Mourilyan Virus Research Articles

A Novel Bunyavirus Discovered in Oriental Shrimp (Penaeus chinensis).

Herein, we describe a novel bunyavirus, oriental wenrivirus 1 (OWV1), discovered in moribund oriental shrimp (Penaeus chinensis) collected from a farm in China in 2016. Like most bunyaviruses, OWV1 particles were enveloped, spherical- to ovoid-shaped, and 80–115 nm in diameter. However, its genome was found to comprise four segments of (-)ssRNA. These included an L RNA segment (6,317 nt) encoding an RNA-directed RNA polymerase (RdRp) of 2,052 aa, an M RNA segment (2,978 nt) encoding a glycoprotein precursor (GPC) of 922 aa, an S1 RNA segment (1,164 nt) encoding a nucleocapsid (N) protein of 243 aa, and an S2 RNA segment (1,382 nt) encoding a putative non-structural (NSs2) protein of 401 aa. All the four OWV1 RNA segments have complementary terminal decanucleotides (5′-ACACAAAGAC and 3′-UGUGUUUCUG) identical to the genomic RNA segments of uukuviruses and similar to those of phleboviruses and tenuiviruses in the Phenuiviridae. Phylogenetic analyses revealed that the RdRp, GPC, and N proteins of OWV1 were closely related to Wēnzhōu shrimp virus 1 (WzSV-1) and Mourilyan virus (MoV) that infect black tiger shrimp (P. monodon). Phylogenetic analyses also suggested that OWV1 could be classified into a second, yet to be established, species of the Wenrivirus genus in the Phenuiviridae. These wenriviruses also clustered with Wenling crustacean virus 7 from shrimps and bunya-like brown spot virus from white-clawed crayfish. Of note there were no homologs of the NSs2 of OWV1 and MoV/WzSV-1 in GenBank, and whether other crustacean phenuiviruses also possess a similar S2 RNA segment warrants further investigation. In addition, we established a TaqMan probe-based reverse-transcription quantitative PCR method for detection of OWV1, and it was detected as 1.17 × 102—1.90 × 107 copies/ng-RNA in gills of 23 out of 32 P. chinensis samples without an obvious gross sign. However, the discovery of OWV1 highlights the expanding genomic diversity of bunyaviruses.

The genomes of Mourilyan virus and Wēnzhōu shrimp virus 1 of prawns comprise 4 RNA segments

Reported here is the complete genome sequence of Mourilyan virus (MoV) that infects giant tiger (Penaeus monodon) and kuruma prawns (P. japonicas) in Australia. Its genome was determined using various PCR strategies based on the sequences of 3 randomly-amplified cDNA clones to its L and M RNA segments discovered in a library generated to determine the genome sequence of gill-associated ronivirus. The sequences of PCR products and clones obtained showed the MoV genome to comprise 4 ssRNA segments (L, M, S1 and S2), as confirmed by Northern blotting using RNA from naïve and MoV-infected prawns, and by Illumina sequence analysis of semi-purified MoV. BLASTn searches identified the MoV L, M and S1 RNA segments to be homologous to Wēnzhōu shrimp virus 1 (WzSV1) segments discovered recently in a P. monodon RNA-Seq library (SRR1745808). Mapping this read library to the MoV S2 RNA segment identified WzSV1 to also possess an equivalent segment. BLASTp searches identified the putative non-structural protein (NSs2; 393–394 aa) encoded in their S2 RNA segments to have no homologs in GenBank. Possibly due to NSs2 being encoded in a discrete RNA segment rather than in ambisense relative to the N protein as in the S RNA segments of other phenuiviruses, each of 6 MoV S1 RNA segment clones sequenced possessed a variable-length (≤ 645 nt) imperfect GA-repeat extending from the N protein stop codon to the more variable ∼90 nt segment terminal sequence. Read mapping of RNA-Seq library SRR1745808 showed the WzSV1 S1 RNA segment to possess a similar GA-repeat. However, paired-read variations hindered definitive assembly of a consensus sequence. All 4 MoV and WzSV1 RNA segments terminated with a 10 nt inverted repeat sequence (5′-ACACAAAGAC.) identical to the RNA segment termini of uukuviruses. Phylogenetic analyses of MoV/WzSV1 RNA-dependant RNA polymerase (L RNA), G1G2 precursor glycoprotein (M RNA) and nucleocapsid (N) protein (S1 RNA) sequences generally clustered them with as yet unassigned crustacean/diptera bunya-like viruses on branches positioned closely to others containing tick-transmitted phenuiviruses. As genome sequences of most phenuiviruses discovered recently have originated from meta-transcriptomics studies, the data presented here showing the MoV and WzSV1 genomes to comprise more than 3 RNA segments, like the plant tenuiviruses, suggests a need to investigate the genomes of these unassigned viruses more closely.

Mourilyan virus pathogenicity in kuruma shrimp (Penaeus japonicus).

The bunyavirus Mourilyan virus (MoV) occurs commonly in Black tiger (Penaeus monodon) and kuruma shrimp (Penaeusjaponicus) farmed in eastern Australia. There is circumstantial evidence of MoV causing mortalities among P.japonicus moved from farm ponds to tanks for rearing as broodstock. To directly assess its pathogenic potential, independent cohorts of pond- (n=24) or tank-reared juvenile (n=21) P.japonicus were challenged intramuscularly with a cephalothorax tissue homogenate of P.monodon containing high loads of MoV (1.48±0.28×108 MoV RNA copies/µg total RNA). In each trial, mortalities accumulated gradually among the saline-injected controls. Mortality onset occurred 12-14days earlier in the pond-reared shrimp, possibly due to them possessing low-level pre-existing MoV infections. Despite the time to onset of mortality differing, Kaplan-Meier survival analyses confirmed mortality rates to be significantly higher in both the pond- (p=.017) and tank-reared shrimp (p=.031) challenged with MoV. RT-qPCR data on shrimp sampled progressively over each trial showed high loads of MoV to establish following challenge and discounted GAV and other endemic viruses from contributing to mortality. Together, the data show that acute MoV infection can adversely compromise the survival of juvenile P.japonicus.

RNA-Binding Domain in the Nucleocapsid Protein of Gill-Associated Nidovirus of Penaeid Shrimp

Gill-associated virus (GAV) infects Penaeus monodon shrimp and is the type species okavirus in the Roniviridae, the only invertebrate nidoviruses known currently. Electrophoretic mobility shift assays (EMSAs) using His6-tagged full-length and truncated proteins were employed to examine the nucleic acid binding properties of the GAV nucleocapsid (N) protein in vitro. The EMSAs showed full-length N protein to bind to all synthetic single-stranded (ss)RNAs tested independent of their sequence. The ssRNAs included (+) and (−) sense regions of the GAV genome as well as a (+) sense region of the M RNA segment of Mourilyan virus, a crustacean bunya-like virus. GAV N protein also bound to double-stranded (ds)RNAs prepared to GAV ORF1b gene regions and to bacteriophage M13 genomic ssDNA. EMSAs using the five N protein constructs with variable-length N-terminal and/or C-terminal truncations localized the RNA binding domain to a 50 amino acid (aa) N-terminal sequence spanning Met11 to Arg60. Similarly to other RNA binding proteins, the first 16 aa portion of this sequence was proline/arginine rich. To examine this domain in more detail, the 18 aa peptide (M11PVRRPLPPQPPRNARLI29) encompassing this sequence was synthesized and found to bind nucleic acids similarly to the full-length N protein in EMSAs. The data indicate a fundamental role for the GAV N protein proline/arginine-rich domain in nucleating genomic ssRNA to form nucleocapsids. Moreover, as the synthetic peptide formed higher-order complexes in the presence of RNA, the domain might also play some role in protein/protein interactions stabilizing the helical structure of GAV nucleocapsids.

Pathogenicity of gill-associated virus and Mourilyan virus during mixed infections of black tiger shrimp (Penaeus monodon)

Gill-associated virus (GAV) and Mourilyan virus (MoV) can occur at very high prevalence in healthy black tiger shrimp (Penaeus monodon) in eastern Australia, and both have been detected in moribund shrimp collected from mid-crop mortality syndrome (MCMS) outbreaks. Experimental evidence presented here indicates that GAV, but not MoV, is the cause of MCMS. Firstly, in healthy P. monodon used for experimental infections, pre-existing MoV genetic loads were very high (mean >10(9) viral RNA copies μg(-1) total RNA) and did not increase significantly following lethal challenge with an inoculum containing both GAV and MoV. In contrast, GAV genetic loads prior to challenge were low (mean ∼10(5) RNA copies μg(-1) total RNA) and increased >10(4)-fold in moribund shrimp. Secondly, dsRNAs targeted to the GAV RNA-dependent RNA polymerase (RdRp) or helicase gene regions reduced GAV genetic loads, delayed the onset of mortalities and improved survival following challenge. In contrast, dsRNA targeted to the MoV RdRp gene (L RNA) was highly effective in reducing MoV genetic loads, but mortality rates were unaffected. Targeting of the MoV S2 RNA, encoding a small non-structural protein (NSs2), a putative supressor of RNA interference, did not reduce the MoV genetic loads or enhance knockdown of GAV when administered simultaneously with dsRNA targeted to the GAV helicase gene. Overall, the data show that P. monodon can tolerate a high-level MoV infection and that mortalities are associated with GAV infection.

Prevalence of shrimp viruses in wild Penaeus monodon from Brunei Darussalam

Over a period of 30 months, wild black tiger shrimp ( Penaeus monodon) were collected from the South China Sea in South-east Asia, in an effort to establish specific pathogen free (SPF) stocks of P. monodon in Brunei Darussalam. The viral status of the broodstock was assessed by real-time and conventional polymerase chain reaction (PCR) using DNA and reverse transcribed RNA extracted from the gill, hepatopancreas and pleopods. Histopathology was performed to confirm PCR results. Over 270 P. monodon were collected, screened and spawned. Of the nine viruses assessed, infectious hypodermal and hematopoietic necrosis virus (IHHNV) was most commonly detected (19.6%), followed by monodon baculovirus (MBV) (7.4%), hepatopancreatic parvovirus (HPV) (3.8%), and Mourilyan virus (MoV) (0.9%). The only multiple viral infections found were a combination of IHHNV and MBV (2.2%). Interestingly, the two most virulent viruses for P. monodon, white spot syndrome virus (WSSV) and yellowhead virus (YHV) were not detected in any shrimp. It is concluded that wild P. monodon from Brunei waters have a low prevalence of viral pathogens, which has permitted the development of SPF stocks as a foundation population for selective breeding.

In situ stress testing to identify Australian black tiger prawns (Penaeus monodon) free of gill‐associated virus and Mourilyan virus

To identify whether black tiger prawns (Penaeus monodon) in the Weipa region of the Gulf of Carpentaria, Queensland, are free of gill-associated virus (GAV) and Mourilyan virus (MoV), which are endemic in P. monodon along the east coast of Queensland. Preliminary screening suggested that Weipa might be a source of P. monodon that are free of GAV and MoV. To assess this, more than 150 prawns captured near Weipa were maintained locally in tanks for 2 weeks and bled three times as a stressor to promote higher-level infections. The existence of GAV and MoV in lymphoid organ tissue was then determined using real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Some prawns were maintained in tanks for an additional 75 days before being tested. Real-time qRT-PCR did not detect GAV in any of 33 pools of RNA isolated from the 166 prawns tested. MoV was detected in five pools of RNA at extremely low viral RNA copy numbers close to the sensitivity threshold of the test. MoV was also detected at a similar low copy number in one of nine pearl-oyster mantle samples used as negative controls. GAV infection is either absent or, like MoV, potentially present at a very low prevalence in juvenile P. monodon inhabiting the inshore waters at Weipa. This region can thus be recommended as a good source of P. monodon certifiable as specific pathogen-free for GAV and MoV, which is desirable for domestication and selective breeding programs in Australia.

A key gene of the RNA interference pathway in the black tiger shrimp, Penaeus monodon: Identification and functional characterisation of Dicer-1

RNA interference (RNAi) is an evolutionarily conserved mechanism by which double-stranded RNA (dsRNA) initiates post-transcriptional silencing of homologous genes. Here we report the amplification and characterisation of a full length cDNA from black tiger shrimp ( Penaeus monodon) that encodes the bidentate RNAase III Dicer, a key component of the RNAi pathway. The full length of the shrimp Dicer (Pm Dcr1) cDNA is 7629 bp in length, including a 5′ untranslated region (UTR) of 130 bp, a 3′ UTR of 77 bp, and an open reading frame of 7422 bp encoding a polypeptide of 2473 amino acids with an estimated molecular mass of 277.895 kDa and a predicted isoelectric point of 4.86. Analysis of the deduced amino acid sequence indicated that the mature peptide contains all the seven recognised functional domains and is most similar to the mosquito ( Aedes aegypti) Dicer-1 sequence with a similarity of 34.6%. Quantitative RT–PCR analysis showed that Pm Dcr1 mRNA is most highly expressed in haemolymph and lymphoid organ tissues ( P < 0.05). However, there was no correlation between Pm Dcr1 mRNA levels in lymphoid organ and the viral genetic loads in shrimp naturally infected with gill-associated virus (GAV) and Mourilyan virus ( P > 0.05). Treatment with synthetic dsRNA corresponding to Pm Dcr1 sequence resulted in knock-down of Pm Dcr1 mRNA expression in both uninfected shrimp and shrimp infected experimentally with GAV. Knock-down of Pm Dcr1 expression resulted in more rapid mortalities and higher viral loads. These data demonstrated that Dicer is involved in antiviral defence in shrimp.

A TaqMan real-time RT-PCR for quantifying Mourilyan virus infection levels in penaeid shrimp tissues

A highly sensitive and specific TaqMan real-time quantitative RT-PCR (qRT-PCR) was developed to detect and quantify Mourilyan virus (MoV), a newly described bunya-like virus of penaeid shrimp. The PCR primers and TaqMan probe targeted a 67-nucleotide (nt) sequence in the MoV M RNA segment. Using dilution series of a 849 nt RNA transcribed in vitro from cDNA clone pMoV4.1, the assay could detect down to a single MoV RNA equivalent, reliably detected 10 RNA copies and had a log linear range up to 1 × 10 9 RNA copies. In experimentally infected Penaeus japonicus shrimp, the test was used to quantify increases in MoV loads over time in hemocytes, lymphoid organ and gills. Sequential increases in MoV RNA copy numbers occurred in lymphoid organ and gill tissues collected at 6, 24 and 48 h post-infection. However, RNA copy numbers decreased slightly in hemocytes sampled at 48 h compared to 24 h. The qRT-PCR data correlated well with amplicon yields generated using a conventional RT-nested PCR targeting the same MoV RNA segment. Moreover, histology and in situ hybridisation using shrimp cephalothorax sections identified increases in lymphoid organ spheroid numbers and confirmed that increases in MoV RNA detected in lymphoid organ tissue were due to expansion in the numbers of infected cells. The qRT-PCR assay should find use in high-throughput screening applications to detect MoV in broodstock and postlarvae used for culture or breeding purposes and for tracking changes in infection levels during shrimp grow-out.

Association of Mourilyan virus with mortalities in farm pond-reared Penaeus ( Marsupenaeus) japonicus transferred to maturation tank systems

A previous study of captive breeding of Penaeus ( Marsupenaeus) japonicus demonstrated that broodstock reared from egg to maturity in controlled environment tanks had significantly higher survival rates than sibling broodstock sourced from farm ponds. The pronounced difference in survival of these siblings prompted the current study of Mourilyan virus (MoV) loads in the tank-reared and pond-reared stocks. We examined variation in MoV prevalence in each of five batches of sibling P. japonicus that had been reared for 6 months in either controlled environment tanks or earthen farm ponds before being transferred to maturation tanks. Screening using RT-nested PCR showed no detectible levels of MoV in any of the batches of 20-day-old postlarvae (PL 20) at the time that the tanks and ponds were stocked. Six months later, at the start of the 56-day maturation phase, MoV remained undetectable in any of the five batches of tank-reared shrimp. However, among the pond-reared shrimp, MoV was variably detected in all five sibling batches. After maturation for 56 days, MoV remained undetectable in all of 15 shrimp sampled from three of the tank-reared batches but was detectable by RT-PCR in 2 of the 10 shrimp sampled in the other two batches. In contrast, MoV was detected by RT-PCR in most (28/30) shrimp from the five sibling batches of P. japonicus that had previously been reared in ponds. The low prevalence of MoV in the tank-reared stocks was associated with high mean survival (± S.E.) (75.81 ± 7.12%). Conversely, the high prevalence of MoV in the pond-reared stocks was associated with low mean survival (11.29 ± 1.42%). The results indicate that P. japonicus broodstock reared in controlled environment systems are less at risk from MoV, due to lower infection rates or a greater capacity to tolerate infection, compared to putative broodstock sourced from farm ponds.

RT-nested PCR detection of Mourilyan virus in Australian Penaeus monodon and its tissue distribution in healthy and moribund prawns

Mourilyan virus (MoV) is a newly identified virus of Penaeus monodon prawns that is genetically related to the Uukuniemi virus and other phleboviruses of the Bunyaviridae. This paper describes an RT-nested PCR test that can reliably detect between 2 and 6 copies of a synthetic MoV RNA. Total RNA isolated from the lymphoid organ, gills and haemocytes of P. monodon with moderate infections gave comparable amplicon yields in the RT-PCR step of the test. However, in prawns with extremely low-level infections, haemocytes and gill tissue proved slightly more reliable in detecting MoV RNA following nested PCR. The distribution of MoV in tissues of healthy and moribund P. monodon was examined by in situ hybridisation (ISH) using a digoxigenin-labelled DNA probe to a approximately 0.8 kb M RNA segment cDNA insert in clone pMoV4.1. The DNA probe targeted a region in the MoV M RNA segment containing a coding sequence with homology to the C-terminus of the G2 glycoprotein of phleboviruses. In healthy prawns harbouring an unapparent MoV infection, ISH signal primarily occurred in the lymphoid organ, where it was more prominent in hypertrophied cells of 'spheroids' than within cells of normal tubules. ISH signal was also sometimes detected in cells of cuticular epithelium, segmental nerve ganglion and the antennal and tegmental glands. MoV was distributed widely throughout these and other cephalothoracic tissues of mesodermal and ectodermal origin in moribund P. monodon following experimental infection or collected from farm pond edges during disease episodes. Transmission electron microscopy of gill of moribund, captive-reared P. monodon identified spherical (approximately 85 nm diameter) to ovoid MoV particles (approximately 85 x 100 nm) in and around highly necrotic cells in which the nucleus and other organelles had disintegrated. MoV virions co-existed with rod-shaped virions of gill-associated virus and were often seen clustered within cytoplasmic vacuoles or associated with the outer rim of concentric ring-shaped structures comprised of endoplasmic membranes likely to represent degenerated Golgi.