1. Introduction

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 ) [5,6,7,8,9, The Malayan pangolin (), a representative mammal species of the order Pholidota, is one of the only eight pangolin species worldwide. Four of them are from Asia (and), whereas another four from Africa (and) [ 1 ]. 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 10 ], 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.

13,14,13,15, 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 15 ]. 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 17 ].

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.