enormous external costs of excessive drinking, excise taxes on alcoholic beverages have been little adjusted for decades, with effective rate of taxation declining over the years. On the other hand, from 2002 to 2009 cigarette tax rates were increased for 88 times by 46 states, D.C., and several US territories. In 2009 the federal tobacco tax was increased from 39 cents to $1.01, partly with the goal of reducing smoking, thus improving health. This difference may be explained by the difference in political influence of the two industries , but thei.e., surplus, to people when the prices they pay for products they enjoy is less than their maximum willingness to pay for them. When taxation increases prices, consumer’s response will likely be reduced consumption, involving changes in private surplus. Two economic models predict this change differently. According to the model of time-inconsistent preferences, persons with risky behaviors will be better off because taxes will serve as a self-control device helping them achieve what they eventually want to do, i.e., quit smoking, drinking or eating excessively. On the other hand, the standard economic model predicts that those with risky behaviors will be worse off because the policy intervention will constrain their rational choices. This loss in private surplus will be due to the fact that consumers will have to pay more for the amount of the product they still continue to consume, and they also will have to consume less of something they enjoy. Comparing changes in private surplus for these risky behaviors can be an important topic for future research. Future research should also address the substitution of one risky behavior for another when taxes were imposed to limit each behavior in isolation. This substitution might lead to higher overall external costs because the external costs of each behavior are different.Taxation has another economic aspect. There is gain, Much interest has been given to possible externalities to employers that stem from medical costs and reduced productivity due to employees’ risky health behaviors. To offset these costs, employers most likely adjust wages; this action eventually would lead individuals with risky health behaviors to partially or fully internalize their higher health care costs –57. ReduAll available studies used data on adult populations with health insurance, including Medicare and Medicaid. Persons between age 18 and 64 years who were obese or who smoked or abused alcohol were more often uninsured than those who did not have these risky behaviors . Being uExternal costs provide one of the strongest economic justifications for government interventions, but there are other justifications, such as public health ethics and altruism ,18,59–63


Condyloma acuminata are soft, skin colored, fleshy warts that are caused by the Human Papilloma Virus (HPV). The disease is highly contagious, can appear singly or in groups, small or large. The incubation period may be from 1–6 months. Although anogenital warts are considered to be sexually transmitted in adults, this may not be the case for children. Genital warts in children may result from several modes of transmission: from the maternal genital tract autoinoculation, from finger warts and nonsexual transmission from members/careers.The presented case is a 13-month-old girl. She was hospitalized at the Clinic of Dermatovenerology in 2001 due to papillomatosis changes on the genital area. The changes had started to appear in the sixth month of life, light purple in color, smooth and combined in a tumorous mass, in the vulva and anal areas.From this case we can come to the conclusion that condyloma acuminate are not only transmitted sexually but through nonsexual ways as well, such is this case, from the infected mother to the infant. Laser therapy is preferred when multiple warts are spread over a large area as though in our case. Venereal warts are caused by the human papilloma virus (HPV) which is a small DNA virus that does not grow in tissue culture but can be identified by electron microscopy .The incubation period may take from one to six months .Although anogenital warts are considered to be sexually transmitted in adults, this may not be the case for children . GenitalHowever, the possibility of sexual abuse must always be borne by mind .The topical treatment of childhood condylomata acuminate includes the application of caustic or irritating agents, such as liquid nitrogen trichloroacetic acid or podophyllum resin. These agents poorly tolerated by a child who may require multiple applications. Vaporization of the warts with a carbon dioxide laser is the newest treatment method. The advantage of using the CO2 laser is the ability to treat a large area without causing a scar, stricture, or narrowing of the lumen. This method tends to eliminate the virus and promote rapid healing with very little scar formation.We present a 13-month-old female patient. She was brought to the outpatient department in Dermatological Clinical Center by her mother with a 7 month history of papillomatosis changes in the anogenital area. The changes had started to appear in the sixth month of life, light purple in color, smooth and combined in a tumor mass, in the vulva and anal areas (Figure The child had been born by vaginal delivery after full term normal pregnancy. From the mother's hetero-anamnesis we learned that the mother has had genital warts during her pregnancy, which have faded out right after the delivery, without previous therapy.Serological tests for syphilis in the infant and her mother gave negative results.Based on the heteroanamnesis from her mother, clinical presentation, laboratory and serological tests for syphilis we made a final diagnosis of condylomata acuminate. After general anesthesia our patient is treated with a carbon dioxide laser. This method is well tolerated and promotes rapid healing with very little scar formation.Sexually transmitted diseases affect the sexually active population being in the reproductive age group and usually are being transmitted in venereal form . Many rePregnant women infected with genital warts can pass them on to their newborns .Generally, in children less than 2 years of age, the mode of transmission is vertical from mother to child during childbirth. There iLaser therapy is preferred when multiple warts are spread over a large area, also useful for treating cervical and vaginal warts, when surgical excision is not possible or would be difficult . As maliThe authors declare that they have no competing interests.MF admit the patient in the clinic, definite the diagnosis and consult the children and plastic surgeon for treatment. AG and LP was a major contributor in writing the manuscript. All together decide for laser treatment. All authors read and approved the final manuscript.Written informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.


RNA interference (RNAi) provides a powerful new means to inhibit viral infection specifically. However, the selection of siRNA-resistant viruses is a major concern in the use of RNAi as antiviral therapeutics. In this study, we conducted a lentiviral vector with a H1-short hairpin RNA (shRNA) expression cassette to deliver small interfering RNAs (siRNAs) into mammalian cells. Using this vector that also expresses enhanced green fluorescence protein (EGFP) as surrogate marker, stable shRNA-expressing cell lines were successfully established and the inhibition efficiencies of rationally designed siRNAs targeting to conserved regions of influenza A virus genome were assessed. The results showed that a siRNA targeting influenza M2 gene (siM2) potently inhibited viral replication. The siM2 was not only effective for H1N1 virus but also for highly pathogenic avian influenza virus H5N1. In addition to its M2 inhibition, the siM2 also inhibited NP mRNA accumulation and protein expression. A long term inhibition effect of the siM2 was demonstrated and the emergence of siRNA-resistant mutants in influenza quasispecies was not observed. Taken together, our study suggested that M2 gene might be an optimal RNAi target for antiviral therapy. These findings provide useful information for the development of RNAi-based prophylaxis and therapy for human influenza virus infection. Influenza A virus (IAV) remains a scourge on human health RNAi is a form of posttranscriptional gene silencing mediated by short double-stranded RNA, known as small interfering RNA (siRNA) IAV is an enveloped, negative-stranded RNA virus. The unique property of single-stranded RNA virus itself makes RNAi an attractive approach for development of anti-avian influenza therapeutics. The single-stranded viral genome, consisting of 8 segments contained at least 10 open reading frames (ORFs), serves as template for both viral genome replication and subgenomic mRNA synthesis. It has been reported that siRNAs respectively targeting to the viral genes of polymerase 1 (PB1), polymerase 2 (PB2), polymerase A (PA), nucleocapsid protein (NP), non-structure proteins (NS1 and NS2), matrix proteins (M1 and M2), especially those specific for NP, PA and PB1, can potently inhibit replication of influenza A viruses In the present study, we identified an effective siRNA targeting M2 gene (siM2), a highly conserved gene in IAV, as compared to a reported effective siRNA targeting NP gene (siNP). We further established cell lines which stably expressing the shRNAs by transducing lentiviral-shRNA vectors to Madin-Darby cannie kidney (MDCK) cells. Using these two cell lines, we evaluated long term antiviral effects of these siRNAs against IAV subtypes H1N1 and H5N1 and further screened the potential siRNA-resistant viral mutations. Our results showed that rationally designed siM2 conferred long term effective inhibition for IAV replication. It was further demonstrated that no siRNA-resistant viral mutation appeared in siM2 targeting sequence even after the virus was cultured in the shRNA expressing stable cell line for 40 passages.Two siRNAs targeting the M2 gene were rationally designed by siRNA target designer  were used as controls. The cell lines were infected with H1N1 virus at a moi of 0.005 and culture supernatants were harvested at indicated time-points to determine the virus titer by plaque assay. As shown in We also measured the accumulation of mRNA for NP and M2 gene in infected MDCK, Mock MDCK, shM2-MDCK and shNP-MDCK cells. The mRNAs were extracted from the cells harvested at 1, 2, 4 and 24 hrs post-infection and tested by real-time RT-PCR. The mRNA expression level is normalized by copy number of β-Actin. The M2 mRNA level in shM2-MDCK cells harvested at 4 hrs and 24 hrs post-infection was significantly lower than those in MDCK, Mock MDCK and shNP-MDCK cells . SimilarTo further confirm whether the suppression of NP mRNA in shM2-MDCK cells indeed affect NP protein expression, the NP protein level was tested by an indirect immunofluorescence assay. As shown in We further tested whether siM2 could also inhibit the replication of a highly pathogenic H5N1 avian influenza virus. As shown in To test if siM2 siRNA-resistant virus mutant would quickly appeared when cultured in shM2-MDCK cells, H5N1 virus was continually cultured in shM2-MDCK cells for 40 passages. Every 10 passages, the culture supernatant was collected and tested by plaque reduction assay. No obvious larger size of plaque was found. Ten plaques with relative larger size were picked to further identify potential mutation in the siRNA targeting region by sequencing. The results showed that no mutation appeared in the siM2 targeting region even after 40 passages of the cultures .The principal finding of this study is that rationally designed siRNA targeting influenza M2 gene (M-950) conferred effective long term inhibition against influenza A virus replication. Such high suppressive effect is not only against H1N1 influenza A virus but also against a highly pathogenic H5N1 subtype. In the previous related studies, Ge and his co-workers In this study we found that siM2 not only decreased the level of M2 mRNA but also the level of NP mRNA, suggesting that siM2 has a broad inhibition manner in the process of influenza virus replication. Ge et al have reported a similar broad inhibition of siRNAs in vitro. Our results showed that H5N1 virus cultured in shM2-MDCK were equally susceptible to siM2 as the original virus even after 40 passages. Moreover, sequencing of siM2 targeted region in 10 such independent plaque purified virus isolates revealed sequence identical to the parental one. The current data have shown no insertion, deletion and nucleotide substitution in the siRNA target sequence, therefore demonstrated siM2 possessed good long term inhibition effect for influenza virus replication without the problem of siRNA resistant mutants.Depending on the stringency of siRNA-target base pairing, siRNA treatment may cause selection of siRNA-resistant viruses, and this has been shown with HIV and HCV Taken together, all the findings about effective RNAi target, lentiviral vector delivery and the establishment of stable shRNA expressing cell lines in our study provide rational information for the development of siRNAs as prophylaxis and therapy for influenza virus infection in humans.50. All experiments with H5N1 virus were performed in BSL-3 laboratory.MDCK and Human embryonic kidney 293T cells were respectively maintained in MEM and DMEM  supplemented with 10% heat-inactivated fetal bovine serum (FBS) and antibiotics (100 U penicillin G/mL and 100 ug streptomycin/mL). Influenza virus strains A/New Caledonia/20/1999 (H1N1) and A/Hong Kong/486/97 (H5N1) used in these experiments were prepared in MDCK cells and virus titers were determined by TCIDhttp://www.promega.com/siRNADesigner/program/). The duplexes of designed and previously reported siRNAs were synthesized by Invitrogene (USA)  were transfected with 20 µg LVTHM or LVTHM-shRNA, 15 µg psAX2 and 6 µg pMD.2G and cultured in DMEM supplemented with 10% FBS and antibiotics. Virus supernatants were collected on day 3 post-transfection, filtered through a 0.45 µm pore-size filter, ultracentrifuged at 40,000 g for 3 hrs at 4°C and resuspended in PBS. Virus stocks were titrated by infecting Hela cells with virus dilutions in DMEM and 8 µg polybrene  ml−1 and analyzed for EGFP expression with a flow cytometer . Data was processed with Cellquest software. Titers of the virus stocks were routinely 107∼108 transduction units (TU) ml−1.Lentiviral vectors with shRNA expression cassette were produced by calcium phosphate-mediated, three-plasmid transfection of 293T cells 5′-ACA GCA GAA TGC TGT GGA T-3′ and siNP- 5′-GGA TCT TAT TTC TTC GGA G-3′, and the reverse primer provided by Ncode™ miRNA Q-RT-PCR kit  and further calculated with normalized Ct values: ΔCt (Mock MDCK−shM2-MDCK or shNP-MDCK).Lentiviral stocks were used to transduce MDCK cells. After 3 days of transduction, the medium containing the lentivirus was replaced with complete culture medium. The transduced MDCK was sub-cultured every 3–4 days for 4 weeks to get stable viral genome integration. Sorting of live GFP positive cells was performed using a FACStar+ instrument  MDCK and the stable shRNA expressing cell lines in 24-well plates were infected with viruses at moi of 0.005∼0.5 (2 µg/mL trypsin was used in the infection process of H1N1). After incubation for 1 hr, the infected medium was removed and MEM without FBS was added. Cell supernatants were collected at different time points. The viral load was detected by hemagglutination (HA) and/or plaque assays as described previously 5′-GAC CAG GAG TGG AGG AAA CA-3′, NP-Reverse: 5′-CGG CCA TAA TGG TCA CTC TT-3′; M2-Forward: 5′-CGT CGC TTT AAA TAC GGT TTG-3′, M2-Reverse: 5′- CGT CAA CAT CCA CAG CAT TC-3′ β-Actin-Forward: 5′- CGT ACC ACT GGC ATC GTG AT-5′, β-Actin-Reverse: 5′-GTG TTG GCG TAC AGG TCT TTG-3′. The reactions were performed at 95°C 10 mins, 40 cycles of 95°C 1 min, 60°C 1 min, 72°C 1 min, followed by melting curve analysis according to instrument documentation (Stratagene M×3000). All reactions were done in triplicates and the results were normalized by β-action.Real-time RT-PCR was carried out as described previously Indirect immunofluorescence assay was performed as described previously 2 flask were infected with H5N1 virus. After cultured for 2 days, the supernatants were harvested. Part of the supernatants was inoculated to shM2-MDCK for next passage of the virus culture, another part was subjected for plaque assay to determine if potential siRNA-resistant virus appeared. Every 10 passages, ten bigger size of plaques in the plaque assay were picked for sequencing to detect any mutation in the siRNA targeting region using a pair of primers: forward, 5′-AAG GCA GAT GGT GCA GGC AAT-3′ and reverse, 5′-TAC TCC AGC TCT ATG CTG ACA-3′.The screening of potential siRNA resistant mutants were performed in our established stable shRNA-expressing cell lines according to previously described protocols Table S1(0.03 MB DOC)Click here for additional data file.


We recently identified 2 Mokola viruses from domestic mammals (a dog and a cat)in South Africa. These cases occurred 8 years after the last reported case ofinfection with this virus. Our findings emphasize the endemicity ofrabies-related lyssaviruses in South Africa and the need to better understandthe epidemiology of Mokola viruses. Mokola virus (MOKV) is classified as genotype (gt) 3 of the genusLyssavirus in the family Rhabdoviridae . Apart from MOKV, the genus Lyssavirus consists of 6gts: classic rabies virus (gt1), Lagos bat virus (gt2), Duvenhage virus (gt4), Europeanbat lyssavirus type 1 (gt5) and type 2 (gt6), and Australian bat lyssavirus (gt7). Somenovel lyssaviruses identified in bat species in the former Soviet Union are consideredputative gts within this genus (Crocidura sp.) inNigeria in 1968. Since then, >20 isolates of thislyssavirus have been found throughout Africa   .We report the identification and characterization of 2 cases of infection with MOKV inSouth Africa. The first was in a domestic dog and is, to our knowledge, the first suchcase in South Africa. The second was in a domestic cat, the host species in which allprevious isolates were found. The cat MOKV isolate belonged to 1 of 2 previouslyidentified South African MOKV phylogenetic lineages, but the dog MOKV isolate appearedto have a different lineage not previously encountered in South Africa or elsewhere inAfrica.Felis domesticus) was adoptedfrom the Society of the Prevention of Cruelty to Animals  and lived with its owner on a farm 23 km outside the city.It had been neutered and had been vaccinated at 10 months of age with an adjuvantedinactivated vaccine against rabies , but nosubsequent vaccinations were given. The cat spent most of the day indoors, but wentout at night and returned in the morning. Unusual behavior was noticed in March2006. It appeared dull and physically unbalanced and its pupils were dilated but itwas not aggressive. The cat was humanely killed, and its brain was sent to theOnderstepoort Veterinary Institute for rabies testing.In October 2004, a 3-month-old kitten (Canis familiaris) was broughtby its owner to a veterinarian in the rural town of Nkomazi . The dog had a temperature of 39.8°C and no appetite. Aftersymptoms were treated, the dog was discharged, but it was brought back 11 days laterbecause it was paralyzed, dehydrated, and had a fixed stare. This animal had neverbeen aggressive to other pets or humans. The dog was humanely killed, and its brainwas sent to the Onderstepoort Veterinary Institute for rabies testing.On June 17, 2005, a 6-month-old puppy ; both methods were successful for the catsample. However, neither method yielded an isolate from the dog sample, despite alyssavirus-specific reaction in the original brain sample by directimmunofluorescent antibody test.Subsequently, antigenic characterization was performed with a panel of 16 monoclonalantibodies to the nucleocapsid protein of rabies virus . Both samples showed reactivity patterns associatedwith MOKV .Final confirmation of MOKV in both case samples was obtained by reversetranscription–PCR, nucleotide sequencing, and phylogenetic analysis asdescribed  showedtInfections with MOKV are rare; only 23 isolates are known. During the past 2 decades,all MOKV isolates have been found in South Africa. Because these viruses are notexclusive to South Africa .Although the epidemiology of MOKV is incomplete, the case for a reservoir host(s)among small terrestrial animals of limited range is supported by our findings.Together with recent isolations of rabies-related lyssaviruses in a human (


In 2004, the raccoon rabies virus variant emerged in Ohio beyond an area where oral rabies vaccine had been distributed to prevent westward spread of this variant. Our genetic investigation indicates that this outbreak may have begun several years before 2004 and may have originated within the vaccination zone. Vulpes vulpes), have led to regional containment and elimination of the rabies virus variants associated with this species in large parts of Canada and Europe –specific rabies virus variant (RRV) expanded substantially from the historically affected southeastern United States to the currently affected eastern North America . These detections raised the question whether current ORV and surveillance strategies are sufficient for containment and reaching the long-term goal of regional elimination of RRV. We used molecular analyses to gain insight into the factors and possible raccoon source populations associated with the breach of the ORV zone in Ohio.During 1999–2004, ORV had apparently limited further spread of the virus . HoweverViral RNA was extracted as described . We amplA Bayesian molecular clock–based method , which suggests a single-source introduction into Ohio. The 2004 lineage was not a direct descendent of any previously sampled lineages, but it shared a common ancestor with another lineage  that contained the viruses responsible for the 1996 Ohio outbreak along with contemporary viruses from western Pennsylvania. No members of either of these lineages had been found east of the ORV barrier ( A), an area dominated by a different group of viruses . The same result was obtained when the larger dataset based on G data only was analyzed  for the concatenated G and N data and 1995  for G data only.Our findings imply that RRV had been circulating undetected among raccoons in the ORV zone, and possibly beyond it, for several years before its detection in 2004. These findings have important implications for the control of wildlife rabies in raccoons through ORV. First, the genetic analyses do not point to a long-distance transmission event to Ohio but rather suggest that the virus was indigenous to the region. In view of potential continued transmission events within the current ORV zone, widening the ORV corridor likely will not prevent such transmission and further spread. Second, the findings suggest that RRV may be able to persist within the ORV zone for several years and thus provide continued risk for eventual spread into unvaccinated raccoon populations. Insufficient levels of immunization among the overall population could contribute to this situation. However, spatial variation in the level of immunization or random fluctuations in the number of infected animals may also enable the virus to persist in parts of the ORV zone. Third, the level of surveillance needed to detect RRV when transmission frequency is low is unclear.Our results indicate that the virus had been present within Ohio for several years when surveillance efforts were relatively low; from January 2000 through June 2004, an average of 71 raccoons were tested each month compared with an average of 139 per month during 1997–1999 . TherefoSamples and GenBank accession nos. used in this study*Maximum-likelihood tree of 67 partial G gene sequences of raccoon rabies virus sampled in or near Ohio, 1987–2004. Sequences from the 2004 outbreak are shown in boldface. Bootstrap values and corresponding Bayesian posterior values (both as percentages) are shown for key nodes. Tree was rooted using a raccoon rabies virus sequence from a Florida raccoon (not shown). Numbers following taxa names indicate the time of sampling, which is expressed relative to the year 1900 . See


Poxviridae consists of large double-stranded DNA containing viruses that replicate exclusively in the cytoplasm of infected cells.  Members of the orthopox genus include variola, the causative agent of human small pox, monkeypox, and vaccinia (VAC), the prototypic member of the virus family.  Within the relatively large (~ 200 kb) vaccinia genome, three classes of genes are encoded: early, intermediate, and late.  While all three classes are transcribed by virally-encoded RNA polymerases, each class serves a different function in the life cycle of the virus.  Poxviruses utilize multiple strategies for modulation of the host cellular environment during infection. In order to understand regulation of both host and virus gene expression, we have utilized genome-wide approaches to analyze transcript abundance from both virus and host cells.  Here, we demonstrate time course infections of HeLa cells with Vaccinia virus and sampling RNA at several time points post-infection.  Both host and viral total RNA is isolated and amplified for hybridization to microarrays for analysis of gene expression.The family  Add 1µg of the aRNA samples into 1.5mL microcentrifuge tubes.do not overdry!Vacuum dry the samples on low or no heat until they are completely dry.  Cap each tube as soon as it is dry - Add 9µl coupling buffer to each tube and resuspend the aRNA by gently vortexing for 1 minute.  Centrifuge briefly to collect the sample in the bottom of the tube, and then let the sample sit on ice.Add 22µl high quality DMSO to each tube of Cy3 or Cy5 dye.  One tube of dye is enough for 2 samples.  The Cy3 dye is for labeling your reference samples, and the Cy5 dye is for labeling your test samples.Vortex the dyes to mix thoroughly.  Keep the dyes in the dark until ready to use.  Do not prepare dye earlier than 1 hour before using.  Make sure no water gets in the dye/DMSO mix at any point.Add 11µl of the prepared DMSO/Cy dye to each sample.  Mix well by vortexing gently.Incubate for 30-45 minutes at room temperature.  Cover the samples with tinfoil or keep them in a drawer to minimize exposure to light.After the incubation, add 4.5µl hydroxlyamine to each sample to quench the reaction.  Mix well by vortexing gently.Incubate for another 15 minutes at room temperature.  Cover the samples with tinfoil or keep them in a drawer to minimize exposure to light.Vortex the RNA binding beads briefly to obtain an even mixture before use.(Table 1)Prepare the aRNA Binding Mix at room temperature. Mix well by vortexing.Aliquote the aRNA Elution Buffer into a 1.5mL tube and incubate at 50-60°C for at least 10 minutes.Add 70µl of the aRNA binding mix to each sample and mix well by pipetting up and down 3-4 times.Transfer the samples from the PCR plate to a 96-well round-bottom plate.Add 50µl of 100% isopropanol to each sample and mix well by pipetting up and down 3-4 times.Gently shake the plate on an orbital shaker for at least 2 minutes to thoroughly mix the samples.Move the plate to a magnetic stand to capture the magnetic beads.  Leave the plate on the stand until the mixture becomes transparent and the binding beads have pelleted.Carefully aspirate the supernatant with a vacuum aspirator without disturbing the magnetic beads.  Alternatively, carefully remove the supernatant with a pipette and discard the supernatant.  The supernatant should be either a bright pink or a bright blue at this point due to the unincorporated dye molecules.Remove the plate from the magnetic stand.Add 100µl aRNA Wash Solution to each well and shake the plate for 1 minute on the orbital shaker at moderate speed.  Beads may not fully disperse at this step.Move the plate to a magnetic stand to capture the magnetic beads.Carefully aspirate the supernatant with a vacuum aspirator without disturbing the magnetic beads.  Alternatively, carefully remove the supernatant with a pipette and discard the supernatant.Remove the plate from the magnetic stand.nd time with 100µl aRNA Wash Solution.Repeat the wash a 2nd wash, dry the beads by shaking the plate for 1 minute on the orbital shaker at the maximum speed.  Do not overdry the samples!After the 2Elute the aRNA from the beads by adding 20µl preheated aRNA Elution Buffer to each sample.Vigorously shake the plate on the orbital shaker for 3 minutes, then check to make sure the magnetic beads are fully dispersed.  If not, continue shaking.Once the magnetic beads have fully dispersed, move the plate to a magnetic stand to capture the magnetic beads.  The supernatant contains the cleaned up, labeled aRNA samples, and should be either a pale pink or a pale blue.Carefully transfer the eluted aRNA to a new PCR plate (or PCR tubes). Check the RNA concentration and the amount of dye in the samples by measuring 1.5µl on a NanoDrop spectrophotometer using the Microarray module.Immediately hybridize the labeled aRNA onto a microarray platform of your choice, or alternatively, you can store the labeled aRNA at –80°C until you are ready for hybridization.*Mix the RNA binding beads with the bead resuspension solution first **Add the isopropanol and mix well before adding the aRNA binding buffer concentrate.When performing the amino allyl coupling, it is critical to resuspend the dye in DMSO shortly (less than 1 hour) before coupling and ensure no water gets into the dye/DMSO mix, as it will react with the active group on the dye.  Do not overdry the RNA (can be dried down to 1-2uL rather than completely dry), and resuspend well in the coupling buffer.  During the coupling reaction, keep the reaction in the dark, with occasional flicking and spin down if desired.The labeled RNA resulting from this protocol can be hybridized to human, viral, or custom microarrays to assess gene expression responses to infected cells in culture.  Microarray platforms vary, so follow manufacturer instructions for preparation of hybridization mixture from labeled probe.1, we were able to classify genes into the general categories of “early” or “late” based on timing of hybridization signal and whether or not viral DNA replication was required for transcript detection.  We observed the expected functional categories of genes in each temporal class  variation as to the exact timing of transcription.Using a custom designed poxvirus array2,3,4.The methods utilized in this work are able to predict virus genes transcribed early or late in the replication cycle, but have more difficulty distinguishing early-only versus genes with an early and late promoter since transcripts with a dual early/late promoter may persist and be detected at late times. In addition, run-through transcription of late viral genes may affect signal at a given probe/spot on the array, as the RNA hybridizing to the array may have come from the designated ORF or an upstream ORF.  Tiling arrays have attempted to resolve this issue, however challenges remain in detecting run through transcription using hybridization based approaches5,6,7,8.  Since the expression of many genes involved in host defense is altered after infection, the contribution of viral genes that counteract host immune responses should therefore be taken into consideration.Host transcriptional patterns can also be assessed using these methods.  However, vaccinia encodes a variety of mechanisms to inhibit host responses, and host transcriptional responses may be diminished compared to other stimuliUtilizing these methods, a map of the transcriptional timing of all viral genes can be identified and used to interrogate functions of unknown viral genes.  In addition, these methods can be utilized to dissect the intricate dialogue between virus and host.  These methods are broadly applicable to other host-pathogen infection systems.  If the pathogen of interest does not have polyadenylated mRNAs, alternative methods can be used to directly label the total RNA, without linear amplification.  By analyzing both host and virus gene expression during synchronous infection, these methods allow us to gain insight into virus interaction with the host cellular environment as well as host counter-defenses against virus infection.