Full Genome Sequence Analysis Research Articles

Infection of bat and human intestinal organoids by SARS-CoV-2.

A novel coronavirus-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-emerged in humans in Wuhan, China, in December 2019 and has since disseminated globally1,2. As of April 16, 2020, the confirmed case count of coronavirus disease 2019 (COVID-19) had surpassed 2 million. Based on full-genome sequence analysis, SARS-CoV-2 shows high homology to SARS-related coronaviruses identified in horseshoe bats1,2. Here we show the establishment and characterization of expandable intestinal organoids derived from horseshoe bats of the Rhinolophus sinicus species that can recapitulate bat intestinal epithelium. These bat enteroids are fully susceptible to SARS-CoV-2 infection and sustain robust viral replication. Development of gastrointestinal symptoms in some patients with COVID-19 and detection of viral RNA in fecal specimens suggest that SARS-CoV-2 might cause enteric, in addition to respiratory, infection3,4. Here we demonstrate active replication of SARS-CoV-2 in human intestinal organoids and isolation of infectious virus from the stool specimen of a patient with diarrheal COVID-19. Collectively, we established the first expandable organoid culture system of bat intestinal epithelium and present evidence that SARS-CoV-2 can infect bat intestinal cells. The robust SARS-CoV-2 replication in human intestinal organoids suggests that the human intestinal tract might be a transmission route of SARS-CoV-2.

Human West Nile Virus Lineage 2 Infection: Epidemiological, Clinical, and Virological Findings.

West Nile virus (WNV) lineage 2 is expanding and causing large outbreaks in Europe. In this study, we analyzed the epidemiological, clinical, and virological features of WNV lineage 2 infection during the large outbreak that occurred in northern Italy in 2018. The study population included 86 patients with neuroinvasive disease (WNND), 307 with fever (WNF), and 34 blood donors. Phylogenetic analysis of WNV full genome sequences from patients’ samples showed that the virus belonged to the widespread central/southern European clade of WNV lineage 2 and was circulating in the area at least since 2014. The incidence of WNND and WNF progressively increased with age and was higher in males than in females. Among WNND patients, the case fatality rate was 22%. About 70% of blood donors reported symptoms during follow-up. Within the first week after symptom onset, WNV RNA was detectable in the blood or urine of 80% of patients, while 20% and 40% of WNND and WNF patients, respectively, were WNV IgM-seronegative. In CSF samples of WNND patients, WNV RNA was typically detectable when WNV IgM antibodies were absent. Blunted or no WNV IgM response and high WNV IgG levels were observed in seven patients with previous flavivirus immunity.

Genetic variability of avian influenza virus subtype H5N8 in Egypt in 2017 and 2018.

Since the incursion of avian influenza virus subtype H5N8 in Egypt in late 2016, it has spread rapidly, causing severe losses in poultry production. Multiple introductions of different reassorted strains were observed in 2017. In this study, a genetic characterization of the HA gene was carried out with 31 isolates selected from different governorates and sectors. Fifteen isolates were selected for NA gene sequence analysis. The HA and NA genes were divided into two subgroups (I and II) with positive selection pressure identified at positions 174 and 29, respectively. The HA gene contained two novel mutations in the antigenic sites, A and E. The HA nucleotide sequence identity ranged from 77 to 90% with different vaccine seeds. Full-genome sequence analysis was carried out for eight viruses, representing different governorates and sectors, to identify the predominant reassorted strain in Egypt. All viruses were similar to a reassorted strain of clade 2.3.4.4b that has been identified in Germany, among other countries. Analysis of these viruses revealed mutations specific to Egyptian strains and not the original virus characterized in 2017 (A/duck/Egypt/F446/2017), with a novel antiviral resistance marker, V27A, indicating resistance to amantadine in the M2 protein of two strains. The results indicate increased variability of circulating H5N8 viruses compared to earlier viruses sequenced in 2016 and 2017. The predominant reassorted virus circulating in 2017 and 2018 originated from an early 2017 strain. It is important to continue this surveillance of avian influenza viruses to monitor the evolution of circulating viruses.

Sequence analysis of the first B5 subgenogroup strain of enterovirus 71 isolated in Korea.

Enterovirus A71 (EV71), the main etiological agent of handfoot- mouth disease (HFMD), circulates in many areas of the world and has caused large epidemics since 1997, especially in the Asia-Pacific region. In this study, we determined the full-genome sequence of CMC718, a newly isolated EV71 strain in Korea. The CMC718 genome was 7,415 nucleotides in length and was confirmed by whole-genome phylogenetic analysis to belong to the B5 genotype. In particular, CMC718 demonstrated maximum identity with strain M988 of the B5 genotype and numerous amino acid variants were detected in the 3D domain of the viral protein P3, which is consistent with the mutation pattern of a B5 strain isolated in 2012–2013. Comparison of the CMC718 sequence with other EV71 reference strains confirmed the relationship and genetic variation of CMC718. Our study was a full-genome sequence analysis of the first EV71 strain of the B5 genotype isolated in South Korea. This information will be a valuable reference for the development of methods for the detection of recombinant viruses, the tracking of infections, and the diagnosis of EV71.

Full genome sequence analysis of a novel adenovirus from a captive polar bear (Ursus maritimus)

The presence of a novel adenovirus (AdV) was detected by PCR and sequencing, in the internal organs of a captive polar bear that had died in the Budapest zoo. The virus content of the samples proved to be high enough to allow for conventional Sanger sequencing on PCR-amplified genomic fragments. With this approach, the sequence of the entire genome of the putative polar bear adenovirus 1 (PBAdV-1) was obtained. Although the genome was found to be short, consisting of 27,952 base pairs merely, with a relatively balanced G + C content of 46.3 %, its organisation corresponded largely to that of a typical mastadenovirus. Every genus-common gene could be identified except that of protein IX. The short E3 region of the PBAdV-1 consisted of two novel, supposedly type-specific ORFs only, whereas no homologue of any of the E3 genes, usually conserved in mastadenoviruses, such as for example that of the 12.5 K protein, were present. In the E4 region, only the highly conserved gene of the 34 K protein was found besides two novel ORFs showing no homology to any known E4 ORFs. In silico sequence analysis revealed putative splicing donor and acceptor sites in the genes of the E1A, IVa2, DNA-dependent DNA polymerase, pTP, 33 K proteins, and also of U exon protein, all being characteristic for mastadenoviruses. Phylogenetic calculations, based on various proteins, further supported that the newly-detected PBAdV is the representative of a new species within the genus Mastadenovirus, and may represent the evolutionary lineage of adenoviruses that coevolved with carnivorans.

First complete genome sequence of garlic virus X infecting Allium sativum- G282 from India

The present report communicates the first full genome sequencing of the Garlic virus X from northern India. The total genome size of Garlic virus X (MK503771) reported in this study is 8458 bp ssRNA. The full genome sequence analysis showed the close relationship of Garlic virus X from India to that of from China, Korea, Australia and Spain. The full genome sequence based study of Indian Garlic virus X reveals the geographical relationship of this virus in India and global origin which may assists in development of control strategy for this virus.

Full genome characterization of 12 citrus tatter leaf virus isolates for the development of a detection assay.

Citrus tatter leaf virus (CTLV) threatens citrus production worldwide because it induces bud-union crease on the commercially important Citrange (Poncirus trifoliata × Citrus sinensis) rootstocks. However, little is known about its genomic diversity and how such diversity may influence virus detection. In this study, full-length genome sequences of 12 CTLV isolates from different geographical areas, intercepted and maintained for the past 60 years at the Citrus Clonal Protection Program (CCPP), University of California, Riverside, were characterized using next generation sequencing. Genome structure and sequence for all CTLV isolates were similar to Apple stem grooving virus (ASGV), the type species of Capillovirus genus of the Betaflexiviridae family. Phylogenetic analysis highlighted CTLV's point of origin in Asia, the virus spillover to different plant species and the bottleneck event of its introduction in the United States of America (USA). A reverse transcription quantitative polymerase chain reaction assay was designed at the most conserved genome area between the coat protein and the 3'-untranslated region (UTR), as identified by the full genome analysis. The assay was validated with different parameters (e.g. specificity, sensitivity, transferability and robustness) using multiple CTLV isolates from various citrus growing regions and it was compared with other published assays. This study proposes that in the era of powerful affordable sequencing platforms the presented approach of systematic full-genome sequence analysis of multiple virus isolates, and not only a small genome area of a small number of isolates, becomes a guideline for the design and validation of molecular virus detection assays, especially for use in high value germplasm programs.

Full genome sequence analysis of a 1-7-4-like PRRSV strain in Fujian Province, China.

PRRS virus (PRRSV) has undergone rapid evolution and resulted in immense economic losses worldwide. In the present study, a PRRSV strain named FJ0908 causing high abortion rate (25%) and mortality (40%) was detected in a swine herd in China. To determine if a new PRRSV genotype had emerged, we characterized the genetic characteristics of FJ0908. Phylogenetic analysis indicated that FJ0908 was related to 1-7-4-like strains circulating in the United States since 2014. Furthermore, the ORF5 sequence restriction fragment length polymorphism (RFLP) pattern of FJ0908 was 1-7-4. Additionally, FJ0908 had a 100 aa deletion (aa329–428) within nsp2, as compared to VR-2332, and the deletion pattern was consistent with most of 1-7-4 PRRSVs. Collectively, the data of this study contribute to the understanding of 1-7-4-like PRRSV molecular epidemiology in China.

Isolation and characterization of an Aves polyomavirus 1 from diseased budgerigars in China

Aves polyomavirus 1 (APV) causes inflammatory disease in psittacine birds, especially in young budgerigar. In this study, an APV virus (SD18 strain) was isolated from a diseased psittacine birds breeding facility. The full genome (4981 bp) of SD18 was determined and analyzed. Phylogenetic analysis of full genome sequences indicated all the APV strains form two groups. The SD18 strain showed close relationship with APV isolated from Poland, however, the other Chinese strains are located in group II, which suggested different genotypes APVs are co-circulating in China. Compared with the consensus sequence of APV full genome, the SD18 strain contains 13 nucleotide mutations, and 2 unique amino acid substitutions (R179M and Q382K) located in VP2/3 and Large T proteins. To explore the pathogenicity of the virus, the SD18 strain was used to challenge 2-week-old budgerigars. All infected birds died no later than 5 days post infection, and virus was detected in multiple organs including brain, heart, ingluvies, liver, and intestine, which indicated that SD18 is fatal and causes systemic infection in young budgerigar. In vitro studies showed that SD18 replicated efficiently in CEF cells and reached the highest viral titers at 9 days post infection. Notably, replication of SD18 stimulated IFN-β response in CEF cells and overexpression of the VP4 or VP4Delta proteins significantly inhibited IFN-β promoter activation, which could be the strategy of APV to escape from the host innate immunity.

A53 MERS-CoV in East African dromedary camels

Human Middle East respiratory syndrome is a zoonotic respiratory disease caused by Middle East respiratory syndrome coronavirus (MERS-CoV) originating from camels in the Arabian Peninsula. While there are a large number of camels in East Africa, often traded to the Arabian Peninsula, no autochthonous human MERS-CoV case is reported in East Africa. Furthermore, there is limited information of MERS-CoV in East Africa. In this study, MERS-CoV in dromedary camels from Ethiopia was detected using RT-qPCR. Next-generation sequencing was used to obtain the full genome of MERS-CoV. MERS-CoV antibodies were also detected through MERS-spike pseudoparticle neutralization assay. Phylogenetic analysis of full-genome sequences and spike-genome antibodies indicates that MERS-CoV in East Africa is genetically distinct from those in the Arabian Peninsula. The results from this study show that MERS-CoV circulating in dromedary camels in East Africa are genetically distinct from those in the Arabian Peninsula. Further studies are needed to evaluate the risk of zoonotic transmission in East Africa.

Comprehensive analysis of full genome sequence and Bd-milRNA/target mRNAs to discover the mechanism of hypovirulence in Botryosphaeria dothidea strains on pear infection with BdCV1 and BdPV1

Pear ring rot disease, mainly caused by Botryosphaeria dothidea, is widespread in most pear and apple-growing regions. Mycoviruses are used for biocontrol, especially in fruit tree disease. BdCV1 (Botryosphaeria dothidea chrysovirus 1) and BdPV1 (Botryosphaeria dothidea partitivirus 1) influence the biological characteristics of B. dothidea strains. BdCV1 is a potential candidate for the control of fungal disease. Therefore, it is vital to explore interactions between B. dothidea and mycovirus to clarify the pathogenic mechanisms of B. dothidea and hypovirulence of B. dothidea in pear. A high-quality full-length genome sequence of the B. dothidea LW-Hubei isolate was obtained using Single Molecule Real-Time sequencing. It has high repeat sequence with 9.3% and DNA methylation existence in the genome. The 46.34 Mb genomes contained 14,091 predicted genes, which of 13,135 were annotated. B. dothidea was predicted to express 3833 secreted proteins. In bioinformatics analysis, 351 CAZy members, 552 transporters, 128 kinases, and 1096 proteins associated with plant-host interaction (PHI) were identified. RNA-silencing components including two endoribonuclease Dicer, four argonaute (Ago) and three RNA-dependent RNA polymerase (RdRp) molecules were identified and expressed in response to mycovirus infection. Horizontal transfer of the LW-C and LW-P strains indicated that BdCV1 induced host gene silencing in LW-C to suppress BdPV1 transmission. To investigate the role of RNA-silencing in B. dothidea defense, we constructed four small RNA libraries and sequenced B. dothidea micro-like RNAs (Bd-milRNAs) produced in response to BdCV1 and BdPV1 infection. Among these, 167 conserved and 68 candidate novel Bd-milRNAs were identified, of which 161 conserved and 20 novel Bd-milRNA were differentially expressed. WEGO analysis revealed involvement of the differentially expressed Bd-milRNA-targeted genes in metabolic process, catalytic activity, cell process and response to stress or stimulus. BdCV1 had a greater effect on the phenotype, virulence, conidiomata, vertical and horizontal transmission ability, and mycelia cellular structure biological characteristics of B. dothidea strains than BdPV1 and virus-free strains. The results obtained in this study indicate that mycovirus regulates biological processes in B. dothidea through the combined interaction of antiviral defense mediated by RNA-silencing and milRNA-mediated regulation of target gene mRNA expression.

Full genome sequence analysis and putative host-shifting of Milk vetch dwarf virus infecting tobacco (Nicotiana tabacum) in China

BackgroundTobacco production in China has been affected by plant viruses with Milk vetch dwarf virus (MDV) as a recent invader posing serious concern. According to most of the studies, MDV mainly infects hosts from Fabaceae family but in our previous study we reported its infection in tobacco plant (Nicotiana tabacum L.) in Shandong province.FindingsIn current study (2016–2017), tobacco plants (Nicotiana tabacum) with severe stunting, yellowing and axillary bunch of new leaves were observed in Zhengning, Gansu province. Isolate GSZN yielded into eight genomic circular single-stranded DNA components while no alphasatellite DNA was obtained. High percent identity of this isolate was recorded in overall nucleotide and amino acid assembly with reported MDV isolates worldwide. Phylogenetic analysis fetched into a separate sub-clade comprising of new isolate along with other tobacco infecting isolates of MDV. While recombination was predicted in DNA-C encoding Clink protein and DNA-U1, which may attribute towards the potential host-shifting phenomenon and ability of this virus to expand its host range.ConclusionTo our knowledge this is the first full genome annotation of a Nanovirus, infecting tobacco in natural field conditions, also this is the first extended analysis on host-shifting behavior of MDV.

An epidemic of chikungunya in northwestern Bangladesh in 2011.

BackgroundIn November 2011, a government hospital physician in Shibganj sub-district of Bangladesh reported a cluster of patients with fever and joint pain or rash. A multi-disciplinary team investigated to characterize the outbreak; confirm the cause; and recommend control and prevention measures.MethodsShibganj's residents with new onset of fever and joint pain or rash between 1 September and 15 December 2011 were defined as chikungunya fever (CHIKF) suspect cases. To estimate the attack rate, we identified 16 outpatient clinics in 16 selected wards across 16 unions in Shibganj and searched for suspect cases in the 80 households nearest to each outpatient clinic. One suspect case from the first 30 households in each ward was invited to visit the nearest outpatient clinic for clinical assessment and to provide a blood sample for laboratory testing and analyses.ResultsWe identified 1,769 CHIKF suspect cases from among 5,902 residents surveyed (30%). Their median age was 28 (IQR:15−42) years. The average attack rate in the sub-district was 30% (95% CI: 27%−33%). The lowest attack rate was found in children <5 years (15%). Anti-CHIKV IgM antibodies were detected by ELISA in 78% (264) of the 338 case samples tested. In addition to fever, predominant symptoms of serologically-confirmed cases included joint pain (97%), weakness (54%), myalgia (47%), rash (42%), itching (37%) and malaise (31%). Among the sero-positive patients, 79% (209/264) sought healthcare from outpatient clinics. CHIKV was isolated from two cases and phylogenetic analyses of full genome sequences placed these viruses within the Indian Ocean Lineage (IOL). Molecular analysis identified mutations in E2 and E1 glycoproteins and contained the E1 A226V point mutation.ConclusionThe consistently high attack rate by age groups suggested recent introduction of chikungunya in this community. Mosquito control efforts should be enhanced to reduce the risk of continued transmission and to improve global health security.

Potential of genotype VII Newcastle disease viruses to cause differential infections in chickens and ducks.

Newcastle disease (ND), caused by ND virus (NDV), is one of the most infectious and economically important diseases of the poultry industry worldwide. While infections are reported in a wide range of avian species, the pathogenicity of chicken-origin virulent NDV isolates in ducks remains elusive. In this study, two NDV strains were isolated and biologically and genetically characterized from an outbreak in chickens and apparently healthy ducks. Pathogenicity assessment indices, including the mean death time (MDT), intracerebral pathogenicity index (ICPI) and cleavage motifs in the fusion (F) protein, indicated that both isolates were velogenic in nature. While these isolates carried pathogenic characteristics, interestingly they showed differential pathogenicity in ducks. The chicken-origin isolate caused high (70%) mortality, whereas the duck-origin virus resulted in low (20%) mortality in 4-week-old ducks. Intriguingly, both isolates showed comparable disease pathologies in chickens. Full-genome sequence analysis showed that the virus genome contains 15192 nucleotides and carried features that are characteristic of velogenic strains of NDV. A phylogenetic analysis revealed that both isolates clustered in class II and genotype VII. However, there were several mutations in the functionally important regions of the fusion (F) and haemagglutinin-neuraminidase (HN) proteins, which may be responsible for the differential pathogenicity of these viruses in ducks. In summary, these results suggest that NDV strains with the same genotype show differential pathogenicity in chickens and ducks. Furthermore, chicken-origin virulent NDVs are more pathogenic for ducks than duck-origin viruses. These findings propose a role for chickens in the evolution of viral pathogenicity and the potential genetic resistance of ducks to poultry viruses.

Molecular evidence for the occurrence of TYLCV on Mentha longifolia in Jeddah, Saudi Arabia.

Begomoviruses are whiteflies transmitted virus causing serious disease in many important plants exhibiting variable symptoms with significant economic loss globally. Mentha is an important crop being grown here in Saudi Arabia for various purposes. The begomovirus associated disease was observed on Mentha crops during field survey which were growing near to tomato field. There is no published report available about the association of begomovirus on Mentha from this region. So, this work was conducted to identify the causative agent associated with yellow vein mosaic disease. Naturally infected samples were collected from various locations and causative agent was identified by PCR using begomovirus specific primers and further cloned and sequenced bidirectionally. The full genome had total 2785 nucleotides while betasatellite molecule had 1365 nucleotides. Based on full-genome sequence analysis, the identity matrix and phylogenetic analysis showed the highest identity (99.6%) with Tomato yellow leaf curl virus (TYLCV) reported from tomato in the Kingdom of Saudi Arabia. The identified begomovirus was observed as isolate of TYLCV.

The Incidence and Genetic Diversity of Apple Mosaic Virus (ApMV) and Prune Dwarf Virus (PDV) in Prunus Species in Australia.

Apple mosaic virus (ApMV) and prune dwarf virus (PDV) are amongst the most common viruses infecting Prunus species worldwide but their incidence and genetic diversity in Australia is not known. In a survey of 127 Prunus tree samples collected from five states in Australia, ApMV and PDV occurred in 4 (3%) and 13 (10%) of the trees respectively. High-throughput sequencing (HTS) of amplicons from partial conserved regions of RNA1, RNA2, and RNA3, encoding the methyltransferase (MT), RNA-dependent RNA polymerase (RdRp), and the coat protein (CP) genes respectively, of ApMV and PDV was used to determine the genetic diversity of the Australian isolates of each virus. Phylogenetic comparison of Australian ApMV and PDV amplicon HTS variants and full length genomes of both viruses with isolates occurring in other countries identified genetic strains of each virus occurring in Australia. A single Australian Prunus infecting ApMV genetic strain was identified as all ApMV isolates sequence variants formed a single phylogenetic group in each of RNA1, RNA2, and RNA3. Two Australian PDV genetic strains were identified based on the combination of observed phylogenetic groups in each of RNA1, RNA2, and RNA3 and one Prunus tree had both strains. The accuracy of amplicon sequence variants phylogenetic analysis based on segments of each virus RNA were confirmed by phylogenetic analysis of full length genome sequences of Australian ApMV and PDV isolates and all published ApMV and PDV genomes from other countries.

An ascovirus isolated from Spodoptera litura (Noctuidae: Lepidoptera) transmitted by the generalist endoparasitoid Meteorus pulchricornis (Braconidae: Hymenoptera).

The family Ascoviridae is a recently described virus family whose members are transmitted by parasitoids and cause chronic and lethal infections in lepidopteran insects. Little is known about the biology and ecology of ascoviruses, and few isolates have been found outside the United States. We report here the isolation of a new ascovirus variant from Spodoptera litura in Japan. Full genome sequence and phylogenetic analyses showed that this virus was closely related to variants in Heliothis virescens ascovirus-3a, and it was named HvAV-3j. HvAV-3j has a DNA genome of 191 718 bp, with 189 putative ORFs and a GC content of 45.6 %, and is highly similar to HvAV-3h, which was isolated in China. In a field survey, the endoparasitoid Meteorus pulchricornis caused a high percentage of parasitization in populations of S. litura larvae, and under laboratory conditions M. pulchricornis was able to transmit HvAV-3j from infected to uninfected larvae by oviposition. Meteorus pulchricornis is thus likely to be a major vector for HvAV-3j transmission in Japan. This species is recognized here for the first time as a vector of ascoviruses that parasitizes a range of host species that extends across families.

ATYPICAL CHLAMYDIACEAE IN WILD POPULATIONS OF HAWKS ( BUTEO SPP.) IN CALIFORNIA.

Chlamydiaceae bacteria infect many vertebrate hosts, and previous reports based on polymerase chain reaction (PCR) assays and serologic assays that are prone to cross-reaction among chlamydial organisms have been used to describe the prevalence of either DNA fragments or antibodies to Chlamydia spp. in wild raptorial populations. This study reports the PCR-based prevalence of Chlamydiaceae DNA that does not 100% match any avian or mammalian Chlamydiaceae in wild populations of hawks in California Buteo species. In this study, multimucosal swab samples ( n = 291) for quantitative PCR (qPCR) and plasma ( n = 78) for serology were collected from wild hawks. All available plasma samples were negative for antibodies using a C. psittaci-specific elementary body agglutination test (EBA; n = 78). For IgY antibodies all 51 available samples were negative using the indirect immunofluorescent assay. The overall prevalence of Chlamydiaceae DNA detection in wild Buteo species sampled was 1.37% (4/291) via qPCR-based analysis. Two fledgling Swainson's hawks ( Buteo swainsoni) and two juvenile red-tailed hawks ( Buteo jamaicensis) were positive by qPCR-based assay for an atypical chlamydial sequence that did not 100% match any known C. psittaci genotype. Positive swab samples from these four birds were sequenced based on the ompA gene and compared by high-resolution melt analysis with all known avian and mammalian Chlamydiaceae. The amplicon sequence did not 100% match any known avian chlamydial sequence; however, it was most similar (98.6%) to C. psittaci M56, a genotype that is typically found in muskrats and hares. Culture and full genome sequence analysis of Chlamydia spp. isolated from diseased hawks will be necessary to classify this organism and to better understand its epizootiology and potential health impact on wild Buteo populations in California.

Spatial transmission of H5N6 highly pathogenic avian influenza viruses among wild birds in Ibaraki Prefecture, Japan, 2016-2017.

From 29 November 2016 to 24 January 2017, sixty-three cases of H5N6 highly pathogenic avian influenza virus (HPAIV) infections were detected in wild birds in Ibaraki Prefecture, Japan. Here, we analyzed the genetic, temporal, and geographic correlations of these 63 HPAIVs to elucidate their dissemination throughout the prefecture. Full-genome sequence analysis of the Ibaraki isolates showed that 7 segments (PB2, PB1, PA, HA, NP, NA, NS) were derived from G1.1.9 strains while the M segment was from G1.1 strains; both groups of strains circulated in south China. Pathological studies revealed severe systemic infection in dead swans (the majority of dead birds and the only species necropsied), thus indicating high susceptibility to H5N6 HPAIVs. Coalescent phylogenetic analysis using the 7 G1.1.9-derived segments enabled detailed analysis of the short-term evolution of these highly homologous HPAIVs. This analysis revealed that the H5N6 HPAIVs isolated from wild birds in Ibaraki Prefecture were divided into 7 groups. Spatial analysis demonstrated that most of the cases concentrated around Senba Lake originated from a single source, and progeny viruses were transmitted to other locations after the infection expanded in mute swans. In contrast, within just a 5-km radius of the area in which cases were concentrated, three different intrusions of H5N6 HPAIVs were evident. Multi-segment analysis of short-term evolution showed that not only was the invading virus spread throughout Ibaraki Prefecture but also that, despite the small size of this region, multiple invasions had occurred during winter 2016-2017.

In Silico Characterisation of a Type D Betaretrovirus from Malayan Flying Fox, Pteropus vampyrus

Bats are well known for their role as reservoirs of viral diseases. Previous studies have reported the presence of endogenous gamma and betaretrovirus in diverse species of bats. Here, we characterize one of these lineages- Pteropus vampyrus endogenous betaretrovirus namely PvEB in depth describing the genomic organization, age estimation and phylogenetic approaches to examine the origin and history of this virus. Phylogenetic analysis of gag, pol and full genome sequence has placed PvEB robustly within the betaretrovirus genus. Subsequently, PvEB was estimated to invade the bat genome since 1.5-8.8mya. The results of bioinformatics analysis were verified through amplification of the PvEB reverse transcriptase gene with the size of 700bp. With these findings together with the previous report, it is evident that PvEB is a member of type D betaretrovirus group.