The Malayan pangolin (Manis javanica
), a representative mammal species of the order Pholidota, is one of the only eight pangolin species worldwide. Four of them are from Asia (M. javanica
, M. pentadactyla
, M. crassicaudata
and M. culionensis
), whereas another four from Africa (M. tricuspis
, M. tetradactyla
, M. gigantea
and M. temminckii
]. Unlike other placental mammals, the skin of pangolins is covered by large and overlapping keratinized scales [2
]. Because of the huge demand for their meat as a delicacy and their scales for use in traditional medicines, pangolins are the most poached and trafficked mammal in the world. That is why all the eight pangolin species are included in the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES). Concerted efforts have been made to conserve and rescue these species in captivity in China because of their threatened status and continuing decline of the population size in the wild. At the same time, poor health condition and low immunity are also important problems for the rescue of pangolins. A previous study reported a complete genome sequence of Parainfluenza Virus 5 (PIV5) from a Sunda Pangolin (the same as Malayan Pangolin) in China, which further broadens the PIV5 infection host spectrum [3
], implicating that pangolins are not only confronted with the potential of great harm from humans, but are also facing the risk of infectious diseases. Recently, a large number of viral metagenomic studies have found pathogenic viruses carried by human, pig, cow, bat, cat, horse, chicken and other animals [4
], some of which successfully isolated new virus strains. However, we still know little about the diseases and their etiologies of rare and threatened terrestrial vertebrate such as pangolins.
Viruses are infectious agents that replicate only inside living cells and have the ability to infect a variety of hosts [11
]. There has been a lot of discussion within the virology community regarding the best method to determine viral infectivity, pathogenicity, and effects on the host microbiome. Virologists use a variety of methods to gain understanding of infection, replication, pathogenicity, and, more recently, the evolution of the viral genome. Unbiased sequencing of nucleic acids from environmental samples has great potential for the discovery and identification of diverse microorganisms [12
]. We know this technique as metagenomics, or random, agnostic or shotgun high-throughput sequencing. In theory, metagenomics techniques enable the identification and genomic characterization of all microorganisms present in a sample with a generic laboratory procedure [16
]. The approach has gained popularity with the introduction of next-generation sequencing (NGS) methods that provide more data in less time at a lower cost than previous sequencing techniques. While initially mainly applied to the analysis of the bacterial diversity, modifications in sample preparation protocols allowed characterization of viral genomes as well. Researchers have seized the opportunity to expand our knowledge in the fields of virus discovery and biodiversity characterization [12
The Guangdong Wildlife Rescue Center received 21 live Malayan pangolins from the Anti-smuggling Customs Bureau on 24 March 2019; most individuals, including adults and subadults, were in poor health, and their bodies were covered with skin eruptions. All these Malayan pangolins were rescued by the Guangdong Wildlife Rescue Center, however, 16 died after extensive rescue efforts. Most of the dead pangolins had a swollen lung which contained a frothy liquid, as well as the symptom of pulmonary fibrosis, and in the minority of the dead ones, we observed hepatomegaly and splenomegaly. We collected 21 organ samples of lung, lymph, and spleen with obvious symptoms from 11 dead Malayan pangolins to uncover the virus diversity and molecular epidemiology of potential etiologies of viruses based on a viral metagenomic study. This study will be beneficial to pangolin disease research and subsequent rescue operation.
Pangolins are important wildlife resources in imminent danger of extinction. Great efforts have been made to rescue trafficked pangolins; however, most of the pangolin individuals intercepted by customs were in a poor health condition, and then dead in a few days. Investigating the potential pathogens carried by pangolins may help to rescue them. Our viral metagenomics analysis revealed a high diversity of viruses carried by dead Malayan pangolins. The Sendai virus and Coronaviruses were dominant virus species conformed by assembled contigs, which might have some relationship with the death of Malayan pangolins. Recently, the prediction of viral zoonosis epidemics has become a major public health issue. A profound understanding of the viral population in key animal species acting as reservoirs represents an important step towards this goal. Bats are natural hosts for a large variety of zoonotic viruses. In a recently study, up to 47 different virus families were detected from bat fecal samples [25
]. Over 130 virus species have been detected in bats as of 2017 [26
], including several emergent human pathogens [27
]. For domesticated animals, virome analysis between sick and health ones could help to find out the pathogens or virus diversity [8
]. Our study showed that viral metagenomics analysis could also work in revealing viral diversity and potential pathogens of rare and threatened terrestrial vertebrates such as pangolins.
The Sendai virus was the most widely distributed pathogens in 11 dead Malayan pangolins, which was one of the potential causes of their death. The whole genome and individual gene phylogenies for Sendai virus sequences assembled in this study all showed that the Sendai virus from Malayan pangolin had the closest relationship with the strain isolated from humans (AB005795.1), which strongly suggests the possibility that the Sendai virus is transmitted between pangolins and humans. Sendai virus is a member of the paramyxovirus subfamily Paramyxovirinae
, genus Respirovirus, members of which primarily infect mammals. The scientific community considers the Sendai virus as the archetype organism of the Paramyxoviridae
family because most of the basic biochemical, molecular and biologic properties of the whole family were derived from its own characteristics [42
]. Sendai virus-associated disease has a worldwide distribution and has been found in mouse colonies in Asia [43
], North America [44
] and Europe [45
], and is responsible for a highly transmissible respiratory tract infection in mice, hamsters, guinea pigs, rats, and occasionally pigs and bats [46
], with infection through both air and direct contact routes. Epizootic infections of mice are usually associated with a high mortality rate, while enzootic disease patterns suggest that the virus is latent and can be cleared over the course of a year. This is the first report of a wild pangolin dying possibly due to Sendai virus infection, which further broadens the Sendai virus infection host spectrum. Because of the lack of healthy individuals as a control, we could not figure out whether the Sendai virus carried by pangolins was caused by infection from other hosts or was inherited.
Besides the Sendai virus, Coronaviruses were also detected as potential pathogens of Malayan pangolins. The phylogeny of Coronavirus sequences assembled and strains from four Coronavirus genera demonstrated complex genetic relationships and high species diversity of the Coronavirus in Malayan pangolins. Coronaviruses can cause a variety of severe diseases including gastroenteritis and respiratory tract diseases, and have been identified in mice, rats, chickens, turkeys, swine, dogs, cats, rabbits, horses, cattle and humans [48
]. Sometimes, but not often, a coronavirus can infect both animals and humans. Human coronaviruses were first described in the 1960s for patients with the common cold. Since then, more have been discovered, including those that cause severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS), two pathogens that can cause fatal respiratory disease in humans [50
]. It was recently discovered that dromedary camels in Saudi Arabia harbor three different human coronaviruses species, including a dominant MERS HCoV lineage that was responsible for the outbreaks in the Middle East and South Korea during 2015 [52
]. The detection of different types of SARS-CoV in this study may also be related to the death of the Malayan pangolins. Considering the outbreak of SARS which was transmitted by masked palm civet from the natural reservoir of bats [29
], Malayan pangolins could be another host with the potential of transmitting the SARS coronavirus to humans. As a consequence, the viral metagenomic study of Malayan pangolin is meaningful both for the conservation of rare wild animals and public health.