Scientists explore how to isolate a bat serpic virus in Japan


In a recent study published in Emerging infectious diseasesThe researchers conducted experiments isolating bat serpic virus using Rhinolophus cornutus Bats from several regions in Japan fell into the same group of viruses associated with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

Study: Isolation of bat SARS-CoV-2 viruses, Japan.  Image credit: Independent Birds/Shutterstock
Stady: Bat virus isolation, Japan. Image credit: Independent Birds/Shutterstock

Human betta is highly pathogenicCorona viruses (β-CoVs) such as severe acute respiratory syndrome coronavirus (SARS-CoV), SARS-CoV-2, and Middle East respiratory syndrome CoV (MERS-CoV) have originated from bats. Therefore, monitoring of β-CoV is essential to improve understanding and assess the potential for spread of β-CoV in humans. Sarpicviruses identified in Asian countries such as China have been reported to be genetically diverse. However, the genetic differences and distribution of bat viruses in Japan are not well described and require further investigation.

The authors of the current study previously showed that a VSV (vesicular stomatitis virus)-based viral pseudotype includes the S protein (spike) of Rc-o319 sarbecovirus from R. atom Only infected Rc-ACE2 (angiotensin-converting enzyme 2)-expressing cells, but not human ACE2 (hACE2)-expressing or other rhinolovos ACE2-expressing cells.

about studying

In this study, researchers report the identification, isolation, and biological and genetic characterization of sarcoid viruses originating from bats in different Japanese locations.

Stool samples were obtained from R. ferrumequinum f R. atom paddle Species in Chiba, Shizuoka and Niigata prefectures. Real-time RT-PCR (reverse transcription polymerase chain reaction) was performed, and the team generated Vero cells expressing RcACE2 (Vero-RcACE2) based on the Vero/transmembrane protein. sIRIN 2 (TMPRSS2).

Next generation sequencing (NGS) analysis was performed to determine the whole genome sequences of all viral isolates, and the sequences have been deposited in GenBank. In addition, a similarity plot analysis of the whole genome sequence was performed using each isolate as a query. A phylogenetic tree of bat sarcoid viruses in Japan was constructed based on whole-genome sequencing with maximum-likelihood analysis and replicates.

The S RBM (receptor binding model) of bat isolates from Japan was aligned with those of other Sarpic viruses. To evaluate the growth kinetics of Sarbic virus isolates from bats in Japan, R. atom Bat isolates Rc-os20, Rc-o319, Rc-mk2 and Rc-kw8 or SARS-CoV-2 (strain B.1.1.7) were inoculated into Vero-RcACE2 [RcACE2, Vero/TMPRSS2 (WT)]Vero-hACE2 (hACE2) or Vero-ACE2KO (ACE2KO) cells and viral titer were determined using plaque assays.

consequences

The isolates showed efficient growth in the expressing cells rhinolovos Maize ACE2 but did not grow well in hACE2-expressing cells, indicating that bat isolates had a narrow range of hosts. RT-PCR analysis enabled the successful detection of the envelope (E) gene sequence of sarcoid viruses in one or two Rhinolophus cornutus samples in each province. Bat viruses have been successfully isolated through the use of Vero-RcACE2 cell cultures, with massive cytopathic effects and syncytial formation from RT-PCR-positive samples of bat feces from each province.

Bat isolates from Shizuoka, Niigata, and Chiba prefectures were modeled as Rc-kw8, Rc-os20, and Rc-mk2, respectively. Moreover, Rc-o319 was isolated by using Vero-RcACE2 cell cultures. On the contrary, everything Specimens of Rhinolophus ferremuquinum It was bat negative which indicates the spread of bat viruses among Rhinolophus cornutus Bats in Japan. Sequences from all bat strains isolated from Japan were highly homogeneous (range 95%–97%). However, Rc-os20 and Rc-mk2 lacked the region coding for ORF8 (Open Reading Frame 8).

The team noted significant similarities between the bat isolates throughout the genome sequence, except for the coding sites of the gene’s S-receptor-binding domain (RBD) and its N-terminal domain (NTD). However, Rc-kw8 and Rc-o319 NTDs were found to be conserved. No significant recombination was observed among bat isolates. In the Phylogenetic analysisthe isolates were observed in a single gene cluster located in a genetic clade that includes SARS-CoV-2-related Sarpic viruses.

The results of the S RBD phylogenetic analysis indicated that the isolates from Japan were placed in the genomic branch of viral organisms using ACE2 orthologues as receptors. Therefore, the team hypothesized that the new strains isolated from Japan use RcACE2 as a receptor. bats efficiently sequestered only Vero-RcACE2 but not Vero-ACE2KO, Vero/TMPRSS2 or Vero-hACE2 cells. The results indicated that the bat isolates relied on RcACE2 for infection.

In contrast, SARS-CoV-2 showed efficient replication in Vero-RcACE2, Vero-hACE2 and Vero/TMPRSS2 cells but did not replicate well in cultures of Vero-ACE2KO cells, suggesting that infection depends on the presence of several ACE2 receptors. , including from Rhinolophus cornutus bat. The results indicated that bat isolates in Japan only used bRcACE2 as a receptor.

The majority of genome sequences between subspecies from Japan were highly conserved, and it may have been since then rhinolovos spp. Bat species migrate relatively short distances and have no cross-contact with other groups of bats. The exceptions were the S regions coding for the RBD and NTD, which showed wide genetic variation due to immunological pressures, suggesting that bats diverged from the ancestral lineage more recently.

In general, the results of the study showed that bat isolates used batACE2 in the form of a receptor without repeats in hACE2-expressing cells, and therefore, it is a distinct species and indicates the scarcity of the possibility of human infection. Sarbecoviruses may mutate and lead to human infection through intermediate host species in livestock or wildlife. Therefore, studies of the epidemiology of sarcoid virus in wild animals, including bats, must be conducted with long-term risk assessments of the possibility of zoonotic transmission.



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