Diversity,origins and virulence of <Emphasis Type="Italic">Avipoxviruses</Emphasis> in Hawaiian Forest Birds

We cultured avian pox (Avipoxvirus spp.) from lesions collected on Hawai‘i, Maui, Moloka‘i, and ‘Oahu in the Hawaiian Islands from 15 native or non-native birds representing three avian orders. Phylogenetic analysis of a 538 bp fragment of the gene encoding the virus 4b core polypeptide revealed two distinct variant clusters, with sequences from chickens (fowlpox) forming a third distinct basal cluster. Pox isolates from one of these two clusters appear closely related to canarypox and other passerine pox viruses, while the second appears more specific to Hawai‘i. There was no evidence that birds were infected simultaneously with multiple pox virus variants based on evaluation of multiples clones from four individuals. No obvious temporal or geographic associations were observed and strict host specificity was not apparent among the 4b-defined field isolates. We amplified a 116 bp 4b core protein gene fragment from an ‘Elepaio (Chasiempis sandwichensis) collected in 1900 on Hawai‘i Island that clustered closely with the second of the two variants, suggesting that this variant has been in Hawai‘i for at least 100 years. The high variation detected between the three 4b clusters provides evidence for multiple, likely independent introductions, and does not support the hypothesis of infection of native species through introduction of infected fowl. Preliminary experimental infections in native Hawai‘i ‘Amakihi (Hemignathus virens) suggest that the 4b-defined variants may be biologically distinct, with one variant appearing more virulent. These pox viruses may interact with avian malaria (Plasmodium relictum), another introduced pathogen in Hawaiian forest bird populations, through modulation of host immune responses.     

Pox infection among captive peacocks.

An outbreak of avian pox was detected among captive peacocks (Pavo cristatus) at Baghdad Zoological Park during spring, 1978. A total of 45 of the 60 birds in the aviary developed pox lesions around the beaks and eyes. Morbidity was 75% and mortality was 13%. A virus isolated from the skin lesions produced large plaques on the chorioallantoic membrane of developing chicken embryos and induced cytopathic effect characteristic for pox viruses in chicken embryo cell cultures. The virus neither haemagglutinated nor haemadsorbed to chicken erythrocytes. It was ether resistant and chloroform sensitive. Chickens inoculated with the virus by scarification developed localized pox-like lesions, while turkeys had only transient swelling of feather follicles at the site of inoculation. Virus partially purified with Genetron 113 was precipitated by antisera to fowlpox and pigeon pox viruses.     

Arthropod-borne disease as a possible factor limiting the distribution of birds

Warner found that arthropod-borne disease in the Hawaiian islands (bird malaria and avian pox in particular) is a factor limiting the distribution of the Drepaniidae. Rowan considers it conceivable that the present distribution of certain African birds (and perhaps some other vertebrates showing similar patterns of occurrence) may have been determined in the same way. This suggestion is supported by the observations of the author in southern Africa in regard to bird malaria and avian pox.     

Genotypic and pathotypic characterization of Newcastle disease viruses from India

Newcastle disease virus (NDV) is an avian paramyxovirus that causes significant economic losses to the poultry industry in most parts of the world. The susceptibility of a wide variety of avian species coupled with synanthropic bird reservoirs has contributed to the vast genomic diversity of this virus as well as diagnostic failures. Since the first panzootic in 1926, Newcastle disease (ND) became enzootic in India with recurrent outbreaks in multiple avian species. The genetic characteristics of circulating strains in India, however, are largely unknown. To understand the nature of NDV genotypes in India, we characterized two representative strains isolated 13 years apart from a chicken and a pigeon by complete genome sequence analysis and pathotyping. The viruses were characterized as velogenic by pathogenicity indices devised to distinguish these strains. The genome length was 15,186 nucleotides (nt) and consisted of six non-overlapping genes, with conserved and complementary 3' leader and 5' trailer regions, conserved gene starts, gene stops, and intergenic sequences similar to those in avian paramyxovirus 1 (APMV-1) strains. Matrix gene sequence analysis grouped the pigeon isolate with APMV-1 strains. Phylogeny based on the fusion (F), and hemagglutinin (HN) genes and complete genome sequence grouped these viruses into genotype IV. Genotype IV strains are considered to have "died out" after the first panzootic (1926-1960) of ND. But, our results suggest that there is persistence of genotype IV strains in India.     

Analysis of <Emphasis Type="Italic">N</Emphasis>-glycans in embryonated chicken egg chorioallantoic and amniotic cells responsible for binding and adaptation of human and avian influenza viruses

The initial step essential in influenza virus infection is specific binding of viral hemagglutinin to host cell-surface glycan receptors. Influenza A virus specificity for the host is mediated by viral envelope hemagglutinin, that binds to receptors containing glycans with terminal sialic acids. Human viruses preferentially bind to α2→6 linked sialic acids on receptors of host cells, whereas avian viruses are specific for the α2→3 linkage on the target cells. Human influenza virus isolates more efficiently infect amniotic membrane (AM) cells than chorioallantoic membrane (CAM) cells. N-glycans were isolated from AM and CAM cells of 10-day-old chicken embryonated eggs and their structures were analyzed by multi-dimensional HPLC mapping and MALDI-TOF-MS techniques. Terminal N-acetylneuraminic acid contents in the two cell types were similar. However, molar percents of α2→3 linkage preferentially bound by avian influenza virus were 27.2 in CAM cells and 15.4 in AM cells, whereas those of α2→6 linkage favored by human influenza virus were 8.3 (CAM) and 14.2 (AM). Molar percents of sulfated glycans, recognized by human influenza virus, in CAM and AM cells were 3.8 and 12.7, respectively. These results have revealed structures and molar percents of N-glycans in CAM and AM cells important in determining human and avian influenza virus infection and viral adaptation.     

The Avian XPR1 Gammaretrovirus Receptor Is under Positive Selection and Is Disabled in Bird Species in Contact with Virus-Infected Wild Mice

Xenotropic mouse leukemia viruses (X-MLVs) are broadly infectious for mammals except most of the classical strains of laboratory mice. These gammaretroviruses rely on the XPR1 receptor for entry, and the unique resistance of laboratory mice is due to two mutations in different putative XPR1 extracellular loops. Cells from avian species differ in susceptibility to X-MLVs, and 2 replacement mutations in the virus-resistant chicken XPR1 (K496Q and Q579E) distinguish it from the more permissive duck and quail receptors. These substitutions align with the two mutations that disable the laboratory mouse XPR1. Mutagenesis of the chicken and duck genes confirms that residues at both sites are critical for virus entry. Among 32 avian species, the 2 disabling XPR1 mutations are found together only in the chicken, an omnivorous, ground-dwelling fowl that was domesticated in India and/or Southeast Asia, which is also where X-MLV-infected house mice evolved. The receptor-disabling mutations are also present separately in 5 additional fowl and raptor species, all of which are native to areas of Asia populated by the virus-infected subspecies Mus musculus castaneus. Phylogenetic analysis showed that the avian XPR1 gene is under positive selection at sites implicated in receptor function, suggesting a defensive role for XPR1 in the avian lineage. Contact between bird species and virus-infected mice may thus have favored selection of mouse virus-resistant receptor orthologs in the birds, and our data suggest that similar receptor-disabling mutations were fixed in mammalian and avian species exposed to similar virus challenges.     

Characterization of canarypox-like viruses infecting endemic birds in the Galápagos Islands

The presence of avian pox in endemic birds in the Galápagos Islands has led to concern that the health of these birds may be threatened by avipoxvirus introduction by domestic birds. We describe here a simple polymerase chain reaction-based method for identification and discrimination of avipoxvirus strains similar to the fowlpox or canarypox viruses. This method, in conjunction with DNA sequencing of two polymerase chain reaction-amplified loci totaling about 800 bp, was used to identify two avipoxvirus strains, Gal1 and Gal2, in pox lesions from yellow warblers (Dendroica petechia), finches (Geospiza spp.), and Galápagos mockingbirds (Nesomimus parvulus) from the inhabited islands of Santa Cruz and Isabela. Both strains were found in all three passerine taxa, and sequences from both strains were less than 5% different from each other and from canarypox virus. In contrast, chickens in Galápagos were infected with a virus that appears to be identical in sequence to the characterized fowlpox virus and about 30% different from the canarypox/Galápagos group viruses in the regions sequenced. These results indicate the presence of canarypox-like viruses in endemic passerine birds that are distinct from the fowlpox virus infecting chickens on Galápagos. Alignment of the sequence of a 5.9-kb region of the genome revealed that sequence identities among Gal1, Gal2, and canarypox viruses were clustered in discrete regions. This indicates that recombination between poxvirus strains in combination with mutation led to the canarypox-like viruses that are now prevalent in the Galápagos.     

Comparisons of Highly Virulent H5N1 Influenza A Viruses Isolated from Humans and Chickens from Hong Kong

Genes of an influenza A (H5N1) virus from a human in Hong Kong isolated in May 1997 were sequenced and found to be all avian-like (K. Subbarao et al., Science 279:393–395, 1998). Gene sequences of this human isolate were compared to those of a highly pathogenic chicken H5N1 influenza virus isolated from Hong Kong in April 1997. Sequence comparisons of all eight RNA segments from the two viruses show greater than 99% sequence identity between them. However, neither isolate’s gene sequence was closely (>95% sequence identity) related to any other gene sequences found in the GenBank database. Phylogenetic analysis demonstrated that the nucleotide sequences of at least four of the eight RNA segments clustered with Eurasian origin avian influenza viruses. The hemagglutinin gene phylogenetic analysis also included the sequences from an additional three human and two chicken H5N1 virus isolates from Hong Kong, and the isolates separated into two closely related groups. However, no single amino acid change separated the chicken origin and human origin isolates, but they all contained multiple basic amino acids at the hemagglutinin cleavage site, which is associated with a highly pathogenic phenotype in poultry. In experimental intravenous inoculation studies with chickens, all seven viruses were highly pathogenic, killing most birds within 24 h. All infected chickens had virtually identical pathologic lesions, including moderate to severe diffuse edema and interstitial pneumonitis. Viral nucleoprotein was most frequently demonstrated in vascular endothelium, macrophages, heterophils, and cardiac myocytes. Asphyxiation from pulmonary edema and generalized cardiovascular collapse were the most likely pathogenic mechanisms responsible for illness and death. In summary, a small number of changes in hemagglutinin gene sequences defined two closely related subgroups, with both subgroups having human and chicken members, among the seven viruses examined from Hong Kong, and all seven viruses were highly pathogenic in chickens and caused similar lesions in experimental inoculations.     

Prevalence of avian pox virus and effect on the fledging success of Laysan Albatross

ABSTRACT.   Avian pox virus ( Poxvirus avium ) is a mosquito-borne disease that occurs worldwide in a variety of bird species, but little is known about its prevalence or effect on seabirds. We monitored prevalence of pox virus and its effect on fledging success of Laysan Albatross ( Phoebastria immutabilis ) on Oahu, Hawaii, from 2003 to 2007. Pox prevalence in albatross chicks averaged 88% in years with high rainfall and 3% in years with low rainfall. Diagnosis of pox virus was clinically confirmed in two birds by Muscovy Duck ( Cairina moschata ) fibrolast cultures. Severity of infection ranged from small wart-like nodules and lesions on the bill, face, eyes, tarsus, and feet, to large tumorous growths that completely covered both eyes and caused deformation of the bill and skull. Most chicks recovered from infection, and the fledging rate in pox epizootic years (82%) did not differ from that in years with low pox prevalence (80%) or the average fledging rate on Midway Atoll (86%). Three chicks with severe infections were resighted as healthy adults on Kauai and Oahu in 2007, confirming postfledging survival of at least some birds. The high recovery rate, fledging success, and postfledging survival indicate that Laysan Albatross have strong immunity to avian pox virus.     

Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts.

The nucleotide and amino acid sequences of 40 influenza virus hemagglutinin genes of the H3 serotype from mammalian and avian species and 9 genes of the H4 serotype were compared, and their evolutionary relationships were evaluated. From these relationships, the differences in the mutational characteristics of the viral hemagglutinin in different hosts were examined and the RNA sequence changes that occurred during the generation of the progenitor of the 1968 human pandemic strain were examined. Three major lineages were defined: one containing only equine virus isolates; one containing only avian virus isolates; and one containing avian, swine, and human virus isolates. The human pandemic strain of 1968 was derived from an avian virus most similar to those isolated from ducks in Asia, and the transfer of this virus to humans probably occurred in 1965. Since then, the human viruses have diverged from this progenitor, with the accumulation of approximately 7.9 nucleotide and 3.4 amino acid substitutions per year. Reconstruction of the sequence of the hypothetical ancestral strain at the avian-human transition indicated that only 6 amino acids in the mature hemagglutinin molecule were changed during the transition between an avian virus strain and a human pandemic strain. All of these changes are located in regions of the molecule known to affect receptor binding and antigenicity. Unlike the human H3 influenza virus strains, the equine virus isolates have no close relatives in other species and appear to have diverged from the avian viruses much earlier than did the human virus strains. Mutations were estimated to have accumulated in the equine virus lineage at approximately 3.1 nucleotides and 0.8 amino acids per year. Four swine virus isolates in the analysis each appeared to have been introduced into pigs independently, with two derived from human viruses and two from avian viruses. A comparison of the coding and noncoding mutations in the mammalian and avian lineages showed a significantly lower ratio of coding to total nucleotide changes in the avian viruses. Additionally, the avian virus lineages of both the H3 and H4 serotypes, but not the mammalian virus lineages, showed significantly greater conservation of amino acid sequence in the internal branches of the phylogenetic tree than in the terminal branches. The small number of amino acid differences between the avian viruses and the progenitor of the 1968 pandemic strain and the great phenotypic stability of the avian viruses suggest that strains similar to the progenitor strain will continue to circulate in birds and will be available for reintroduction into humans.     

Emergence of a Novel Avian Pox Disease in British Tit Species

Avian pox is a viral disease with a wide host range. In Great Britain, avian pox in birds of the Paridae family was first diagnosed in a great tit (Parus major) from south-east England in 2006. An increasing number of avian pox incidents in Paridae have been reported each year since, indicative of an emergent infection. Here, we utilise a database of opportunistic reports of garden bird mortality and morbidity to analyse spatial and temporal patterns of suspected avian pox throughout Great Britain, 2006–2010. Reports of affected Paridae (211 incidents) outnumbered reports in non-Paridae (91 incidents). The majority (90%) of Paridae incidents involved great tits. Paridae pox incidents were more likely to involve multiple individuals (77.3%) than were incidents in non-Paridae hosts (31.9%). Unlike the small wart-like lesions usually seen in non-Paridae with avian pox in Great Britain, lesions in Paridae were frequently large, often with an ulcerated surface and caseous core. Spatial analyses revealed strong clustering of suspected avian pox incidents involving Paridae hosts, but only weak, inconsistent clustering of incidents involving non-Paridae hosts. There was no spatial association between Paridae and non-Paridae incidents. We documented significant spatial spread of Paridae pox from an origin in south-east England; no spatial spread was evident for non-Paridae pox. For both host clades, there was an annual peak of reports in August/September. Sequencing of the avian poxvirus 4b core protein produced an identical viral sequence from each of 20 great tits tested from Great Britain. This sequence was identical to that from great tits from central Europe and Scandinavia. In contrast, sequence variation was evident amongst virus tested from 17 non-Paridae hosts of 5 species. Our findings show Paridae pox to be an emerging infectious disease in wild birds in Great Britain, apparently originating from viral incursion from central Europe or Scandinavia.     

Transmission of avian pox from starlings to Rothchild's mynahs

Several wild species of birds, including starlings (Sturnus vulgaris), house sparrows (Passer domesticus) and pigeons (Columba livia) gained access to an aviary housing Rothchild's mynahs (Leucospar rothchildii) and over 100 additional birds representing a variety of species. Six of approximately 15 mynahs became infected with avian pox and all of them died. None of the other birds in the aviary developed lesions. Pox virus was isolated from mynah facial lesions on chicken chorioallantoic membrane and in duck embryo fibroblast cell culture. It did not produce lesions in white Leghorn chickens, but did produce lesions in 4 of 11 wild starlings captured outside the aviary. Results indicated the agent was an indigenous starling pox capable of infecting and producing disease in mynah birds. Destruction of the captive starlings and isolation of the remaining mynahs immediately stopped the mortality.     

Characterization of highly pathogenic H5N1 avian influenza A viruses isolated from South Korea

An unprecedented outbreak of H5N1 highly pathogenic avian influenza (HPAI) has been reported for poultry in eight different Asian countries, including South Korea, since December 2003. A phylogenetic analysis of the eight viral genes showed that the H5N1 poultry isolates from South Korea were of avian origin and contained the hemagglutinin and neuraminidase genes of the A/goose/Guangdong/1/96 (Gs/Gd) lineage. The current H5N1 strains in Asia, including the Korean isolates, share a gene constellation similar to that of the Penfold Park, Hong Kong, isolates from late 2002 and contain some molecular markers that seem to have been fixed in the Gs/Gd lineage virus since 2001. However, despite genetic similarities among recent H5N1 isolates, the topology of the phylogenetic tree clearly differentiates the Korean isolates from the Vietnamese and Thai isolates which have been reported to infect humans. A representative Korean isolate was inoculated into mice, with no mortality and no virus being isolated from the brain, although high titers of virus were observed in the lungs. The same isolate, however, caused systemic infections in chickens and quail and killed all of the birds within 2 and 4 days of intranasal inoculation, respectively. This isolate also replicated in multiple organs and tissues of ducks and caused some mortality. However, lower virus titers were observed in all corresponding tissues of ducks than in chicken and quail tissues, and the histological lesions were restricted to the respiratory tract. This study characterizes the molecular and biological properties of the H5N1 HPAI viruses from South Korea and emphasizes the need for comparative analyses of the H5N1 isolates from different countries to help elucidate the risk of a human pandemic from the strains of H5N1 HPAI currently circulating in Asia.     

Are seals frequently infected with avian influenza viruses?

Influenza A virus isolates of the H4N5 subtype (which has previously been detected only in birds) were recovered from harbor seals dying of viral pneumonia on the New England coast from June 1982 through March 1983. When these isolates were compared with other mammalian and avian viruses in serological assays and RNA-RNA competitive hybridization, it was found that the seal viruses were most closely related antigenically and genetically to recent avian virus strains and were readily distinguishable from mammalian viruses, including H7N7 isolates recovered from seals in 1980. Unlike any previous isolates from mammals, these recent seal viruses replicate in the intestinal tracts of ducks, a characteristic of avian viruses. The association of avian viruses with influenza outbreaks in seals suggests that transmission of avian viruses to seals is occurring in nature. Potentially, this may be an example of the adaptation of avian viruses to mammals, which would represent an intermediate step in the evolution of new mammalian strains.     

Do crocodilians get the flu? Looking for influenza A in captive crocodilians

It is well established that several wild aquatic bird species serve as reservoirs for the influenza A virus. It has also been shown that the influenza A virus can be transmitted to mammalian species such as tigers and domestic cats and dogs through ingestion of infected birds. Another group of animals that should also be considered as potential hosts for the influenza A virus are the crocodilians. Many crocodilian species share aquatic environments with wild birds that are known to harbor influenza viruses. In addition, many large crocodilians utilize birds as a significant food source. Given these factors in addition to the close taxonomic proximity of aves to the crocodilians, it is feasible to ask whether crocodilian species may also harbor the influenza A virus. Here we analyzed 37 captive crocodilians from two locations in Florida (plus 5 wild bird fecal-samples from their habitat) to detect the presence of influenza A virus. Several sample types were examined. Real-time RT-PCR tests targeting the influenza A matrix gene were positive for four individual crocodilians--Alligator sinensis, Paleosuchus trigonatus, Caiman latirostris and Crocodylus niloticus. Of the seven serum samples tested with the avian influenza virus agar gel immunodiffusion assay, three showed a nonspecific reaction to the avian influenza virus antigen-A. sinensis, P. trigonatus and C. niloticus (C. latirostris was not tested). Viable virus could not be recovered from RT-PCR-positive samples, although this is consistent with previous attempts at viral isolation in embryonated chicken eggs with crocodilian viruses.     

Influenza a viruses from wild birds in Guatemala belong to the North American lineage

The role wild bird species play in the transmission and ecology of avian influenza virus (AIV) is well established; however, there are significant gaps in our understanding of the worldwide distribution of these viruses, specifically about the prevalence and/or significance of AIV in Central and South America. As part of an assessment of the ecology of AIV in Guatemala, we conducted active surveillance in wild birds on the Pacific and Atlantic coasts. Cloacal and tracheal swab samples taken from resident and migratory wild birds were collected from February 2007 to January 2010.1913 samples were collected and virus was detected by real time RT-PCR (rRT-PCR) in 28 swab samples from ducks (Anas discors). Virus isolation was attempted for these positive samples, and 15 isolates were obtained from the migratory duck species Blue-winged teal. The subtypes identified included H7N9, H11N2, H3N8, H5N3, H8N4, and H5N4. Phylogenetic analysis of the viral sequences revealed that AIV isolates are highly similar to viruses from the North American lineage suggesting that bird migration dictates the ecology of these viruses in the Guatemalan bird population.     

Newcastle disease virus-based live attenuated vaccine completely protects chickens and mice from lethal challenge of homologous and heterologous H5N1 avian influenza viruses

H5N1 highly pathogenic avian influenza virus (HPAIV) has continued to spread and poses a significant threat to both animal and human health. Current influenza vaccine strategies have limitations that prevent their effective use for widespread inoculation of animals in the field. Vaccine strains of Newcastle disease virus (NDV), however, have been used successfully to easily vaccinate large numbers of animals. In this study, we used reverse genetics to construct a NDV that expressed an H5 subtype avian influenza virus (AIV) hemagglutinin (HA). Both a wild-type and a mutated HA open reading frame (ORF) from the HPAIV wild bird isolate, A/Bar-headed goose/Qinghai/3/2005 (H5N1), were inserted into the intergenic region between the P and M genes of the LaSota NDV vaccine strain. The recombinant viruses stably expressing the wild-type and mutant HA genes were found to be innocuous after intracerebral inoculation of 1-day-old chickens. A single dose of the recombinant viruses in chickens induced both NDV- and AIV H5-specific antibodies and completely protected chickens from challenge with a lethal dose of both velogenic NDV and homologous and heterologous H5N1 HPAIV. In addition, BALB/c mice immunized with the recombinant NDV-based vaccine produced H5 AIV-specific antibodies and were completely protected from homologous and heterologous lethal virus challenge. Our results indicate that recombinant NDV is suitable as a bivalent live attenuated vaccine against both NDV and AIV infection in poultry. The recombinant NDV vaccine may also have potential use in high-risk human individuals to control the pandemic spread of lethal avian influenza.     

Complete nucleotide sequence of the influenza A/PR/8/34 virus NS gene and comparison with the NS genes of the A/Udorn/72 and A/FPV/Rostock/34 strains.

The nucleotide sequence of the NS gene of the human influenza virus A/PR/8/34 was determined and found to be the same length (890 nucleotides) as the NS gene of another human influenza virus A/Udorn/72 and of the avian isolate A/FPV/Rostock/34. Comparison of the sequences of the NS genes of the two human influenza viruses shows an 8.9% difference whereas the NS gene of the avian isolate differs by only 8% from that of the human strain A/PR/8/34. The extensive sequence similarity among these three genes does not support the notion of species specific homology groups among NS genes of avian and human influenza virus strains. The primary sequence of the A/PR/8/34 NS gene is consistent with the findings that the influenza virus NS gene may code for two overlapping polypeptides. In addition, an open reading frame potentially coding for a polypeptide 167 amino acids in length was found in the negative strand RNA of the A/PR/8/34 virus NS gene.     

Viruses budding from either the apical or the basolateral plasma membrane domain of MDCK cells have unique phospholipid compositions.

Influenza virus and vesicular stomatitis virus (VSV) obtain their lipid envelope by budding through the plasma membrane of infected cells. When monolayers of Madin-Darby canine kidney (MDCK) cells, a polarized epithelial cell line, are infected with fowl plague virus (FPV), an avian influenza virus, or with VSV, new FPV buds through the apical plasma membrane whereas VSV progeny is formed by budding through the basolateral plasma membrane. FPV and VSV were isolated from MDCK host cells prelabeled with [32P]orthophosphate and their phospholipid compositions were compared. Infection was carried out at 31 degrees C to delay cytopathic effects of the virus infection, which lead to depolarization of the cell surface. 32P-labeled FPV was isolated from the culture medium, whereas 32P-labeled VSV was released from below the cell monolayer by scraping the cells from the culture dish 8 h after infection. At this time little VSV was found in the culture medium, indicating that the cells were still polarized. The phospholipid composition of the two viruses was distinctly different. FPV was enriched in phosphatidylethanolamine and phosphatidylserine and VSV in phosphatidylcholine, sphingomyelin, and phosphatidylinositol. When MDCK cells were trypsinized after infection and replated, non-infected control cells attached to reform a confluent monolayer within 4 h, whereas infected cells remained in suspension. FPV and VSV could be isolated from the cells in suspension and under these conditions the phospholipid composition of the two viruses was very similar. We conclude that the two viruses obtain their lipids from the plasma membrane in the same way and that the different phospholipid compositions of the viruses from polarized cells reflect differences in the phospholipid composition of the two plasma membrane domains.