Plum Island Animal Disease Center

Foot-and-mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals. The disease was initially described in the 16th century and was the first animal pathogen identified as a virus. Recent FMD outbreaks in developed countries and their significant economic impact have increased the concern of governments worldwide. This review describes the reemergence of FMD in developed countries that had been disease free for many years and the effect that this has had on disease control strategies. The etiologic agent, FMD virus (FMDV), a member of the Picornaviridae family, is examined in detail at the genetic, structural, and biochemical levels and in terms of its antigenic diversity. The virus replication cycle, including virus-receptor interactions as well as unique aspects of virus translation and shutoff of host macromolecular synthesis, is discussed. This information has been the basis for the development of improved protocols to rapidly identify disease outbreaks, to differentiate vaccinated from infected animals, and to begin to identify and test novel vaccine candidates. Furthermore, this knowledge, coupled with the ability to manipulate FMDV genomes at the molecular level, has provided the framework for examination of disease pathogenesis and the development of a more complete understanding of the virus and host factors involved.

OPINION article Front. Microbiol., 10 May 2019Sec. Virology Volume 10 - 2019 | https://doi.org/10.3389/fmicb.2019.01057

Here we present complete genome sequences, including a comparative analysis, of 103 isolates of foot-and-mouth disease virus (FMDV) representing all seven serotypes and including the first complete sequences of the SAT1 and SAT3 genomes. The data reveal novel highly conserved genomic regions, indicating functional constraints for variability as well as novel viral genomic motifs with likely biological relevance. Previously undescribed invariant motifs were identified in the 5' and 3' untranslated regions (UTR), as was tolerance for insertions/deletions in the 5' UTR. Fifty-eight percent of the amino acids encoded by FMDV isolates are invariant, suggesting that these residues are critical for virus biology. Novel, conserved sequence motifs with likely functional significance were identified within proteins L(pro), 1B, 1D, and 3C. An analysis of the complete FMDV genomes indicated phylogenetic incongruities between different genomic regions which were suggestive of interserotypic recombination. Additionally, a novel SAT virus lineage containing nonstructural protein-encoding regions distinct from other SAT and Euroasiatic lineages was identified. Insights into viral RNA sequence conservation and variability and genetic diversity in nature will likely impact our understanding of FMDV infections, host range, and transmission.

A DNA sequence coding for the immunogenic capsid protein VP 3 of foot-and-mouth disease virus A 12 , prepared from the virion RNA, was ligated to a plasmid designed to express a chimeric protein from the Escherichia coli tryptophan promoter-operator system. When Escherichia coli transformed with this plasmid was grown in tryptophan-depleted media, approximately 17 percent of the total cellular protein was found to be an insoluble and stable chimeric protein. The purified chimeric protein competed equally on a molar basis with VP 3 for specific antibodies to foot-and-mouth disease virus. When inoculated into six cattle and two swine, this protein elicited high levels of neutralizing antibody and protection against challenge with foot-and-mouth disease virus.

The family Asfarviridae includes the single species African swine fever virus, isolates of which have linear dsDNA genomes of 170-194 kbp. Virions have an internal core, an internal lipid membrane, an icosahedral capsid and an outer lipid envelope. Infection of domestic pigs and wild boar results in an acute haemorrhagic fever with transmission by contact or ingestion, or by ticks of the genus Ornithodoros. Indigenous pigs act as reservoirs in Africa, where infection is endemic, and from where introductions occur periodically to Europe. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Asfarviridae, which is available at www.ictv.global/report/asfarviridae.

Lumpy skin disease virus (LSDV), a member of the capripoxvirus genus of the Poxviridae, is the etiologic agent of an important disease of cattle in Africa. Here we report the genomic sequence of LSDV. The 151-kbp LSDV genome consists of a central coding region bounded by identical 2.4 kbp-inverted terminal repeats and contains 156 putative genes. Comparison of LSDV with chordopoxviruses of other genera reveals 146 conserved genes which encode proteins involved in transcription and mRNA biogenesis, nucleotide metabolism, DNA replication, protein processing, virion structure and assembly, and viral virulence and host range. In the central genomic region, LSDV genes share a high degree of colinearity and amino acid identity (average of 65%) with genes of other known mammalian poxviruses, particularly suipoxvirus, yatapoxvirus, and leporipoxviruses. In the terminal regions, colinearity is disrupted and poxvirus homologues are either absent or share a lower percentage of amino acid identity (average of 43%). Most of these differences involve genes and gene families with likely functions involving viral virulence and host range. Although LSDV resembles leporipoxviruses in gene content and organization, it also contains homologues of interleukin-10 (IL-10), IL-1 binding proteins, G protein-coupled CC chemokine receptor, and epidermal growth factor-like protein which are found in other poxvirus genera. These data show that although LSDV is closely related to other members of the Chordopoxvirinae, it contains a unique complement of genes responsible for viral host range and virulence.

Sheeppox virus (SPPV) and goatpox virus (GTPV), members of the Capripoxvirus genus of the Poxviridae, are etiologic agents of important diseases of sheep and goats in northern and central Africa, southwest and central Asia, and the Indian subcontinent. Here we report the genomic sequence and comparative analysis of five SPPV and GTPV isolates, including three pathogenic field isolates and two attenuated vaccine viruses. SPPV and GTPV genomes are approximately 150 kbp and are strikingly similar to each other, exhibiting 96% nucleotide identity over their entire length. Wild-type genomes share at least 147 putative genes, including conserved poxvirus replicative and structural genes and genes likely involved in virulence and host range. SPPV and GTPV genomes are very similar to that of lumpy skin disease virus (LSDV), sharing 97% nucleotide identity. All SPPV and GTPV genes are present in LSDV. Notably in both SPPV and GTPV genomes, nine LSDV genes with likely virulence and host range functions are disrupted, including a gene unique to LSDV (LSDV132) and genes similar to those coding for interleukin-1 receptor, myxoma virus M003.2 and M004.1 genes (two copies each), and vaccinia virus F11L, N2L, and K7L genes. The absence of these genes in SPPV and GTPV suggests a significant role for them in the bovine host range. SPPV and GTPV genomes contain specific nucleotide differences, suggesting they are phylogenetically distinct. Relatively few genomic changes in SPPV and GTPV vaccine viruses account for viral attenuation, because they contain 71 and 7 genomic changes compared to their respective field strains. Notable genetic changes include mutation or disruption of genes with predicted functions involving virulence and host range, including two ankyrin repeat proteins in SPPV and three kelch-like proteins in GTPV. These comparative genomic data indicate the close genetic relationship among capripoxviruses, and they suggest that SPPV and GTPV are distinct and likely derived from an LSDV-like ancestor.

Although North American and European serotypes of porcine reproductive and respiratory syndrome virus (PRRSV) are recognized, only the genome of the European Lelystad strain (LV) has been sequenced completely. Here, the genome of the pathogenic North American PRRSV isolate 16244B has been sequenced and compared with LV. The genomic organization of 16244B was the same as LV but with only 63.4% nucleotide identity. The 189 nucleotide 5' non-coding region (NCR) of 16244B was distinct from the LV NCR, with good conservation (83%) only over a 43 base region immediately upstream of open reading frame (ORF) 1a. Major differences were found in the region encoding the non-structural part of the ORF1a polyprotein, which shared only 47% amino acid identity over 2503 residues of the six non-structural proteins (Nsps) encoded. Nsp2, thought to have a species-specific function, showed the greatest divergence, sharing only 32% amino acid identity with LV and containing 120 additional amino acids in the central region. Nsps encoded by the 5'-proximal and central regions of ORF1b had from 66 to 75% amino acid identity; however, the carboxy-terminal protein CP4 was distinct (42% identity). The ORF 1a-1b frameshift region of 16244B had 98% nucleotide identity with LV. Consistent with previous reports for North American isolates, the six structural proteins encoded were 58 to 79% identical to LV proteins. The 3' NCR (150 nucleotides) was 76% identical between isolates. These genomic differences confirm the presence of distinct North American and European PRRSV genotypes.

UNLABELLED: African swine fever virus (ASFV) is the etiological agent of a contagious and often lethal disease of domestic pigs that has significant economic consequences for the swine industry. The control of African swine fever (ASF) has been hampered by the unavailability of vaccines. Experimental vaccines have been developed using genetically modified live attenuated ASFVs where viral genes involved in virus virulence were removed from the genome. Multigene family 360 (MGF360) and MGF505 represent a group of genes sharing partial sequence and structural identities that have been connected with ASFV host range specificity, blocking of the host innate response, and virus virulence. Here we report the construction of a recombinant virus (ASFV-G-ΔMGF) derived from the highly virulent ASFV Georgia 2007 isolate (ASFV-G) by specifically deleting six genes belonging to MGF360 or MGF505: MGF505-1R, MGF360-12L, MGF360-13L, MGF360-14L, MGF505-2R, and MGF505-3R. ASFV-G-ΔMGF replicates as efficiently in primary swine macrophage cell cultures as the parental virus. In vivo, ASFV-G-ΔMGF is completely attenuated in swine, since pigs inoculated intramuscularly (i.m.) with either 10(2) or 10(4) 50% hemadsorbing doses (HAD50) remained healthy, without signs of the disease. Importantly, when these animals were subsequently exposed to highly virulent parental ASFV-G, no signs of the disease were observed, although a proportion of these animals harbored the challenge virus. This is the first report demonstrating the role of MGF genes acting as independent determinants of ASFV virulence. Additionally, ASFV-G-ΔMGF is the first experimental vaccine reported to induce protection in pigs challenged with highly virulent and epidemiologically relevant ASFV-G. IMPORTANCE: The main problem for controlling ASF is the lack of vaccines. Studies focusing on understanding ASFV virulence led to the production of genetically modified recombinant viruses that, while attenuated, are able to confer protection in pigs challenged with homologous viruses. Here we have produced an attenuated recombinant ASFV derived from highly virulent ASFV strain Georgia (ASFV-G) lacking only six of the multigene family 360 (MGF360) and MGF505 genes (ASFV-G-ΔMGF). It is demonstrated, by first time, that deleting specific MGF genes alone can completely attenuate a highly virulent field ASFV isolate. Recombinant virus ASFV-G-ΔMGF effectively confers protection in pigs against challenge with ASFV-G when delivered once via the intramuscular (i.m.) route. The protection against ASFV-G is highly effective by 28 days postvaccination. This is the first report of an experimental vaccine that induces solid protection against virulent ASFV-G.

Currently, there is no commercially available vaccine against African swine fever. Outbreaks of this disease are devastating the swine industry from Central Europe to East Asia, and they are being caused by circulating strains of African swine fever virus derived from the Georgia 2007 isolate. Here, we report the discovery of a previously uncharacterized virus gene, which when deleted completely attenuates the Georgia isolate. Importantly, animals infected with this genetically modified virus were protected from developing ASF after challenge with the virulent parental virus. Interestingly, ASFV-G-ΔI177L confers protection even at low doses (10 2 HAD 50 ) and remains completely attenuated when inoculated at high doses (10 6 HAD 50 ), demonstrating its potential as a safe vaccine candidate. At medium or higher doses (10 4 HAD 50 ), sterile immunity is achieved. Therefore, ASFV-G-ΔI177L is a novel efficacious experimental ASF vaccine protecting pigs from the epidemiologically relevant ASFV Georgia isolate.

Virus particles morphologically similar to caliciviruses and rotaviruses were detected by electron microscopy (EM) in the intestinal contents of a 27-day-old diarrheic nursing pig. A third small spherical 23-nm virus-like particle was also observed. Calicivirus-like particles averaged 33 nm in diameter. Similar to rotaviruses, rotavirus-like particles were present as single-capsid 55-nm forms or double-capsid 70-nm particles. Most gnotobiotic pigs orally exposed to samples containing these three viruses developed diarrhea and villous atrophy of the small intestine, and all shed the three viruses in their intestinal contents. Attempts to propagate these viruses in cell culture were unsuccessful. The antigenic relationship of the rotavirus-like particles to known rotaviruses was explored by immune EM and immunofluorescent staining. By these techniques, the rotavirus-like particles did not cross-react with antisera to porcine, bovine, or human rotaviruses or to reovirus type 3. Antisera from gnotobiotic pigs exposed to all three viruses had enzyme-linked immunosorbent assay and virus neutralization titers of <4 against porcine rotavirus. Previous infection of gnotobiotic pigs with the mixture containing rotavirus-like particles failed to protect them against a subsequent challenge with porcine rotavirus. The antigenic relationship of the calicivirus-like particles to known caliciviruses was investigated by immune EM and virus neutralization. By these tests, the calicivirus-like particles did not react with antisera against feline calicivirus strain 255 or M-8. In a study conducted at Plum Island Animal Disease Center, antiserum against the three combined agents did not specifically neutralize any serotype of swine vesicular exanthema virus.

Suppression of host protein synthesis in cells infected by poliovirus and certain other picornaviruses involves inactivation of the cap-binding protein complex. Inactivation of this complex has been correlated with the proteolytic cleavage of p220, a component of the cap-binding protein complex. Since picornaviral RNA is not capped, it continues to be translated as the cap-binding protein complex is inactivated. The cleavage of p220 can be induced to occur in vitro, catalyzed by extracts from infected cells or by reticulocyte lysates translating viral RNA. Expression of polioviral protease 2A is sufficient to induce p220 cleavage, and the presence in 2A of an 18-amino-acid sequence representing a putative cysteine protease active site correlates with the ability of different picornaviruses to induce p220 cleavage. Foot-and-mouth disease virus (FMDV) infection induces complete cleavage of p220, yet the FMDV genome codes for a 2A protein of only 16 amino acids, which does not include the putative cysteine protease active site. Using cDNA plasmids encoding various regions of the FMDV genome, we have determined that the leader protein is required to initiate p220 cleavage. This is the first report of a function for the leader protein, other than that of autocatalytic cleavage from the FMDV polyprotein.

Here we present the genomic sequence, with analysis, of a pathogenic fowlpox virus (FPV). The 288-kbp FPV genome consists of a central coding region bounded by identical 9.5-kbp inverted terminal repeats and contains 260 open reading frames, of which 101 exhibit similarity to genes of known function. Comparison of the FPV genome with those of other chordopoxviruses (ChPVs) revealed 65 conserved gene homologues, encoding proteins involved in transcription and mRNA biogenesis, nucleotide metabolism, DNA replication and repair, protein processing, and virion structure. Comparison of the FPV genome with those of other ChPVs revealed extensive genome colinearity which is interrupted in FPV by a translocation and a major inversion, the presence of multiple and in some cases large gene families, and novel cellular homologues. Large numbers of cellular homologues together with 10 multigene families largely account for the marked size difference between the FPV genome (260 to 309 kbp) and other known ChPV genomes (178 to 191 kbp). Predicted proteins with putative functions involving immune evasion included eight natural killer cell receptors, four CC chemokines, three G-protein-coupled receptors, two beta nerve growth factors, transforming growth factor beta, interleukin-18-binding protein, semaphorin, and five serine proteinase inhibitors (serpins). Other potential FPV host range proteins included homologues of those involved in apoptosis (e.g., Bcl-2 protein), cell growth (e.g., epidermal growth factor domain protein), tissue tropism (e.g., ankyrin repeat-containing gene family, N1R/p28 gene family, and a T10 homologue), and avian host range (e.g., a protein present in both fowl adenovirus and Marek's disease virus). The presence of homologues of genes encoding proteins involved in steroid biogenesis (e.g., hydroxysteroid dehydrogenase), antioxidant functions (e.g., glutathione peroxidase), vesicle trafficking (e.g., two alpha-type soluble NSF attachment proteins), and other, unknown conserved cellular processes (e.g., Hal3 domain protein and GSN1/SUR4) suggests that significant modification of host cell function occurs upon viral infection. The presence of a cyclobutane pyrimidine dimer photolyase homologue in FPV suggests the presence of a photoreactivation DNA repair pathway. This diverse complement of genes with likely host range functions in FPV suggests significant viral adaptation to the avian host.

The amino acid sequence Arg-Gly-Asp (RGD) is highly conserved on the VP1 proteins of different serotypes and subtypes of foot-and-mouth disease virus (FMDV) and is essential for cell attachment. This sequence is also found in certain extracellular matrix proteins that bind to a family of cell surface receptors called integrins. Within the Picornaviridae family, enterovirus coxsackievirus A9 also has an RGD motif on its VP1 capsid protein and has recently been shown to utilize the vitronectin receptor integrin alpha V beta 3 as a receptor on monkey kidney cells. Competition binding experiments between type A12 FMDV and coxsackievirus A9 using BHK-21 and LLC-MK2 cells revealed shared receptor specificity between these two viruses. Polyclonal anti-serum to the vitronectin receptor and a monoclonal antibody to the alpha V subunit inhibited both FMDV binding and plaque formation, while a monoclonal antibody to the beta 3 subunit inhibited virus binding. In contrast, antibodies to the fibronectin receptor (alpha 5 beta 1) or to the integrin (alpha V beta 5) had no effect on either binding or plaque formation. These data demonstrate that the alpha V beta 3 vitronectin receptor can function as a receptor for FMDV.

Isolates of foot-and-mouth disease virus (FMDV) exist as complex mixtures of variants. Two different serotype O1 Campos preparations that we examined contained two variants with distinct plaque morphologies on BHK cells: a small, clear-plaque virus that replicates in BHK and CHO cells, and a large, turbid-plaque virus that only grows in BHK cells. cDNAs encoding the capsids of these two variants were inserted into a genome-length FMDV type A12 infectious cDNA and used to produce chimeric viruses that exhibited the phenotype of the original variants. Analyses of these viruses, and hybrids created by exchanging portions of the capsid gene, identified codon 56 in VP3 (3056) as the critical determinant of both cell tropism and plaque phenotype. Specifically, the CHO growth/clear-plaque phenotype is dependent on the presence of the highly charged Arg residue at 3056, and viruses with this phenotype and genotype were selected during propagation in tissue culture. The genetically engineered Arg 3056 virus was highly attenuated in bovines, but viruses recovered from animals inoculated with high doses of this virus had lost the ability to grow in CHO cells and contained either an uncharged residue at 3056 or a negatively charged Glu substituted for a Lys at a spatially and antigenically related position on VP2 (2134). Comparison of these animal-derived viruses to other natural and engineered viruses demonstrated that positively charged residues are required at both 2134 and 3056 for binding to heparin. Taken together, these results indicate that in vitro cultivation of FMDV type O selects viruses that bind to heparin and that viruses with the heparin-binding phenotype are attenuated in the natural host.

Bovine papular stomatitis virus (BPSV) and orf virus (ORFV), members of the genus Parapoxvirus of the Poxviridae, are etiologic agents of worldwide diseases affecting cattle and small ruminants, respectively. Here we report the genomic sequences and comparative analysis of BPSV strain BV-AR02 and ORFV strains OV-SA00, isolated from a goat, and OV-IA82, isolated from a sheep. Parapoxvirus (PPV) BV-AR02, OV-SA00, and OV-IA82 genomes range in size from 134 to 139 kbp, with an average nucleotide composition of 64% G+C. BPSV and ORFV genomes contain 131 and 130 putative genes, respectively, and share colinearity over 127 genes, 88 of which are conserved in all characterized chordopoxviruses. BPSV and ORFV contain 15 and 16 open reading frames (ORFs), respectively, which lack similarity to other poxvirus or cellular proteins. All genes with putative roles in pathogenesis, including a vascular endothelial growth factor (VEGF)-like gene, are present in both viruses; however, BPSV contains two extra ankyrin repeat genes absent in ORFV. Interspecies sequence variability is observed in all functional classes of genes but is highest in putative virulence/host range genes, including genes unique to PPV. At the amino acid level, OV-SA00 is 94% identical to OV-IA82 and 71% identical to BV-AR02. Notably, ORFV 006/132, 103, 109, 110, and 116 genes (VEGF, homologues of vaccinia virus A26L, A33R, and A34R, and a novel PPV ORF) show an unusual degree of intraspecies variability. These genomic differences are consistent with the classification of BPSV and ORFV as two PPV species. Compared to other mammalian chordopoxviruses, PPV shares unique genomic features with molluscum contagiosum virus, including a G+C-rich nucleotide composition, three orthologous genes, and a paucity of nucleotide metabolism genes. Together, these data provide a comparative view of PPV genomics.

Here we present the first complete genomic sequence, with analysis, of a very virulent strain of Marek's disease virus serotype 1 (MDV1), Md5. The genome is 177,874 bp and is predicted to encode 103 proteins. MDV1 is colinear with the prototypic alphaherpesvirus herpes simplex virus type 1 (HSV-1) within the unique long (UL) region, and it is most similar at the amino acid level to MDV2, herpesvirus of turkeys (HVT), and nonavian herpesviruses equine herpesviruses 1 and 4. MDV1 encodes 55 HSV-1 UL homologues together with 6 additional UL proteins that are absent in nonavian herpesviruses. The unique short (US) region is colinear with and has greater than 99% nucleotide identity to that of MDV1 strain GA; however, an extra nucleotide sequence at the Md5 US/short terminal repeat boundary results in a shorter US region and the presence of a second gene (encoding MDV097) similar to the SORF2 gene. MD5, like HVT, encodes an ICP4 homologue that contains a 900-amino-acid amino-terminal extension not found in other herpesviruses. Putative virulence and host range gene products include the oncoprotein MEQ, oncogenicity-associated phosphoproteins pp38 and pp24, a lipase homologue, a CxC chemokine, and unique proteins of unknown function MDV087 and MDV097 (SORF2 homologues) and MDV093 (SORF4). Consistent with its virulent phenotype, Md5 contains only two copies of the 132-bp repeat which has previously been associated with viral attenuation and loss of oncogenicity.

In 1898, foot-and-mouth disease (FMD) earned a place in history as the first disease of animals shown to be caused by a virus. Yet, despite over a century of active investigation and elucidation of many aspects of FMD pathogenesis, critical knowledge about the virus-host interactions is still lacking. The aim of this review is to provide a comprehensive overview of FMD pathogenesis in cattle spanning from the earliest studies to recently acquired insights emphasizing works which describe animals infected by methodologies most closely resembling natural infection (predominantly aerosol or direct/indirect contact). The three basic phases of FMD pathogenesis in vivo will be dissected and characterized as: (i) pre-viraemia characterized by infection and replication at the primary replication site(s), (ii) sustained viraemia with generalization and vesiculation at secondary infection sites and (iii) post-viraemia/convalescence including resolution of clinical disease that may result in long-term persistent infection. Critical evaluation of the current status of understanding will be used to identify knowledge gaps to guide future research efforts.

A system has been developed for generating chimeric yellow fever/Japanese encephalitis (YF/JE) viruses from cDNA templates encoding the structural proteins prM and E of JE virus within the backbone of a molecular clone of the YF17D strain. Chimeric viruses incorporating the proteins of two JE strains, SA14-14-2 (human vaccine strain) and JE Nakayama (JE-N [virulent mouse brain-passaged strain]), were studied in cell culture and laboratory mice. The JE envelope protein (E) retained antigenic and biological properties when expressed with its prM protein together with the YF capsid; however, viable chimeric viruses incorporating the entire JE structural region (C-prM-E) could not be obtained. YF/JE(prM-E) chimeric viruses grew efficiently in cells of vertebrate or mosquito origin compared to the parental viruses. The YF/JE SA14-14-2 virus was unable to kill young adult mice by intracerebral challenge, even at doses of 10(6) PFU. In contrast, the YF/JE-N virus was neurovirulent, but the phenotype resembled parental YF virus rather than JE-N. Ten predicted amino acid differences distinguish the JE E proteins of the two chimeric viruses, therefore implicating one or more residues as virus-specific determinants of mouse neurovirulence in this chimeric system. This study indicates the feasibility of expressing protective antigens of JE virus in the context of a live, attenuated flavivirus vaccine strain (YF17D) and also establishes a genetic system for investigating the molecular basis for neurovirulence determinants encoded within the JE E protein.

The complete genome of Mycoplasma gallisepticum strain R(low) has been sequenced. The genome is composed of 996,422 bp with an overall G+C content of 31 mol%. It contains 742 putative coding DNA sequences (CDSs), representing a 91 % coding density. Function has been assigned to 469 of the CDSs, while 150 encode conserved hypothetical proteins and 123 remain as unique hypothetical proteins. The genome contains two copies of the rRNA genes and 33 tRNA genes. The origin of replication has been localized based on sequence analysis in the region of the dnaA gene. The vlhA family (previously termed pMGA) contains 43 genes distributed among five loci containing 8, 2, 9, 12 and 12 genes. This family of genes constitutes 10.4% (103 kb) of the total genome. Two CDSs were identified immediately downstream of gapA and crmA encoding proteins that share homology to cytadhesins GapA and CrmA. Based on motif analysis it is predicted that 80 genes encode lipoproteins and 149 proteins contain multiple transmembrane domains. The authors have identified 75 proteins putatively involved in transport of biomolecules, 12 transposases, and a number of potential virulence factors. The completion of this sequence has spawned multiple projects directed at defining the biological basis of M. gallisepticum.