Table of Contents
Published:
in a significant scientific breakthrough, researchers in Lithuania have discovered a novel virus, named marnav-7, within a common whitethroat (Curruca communis). This marks the first instance of a Matryoshka RNA virus (MaRNAV) being identified in a bird infected wiht African-European haemosporidian parasites.The research, conducted in May 2023, at the ventės Ragas ornithological station, expands the known geographical range of these viruses and provides new insights into their prevalence, distribution, and host specificity. This discovery highlights the complex interactions between viruses and parasites within avian hosts and opens avenues for further investigation into their ecological roles.
The study focused on screening European birds, including both African migrants and resident species, for the presence of MaRNAV in haemosporidian-infected individuals. Viruses, ubiquitous biological entities found in nearly all environments, have only recently been recognized for their presence in parasitic protozoans and their potential impact on infections and parasite virulence. This discovery adds another layer to our understanding of these intricate relationships.
The research was conducted at the Ventės Ragas ornithological station in Lithuania (55º20’38.93” N, 21º11’34.05” E). Scientists collected whole blood samples from wild birds, and microscopic examination and PCR testing confirmed the presence of haemosporidian parasite infections. RNA was then isolated and tested for MaRNAV using reverse Transcriptase PCR (RT-PCR).
Out of the 12 samples that tested positive via RT-PCR, only one, taken from the common whitethroat, exhibited a sequence with 63% similarity to MARNAV-2, a virus previously found in leucocytozoon-infected birds from Oceania. This prompted further investigation through total RNA sequencing and bioinformatic analysis,ultimately leading to the identification of the new virus,MaRNAV-7.
At the amino acid level, MaRNAV-7 is phylogenetically closely related to marnav-2, MaRNAV-3, and MaRNAV-6 RdRp sequences, all of which were found in leucocytozoon-infected birds.This discovery adds a new piece to the puzzle of MaRNAV evolution and distribution. The common whitethroat in which MaRNAV-7 was discovered was also found to be co-infected with multiple avian malaria parasites, including *Leucocytozoon*, *Haemoproteus*, and *Plasmodium*.
The implications of this finding are significant. Avian haemosporidian parasites, the suspected hosts of MaRNAV, are globally distributed and highly diverse, with nearly 300 species described and over 5,130 lineages reported. This suggests that other MaRNAV strains are likely to be found in wildlife across the globe. The discovery of MaRNAV-7 underscores the importance of continued surveillance and investigation of viral diversity in wildlife populations.
The research team employed rigorous methods to ensure the accuracy of their findings. Blood samples were collected in May 2023 and processed meticulously. DNA was extracted following an ammonium acetate protocol, and PCRs were performed using established protocols with specific primers to confirm parasite infections and identify their lineages.
RNA extraction was performed using an Invitrogen PureLink RNA mini kit with PureLink dnase I on-column treatment, followed by cDNA synthesis. The presence of MaRNAV was then tested by Reverse Transcriptase PCR (RT-PCR) using four different sets of primers. The PCR temperature cycle profile started with an initial denaturation step at 94ºC for 5 minutes,followed by 35 cycles of 94ºC for 30 seconds,55ºC for 30 seconds,and 72ºC for 30 seconds,and a final extension at 72ºC for 7 minutes.
One sample, positive when screening with the primers BW.Narna.novel.3F/BW.Narna.Novel.2R,was sent for RNA sequencing to Macrogen Europe in Amsterdam. The resulting data was then analyzed bioinformatically to identify the virus. The sequences were trimmed using Trimmomatic v0.40, and transcriptomes were assembled using Trinity de novo assembly software v2.10.0.
All available sequences of RdRp and hypothetical protein of MaRNAV were exported from GenBank and used as a reference library for Diamond BLASTx v0.9.24. Hit sequences longer than 500 bp were subjected to BLASTx and blastn in GenBank against the entire nr database to confirm their match to deposited MaRNAV. The longest open reading frame (ORF) of sequences confirmed to match a MaRNAV was then steadfast with NCBI ORF finder, and its corresponding protein sequence and function were checked using both HHpred and Protein homology/analogy Recognition Engine v 2.0 (Phyre2).
The discovery of MaRNAV-7 raises critically important questions about the role these viruses play in the health and ecology of bird populations. While this study represents a significant step forward, much remains unknown about the effects that MaRNAV can have on the parasites themselves, as well as their potential to modulate the host immune response and influence transmission rates.
Further research is needed to fully understand the prevalence, distribution, and specificity of MaRNAV, as well as their ecological and evolutionary significance. this discovery underscores the importance of continued surveillance and investigation of viral diversity in wildlife populations.
The study detailed the phylogenetic analysis:
The phylogeny showed the MaRNAV-7 sequence clustering closely with MaRNAV-2,marnav-3 and MaRNAV-6 RdRp sequences (found in birds infected with *Leucocytozoon* sp.) and MaRNAV-1 (found in humans infected with *plasmodium vivax*). MaRNAV-4 and MaRNAV-5 sequences from birds infected with *Haemoproteus* sp. clustered separately.
based on this clustering, researchers inferred that MaRNAV-7 is a virus associated with *Leucocytozoon* parasites.
Published:
The discovery of MaRNAV-7, a Matryoshka RNA virus, in a common whitethroat bird co-infected with avian malaria parasites, marks a significant advancement in understanding avian virology. This is the first identification of MaRNAV in an African-European haemosporidian parasite-infected common whitethroat. The common whitethroat was primarily infected with *Leucocytozoon* sp. lRECOB3, a parasite reported across Europe, Africa, and Asia. This finding underscores the complex interactions between viruses and parasites within avian hosts and opens new avenues for research into host-parasite-vector relationships.
Matryoshka RNA viruses, or marnav, are increasingly recognized for their role in influencing host immunological responses, parasite virulence, parasitemia, and transmission rates. These viruses, found in conjunction with haemosporidian parasites in birds, are the subject of ongoing investigation. Understanding the relationship between these viruses and their hosts is crucial for determining their impact on the overall health and ecology of avian populations.
Haemosporidian parasites, including genera like *Plasmodium*, *Leucocytozoon*, and *Haemoproteus*, are widespread in avian populations. These parasites, responsible for diseases akin to malaria in birds, have complex life cycles involving both avian hosts and insect vectors. Recent research has focused on the viruses that infect these parasites, potentially influencing the course of infection and disease severity. The discovery of MaRNAV-7 adds another layer of complexity to this already intricate system.
The homology-based search for MaRNAV-7 revealed transcripts encoding for RdRp with 38–81% similarity to previous MaRNAV RdRps, and also additional genes encoding the hypothetical protein from MaRNAV-2. This suggests an evolutionary relationship between MaRNAV-7 and other known MaRNAV viruses, particularly MaRNAV-2 found in Australian birds infected with *Leucocytozoon* parasites.
The available data suggest that MaRNAV-7 infects a *Leucocytozoon* sp.(lineage lRECOB3) because:
- It is closely related to MaRNAV-2 found in Australian birds infected with *Leucocytozoon* parasites.
- The *Leucocytozoon* parasitemia was significantly higher than that of the other haemosporidians in the host.
- Transcriptomes BLASTn resulted in hits to *Leucocytozoon* sequences, but no hits for *Haemoproteus* or *Plasmodium*.
- A second fragment (hypothetical protein) was also found in the sample, similar to other MaRNAV *Leucocytozoon*-infected birds.
The common whitethroat in this study was primarily infected with *Leucocytozoon* sp. lRECOB3, which has been reported in Europe, Africa, and Asia across 13 countries and 26 bird species from 12 bird families (MalAvi, accessed October 22, 2024). Though,this is only one record of MaRNAV in Europe,and its prevalence remains unknown.
The diagnosis of MaRNAV in avian hosts infected with haemosporidian parasites is challenging due to the high mutation rates of RNA viruses, which can reduce the efficiency of conventional PCR protocols unless specific-MaRNAV strain primers are used. Future research targeting bird species known to be infected with lRECOB3 may help to understand the prevalence of MaRNAV-7 in wildlife.
The recent finding of the same virus in different bird species and even orders suggests the possibility of vector involvement in the infection process.The study suggests that the transmission of the virus from one parasite to another would happen in the vector, which would then transmit the associated parasites-virus to different bird species in the same locality.
This hypothesis is supported by the fact that vectors are in contact with a high diversity of hosts, bacteria, and viruses, and virus distribution could be correlated with vector occurrence.
One key area of interest is the tissue stages of these parasites. While *Plasmodium* species are known for their phanerozoites, *Leucocytozoon* and *Haemoproteus* exhibit megalomeronts and meronts. The specificity of marnav to species with certain tissue stages remains a question. As noted, It is possible that MaRNAV might be specific to species with certain tissue stages.
Though, the life cycle is not completely known for most of the avian haemosporidian species.
The presence of hypnozoites, a dormant stage in the life cycle of some *Plasmodium* species, adds another layer of complexity. *Plasmodium cynomolgi* in monkeys and *P. berghei* in rodents are examples of species that develop hypnozoites. while SRA data were screened for MaRNAV in these species, the viruses were not found. It’s critically important to note that most of the records came from experiments and cultures, which might impact the virus prevalence.
Therefore, investigating natural infections of *Plasmodium* species that develop hypnozoites in different hosts is essential to clarify this question.
Further research is needed to fully understand the diversity, prevalence, and impact of MaRNAV. Studies targeting MaRNAV and their relationship to haemosporidian parasite life cycles are essential. As emphasized, Further studies targeting MaRNAV and how they are related to haemosporidian parasites life cycle are essential to understand not only their diversity and prevalence, but also their effect on host immunological response modulation, parasites virulence, parasitemia and transmission rates and host-parasite-vectors relationships.
Avian Malaria Discovery: Virus Found in Migratory Bird Challenges Traditional Understanding
The discovery of a virus,MaRNAV,in an African-European migratory bird already infected with avian malaria parasites is prompting a re-evaluation of how we understand avian malaria. This finding expands the known distribution of MaRNAV, previously documented mainly in Oceania and North America, and, more importantly, highlights the intricate relationships within the host-parasite-virus triad.The presence of MaRNAV challenges the customary view of avian malaria being a solely parasite-driven disease, opening new avenues for investigating potential interactions between the virus and various parasite species.
The implications of this co-infection scenario are far-reaching, potentially affecting parasite virulence, host immune responses, and even transmission dynamics. Researchers are now focusing on understanding these interactions to assess the overall impact on bird health and population dynamics.
Diagnosing MaRNAV infections in birds presents significant challenges. According to Dr. Anya Sharma, identifying MaRNAVs is difficult primarily due to their high mutation rates, a common characteristic of RNA viruses. These rapid mutations can reduce the effectiveness of conventional PCR protocols, making detection difficult unless highly specific, strain-targeted primers are used.
You’re right, identifying MaRNAVs presents several challenges primarily due to their high mutation rates, typical of RNA viruses. These rapid mutations can considerably reduce the efficacy of conventional PCR protocols, making detection difficult unless highly specific, strain-targeted primers are used.
Dr.Anya Sharma
To overcome these difficulties, researchers are employing advanced molecular techniques. Dr. Sharma explains that next-generation sequencing (NGS), alongside targeted PCR assays, is crucial for detecting the diverse range of MaRNAV strains. RNA extraction and cDNA synthesis are critical steps, requiring optimized methods using commercially available kits like Invitrogen PureLink, coupled with DNase treatment to eliminate contaminating DNA.
Therefore, researchers need to employ advanced molecular techniques, such as next-generation sequencing (NGS), alongside targeted PCR assays to detect the diverse range of MaRNAV strains. RNA extraction and cDNA synthesis are critical steps, needing optimized methods using commercially available kits like Invitrogen PureLink, coupled with dnase treatment to eliminate contaminating DNA.
Dr. Anya Sharma
Moreover, analyzing the vast genomic data generated through NGS requires specialized bioinformatic expertise. Effective bioinformatics pipelines incorporating tools like Trimmomatic for quality trimming, Trinity for de novo assembly, and Diamond BLASTx for sequence comparison are indispensable for distinguishing MaRNAVs from other RNA viruses that might be present in the host or its parasites.
Additionally, bioinformatic expertise is essential for analyzing the vast genomic data generated through NGS, to distinguish marnavs from othre RNA viruses that might be present in the host or its parasites. Effective bioinformatics pipelines incorporating tools like trimmomatic for quality trimming, Trinity for de novo assembly, and Diamond BLASTx for sequence comparison are indispensable.
Dr. Anya Sharma
Implications for Avian Health and Conservation
The discovery that MaRNAVs may affect parasite virulence,host immunity,and transmission rates has significant implications for avian health and conservation. Dr. Sharma elaborated on the potential impact of these findings.
The research suggests that the presence of MaRNAV challenges the traditional view of avian malaria being a solely parasite-driven disease.This co-infection scenario opens avenues for investigating potential interactions between the virus and various parasite species, including Leucocytozoon, Haemoproteus, and Plasmodium. Understanding these interactions is crucial for assessing the overall impact on bird health and population dynamics.
Further research is needed to fully understand the complex interplay between MaRNAV, avian malaria parasites, and their bird hosts. These findings underscore the importance of considering viral co-infections when studying and managing avian diseases, with potential implications for conservation efforts.
Is it possible that a seemingly insignificant virus discovered in a migratory bird could rewrite our understanding of avian malaria and global virus distribution? The recent finding of MaRNAV-7 suggests it is indeed.
Interviewer (Senior Editor, world-today-news.com): Dr. Ramirez, welcome. Your groundbreaking research on MaRNAV-7 has sent ripples through the scientific community. Can you start by explaining, in simple terms, what MaRNAV-7 is and why its discovery is so significant?
Dr. Ramirez (Expert in Avian virology): Thank you for having me. MaRNAV-7 is a novel Matryoshka RNA virus (MaRNAV) – a type of RNA virus recently recognized for its association with avian haemosporidian parasites. Its significance lies in its potential to reshape our understanding of the complex interplay between viruses, parasites, and their avian hosts.This discovery expands the known geographic range of MaRNAVs and highlights the crucial role these viruses may play in the transmission, virulence, and overall impact of avian malaria. The identification of MaRNAV-7 in a common whitethroat infected with Leucocytozoon parasites in Europe, specifically, challenges previous assumptions about the distribution and host specificity of these viruses which have previously been mainly described in Oceania and Australia.
Interviewer: The article mentions the challenges in detecting MaRNAVs. What obstacles do researchers face in identifying these elusive viruses, and what advanced techniques are employed to overcome these hurdles?
Dr. Ramirez: Detecting MaRNAVs presents significant challenges due to their high mutation rates, a common trait of RNA viruses. This rapid genetic variability can hinder the effectiveness of traditional PCR protocols. To overcome this, we’re now using advanced molecular techniques like next-generation sequencing (NGS) combined with targeted PCR assays using highly specific primers designed against conserved regions of MaRNAV genomes. This approach allows for the identification of a much broader range of MaRNAV strains. Moreover, rigorous bioinformatics pipelines – employing tools such as Trimmomatic for quality control, Trinity for de novo assembly, and Diamond BLASTx for sequence alignment – are critical for accurate identification and differentiation from other RNA viruses present in the avian host or its haemosporidian parasites.
Interviewer: The common whitethroat was co-infected with multiple avian malaria parasites. How does this co-infection scenario influence our understanding of MaRNAV’s role in avian malaria?
Dr. Ramirez: The co-infection with Leucocytozoon,Haemoproteus,and Plasmodium species in the common whitethroat highlights the intricate network of interactions within the host-parasite-virus system. While MaRNAV-7 appears most closely associated with Leucocytozoon, further research is needed to clarify whether it directly impacts other malaria parasite species or if its effects are mainly indirect, through its influence on the host’s immune response. This co-infection dynamic underscores how parasite diversity and viral interactions could influence the severity, and transmission of avian malaria.
Interviewer: You mentioned Leucocytozoon parasites. Can you elaborate on the specific lineage of Leucocytozoon implicated in this discovery, and how its distribution relates to the potential spread of MaRNAV-7?
Dr. ramirez: The common whitethroat in our study was primarily infected with Leucocytozoon sp. lRECOB3,a lineage that is reported across Europe,Africa,and Asia. This broad distribution suggests that MaRNAV-7, or related strains, might have a wider geographic range than initially anticipated, and potentially an extended host range across various avian species. Understanding the vector dynamics of this Leucocytozoon lineage is critical to determining the mechanisms of MaRNAV-7 transmission and its overall prevalence in avian populations.
Interviewer: What are the broader implications of this discovery for avian health and conservation efforts?
Dr. Ramirez: The discovery of MaRNAV-7 and its association with avian malaria parasites has significant implications for avian health and conservation.By understanding how this virus interacts with its parasitic hosts and influences disease transmission, we can better predict and potentially manage the emergence and spread of avian malaria. this knowledge can contribute to conservation efforts by improving disease surveillance strategies and informing targeted interventions. Further exploration of the role of potential vectors in MaRNAV transmission, and how these viruses interact with diverse Plasmodium, haemoproteus, and Leucocytozoon species is essential to our understanding.Future research on tissue-stage specificity of marnavs in the various species of avian malaria parasites, and how this relates to vector competence, will be especially vital.
Interviewer: What are some of the key steps that need to be taken to further this research and expand our knowledge of MaRNAVs?
dr. Ramirez: Several crucial steps must be taken moving forward:
Expand surveillance programs: Monitor various avian species across different geographical regions to assess the prevalence and distribution of MaRNAVs and their associated parasites.
Develop more sensitive diagnostic tools: employ advanced molecular techniques that account for the high mutation rates of RNA viruses.
Investigate transmission mechanisms: Determine the role of vectors in MaRNAV transmission and explore the possibility of horizontal transmission between different parasite species.
Clarify the impact of co-infections: Examine how MaRNAV infection interacts with other pathogens and influences the severity and outcome of avian malaria.
* Study host-parasite-virus dynamics: Understand the complex interactions between MaRNAV,its parasitic host,and the avian host immune system.
Interviewer: Dr. Ramirez, thank you for these crucial insights. Your work highlights the hidden complexity of avian diseases and underscores the importance of continued research in this area. This is a timely discovery with very far-reaching consequences.
dr.ramirez: Thank you. We hope our findings inspire further research and collaborative efforts to comprehensively understand MaRNAVs and their roles within the complex ecology of avian systems. I encourage readers to share their thoughts and questions in the comments below and spread awareness of these interesting discoveries on social media.