Researchers discover a startling diversity of viruses in Japanese bats, with over 60% infected, including novel strains, highlighting the urgent need for viral surveillance to prevent future pandemics.

Study: Detection of various DNA and RNA viruses in bats in Yamaguchi Prefecture, Japan. Image Credit: Agami Photo Agency / Shutterstock

In a recent study published in the journal Microbes and Infection, researchers conducted an intensive survey of bat-derived DNA and RNA viruses from the Yamaguchi prefecture, Japan. They sequenced tissue from more than 130 bats (4 species) from four sampling locations and found that more than 60% (n = 80) of them were infected with at least one strain of potentially zoonotic viruses.

Alarmingly, the study revealed significant genetic divergence in some viral strains, particularly adenoviruses, and astroviruses, indicating that these viruses may be evolving rapidly. Notably, the study identified ten novel herpesvirus strains, reported the first occurrence of astrovirus in Japanese bats, and revealed numerous representatives of adenovirus and coronavirus families.

These findings highlight the hitherto unknown diversity of viral pathogens in Japanese bats and their surprisingly high prevalence across sampled bat species. The detection of viral sequences that were genetically identical to strains found in China and Korea underscores a potential connection between Japanese and mainland Asian bats despite the general belief that these populations do not intermingle.

The study found viral sequences identical to those of Chinese and Korean origins, suggesting limited chiropteran migration between these landmasses. However, the possibility of occasional bat migration or viral exchange through other mechanisms, such as human or animal movement, cannot be ruled out.

Additional research to characterize these viruses’ life histories and pathophysiologies is imperative before action plans against zoonotic transmission can be formulated, thereby preventing future bat-transmitted disease outbreaks.

Background

Bats are speciose (>1470 species) and highly successful members of the order Chiroptera, representing at least 20% of all known mammalian species. Unlike other mammals, bats are capable of true flight, with some species known to embark on seasonal migrations of hundreds of miles. These fascinating animals are almost ubiquitous in their distribution, found on every major landmass except the Arctic and Antarctic zones.

Unfortunately, bats are also known reservoirs (natural hosts) for more than 20,000 viral species (33 families), some of which cause zoonotic diseases, including rabies, Nipah, Hendra, Ebola, and coronavirus.

Recent research suggests that the coronavirus disease 2019 (COVID-19) outbreak, arguably the worst pandemic in human history, originated in Chinese bats. Therefore, monitoring bats and their viral parasites is imperative to prepare for future zoonotic outbreaks.

While bats in China and the Western world have been the subject of extensive research, Japanese bats have been comparatively ignored. Hitherto, the limited number of bat-derived virus investigations have only revealed 109 strains (9 families) from the country, a probable severe underestimate of these viral carriers’ true pathogenic potential and diversity.

This limited research also leaves gaps in understanding how virus strains in Japanese bats genetically differ from those found in other regions, particularly Southeast Asia. Unraveling the viral populations in Japanese bats would allow the island nation and (given bats’ long-range dispersal ability) the rest of the SE Asian and Australasian region to better prepare for future zoonotic events.

About the study

The present study comprised a year-long (2021-22) intensive investigation of DNA and RNA viruses inhabiting Japanese bats (n = 132, 4 species) from four locations within the Yamaguchi prefecture, Japan. Bats were captured in nets, and morphological examinations were conducted to classify individuals as Miniopterus fuliginosus, Myotis macrodactylus, Rhinolophus ferrumequinum, or Rhinolophus cornutus.

Of the 132 individuals captured, 85 were euthanized and harvested for lung (n = 40), brain (n = 40), and rectal swab samples (n = 85). Additionally, oral swab samples were collected from all 132 captured individuals. DNA extracted from samples was amplified using nested polymerase chain reaction (PCR) to identify and characterize DNA viruses.

Similarly, RNA was extracted and amplified using reverse transcription (RT) -PCR followed by nested or semi-nested PCR to identify RNA viruses.  While 178 samples were screened for the presence of calicivirus, paramyxovirus, flavivirus, and rotavirus, all were negative for these pathogens.

Finally, the Molecular Evolutionary Genetics Analysis (MEGA) software package was used for sequence cleaning and alignment (ClustalW). Cleaned sequences were compared with preexisting genetic data from the GenBank database. Neighbor-joining (NJ) trees were constructed for phylogenetic analysis using MEGA, with visualization done in R Software.

Study findings

Of the 297 samples collected, 124 (~42%) were positive for viral nuclear material. Notably, more than 60% of screened bats (80 of 132) were found to be infected with one or more virus strains (36% of infected bats presented multiple viral strains). Herpesvirus was the most prevalent viral family, infecting all sampled bat species and present in 42.4% (56/132) of screened oral swabs.

In particular, the herpesvirus strains identified in this study displayed phylogenetic similarities to previously identified strains from China and Spain, suggesting a more complex evolutionary history of these viruses than previously thought. Astroviruses (15.2%) and coronaviruses (9.4%) were represented in all sampled species except R. cornutus. Adenovirus was the rarest viral family observed in only five R. ferrumequinum and My. macrodactylus individuals.

Herpesviruses isolated in this study were found to belong to subfamilies Betaherpesvirinae and Gammaherpesvirinae, 10 of which were novel to science. Bats infected with herpesviruses often presented multiple strains (a ‘superinfection’).

“In the present study, we also observed some mixed peaks in the Sanger sequencing chromatograms, precluding the accurate determination of nucleotide sequences in these samples (data not shown). Considered together, these lines of evidence strongly suggest that bats may be superinfected with multiple herpesviruses, meaning that the detection of bat herpesviruses by sequencing may be complicated by the presence of mixed sequences derived from distinct viruses.”

Surprisingly, each of the five animals depicting adenovirus infections presented a unique viral strain, all aligned with sequences from the genus Mastadenovirus. These adenoviruses showed significant genetic divergence from previously sequenced Japanese bat-derived viruses, suggesting a rapid evolutionary process, possibly driven by host-specific factors.  This study is also the first report of astroviruses from Japanese bats.

Since adenoviruses and astroviruses are often implicated in mammalian interspecies transmission (including humans), additional research on these strains’ pathogenicities, transmission routes, and transmission cycles is required to help prepare policymakers and clinicians for future viral outbreaks.

“To date, many coronaviruses belonging to the genus Betacoronavirus, including merbecoviruses isolated from Vespertilio sinensis and Eptesicus japonensis and sarbecoviruses isolated from R. cornutus, have been detected in eastern Japan. In the present study, only alphacoronaviruses were detected in R. cornutus, although this correlation may reflect the small sample size employed in our study. Further investigation will be needed to identify possible infection of bats by betacoronaviruses, especially sarbecovirus and merbecovirus in the western part of Japan.”

Conclusions

The present study reveals an unexpectedly high prevalence (>60%) of zoonotic viral infections among Japanese bats from the Yamaguchi prefecture. At least 10 novel species of herpesvirus and five novel strains of adenovirus were obtained from genetic analysis, some of which displayed genetic sequences identical to those found in mainland Asia, raising questions about bat migration patterns or alternative routes of viral transmission. The rapid evolution of these viruses highlights the need for routine monitoring and screening of bat populations and parallel epidemiological research on identified strains to help prevent or address future epi- or pandemic viral outbreaks in bats, mammals, and humans.