Many illnesses, including influenza, hemorrhagic fever, and rabies, are caused by a group of viruses called negative-sense RNA viruses. The genetic information—or genome—of these viruses is encoded in strands of RNA that must be copied before they can be translated into the proteins needed to build new viruses. It is currently known that there are at least eight different families of these viruses, which have a wide range of shapes and sizes and arrange their RNA in different ways.

Insects, spiders, and other arthropods carry many different RNA viruses. Many of these viruses have not previously been studied, and those that have been studied so far are mainly those that cause diseases in humans and other vertebrates. Researchers therefore only know a limited amount about the diversity of the negative-sense RNA viruses that arthropods harbor and how these viruses evolved. Studying how viruses evolve helps scientists to understand what makes some viruses deadly and others harmless and can also help develop treatments or vaccines for the diseases caused by the viruses.

Li, Shi, Tian, Lin, Kang et al. collected 70 species of insects, spiders, centipedes, and other arthropods in China and sequenced all the negative-sense RNA viruses in the creatures. This revealed an enormous number of negative-sense RNA viruses, including 112 new viruses. Many of the newly discovered arthropod viruses appear to be the ancestors of disease-causing viruses, including influenza viruses and the filoviruses—the group that includes the Ebola virus. Indeed, it appears that arthropods host many—if not all—of the negative-sense RNA viruses that cause disease in vertebrates and plants.

While documenting the new RNA viruses and how they are related to each other, Li et al. found many different genome structures. Some genomes were segmented, which may play an important role in evolution as segments can be easily swapped to create new genetic combinations. Non-segmented and circular genomes were also found. This genetic diversity suggests that arthropods are likely to have played a key role in the evolution of new viruses by acting as a site where many different viruses can interact and exchange genetic information.

Negative-sense RNA viruses are important pathogens that cause a variety of diseases in humans including influenza, hemorrhagic fever, encephalitis, and rabies. Taxonomically, those negative-sense RNA viruses described to date comprise at least eight virus families and four unassigned genera or species (King et al., 2012). Although they share (i) a homologous RNA-dependent RNA polymerase (RdRp), (ii) inverted complementary genome ends, and (iii) an encapsidated negative-sense RNA genome, these viruses display substantial diversity in terms of virion morphology and genome organization (King et al., 2012). One key aspect of genome organization is the number of distinct segments, which is also central to virus classification. Among negative-sense RNA viruses, the number of segments varies from one (order Mononegavirales; unsegmented) to two (family Arenaviridae), three (Bunyaviridae), three-to-four (Ophioviridae), and six-to-eight (Orthomyxoviridae) and is further complicated by differences in the number, structure, and arrangement of the encoded genes.

Despite their diversity and importance in infectious disease, the origins and evolutionary history of the negative-sense RNA viruses are largely obscure. Arthropods harbor a diverse range of RNA viruses, which are often divergent from those that infect vertebrates (Marklewitz et al., 2011, 2013; Cook et al., 2013; Ballinger et al., 2014; Qin et al., 2014; Tokarz et al., 2014a, 2014b). However, those arthropod viruses sampled to date are generally those that have a relationship with vertebrates or are known to be agents of disease (Junglen and Drosten, 2013). To determine the extent of viral diversity harbored by arthropods, as well as their evolutionary history, we performed a systematic survey of negative-sense RNA viruses using RNA sequencing (RNA-seq) on a wide range of arthropods.

Our study suggests that arthropods are major reservoir hosts for many, if not all, of the negative-sense RNA viruses in vertebrates and plants, and hence have likely played a major role in their evolution. This is further supported by the high abundance of viral RNA in the arthropod transcriptome, as well as by the high frequencies of endogenous copies of these viruses in the arthropod genome, greatly expanding the known biodiversity of these genomic ‘fossils’ (Katzourakis and Gifford, 2010; Cui and Holmes, 2012). The often basal position of the arthropod viruses in our phylogenetic trees is also compatible with the idea that the negative-sense RNA viruses found in vertebrates and plants ultimately have their ancestry in arthropods, although this will only be confirmed with a far wider sample of virus biodiversity.

The rich genetic and phylogenetic diversity of arthropod RNA viruses may in part reflect the enormous species number and diversity of arthropods, and that they sometimes live in large and very dense populations that provide abundant hosts to fuel virus transmission. Furthermore, arthropods are involved in almost all ecological guilds and actively interact with other eukaryotes, including animals, plants, and fungi, such that it is possible that they serve as both sources and sinks for viruses present in the environment. In addition, not only were diverse viruses present, but they were often highly abundant. For example, in the pool containing 12 individuals (representing two species) from the Gerridae (Water striders) collected at the same site, we identified at least five negative-sense RNA viruses whose TPM values are well above 100, and where the viral RNA collectively made up more than 50% of the host total RNA (rRNA excluded). Determining why arthropods are able to carry such a large viral diversity and at such frequencies clearly merits further investigation.

The viruses discovered here also exhibited a huge variation in level of abundance. It is possible that this variation is in part due to the stage or severity of infection in individual viruses and may be significantly influenced by the process of pooling, since most of our libraries contain an uneven mixture of different host species or even genera. In addition, it is possible that some low abundance viruses may in fact be derived from other eukaryotic organisms present in the host sampled, such as undigested food or prey, gut micro flora, and parasites. Nevertheless, since the majority of the low abundance viruses appear in the same groups as the highly abundant ones in our phylogenetic analyses, these viruses are most likely associated with arthropods.

Viral infections in vertebrates and plants can be divided into two main categories: (i) arthropod-dependent infections, in which there is spill-over to non-arthropods but where continued virus transmission still requires arthropods, and (ii) arthropod-independent infections, in which the virus has shifted its host range to circulate among vertebrates exclusively (Figure 12). The first category of infections is often associated with major vector-borne diseases (Zhang et al., 2011, 2012). Given the biodiversity of arthropod viruses documented here, it seems likely that arthropod-independent viruses were ultimately derived from arthropod-dependent infections, with subsequent adaptation to vertebrate-only transmission (Figure 12).

Figure 12

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One of the most notable discoveries was that of a novel family, the Chuviridae. The identification of this diverse virus family provides a new perspective on the evolutionary origins of segmented and unsegmented viruses. In particular, the chuviruses occupy a phylogenetic position that is in some sense ‘intermediate’ between the segmented and unsegmented negative-sense RNA viruses and display genomic features of both. Indeed, our phylogenetic analysis reveals that genome segmentation has evolved multiple times within the diversity of chuviruses (Figure 7), such that this trait appears to be more flexible than previously anticipated. In addition, the majority of the chuviruses possess circular genomes. To date, the only known circular RNA virus is (hepatitis) deltavirus, although this potentially originated from the human genome (Salehi-Ashtiani et al., 2006) and requires hepatitis B virus for successful replication. As such, the chuviruses may represent the first report of autonomously replicating circular RNA viruses, which opens up an important line of future research.

Our results also provide insights into the evolution of genome segmentation. Within the bunya-arena-like viruses (Figures 3C, 4), the three-segment structure is the most common, with the viral polymerase, nucleoprotein, and surface glycoproteins present on different segments. Notably, our phylogenetic analysis seemingly revealed independent occurrences of both increasing (Tenuivirus and Emaravirus) and decreasing (Arenavirus) segment numbers from the three-segment form (Figure 4). Independent changes of genome segmentation numbers are also observed in the mononegavirales-like viruses (Figure 4) and, more frequently, in the chuviruses (Figure 7A). Consequently, the number of genome segments appears to be a relatively flexible trait at a broad evolutionary scale, although the functional relevance of these changes remains unclear. While the segmented viruses (bunya-arenaviruses, orthomyxoviruses, and ophioviruses) appear to be distinct from the largely unsegmented mononegavirales-like viruses in our phylogenetic analysis, this may be an artifact of under-sampling, especially given that only a tiny fraction of eukaryotes have been sampled to date. With a wider sample of eukaryotic viruses it will be possible to more accurately map changes in segment number onto phylogenetic trees and in so doing come to a more complete understanding of the patterns and determinants of the evolution of genome segmentation.

In sum, our results highlight the remarkable diversity of arthropod viruses. Because arthropods interact with a wide range of organisms including vertebrate animals and plants, they can be seen as the direct or indirect source of many clinically or economically important viruses. The viral genetic and phenotypic diversity documented in arthropods here therefore provides a new perspective on fundamental questions of virus origins, diversity, host range, genome evolution, and disease emergence.

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Author details

1. Ci-Xiu Li

   1. State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, China
   2. Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Hangzhou, China

   Contribution

   C-XL, Acquisition of data, Analysis and interpretation of data

   Contributed equally with

   Mang Shi, Jun-Hua Tian, Xian-Dan Lin and Yan-Jun Kang

   Competing interests

   The authors declare that no competing interests exist.

2. Mang Shi

   1. State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, China
   2. Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Hangzhou, China
   3. Marie Bashir Institute for Infectious Diseases and Biosecurity, Charles Perkins Centre, School of Biological Sciences and Sydney Medical School, The University of Sydney, Sydney, Australia

   Contribution

   MS, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Contributed equally with

   Ci-Xiu Li, Jun-Hua Tian, Xian-Dan Lin and Yan-Jun Kang

   Competing interests

   The authors declare that no competing interests exist.

3. Jun-Hua Tian

   Wuhan Center for Disease Control and Prevention, Wuhan, China

   Contribution

   J-HT, Acquisition of data, Analysis and interpretation of data, Contributed unpublished essential data or reagents

   Contributed equally with

   Ci-Xiu Li, Mang Shi, Xian-Dan Lin and Yan-Jun Kang

   Competing interests

   The authors declare that no competing interests exist.

4. Xian-Dan Lin

   Wenzhou Center for Disease Control and Prevention, Wenzhou, China

   Contribution

   X-DL, Acquisition of data, Analysis and interpretation of data, Contributed unpublished essential data or reagents

   Contributed equally with

   Ci-Xiu Li, Mang Shi, Jun-Hua Tian and Yan-Jun Kang

   Competing interests

   The authors declare that no competing interests exist.

5. Yan-Jun Kang

   1. State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, China
   2. Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Hangzhou, China

   Contribution

   Y-JK, Acquisition of data, Analysis and interpretation of data, Contributed unpublished essential data or reagents

   Contributed equally with

   Ci-Xiu Li, Mang Shi, Jun-Hua Tian and Xian-Dan Lin

   Competing interests

   The authors declare that no competing interests exist.

6. Liang-Jun Chen

   1. State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, China
   2. Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Hangzhou, China

   Contribution

   L-JC, Acquisition of data, Analysis and interpretation of data, Contributed unpublished essential data or reagents

   Competing interests

   The authors declare that no competing interests exist.

7. Xin-Cheng Qin

   1. State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, China
   2. Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Hangzhou, China

   Contribution

   X-CQ, Acquisition of data, Analysis and interpretation of data, Contributed unpublished essential data or reagents

   Competing interests

   The authors declare that no competing interests exist.

8. Jianguo Xu

   1. State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, China
   2. Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Hangzhou, China

   Contribution

   JX, Acquisition of data, Analysis and interpretation of data, Contributed unpublished essential data or reagents

   Competing interests

   The authors declare that no competing interests exist.

9. Edward C Holmes

   1. State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, China
   2. Marie Bashir Institute for Infectious Diseases and Biosecurity, Charles Perkins Centre, School of Biological Sciences and Sydney Medical School, The University of Sydney, Sydney, Australia

   Contribution

   ECH, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

10. Yong-Zhen Zhang

    1. State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, China
    2. Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Hangzhou, China

    Contribution

    Y-ZZ, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents

    For correspondence

    zhangyongzhen@icdc.cn

    Competing interests

    The authors declare that no competing interests exist.

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