Poxviridae Classification Essay

1. Introduction

Poxviruses are among the best known and most feared viruses. The Poxviridae family is currently divided in two subfamilies, named Entomopoxvirinae (insect-infecting viruses) and Chordopoxvirinae (vertebrate-infecting viruses), wherein the first is composed of three genera, and the latter contains 10 genera, in addition to two viral species that have yet to be classified into each subfamily [1]. While the entomopoxviruses have been poorly investigated over the years, the chordopoxviruses are among the most studied groups in virology, due to the medical and veterinary relevance of many of their members. Among the chordopoxviruses, the Variola virus (VARV_abbreviations are shown in Supplementary Table S1) is one of the most well-known species. VARV is the agent of smallpox, a disease that has plagued humanity for centuries, until it was considered eradicated by the World Health Organization in 1980 after a successful global vaccination and surveillance campaign [2,3,4]. Other chordopoxviruses, such as vaccinia virus (VACV), cowpox virus (CPXV) and monkeypox virus (MPXV), are responsible for several outbreaks of exantematic diseases around the world, both in humans and other animals (e.g., bovines and equids), and are considered emergent zoonotic viral diseases [5,6]. Furthermore, studies with poxviruses have been pivotal for the advancement of other areas of knowledge, especially in cell biology, vaccinology, and virotherapy, where it was possible to elucidate many important metabolic pathways for immune response and the development of different strategies of immunization against infectious diseases [7,8,9,10].

The Poxviridae family consists of large double-stranded DNA viruses, which replicate entirely in the cytoplasm of host cells [11]. The poxviruses have a complex structure and an extensive linear genome ranging from 128 to 365 kbp (Genera Parapoxvirus and Avipoxvirus, respectively), which code for over 200 genes [12,13,14]. Chordopoxvirinae is divided in two clusters that are well-resolved phylogenetically. The first of the two clusters corresponds to the Orthopoxvirus (OPV) genus. The second cluster formed by the genera Yatapoxvirus, Leporipoxvirus, Capripoxvirus, Cervidpoxvirus, and Suipoxvirus forms a sister clade to orthopoxviruses, and the former can be classified as “clade II” poxviruses [15,16]. The origin and evolution of poxviruses are still blurred. Although there is some strong evidence suggesting that these viruses emerged thousands of years ago, their genome has evolved through the gain and loss of genes, especially through gene duplication and horizontal gene transfer (HGT) [15,16,17]. Many of the genes present in the poxvirus genome are not essential to viral replication in cell culture, but are important to the modulation of the host antiviral response, and thus are considered virulence genes [18,19]. Some of these genes impact viral replication only in a set of cell lineages that originated on different tissues or host species. These genes act on poxvirus-specific differences in tropism and host range, and have been referred to as host range genes [18,19,20]. All poxviruses are predicted to encode a unique collection of host range genes; however, only the genera Orthopoxvirus and Leporipoxvirus have been observed in many of the biological studies so far [13]. Known poxvirus host range genes are currently grouped into 12 distinct classes, some of which have only one gene (e.g., K3L, E3L, K1L, others), and others exhibiting many members (e.g., serpins, C7L family, TNFRII family, others), which likely result from lineage duplication events [20]. Some of these factors were functionally characterized using in vitro models and gene knockout analysis, which is associated mostly with the manipulation of diverse cellular targets, including cellular kinases and phosphatases, apoptosis, and many antiviral pathways [19,21]. In the absence of these genes, viruses lose the ability to infect certain cell lineages, whereas infection is efficiently established in the presence of the genes. Several in vivo investigations showed that some factors impact viral pathogenicity, although the model animals were still infected [22,23,24].

Historically, these genes have been referred to as host range genes when considering only the cells as hosts, i.e., not considering the animals that are actually infected by the viruses [18,19,20]. Would it be possible to extrapolate the restriction of viral replication in a specific cell lineage in an animal, which is a far more complex organism? Some works have suggested a direct association between the diversity of host range factors and the amount of host species for different poxviruses, but this association is still under debate [18,19]. In view of these intriguing questions, we sought to establish the natural hosts for the poxviruses officially assigned to viral species and recognized by the International Committee on Taxonomy of Viruses (ICTV) [1]. Based on the available data so far, we performed an extensive search for different host range genes according to those that were described previously [20], reviewing the main features of each class of host range factors. In light of the data presented here, we could not associate a diversity of host range factors with the amount of hosts, which lead us to discuss the assertiveness of the term “host range genes”. Finally, we analyzed the evolutionary history of these genes, reaffirming the occurrence of HGT for some elements, as previously suggested [16,17,20].

Among the nucleocytoplasmic large DNA viruses (NCLDV), the poxviruses are those with the widest host range, which are able to infect different groups of insects and vertebrates [25]. However, when we look specifically at each member of the Poxviridae family, the host spectrum is variable, wherein some viruses can infect a large range of hosts [25,26,27], while others are restricted to only one host species [3,28,29]. To have a clear view of the host range of poxviruses, we performed an extensive search to define the natural hosts for each member of the Poxviridae family, and we presented this relationship in a network graph. In this analysis, we searched only the hosts for viruses that are currently classified into viral species by the ICTV, since it presents the most up-to-date dataset of known viral species, and gathers and reflects the diversity of the circulating viruses in nature. We defined hosts as those organisms in which consistent evidence was available related to viral detection in a given species by isolation, serology, and molecular detection. In this view, we seek to associate hosts at the lowest possible taxonomical level.

The ICTV currently recognizes a total of 71 species of poxvirus, with 30 belonging to the Entomopoxvirinae subfamily, and 41 to the Chordopoxvirinae subfamily (Supplementary Table S2). The known entomopoxviruses infect Pterygota subclass members (winged insects) from the orders Diptera, Coleoptera, and Hymenoptera, but mainly Lepidoptera and Orthoptera (Figure 1).

For most of the viral species of this group, it was not possible to determine the virus hosts beyond the order taxonomic level. For the remaining viral species, we determined the hosts at the genus or species level. Corroborating the previous descriptions in the literature, entomopoxviruses tend to exhibit a fairly narrow host range, but the species of Betaentomopoxvirus genus can infect distant hosts, which suggests that large host shifts can occur. This view of entomopoxvirus hosts is likely a consequence of the lack of host range studies that have been performed on these viruses [30,31]. Recently, a study showed that genes involved in lateral gene transfer (LGT) events among entomopoxviruses species conferred a possible adaptation to both specific and distantly related hosts [32]. It is possible that with the advancement of metaviromic approaches, we will be able to move forward in our comprehension of the diversity and host range of entomopoxviruses, uncover new viruses in different and unexplored hosts, and therefore improve the network presented here [33].

In contrast, the chordopoxviruses are the targets of intense investigation because of the clinical relevance of many of its members to humans and domesticated animals of economic importance [34,35,36,37,38]. The known chordopoxviruses infect mammals (29 viral species), birds (10 viral species), and reptiles (one viral species), and viruses do not cross this host barrier, i.e., viruses infecting mammals do not infect birds, and vice versa (Figure 1). Interestingly, an avipoxvirus was isolated once from a terminally ill rhinoceros in 1969 and characterized as an atypical fowlpox virus, thus raising questions about the host restriction of avipoxviruses [39]. However, since it was an isolated case and there are no other descriptions of an avipoxvirus infecting a non-avian host, it is still uncertain whether these viruses can efficiently cross the host barrier. Differently from entomopoxviruses, there were only four chordopoxviruses for which we could not define the hosts at the genus or species level (4/40 = 10%). One of these viruses is the yokapox virus (YOKV), a poxvirus isolated from a mosquito pool over 40 years ago whose natural host is probably a mammal. However, further investigation is required to better understand the biology of this virus [40]. Similar to YOKV, it is possible that other hosts are associated with known chordopoxviruses, but these relationships need to be further investigated. Most of the chordopoxviruses are associated with only one host genus (25/40 = 62.5%), which suggests a restricted host range for these viruses (Figure 1). Interestingly, there is a trend among large DNA viruses to exhibit a narrow host range, but some viruses can infect a broader range of hosts, such as VACV and MPXV, which have been associated with outbreaks involving humans and cattle. For these viruses, it is likely that rodents act as reservoir hosts of these viruses [41,42,43]. The CPXV is the most prominent member in the group, since it presents by far the widest host spectrum among the poxviruses, being able to infect at least 27 different groups of hosts, including humans, cattle, equids, and felines (Figure 1). Recent data suggests that Cowpox virus is comprised of at least five different viral species [44]. Assuming this, it is likely that the hosts defined here of what we now consider as Cowpox virus are actually a host compilation of hosts for at least five distinct viral species.

After the eradication of smallpox in 1980, several poxvirus outbreaks have been reported around the world that are related to different viruses, driving a huge effort to identify these viruses and their possible natural and reservoir hosts [45,46,47,48]. At least 11 poxvirus species have been known to cause human infections to date: CPXV, VACV, MPXV, VARV, Molluscum contagiosum virus (MOCV), Orf virus (ORFV), Camelpox virus (CMLV), Yaba monkey tumor virus (YMTV), Tanapox virus (TPV), Bovine papular stomatitis virus (BPSV), and Pseudocowpox virus (PCPV) [49,50] (Figure 1). Among them, only VARV and MOCV have humans as the sole host, while the other poxviruses are emerging zoonoses that affect different groups of animals, such as cattle, rodents, primates, and others (Figure 1) [3,28]. The main clinical feature of poxvirus infection is skin lesions, which can vary from small pearly papules in MOCV infection to large crusts and generalized pustules in VARV infection. However, other symptoms are common, including fever, headache, and rash. Similar clinical signals are verified in other animals infected by these viruses. Cases of human infections by some poxviruses, such as VACV, CPXV, and MPXV, have been constantly reported [3,26,28,35,51]. Differently, skin lesions related to CMLV have only been observed during the last few years, which opens important questions regarding the expanding host range of this virus [52]. The Parapoxvirus genus comprises four viral species that are distributed worldwide and mainly infect domestic ruminants and a broader host range, which includes camels, seals, deer species, and humans [53]. In this context, it is possible that other known poxviruses might affect humans. An example of this is the isolation of a strain of Ectromelia virus (ECTV) from the throat swabs of an affected man in China during an outbreak of erythromelalgia [54]. It is still uncertain whether this virus is a true human pathogen, since ECTV has only been found in Mus sp. (Figure 1), causing mousepox disease [55]. If this association is confirmed, it will be another example of host range expansion in poxviruses. Furthermore, one must have in mind that a virus can infect a host without causing any disease. Although the majority of poxviruses isolation reports are related to any clinical manifestation in a given host, some viruses have already been identified in known hosts without presenting any clinical manifestation, such as the group 1 of Brazilian VACV strains [41]. Moreover, there are descriptions of poxvirus detection in cattle, but the hosts had no clinical signals [56]. There is also a description of the isolation of a parapoxvirus from an apparently healthy red deer in the Bavarian Alps [57], which reinforces that poxviruses are related to hosts exhibiting no disease. In this context, we might expect that the network presented here is likely more interconnected, but more recurrent data regarding poxvirus’ detection in healthy organisms will be needed.

Based on currently available data, it appears that certain poxviruses have a broad host range, such as VACV, MPXV, and CPXV. However, these viruses are the most studied within the Poxviridae family, which is the reason why we recognize more host species for them compared with other viruses, and especially compared with entomopoxviruses. In contrast, other viruses that are also the targets of intense research exhibit a very restricted host range, as is the case with VARV and MOCV (both infect only humans). The reason for such a difference is still a mystery, but some studies have suggested that the answer might lie in the genetic diversity between those viruses, namely, the diversity and abundance of the referred host range genes [18,19].

Abstract

Because they were the largest of all viruses and could be visualised with a light microscope, the poxviruses were the first viruses to be intensively studied in the laboratory. It was clear from an early date that they caused important diseases of humans and their domestic animals, such as smallpox, cowpox, camelpox, sheeppox, fowlpox and goatpox. This essay recounts some of the early history of their recognition and classification and then expands on aspects of research on poxviruses in which the author has been involved. Studies on the best-known genus, Orthopoxvirus, relate to the use of infectious ectromelia of mice as a model for smallpox, embracing both experimental epidemiology and pathogenesis, studies on the genetics of vaccinia virus and the problem of non-genetic reactivation (previously termed ‘transformation’) and the campaign for the global eradication of smallpox. The other group of poxviruses described here, the genus Leporipoxvirus, came to prominence when the myxoma virus was used for the biological control of Australian wild rabbits. This provided a unique natural experiment on the coevolution of a virus and its host. Future research will include further studies of the many immunomodulatory genes found in all poxviruses of vertebrates, since these provide clues about the workings of the immune system and how viruses have evolved to evade it. Some of the many recombinant poxvirus constructs currently being studied may come into use as vaccines or for immunocontraception. A field that warrants study but will probably remain neglected is the natural history of skunkpox, raccoonpox, taterapox, yabapox, tanapox and other little-known poxviruses. A dismal prospect is the possible use of smallpox virus for bioterrorism.

Orthopoxvirus, Ectromelia virus, Smallpox, Myxomatosis, Poxvirus, Australia, Smallpox eradication, Biocontrol

1 Introduction

In accordance with the request from the editors, this essay will concentrate on the two fields of virology on which my work has focused over the last 50 years: the orthopoxviruses, principally ectromelia, vaccinia and variola viruses, and the leporipoxviruses, principally myxoma virus. With colleagues as co-authors, I have written books on both of these topics [1–3]; in this essay I will try to summarise the ‘ancient history’ of the subject, to extract the highlights of work with which I have had some connection, and briefly to look into the unknown future to see what it may hold.

2 Early history of poxviruses

Smallpox, caused by an orthopoxvirus, was once the most serious disease of humankind. Unlike malaria, it was not limited by climate, and unlike plague, it was always present. The agent that caused it was the first virus to be seen with a microscope [4], it provided the first example of inoculation with a virus as a preventative measure against the disease caused by that agent [5], it was the first disease against which an effective vaccine was used [6], the group to which it belonged was the first to be correctly classified as what came to be known as a family [7] and it was the first human disease to be eradicated globally [5].

The virus used to prevent smallpox (vaccinia virus) became a model for early biological and biochemical studies of viruses [8]. It was the first animal virus to be purified sufficiently to show that it contained DNA but not RNA [9] and to be visualised by electron microscopy [10]. It also provided an early model for assay methods comparable with colony counts for bacteria, namely an assay for infectivity by pock counts on the chorioallantoic membrane of the developing chick embryo [11, 12]. More recently, it was the first virus to be used as a vector for carrying foreign genes into animals in such a way that the proteins for which they coded were expressed [13–15].

The model virus of the other genus on which this review will focus, Leporipoxvirus, also has a history going back to the early days of virology. In 1893 Guiseppe Sanarelli, an Italian bacteriologist who had worked in the Pasteur Institute in Paris, went to Montevideo in Uruguay to establish an Institute of Experimental Hygiene. In 1896 a mysterious and lethal infectious disease broke out among his laboratory rabbits, which had been imported from Brazil, from which he recovered a non-cultivable and invisible infectious agent which he named the ‘myxomatogene virus’ [16]. It shared with foot-and-mouth disease virus the distinction of being the first virus of vertebrates (in the modern sense of ‘virus’) to be described as such. Until the mid-1930s myxomatosis excited little interest except among people in Brazil and California who were trying to farm European rabbits, although the Brazilian microbiologist H.B. Aragão (quoted in [3]) suggested in 1918 that it might be used to control pest rabbits in Australia. In later studies Aragão went on to show that the myxoma virus was morphologically similar to the variola virus [17], that its natural host was the Brazilian forest rabbit and that it could be transmitted mechanically by mosquitoes and fleas [18]. It was eventually imported into Australia in the mid-1930s and, after laboratory and field studies that extended over several years, it was released among Australian wild rabbits in the early 1950s. Two years after its spread in Australia it was illegally introduced in France and spread widely in Europe, to the dismay of rabbit farmers and hunters but the delight of foresters and other farmers [3]. It proved to be the most effective biological control method for a vertebrate pest that has ever been discovered.

3 Classification of poxviruses

The earliest classifications of viruses were based on disease symptoms. Certain diseases of humans, cow, sheep, horse and pig were classed as ‘poxes’ because disease with which they were associated were characterised by pocks on the skin. As it turned out, several of these diseases were indeed caused by poxviruses, but the deficiencies of a classification based on clinical symptoms was highlighted by the inclusion of chickenpox and the ‘great pox’ (syphilis) in the same group as the smallpox virus. Of course, the fallacies of such a method of classification of viruses (as distinct from diseases) are even more apparent with the terms ‘hepatitis viruses’, ‘encephalitis viruses’ and ‘haemorrhagic fever viruses’.

Being the largest of all animal viruses and visible in stained smears by light microscopy, the poxviruses were the first ‘group’ of viruses to be described, i.e. viruses that were not serologically related but appeared to have a more general resemblance, in size and certain other characteristics. As it turned out, this seemingly superficial resemblance has proved to be remarkably robust in terms of the characteristics now used to classify viruses. Aragão [17] pointed out the resemblance between the viruses of ‘variola, myxoma, bird-epithelioma, molluscum contagiosum, etc.’ and in 1933 Goodpasture [19] formally proposed that vaccinia-variola, fowlpox, horsepox, sheeppox, goatpox, swinepox and molluscum contagiosum viruses should be grouped together as the genus Borreliota.

Viral classification took a step forward in 1948 when Holmes (a plant virologist) published a comprehensive classification of viruses in the standard book on the classification of bacteria [20]. Technically, it was a step backwards, because it placed major reliance on the symptomatology of disease. However, this ‘nomenclatural bombshell’ stimulated others to study viral classification seriously and after several discussion papers [21, 22] in which Sir Christopher Andrewes (then the leading virologist in the UK) figured prominently, the Fifth International Congress for Microbiology in 1950 gave serious consideration to questions of viral classification and nomenclature. These were followed up at the next Congress, in 1953, by the establishment of study groups to consider five groups of viruses, of which the poxviruses were one. On behalf of the Poxvirus Study Group, in 1957 Fenner and Burnet [23] published a short description of the poxviruses of vertebrates (which are the only ones to be discussed in this essay) that has remained the basis of subsequent classification in respect of the criteria used and subdivisions adopted. The basic features of the family are the large brick-shaped or ovoid virions, with a genome consisting of a single linear molecule of covalently closed, double-stranded DNA, between 130 and 220 kb in length. Unlike most other DNA viruses, poxviruses replicate in the cytoplasm of the cell. The family Poxviridae is subdivided into two subfamilies, Chordopoxvirinae and Entomopoxvirinae, found in vertebrates and insects, respectively. Viruses of the subfamily Chordopoxvirinae are subdivided into eight genera, distinguished from each other primarily by serologic cross-reaction and cross-protection (Table 1).

Table 1

Classification of the poxviruses of vertebrates

Family: Poxviridae; subfamily: Chordopoxvirinae 
Genus prototype virus 
Orthopoxvirusvaccinia virus 
Parapoxviruspseudocowpox virus 
Capripoxvirussheep pox virus 
Suipoxvirusswinepox virus 
Leporipoxvirusmyxoma virus 
Avipoxvirusfowlpox virus 
Yatapoxvirustanapoxvirus 
Molluscipoxvirusmolluscum contagiosum virus 
Family: Poxviridae; subfamily: Chordopoxvirinae 
Genus prototype virus 
Orthopoxvirusvaccinia virus 
Parapoxviruspseudocowpox virus 
Capripoxvirussheep pox virus 
Suipoxvirusswinepox virus 
Leporipoxvirusmyxoma virus 
Avipoxvirusfowlpox virus 
Yatapoxvirustanapoxvirus 
Molluscipoxvirusmolluscum contagiosum virus 

View Large

4 The genus Orthopoxvirus

The genus Orthopoxvirus, with which the first section of this essay is concerned, contains 10 species (Table 2). The four species with which I have worked will be discussed: ectromelia, vaccinia, variola and monkeypox viruses.

Table 2

Species of the genus Orthopoxvirus

Species Host range in laboratory animals Animals found naturally infected Geographic range of natural infections 
Camelpox virus narrow camels Africa and Asia 
Cowpox virus broad numerous: carnivores, cow, elephant, humans, rats; natural hosts: gerbils, other rodents Europe and former USSR 
Ectromelia virus narrow mice; natural host unknown, possibly voles Europe 
Monkeypox virus broad apes, monkeys, squirrels, humans; natural hosts, squirrels western and central Africa 
Raccoonpox virus (?) broad raccoon USA 
Tatera poxvirus narrow Tatera kempi (a gerbil) Western Africa 
Uasin Gishu poxvirus medium horse (natural host unknown) Kenya, Zambia 
Vaccinia virus (smallpox vaccine virus) broad numerous: buffalo, cow, man, pig, rabbitaIndia (buffalopox); Europe and USA (rabbitpox) 
Variola virus narrow humans (now eradicated) formerly worldwide 
Vole poxvirus (?) broad voles USA 
Species Host range in laboratory animals Animals found naturally infected Geographic range of natural infections 
Camelpox virus narrow camels Africa and Asia 
Cowpox virus broad numerous: carnivores, cow, elephant, humans, rats; natural hosts: gerbils, other rodents Europe and former USSR 
Ectromelia virus narrow mice; natural host unknown, possibly voles Europe 
Monkeypox virus broad apes, monkeys, squirrels, humans; natural hosts, squirrels western and central Africa 
Raccoonpox virus (?) broad raccoon USA 
Tatera poxvirus narrow Tatera kempi (a gerbil) Western Africa 
Uasin Gishu poxvirus medium horse (natural host unknown) Kenya, Zambia 
Vaccinia virus (smallpox vaccine virus) broad numerous: buffalo, cow, man, pig, rabbitaIndia (buffalopox); Europe and USA (rabbitpox) 
Variola virus narrow humans (now eradicated) formerly worldwide 
Vole poxvirus (?) broad voles USA 

View Large

5 Studies with ectromelia virus

Because of the absence of work with the ectromelia virus in many countries it is convenient to summarise this account in three time periods: early and medium-term, which are almost restricted to work done in Australia, and the modern era, during which the advantages in the mouse as a model virus-host system are being increasingly exploited.

5.1 Early work (1930–1948)

The ectromelia virus was discovered in 1930 by Marchal [24]. Being a virus infection that was naturally transmitted from one mouse to another, it was immediately used by Greenwood et al. [25] to expand their long-term experiments on experimental epidemiology, previously restricted to the bacterial diseases, mouse typhoid and mouse pasteurellosis.

After the discovery of haemagglutination by influenza virus [26], Burnet made this the main focus of virological work in the Walter and Eliza Hall Institute [27]. Being a ‘collector’ by nature (beetles in his childhood), he examined as many viruses as were available in Australia to see how common this phenomenon was. In addition to showing that Newcastle disease and mumps viruses, like influenza virus, caused haemagglutination and subsequently eluted from the red cells [28, 29], he found that ectromelia and vaccinia viruses caused a rather different kind of haemagglutination and did not elute. Serological and cross-protection tests then showed that ectromelia virus was a member of the vaccinia-variola virus group, i.e. an Orthopoxvirus[30].

0 thoughts on “Poxviridae Classification Essay”

    -->

Leave a Comment

Your email address will not be published. Required fields are marked *