Virus Games
G. L. Sheerin
Synergy Books
PO Box 80107, Austin, Texas, 78758
Phenix & Phenix (publicity)
2100 Kramer Lane, Suite 300, Austin, Texas 78758
9781934454046, $11.95, www.synergybooks.net
The most hardcore of technophobes–he wanted nothing to computers, barely tolerating the sight of them. "Virus Games: Peter’s Packet Series 00000001" is the tale of Peter, and how his anti-computer attitude was suddenly changed simply by nearly getting killed by a bolt of lightning–and finds he can now see into cyberspace. He finds himself befriending packets of data, and his love for the world of the PC grows–granting him a dilemma when a super virus begins to appear on the net and starts to cause some trouble for his new acquaintances. "Virus Games: Peter’s Packet Series 00000001" is a deftly written work of Science fiction, clever in it’s pose, and highly recommended for fans of the genre and community library collections catering to those fans.
COPYRIGHT 2008 Midwest Book Review
COPYRIGHT 2008 Gale, Cengage Learning
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Avian influenza virus.
Ed. by Erica Spackman.
Humana Press Inc.
2008
141 pages
$99.50
Hardcover
Methods in molecular biology; 436
SF995
US scientists provide laboratory protocols for investigating the diagnosis of bird flu and the virus that causes it, emphasizing recent molecular procedures used for basic and applied research. Among their topics are extracting virus RNA from tissue and swab material, isolating and propagating the virus in chicken eggs, wild bird surveillance, and reverse genetics.
([c]20082005 Book News, Inc., Portland, OR)
COPYRIGHT 2008 Book News, Inc.
COPYRIGHT 2008 Gale, Cengage Learning
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Measles virus nucleoprotein.
Ed. by Sonia Longhi.
Nova Biomedical Books
2007
163 pages
$79.00
Hardcover
Intrinsically disordered proteins
QR201
Longhi (biological macromolecules, UMR 6098 CNRS, University Aix- Marseille I and II, Luminy Campus, France) collects recent research from France, the UK, and the US on the measles virus nucleoprotein. The book focuses on the main structural information available on the nucleoprotein, showing that it consists of a structured core (NCORE) and an intrinsically disordered C-terminal domain (NTAIL). The functional implications of the disordered nature of NTAIL are discussed in light of the ability of disordered regions to establish interactions with multiple partners, thus leading to multiple biological effects. The book consists of five chapters. Two chapters are devoted to the general functions of the nucleoprotein in transcription and replication and to a detailed overview of its structural properties. The remaining three chapters focus on the functional relevance of the interaction between NTAIL and its various intracellular and extracellular partners. B&w and color images are included.
([c]20082005 Book News, Inc., Portland, OR)
COPYRIGHT 2008 Book News, Inc.
COPYRIGHT 2008 Gale, Cengage Learning
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Food-borne viruses; progress and challenges.
Ed. by Marion P.G. Koopmans et al.
ASM Press
2008
245 pages
$99.95
Hardcover
Emerging issues in food safety
QR201
Virologists mostly from Europe but also India and the US review the current understanding of viruses in food products, illnesses they can cause, and how to combat both. Among their topics are enterically transmitted hepatitis, viral evolution and its relevance for food-borne virus epidemiology, and using the codex risk analysis framework to reduce risks associated with viruses in food.
([c]20082005 Book News, Inc., Portland, OR)
COPYRIGHT 2008 Book News, Inc.
COPYRIGHT 2008 Gale, Cengage Learning
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Phylogenetic analysis of influenza A viruses (H5N1) isolated from Kuwait in 2007 show that (H5N1) sublineage clade 2.2 viruses continue to spread across Europe, Africa, and the Middle East. Virus isolates were most closely related to isolates from central Asia and were likely vectored by migratory birds.
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Highly pathogenic avian influenza (HPAI) virus (H5N1) has been endemic in poultry in Asia since 2003 (1,2). From 2002 through 2005, influenza virus (H5N1) has also been sporadically isolated from dead wild birds in Hong Kong Special Administrative Region, People’s Republic of China; however, these birds were considered dead-end hosts of viruses acquired from poultry (3,4). In April 2005, an influenza (H5N1) outbreak was detected in bar-headed geese (Anser indicus) at Qinghai Lake in western China (5). Following this outbreak, the Qinghai-like (clade 2.2) influenza virus (H5N1) lineage was detected in wild birds and domestic poultry in countries in central Asia, the Middle East, Europe, and Africa (6-10). The source of these introductions, while still debated, is likely through bird migration, although in some instances, the role of the poultry trade has not been ruled out (6-12).
The clade 2.2 influenza (H5N1) viruses continue to be detected throughout these regions; 69 human cases with 31 deaths were reported from Azerbaijan, Djibouti, Egypt, Iraq, Nigeria, Pakistan, and Turkey from January 2006 through December 2007 (13). Since early 2007, the Qinghai-like influenza (H5N1) lineage has continued its geographic spread and has been reported from more than 40 countries in Eurasia and Africa (6). The continued detection of these viruses in Africa, Europe, and the Middle East from mid-2006 onward suggests that the virus may now be endemic in these regions.
The Study
On February 13, 2007, the Public Authority for Agriculture and Fisheries of Kuwait reported the initial outbreak of influenza (H5N 1) in poultry in the A1 Wafrah farm area in southern Kuwait. Subsequently, 131 influenza virus (H5N1)–infected poultry were confirmed from 20 farms throughout the country (Figure 1, panel A). The disease resulted in high mortality rates among infected flocks, especially in the commercial broiler farms in Al-Wafrah and among poultry raised in privately owned residential homes and backyard farms. Disease control measures were implemented beginning February 18, 2007, including control of poultry movement, vaccination, disinfection of infected premises, and culling of [approximately equal to] 500,000 birds. The final case of subtype H5N1 was detected on April 20, 2007, and all restrictions were lifted on May 12, 2007. Kuwait was declared free of highly pathogenic avian influenza (HPAI) (H5N1) on July 21, 2007.
[FIGURE 1 OMITTED]
During these outbreaks, 20 samples were collected from small backyard farms in the Al Sulaibiya area (Figure 1, panel A). Among those samples, 10 throat and cloacal swabs were collected from chickens that showed signs of disease; 10 more samples were collected from internal organs (liver and spleen) of dead chickens. Seven of the 10 organ samples tested positive for subtype H5N1 by using the TaqMan Influenza A/H5 Detection Kit v 1.0 on the 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions.
We sequenced the complete genome of these 7 subtype H5N1 strains isolated from poultry outbreaks in Kuwait during 2007. All sequences that were generated in this study have been deposited in GenBank (accession nos. CY029945-CY030000). To understand the developments of influenza A virus (H5N1) in Kuwait, we characterized and phylogenetically analyzed all 8 gene segments of these 7 viruses with all available influenza (H5N1) viruses previously isolated from Africa, Eurasia, Southeast Asia, and southern China, and with reference viruses belonging to each subtype H5N1 clade. Sequence assembly, editing, multiple sequence alignment, neighbor-joining, and Bayesian phylogenetic analyses were conducted as previously described (11).
Phylogenetic analysis of the hemagglutinin (HA) genes showed that all 7 subtype H5N1 isolates were derived from the Goose/Guangdong-like lineage and clustered together with other Qinghai-like (clade 2.2) viruses (Figure 2). The Kuwait isolates were most closely related to viruses from Germany and Krasnodar, in southwest Russia, which were also isolated in 2007 (Figure 1, panel B). Those viruses were mostly isolated from wild bird species (swan and grebe), although a single isolate was from chicken in Krasnodar. This group of viruses was in turn related to 2006 isolates from diverse geographic areas such as Afghanistan, Mongolia, and Siberian Russia (Figure 1, panel B). Phylogenetic analyses of the neuraminidase gene and all internal gene segments (data not shown) show that all of the viruses belong to subtype H5N1, genotype Z, and maintain phylogenetic relationships similar to the HA tree.
The HA protein of all 7 isolates maintained the motif of multiple basic amino acids (QGERRRKKR/G) at the HA-connecting peptide, a feature that is characteristic of HPAI virus. The receptor-binding pocket of HA1 retains Gln 222 and Gly 224 (H5 numbering) that preferentially binds avian-like [alpha]2,3-NeuAcGal linkages. However, a single Glu212Lys substitution occurred in the HA receptor binding site in all 7 Kuwait isolates, which has also been observed in all clade 2.2 influenza (H5N1) viruses characterized to date. The biological implications of this mutation remain to be investigated. None of the isolates had mutations in the M2 ion channel or the neuraminidase, conferring resistance to amantadine and oseltamivir, respectively. All isolates possessed Lys at position 627 of the PB2 gene, which is associated with increased virulence in mammals and is present in all known clade 2.2 viruses. Other virulence mutations were not recognized in any of the viruses characterized in this study.
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In 2004, canine influenza virus subtype H3N8 emerged in greyhounds in the United States. Subsequent serologic evidence indicated virus circulation in dog breeds other than greyhounds, but the virus had not been isolated from affected animals. In 2005, we conducted virologic investigation of 7 nongreyhound dogs that died from respiratory disease in Florida and isolated influenza subtype H3N8 virus. Antigenic and genetic analysis of A/canine/Jacksonville/2005 (H3N8) and A/canine/Miami/2005 (H3N8) found similarity to earlier isolates from greyhounds, which indicates that canine influenza viruses are not restricted to greyhounds. The hemagglutinin contained 5 conserved amino acid differences that distinguish canine from equine lineages. The antigenic homogeneity of the canine viruses suggests that measurable antigenic drift has not yet occurred. Continued surveillance and antigenic analyses should monitor possible emergence of antigenic variants of canine influenza virus.
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Influenza A viruses (family Orthomyxoviridae) are known to cause acute respiratory disease in humans, horses, pigs, and domestic poultry (1,2). Influenza A virus subtype H3N8 has recently emerged as a respiratory pathogen in dogs, associated with outbreaks of acute respiratory disease in racing greyhounds (3). The disease is caused by a novel virus closely related to contemporary equine influenza A virus subtype H3N8. These viruses share [greater than or equal to] 96% nucleotide sequence identity, which suggests direct transmission of the entire virus from horses to dogs without reassortment with other strains (3).
Canine influenza virus (CIV) was first identified in racing greyhounds in Florida in January 2004 and was later associated with respiratory disease outbreaks in racing greyhounds in 9 states from 2004 through 2006 (3,4). Most affected greyhounds had clinical signs associated with virus infection of the upper respiratory tract–cough for 10-30 days, nasal discharge, low-grade fever–followed by recovery. However, some dogs died peracutely with extensive hemorrhage in the lungs, mediastinum, and pleural cavity. Histologic examination showed tracheitis, bronchitis, bronchiolitis, and suppurative bronchopneumonia associated with extensive erosion of epithelial cells and infiltration with neutrophils. The isolation of 4 closely related influenza A subtype H3N8 viruses from dogs that died in different geographic locations over a 25-month period, together with substantial serologic evidence of widespread infection among racing greyhounds in 9 states, suggested sustained CIV circulation in this population by dog-to-dog transmission (3,4).
The first evidence of CIV infection in dogs other than greyhounds came from serologic testing of dogs with acute respiratory disease in shelters, boarding kennels, and veterinary clinics in Florida and New York in 2004 and 2005 (3). Since August 2005, a national syndromic serosurvey for canine influenza has been conducted on >5,000 samples collected from nongreyhound dogs with compatible clinical signs (Cornell University College of Veterinary Medicine, http://diaglab.vet.cornell.edu/issues/civ-stat.asp). As of April 2008, seropositive dogs have been identified in 25 states and the District of Columbia.
In April and May 2005, an outbreak of respiratory disease occurred in dogs housed in a shelter facility in northeastern Florida (3). The outbreak involved at least 58 dogs, ranging in age from 3 months to 9 years, and included purebred dogs as well as mixed breeds; 6 were euthanized. In May 2005, a respiratory disease outbreak occurred among [approximately equal to] 40 pet dogs at a veterinary clinic in southeastern Florida; 1 died. We performed molecular analyses on 2 influenza A subtype H3N8 viruses isolated from these 7 nongreyhound dogs that died and genetically and antigenically compared them with influenza (H3N8) viruses from racing greyhounds.
Materials and Methods
Specimen Collection
Postmortem examinations were performed on the 6 mixed-breed shelter dogs that died in April and May 2005 and on the 1 pet Yorkshire terrier that died in the veterinary clinic in May 2005. Tissues were fixed in 10% neutral buffered formalin and embedded in paraffin; 5-[micro]m sections were stained with hematoxylin and eosin for histopathologic diagnosis. Unfixed tissues for virologic and molecular analyses were stored at -80[degrees]C.
RNA Extraction
Frozen hmg tissues from each dog were thawed and homogenized in lysis buffer containing [beta]-mercaptoethanol by using a disposable tissue grinder (Kendall, Lifeline Medical Inc., Danbury, CT, USA). Total RNA was extracted by using a commercial kit (RNeasy Mini Kit, QIAGEN Inc., Valencia, CA, USA) according to manufacturer’s instructions and eluted in a final volume of 60 [micro]L of buffer. Total RNA was also extracted from lung tissue collected from specific-pathogen-free dogs without respiratory disease.
Real-Time Reverse Transcription-PCR
A single-step quantitative real-time reverse transcription-PCR (RT-PCR) was performed on total RNA extracted from the canine tissue samples by using the QuantiTect Probe RT-PCR Kit containing ROX as a passive reference dye (QIAGEN). Briefly, 2 primer-probe sets were used for detection of influenza A sequences in each sample (Table 1). One primer-probe set was selective for canine influenza subtype H3 gene sequences. The other primer-probe set targeted a highly conserved region of the matrix (M) gene of type A influenza virus. The sequence of the M probe contained 3 locked nucleic acids. For each real-time RT-PCR, 5 [micro]L of total RNA was added to a reaction mixture containing 12.5 [micro]L of 2x QuantiTect Probe RT-PCR Master Mix, 0.25 [micro]L of QuantiTech RT Mix (both QIAGEN), forward and reverse primers (0.4 [micro]mol/L final concentration for each), probe (0.1 [micro]mol/L final concentration), and RNase-free water in a final volume of 25 [micro]L. Real-time PCR for eukaryotic 18S rRNA was performed by using commercially available assay reagents (VIC/TAMRA; TaqMan, Applied Biosystems, Foster City, CA, USA), according to manufacturer’s instructions for detection of endogenous 18S rRNA, as an internal control for RNA extraction from the tissues.
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To investigate the presence of Lagos bat virus (LBV)-specific antibodies in megachiroptera from West Africa, we conducted fluorescent antibody virus neutralization tests. Neutralizing antibodies were detected in Eidolon helvum (37%), Epomophorus gambianus (3%), and Epomops buettikoferi (33%, 2/6) from Ghana. These findings confirm the presence of LBV in West Africa.
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Bats host a range of lyssaviruses, depending on their species and locality. The genus Lyssavirus is differentiated into 7 genetically divergent genotypes: classical rabies virus (genotype 1), Lagos bat virus (LBV; genotype 2), Mokola virus (MOKV; genotype 3), Duvenhage virus (genotype 4), European bat lyssavirus (genotypes 5 and 6), and Australian bat lyssavirus (genotype 7) (1). All but MOKV have been isolated from bats.
LBV and MOKV are each distributed in Africa and are members of phylogroup 2 within the genus Lyssavirus (1). Because LBV isolates (2) from African bats are increasing, our goal was to determine the prevalence of virus neutralizing antibodies against LBV in bat populations in West Africa.
The Study
Bats were sampled in January and May 2007 at 6 sites in Ghana: the center of Accra (urban habitat); the wooded outskirts of Accra (savannah habitat); and forested habitats at Pra, Kibi, Adoagyiri, and Oyibi (a plantation with woodland/forest border). Bats were captured by using 6-18-m mist nets; roosting Eidolon helvum were captured by using nets on poles. A sample size of 59 would provide 95% confidence of finding at least 1 LBV-seropositive bat in a large population (>5,000), given a seroprevalence of 5% and assuming random sampling (3). Species were identified by using a dichotomous key (4). Captured bats were manually restrained and anesthetized by intravenous injection; [approximately equal to] 0.2-1.0 mL of blood was collected from the propatagial vein before the bat was released. Blood was centrifuged in the field at ambient temperature at 3,000 rpm for 15 rain. Serum was heat treated at 56[degrees]C for 30 min and frozen at -70[degrees]C.
Two species, Epomophorus gambianus and E. helvum, were caught in sufficient numbers (117 and 66, respectively) for reasonable inferences to be made about LBV seroprevalence rates (Table). A standard approach was used to calculate 95% confidence intervals (CIs) for seroprevalence (3). Because of the relatively short distances between study sites and the likelihood of bats mixing between these sites, bats of each species were considered to belong to single populations. All but 3 E. helvum were derived from a colony in Accra, whereas E. gambianus were derived from all habitat types.
Bat serum samples were tested for virus neutralizing antibody against classical rabies virus (challenge virus standard) by using a standard fluorescent antibody virus neutralization (FAVN) test (5). Antibodies to LBV were measured by using a modified FAVN test (6). Because positive bat antiserum from naturally infected bats was not available, for positive controls we used human rabies immunoglobulin, LBV-positive rabbit serum, and serum from mice vaccinated with human diploid cell vaccine. Negative controls were negative rabbit and mouse serum. All samples were analyzed in duplicate and serially diluted by using a 3-fold series (representing reciprocal titers of 9, 27, 81, and 243-19,683) (6).
The modified FAVN test requires a cut-off threshold, which in prior bat lyssavirus studies has been a titer of 27, to avoid false-positive results (6, 7). The first 121 samples collected were tested against the challenge virus standard; no tested bat was seropositive at 1:3 dilutions. A mean titer >9 was considered positive for LBV (Figure 1; [8]).
Levels of specific virus neutralizing antibodies against LBV were higher in E. helvum (seroprevalence 37%, 95% C124%-49%) than in E. gambianus (3%, 95% CI 0%-7%). Of 6 Epomops buettikoferi, 2 were seropositive (30%, 95% CI 0%-70%). No sex differences in E. helvum seroprevalence were evident ([chi square] 1.0, p > 0.9).
Because of the high level of seropositivity in E. helvum, we attempted to determine a possible case reproduction rate ([R.sub.0]) for LBV infection in this species by using the equation [R.sub.0] = 1/[x.sup.*], where [x.sup.*] = proportion of susceptible hosts in a population (9). We assumed that infection with each virus within the bat populations is endemic, stable, and randomly dispersed; that all seropositive animals have lifelong immunity that is detectable serologically; and that seropositivity is to 1 virus. On the basis of these assumptions, [R.sub.0] = 1.6 (95% CI 1.3-2.0).
Conclusions
We found antibodies against LBV in healthy E. helvum bats in Ghana. Previous studies have suggested that healthy bats develop antibodies to other lyssavirus infections (7,10,11), which may reflect past exposure, rather than survival from clinical disease. LBV likely co-evolved with its natural megachiropteran host until a genetic stasis had been reached in which the virus-host relationship was in equilibrium. This situation would result in high seroprevalence rates after a wave of virus circulation in a colony. Nine seropositive bats (8 E. helvurn, 1 E. buettikoferi) were apparently healthy pregnant females. These results support theories that lyssaviruses are endemic within specific bat populations, that they may not cause high mortality rates, that exposure rates of LBV between megachiroptera in Old World African bats are high, and that bats may breed successfully after LBV exposure (7,8). The number of high reciprocal titers against LBV (Figure 1) and the history of LBV isolation in E. helvum suggest that LBV circulates in megachiroptera in Ghana. However, further work is needed to determine the specific phylogroup 2 virus and its prevalence within specific bat populations.
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We examined West Nile virus (WNV) seroprevalence in wild mammals along a forest-to-urban gradient in the US mid-Atlantic region. WNV antibody prevalence increased with age, urbanization, and date of capture for juveniles and varied significantly between species. These findings suggest several requirements for using mammals as indicators of transmission.
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West Nile virus (WNV) is maintained in an enzootic bird-mosquito-bird cycle and is transmitted by numerous mosquito species, including many that feed on mammals (1). Several mammal species have been found to be naturally exposed to WNV, and it has been suggested that wild mammals could be used as indicators of transmission (2-4). WNV seroprevalence in wild mammals will be a useful indicator of WNV activity only if it differs between sites, if it reflects within-season transmission, and if other key confounding factors are accounted for.
To test 4 hypotheses about the exposure of mammals to WNV, we examined WNV seroprevalence in wild mammals in the eastern United States. First, we predicted that WNV seroprevalence would differ significantly among species because of differences in mosquito preferences, mammal behavior and survival, and other factors (2, 3). Second, we predicted that seroprevalence would be higher for adults than for juveniles because adults have been exposed to WNV for at least 1 additional year. Third, we predicted that WNV exposure would increase with the date of capture over the transmission season because peak transmission occurs during late summer. Finally, we predicted that WNV seroprevalence would vary among sites and increase with urbanization because the abundance of Culex pipiens, the dominant enzootic vector in this region (1), increases with human population density (4).
The Study
We trapped mammals at 7 sites along a forest-to-urban gradient in Maryland and Washington, DC, USA, from early June to late September 2005 and in April 2006. The sites included 1 forested area (Smithsonian Environmental Research Center, Edgewater, MD), 2 large wooded parks (Rock Creek Park, Rockville, MD; Fort Dupont Park, Washington, DC), 2 residential neighborhoods (Takoma Park, MD; Bethesda, MD), and 2 urban areas (Baltimore, MD; Washington, DC).
We quantified the land use around each site by calculating an urbanization index (UI) within a 1,000-m radius as follows:
UI = (100% - % tree cover + % impervious surface)/2
Impervious land and forest cover were estimated by using multitemporal (leaf-on and leaf-off) compilations of Landsat satellite images at 30-m spatial resolution, higher resolution satellite imagery, and digital orthophotography (5).
We ran trap lines of Tomahawk (models 201, 203, 204, 207; Tomahawk Live Trap Company, Tomahawk, WI, USA) and Sherman (model LFAHD; H.B. Sherman Traps, Inc., Tallahassee, FL, USA) traps for 2-5 days and nights at each site. Captured animals were chemically restrained and tagged, and age was determined by using body mass and/or reproductive characters (6). Blood samples (0.1 mL) were obtained, dispensed into tubes containing 0.9 mL BA-1 medium, and placed on ice packs until storage at -80[degrees]C. Blood samples were allowed to clot before antibody assays were run. We assayed the blood samples for neutralizing antibodies to WNV and Powassan virus (but not St. Louis encephalitis virus, which was absent in the local bird community at these sites [7]) by using the plaque-reduction neutralization test (8) at a 1:10 dilution, with 80% and 90% neutralization of plaques as cutoffs. We examined variation in WNV antibody prevalence by using binary logistic regression with species and age as categorical factors and capture date and urbanization index as covariates. We used October 15, 2005, as the capture date for the April 2006 samples because the abundance of WNV-infected mosquitoes falls precipitously after this date (9).
We obtained 244 samples from 11 mammal species (Table 1). The probability of being WNV antibody–positive varied significantly among species, was significantly higher for adults, increased with capture date for juveniles, and increased with the urbanization index (Table 2). The higher seroprevalence in samples collected in April 2006 showed that WNV exposure of juvenile eastern gray squirrels (Sciurus carolinensis) continued after the last trapping periods in September 2005 (Table 2).
Seroprevalence rates were highest (and not significantly different) in 4 peridomestic species: eastern gray squirrels, Virginia opossums (Didelphis virginiana), raccoons (Procyon lotor), and Norway rats (Rattus norvegicus) (Tables 1, 2). Eastern gray squirrels were 5.5x more likely than eastern chipmunks (Tamias striatus) to have WNV antibodies and 4.5x more likely than Peromyscus leucopus; both differences were significant (Table 2).
Conclusions
Previous research on the exposure of mammals to WNV has shown patterns of antibody prevalence across several states and species (2,3,10-12). However, few studies have tested for statistical differences in the factors that influence the exposure of mammals to WNV, which thus would establish their usefulness as indicators of variation in WNV transmission. We found significant effects of age, species, site