Genetic Characterization of Palyam Serogroup Viruses Isolated in Japan from 1984 to 2018 and Development of a Real-Time RT-PCR Assay for Broad Detection of Palyam Serogroup Viruses and Specific Detection of Chuzan (Kasba) and D’Aguilar Viruses

We performed whole genome sequencing (WGS) of 15 Palyam serogroup virus (PALV) strains isolated from cattle or Culicoides biting midges in Japan from 1984 to 2018. We found that the PALV strains consisted of Chuzan (Kasba) virus (CHUV), D‘Aguilar virus (DAGV), Bunyip Creek virus, and another PALV, Marrakai virus (MARV). The Japanese MARV strains isolated in 1997 were closely related to Australian PALV strains isolated in 1968–1976 in genome segments 2 and 10, but they were most closely related to other Japanese PALV strains in the other genome segments. Our data suggest that the Japanese MARV strains were reassortant viruses between Asian and Australian PALVs. In addition to the WGS, we developed a real-time reverse-transcription polymerase chain reaction assay that can broadly detect PALV and specifically detect CHUV and DAGV, utilizing the data obtained by the WGS in this study. We detected the DAGV gene in bovine stillborn fetuses and congenitally abnormal calves in 2019 using the newly developed assay. To our knowledge, this is the first report of isolation of MARV outside of Australia and the first report of detection of PALV in bovine fetuses or calves with congenital abnormality outside of Africa.


Introduction
Orbivirus palyamense is a member of the genus Orbivirus belonging to the family Sedoreoviridae.This virus is commonly described as the Palyam serogroup, and 13 serotypes have been recognized to date: Palyam, Kasba, Vellore, Abadina, D'Aguilar, Nyabira, CSIRO Village, Marrakai, Gweru, Bunyip Creek, Petero, Marondera, and Kindia [1].The virus is transmitted by arthropod vectors, and some serotypes of this virus are associated with abortion and teratology in cattle and possibly other ruminants [1].Chuzan virus (CHUV) was originally isolated from bovine blood and Culicoides oxystoma in Japan in 1985 and later identified as Kasba virus [2,3].CHUV causes Chuzan disease in cattle, which is characterized by congenital abnormalities with hydranencephaly-cerebellar hypoplasia (HCH) syndrome in calves and affected calves can cause impairment of mobility and neurological symptoms [4][5][6].An epidemic of Chuzan disease occurred in Japan between November 1987 and April 1988, affecting at least 2463 calves [5,6].Additionally, another Palyam serogroup virus (PALV), D'Aguilar virus (DAGV), is thought to be involved in bovine stillbirths and congenital abnormalities in calves [7].
PALVs have been isolated in Africa (South Africa, Zimbabwe, and the Central African Republic), Asia (India, Japan, and China), and Australia [1,15,16].In Japan, the whole genome sequence of the prototype strain of CHUV has been determined [9], but sequence data of other PALV strains are limited to partial sequences, and the relationship between Japanese and foreign PALV strains is not well understood.Therefore, the first objective of this study was to analyze whole genome sequences (WGS) of Japanese PALV strains isolated from cattle or Culicoides biting midges in Japan between 1984 and 2018.
A conventional reverse-transcription polymerase chain reaction (RT-PCR) assay has been used to diagnose congenital abnormalities in cattle and identify viruses isolated from sentinel cattle or Culicoides biting midges in Japan [17].However, no PALV has been detected in affected fetuses or calves, likely because most of the PALV in affected fetuses or calves is eliminated during the several months between when the dams are infected with CHUV or DAGV and when abnormal calving occurs.Therefore, diagnosis is usually based on the detection of neutralizing antibodies in serum from calves that have not ingested colostrum, pathological findings of the fetuses or calves, and seroprevalence of sentinel cattle.A problem with the diagnosis is that if an affected calf acquires maternal antibodies by ingesting colostrum, it is not possible to distinguish whether the neutralizing antibodies originate from the dam or are produced by the calf, making it difficult to identify the calf's infection.To solve this problem, it is necessary to detect the virus, rather than neutralizing antibodies, in the affected calves, and it is thus necessary to develop a highly sensitive assay that can detect PALV, especially CHUV and DAGV.To date, no real-time RT-PCR assay to detect PALV has been reported in a peer-reviewed journal, although some have been developed [18,19].Since many PALVs have been isolated in Japan, we believe that developing a real-time RT-PCR assay utilizing the sequence data of Japanese PALV strains, as well as PALVs isolated in other countries, will provide higher sensitivity and specificity.Thus, the second objective was to develop a real-time RT-PCR assay that can detect CHUV, DAGV, and other PALVs utilizing the sequence data of Japanese PALV strains to be analyzed in this study.
Overall, this study aims to fill the knowledge gaps in the genetic characterization of Japanese PALV by the WGS and to develop a real-time RT-PCR assay for the detection of PALV with high sensitivity and specificity.The successful completion of these objectives will significantly advance our understanding of PALV epidemiology and enhance our ability to diagnose PALV-related diseases in cattle.

Viruses
The fifteen Japanese PALV strains used for genetic analysis in this study are listed in Table 1.The strains were originally isolated from bovine erythrocytes or Culicoides biting midges between 1984 and 2018 in four prefectures in Kyushu Island and Okinawa Prefecture in southwestern Japan (Figure S1).Additionally, two more Japanese PALV strains  CSIRO 11 andCSIRO 82 isolated in 1968-1981) and a Zimbabwean PALV strain (VRL792/73 isolated in 1973) were used for developing a real-time RT-PCR assay.All the strains were propagated in hamster lung (HmLu-1) cell cultures until approximately 90% of the monolayer showed cytopathic effects (CPE).

Full-Length Amplification of cDNAs (FLAC), Next-Generation Sequencing (NGS) and Sanger Sequencing
For whole genome sequencing (WGS), the 15 Japanese PALV strains were propagated in HmLu-1 cell cultures.We then extracted viral dsRNA from the infected cell cultures and ligated an anchor primer (p-GACCTCTGAGGATTCTAAAC/iSp9/TCCAGTTTAGAATCC-OH) for full-length amplification of cDNAs (FLAC) to the 3 -termini of the dsRNA using T4 RNA ligase 1 (New England BioLabs, Ipswich, MA, USA) as described [20,21].The viral dsRNA ligated to the anchor primer was purified by using the Monarch RNA Cleanup Kit (New England BioLabs) and then subjected to double-stranded cDNA synthesis by using the NEBNext RNA Ultra First Strand Synthesis Module and the NEBNext RNA Ultra Second Strand Synthesis Module (New England BioLabs).We used the cDNA to prepare libraries for the next-generation sequencing (NGS) process using the TruSeq DNA Nano LT Library Prep Kit (Illumina, San Diego, CA, USA).We then loaded the libraries on an iSeq 100 i1 Reagent (Illumina) and sequenced on iSeq 100 (Illumina).The sequence reads were subjected to de novo assembly by using CLC Genomics Workbench 12 (Qiagen, Hilden, Germany), and the possible viral sequences were identified from contigs to compare sequences in GenBank using BLASTx.We amplified probable gaps between the obtained contigs by RT-PCR and sequenced the products by using BigDye Terminator v 3.1 (Thermo Fisher Scientific, Waltham, MA, USA) on an ABI 3100-Avanti Genetic Analyzer (Thermo Fisher Scientific).

Phylogenetic Analysis
To analyze the phylogenetic relationships among PALV strains, we aligned the coding sequence of each genome segment using CLUSTAL W (version 2.1) [22] and constructed phylogenetic trees with MEGA X software (version 10.2.6) using the method of maximum likelihood [23]; we evaluated the reliability of the branching orders by the bootstrap test (1000 replicates).We used GENETYX version 15 (Genetyx, Tokyo, Japan) to calculate sequence identities among the strains.The nucleotide sequence data reported herein have been deposited in the DNA Data Bank of Japan (DDBJ) under accession numbers LC601654-LC601657, LC601662-LC601669, LC601674-LC601685, and LC818215-LC818340.

Primer and Probe Design for a Real-Time RT-PCR Assay
The sequences of the primers and probes for the real-time RT-PCR assay are shown in Table 2. Two sets of primers and a probe were designed for the broad-range detection of PALV: a set of primers (PALV/S9/182F and PALV/S9/246R) and a probe (PALV/S9/204P) based on conserved regions of PALV Seg-9 (Pan-PALV-1 set), and another set of primers (PALV/S7/1047F and PALV/S7/1109R) and a probe (PALV/S7/1067P) based on conserved regions of PALV Seg-7 (Pan-PALV-2 set).Additionally, two other sets of primers and a probe were designed for specific detection of CHUV or DAGV: a set of primers (CHUV/S2/316F and CHUV/S2/443R) and a probe (CHUV/S2/365P) based on conserved regions of CHUV Seg-2 (Chuzan-specific set), and another set of primers (DAGV/S2/10F and DAGV/S2/99R) and a probe (DAGV/S2/49P) based on conserved regions of DAGV Seg-2 (D'Aguilar-specific set).All the primers and probes were designed using the sequence data obtained by the WGS in this study and other PALV sequence data available in GenBank.The sequences of the primers and probes were prescreened with a free webbased tool for analyzing primers, Net Primer (Premier Biosoft International, Palo Alto, CA, USA), for the real-time RT-PCR assays to work properly.The sequences of the primers and probes were also checked with the Basic Local Alignment Search Tool (BLAST) to prevent non-specific reactions.

A Real-Time RT-PCR Assay
The conditions for a real-time RT-PCR assay were optimized with the SuperScript III Platinum One-Step Quantitative RT-PCR System (Invitrogen/Life Technologies, Carlsbad, CA, USA) and a real-time PCR system, MyiQ 2 (BIO-RAD, Hercules, CA, USA).For each RNA sample, 2 µL was mixed with 23 µL of reaction mix containing 12.5 µL of 2× Reaction Mix (included in the kit), 10 pmol of each primer, 3 pmol of each probe, 0.5 µL of SuperScript III RT/Platinum Taq Mix (kit), 0.5 µL of RNase OUT Recombinant Ribonuclease Inhibitor (Invitrogen) and nuclease-free water.Since the MyiQ 2 can detect two excited fluorophores, the assays were designed to be multiplex assays to detect 6-carboxyfluorescein (FAM) and 6-VIC or hexachloro-6-carboxyfluorescein (HEX) fluorophores in each tube.The probes of Pan-PALV-1 and Chuzan-specific sets (PALV/S9/204P and CHUV/S2/365P) were both labeled with FAM at the 5 end, the probe of Pan-PALV-2 set (PALV/S7/1067P) was labeled with VIC at the 5 end, and the probe of D'Aguilar-specific set (DAGV/S2/49P) was labeled with HEX at the 5 end, respectively.A primer and probe set for an internal control gene, the bovine β-actin gene (β-actin set), was also used for bovine samples [24], and 5 pmol of each primer and 2.5 pmol of the probe were used per reaction.Both FAM-labeled and HEX-labeled probes for the bovine β-actin gene were prepared, and one of them was selected depending on the fluorophore of the other probe used in the multiplex assays.Probes of the Pan-PALV-1 and Pan-PALV-2 sets (PALV/S9/204P and PALV/S7/1067P) were labeled with minor groove binder-non-fluorescent quencher (MGB-NFQ) at the 3 end, and the other probes were labeled with the dark quencher, BHQ-1, at the 3 end.The conditions for the assays were as follows: 50 • C for 15 min for reverse transcription (RT), 95 • C for 2 min for inactivation of the RT enzyme and initial denaturation, 40 cycles of 95 • C for 15 s (denaturation), and 60 • C for 30 s (annealing and extension).

Evaluation of Analytical Specificity and Sensitivity of the Real-Time RT-PCR Assay
The analytical specificity of the assays was determined by using RNA samples extracted from 17 Japanese PALV strains, four Australian PALV strains, a Zimbabwean PALV strain and six other arboviruses (Akabane, Aino, Peaton, bluetongue, epizootic hemorrhagic disease and bovine ephemeral fever viruses).Viral RNA was extracted from the supernatant of the virus-infected cell cultures using a High Pure Viral RNA Kit (Roche Diagnostics, Basel, Switzerland).The RNA samples were tested in duplicate in multiplex assays with a combination of Pan-PALV-1 and Pan-PALV-2 sets and another combination of Chuzan-and D'Aguilar-specific sets, respectively.
In order to produce standard RNA for the real-time RT-PCR assay, artificial RNA templates were synthesized containing target sequences by molecular cloning and following in vitro transcription as previously described [25].CHUV strain ON-1/E/02, DAGV strain ON-1/E/18 and Bunyip Creek virus (BCV) strain ON-14/E/17 were selected for the synthesis, and cDNA containing target sequences was amplified by RT-PCR with the QIAGEN OneStep RT-PCR Kit (Qiagen).The primers used for the RT-PCR are shown in Table 2.The products of the in vitro transcription were purified with an RNeasy Mini Kit (Qiagen), and the concentration and purity of the purified RNA were measured with a NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA).After the measurement, 10-fold dilutions were prepared as 1 × 10 9 to 1 × 10 1 copies/2 µL and used as standards for validation of the real-time RT-PCR assay.
The sensitivity of the assay was also examined by using spiked samples.To prepare spiked samples, 50 µL of propagated virus (cell culture supernatant; CHUV strain ON-1/E/02 at 1.58 × 10 6 TCID 50 /mL, DAGV strain ON-1/E/18 at 1.32 × 10 5 TCID 50 /mL, and BCV strain ON-14/E/17 at 1.00 × 10 6 TCID 50 /mL) was added to 950 µL of EDTA-treated whole blood or brain homogenate of a healthy cow (PALV-free) and then mixed well.Next, the samples were centrifuged, and the supernatant was collected for RNA extraction.In parallel, 50 µL of each virus was mixed with 950 µL of Eagle's MEM.Then, RNA was extracted from both the supernatant and the MEM using the High Pure Viral RNA Kit (Roche Diagnostics).It was then used as a template for the real-time RT-PCR assay.

Validation of the Real-Time RT-PCR Assay Using Field-Collected Samples
The diagnostic specificity of the assay was examined by using PALV-free bovine blood and tissue samples.RNA was extracted from 31 tissue samples from 22 cattle and 11 blood cell samples from 11 cattle using the High Pure Viral RNA kit (Roche Diagnostics) and used as templates for the real-time RT-PCR assay.The diagnostic sensitivity of the assay was examined using blood and tissue samples collected from stillborn fetuses and newborn calves with Chuzan disease-like congenital abnormalities in Japan from February 2019 to April 2019.A main feature in the stillborn fetuses was cerebral defects, and the main features in the calves were inability to stand, blindness and circling.Ten blood samples and 75 tissue samples from a total of 16 cattle were subjected to viral RNA extraction using High Pure Viral RNA Kit (Roche Diagnostics).The RNA samples were used as templates for the real-time RT-PCR assay, and all the samples were tested in duplicate.

Diagnostic Specificity and Sensitivity of the Real-Time RT-PCR Assay
All the PALV-free tissue homogenate and blood cells tested negative using the Pan-PALV-1, Pan-PALV-2, Chuzan-specific, and D'Aguilar-specific sets.On the other hand, all the samples tested positive using the β-actin set, and the Ct values were 19.96-31.64.The concentration of the RNA was 61.3-969.2ng/µL.
The real-time RT-PCR assay was performed using the field samples collected from stillborn fetuses and newborn calves with congenital abnormalities, and 22, 34, and 29 samples tested positive using Pan-PALV-1, Pan-PALV-2, and D'Aguilar-specific sets, respectively.The tissue homogenate and blood cells that tested positive using the Pan-PALV-1 set had Ct values of 29.75-37.48and 29.81-37.48,and Ct values obtained using the Pan-PALV-2 set were 29.99-38.51and 30.33-37.37,respectively.For tissue homogenate and blood cells that tested positive using the D'Aguilar-specific set, Ct values were 28.42-38.26and 28.94-38.43,respectively.All the samples collected from the stillborn fetuses and newborn calves tested negative using a Chuzan-specific set.Of the 16 cattle tested, one tested positive for PALV only, another tested positive for DAGV only, and the remaining 14 tested positive for both PALV and DAGV (Table 6).The concentration of the RNA was 50.0-830.5ng/µL.

Discussion
Genetic analysis of the complete coding region of Seg-2 in the present study revealed that the Japanese PALV strains were classified into four serotypes: CHUV, DAGV, and BCV, which we have previously identified [7,26], plus MARV.MARV is a member of PALV, identified in Australia in 1974-1976 from mosquitoes and Culicoides biting midges [27].Our data suggest that MARV had been introduced to Asia in the past and was present in Japan at least in 1997.Although it has been suggested that MARV isolated from Culicoides biting midges in Australia in 1975 may have resulted from the reassortment of some PALVs in Australia and India [11] to our knowledge, this is the first case of MARV isolation outside Australia and the first case of MARV isolation from cattle.
Genetic analysis of the complete coding region of Seg-2 in the present study also revealed that seven Japanese PALV strains isolated in 1987-2018 showed slightly higher identities to the NYAV prototype strain VRL792/73 (95.66-96.67%)than to the DAGV prototype strains B8112 (94.45-94.85%)at the amino acid level.Considering these identities, the seven Japanese PALV strains may be classified as NYAV; however, to begin with, the prototype strains of NYAV and DAGV are highly identical at VP2, with 95.96% identity.If multiple virus strains show such a high identity, they can be classified as the same serotype in another orbivirus, epizootic hemorrhagic disease virus [28].In addition, the VP2 identity between CHUV and Kasba virus, which were reported to be serologically identical [3], is 95.50%.Therefore, the seven Japanese PALV strains analyzed in this study, KY-115, ON91-5, ON-1/E/00, ON-5/E/12, KSB-1/C/13, ON-3/E/17, and ON-1/E/18, could be classified as either NYAV or DAGV.However, the seven Japanese PALV strains were more closely related to other Asian/Australian PALV strains, not African PALV strains, in the phylogenetic analysis based on all the genome segments other than Seg-2 and 6.In addition, the prototype strains of NYAV and DAGV were sorted into Afrotropical and Australasian groups in the phylogenetic analysis based on the concatenated dataset, respectively [11]; thus, it would be more appropriate to classify the seven Japanese PALV strains as DAGV rather than NYAV.
Our data obtained by WGS suggest that PALV forms a gene pool in Japan, China, and India and has occasionally caused reassortment between Asian and Australian PALVs in the past.The Japanese PALV strains sequenced in this study and Chinese PALV strains were closely related to the Kasba and Vellore viruses isolated in India in 1956-1957 in genome segments encoding viral proteins other than outer-capsid.On the other hand, for Seg-7 and/or 10, some Japanese strains, BCV strain ON-14/E/17 and MARV strains KSB-30/C/97 and MZ-16/E/97, are closely related to Australian PALV strains isolated in 1968-1976: the CSIRO Village virus strain CSIRO 11, the BCV strain CSIRO 87, and the DAGV strain B8112.Therefore, it is presumed that these Japanese BCV and MARV strains are reassortant viruses that have acquired Seg-7 and/or Seg-10 as well as Seg-2 from the Australian PALV strains.Another characteristic of the Japanese PALV strains revealed by the WGS was that they formed two groups with partial correlations of serotypes in the phylogenetic tree based on Seg-6 (Figure S6).This result was similar to that of the phylogenetic analysis of Seg-6 in Japanese BTV and EHDV strains [17,29].
The newly developed real-time RT-PCR assay is capable of broad detection of PALV and specific detection of CHUV and DAGV, and it has sufficient sensitivity for diagnosis.Using this assay, the DAGV gene was detected in blood and tissue samples collected from bovine stillborn fetuses and calves with congenital abnormalities in Japan from February 2019 to April 2019.In the area where the samples were collected, seroconversion to DAGV was observed in sentinel cattle in the summer of 2018 [30], suggesting that DAGV infection of dams may have caused stillbirths and congenital abnormalities in calves.Since congenital abnormalities in calves occur approximately five months after CHUV infection in the dams in Chuzan disease [4][5][6], it is assumed that DAGV also has a similar time lag between infection and disease occurrence.It is thus difficult to isolate CHUV or DAGV or to detect viral genes by conventional RT-PCR assays from the affected fetuses and calves.All the bovine fetuses and calves sampled in this study tested negative for PALV by a conventional multiplex RT-PCR assay for arboviruses [7]; however, they tested positive for PALV and/or DAGV by the newly developed real-time RT-PCR assay.To our knowledge, this is the first report of PALV gene detection in bovine stillborn fetuses or calves with congenital abnormalities.
The proposed use of the real-time RT-PCR assay is shown in Figure 3. First, a multiplex real-time RT-PCR assay is performed using Pan-PALV-1 and β-actin sets and Pan-PALV-2 and β-actin sets, respectively (first test).If samples test positive for PALV, a multiplex real-time RT-PCR assay is performed using Chuzan-specific and D'Aguilar-specific sets as a second test to check the presence of CHUV and DAGV.If a sample tests positive for PALV by the 1st test but negative for CHUV or DAGV by the second test, metagenomic virus detection is recommended.Since much remains unknown about the risk of clinical disease due to PALV infection in cattle, it is expected that this assay will help to further clarify the actual status of clinical cases caused by PALV.

Conclusions
This study provides a comprehensive genetic characterization of PALVs isolated in Japan from 1984 to 2018, revealing the presence of MARV outside Australia for the first time and the possibility of reassortment events among PALVs in the Asia-Pacific region.In addition, we developed a sensitive and specific real-time RT-PCR assay capable of broad detection of PALV and specific identification of CHUV and DAGV.This assay suc-

Conclusions
This study provides a comprehensive genetic characterization of PALVs isolated in Japan from 1984 to 2018, revealing the presence of MARV outside Australia for the first

3. 3 . 16 Figure 1 .Table 3 .
Figure 1.Phylogenetic profile showing the relationships among the Palyam serogroup virus (PALV) strains based on the complete coding region of genome segment 2. The Japanese PALV strains sequenced in this study are underlined.Serotypes Chuzan, Marrakai, Bunyip Creek, and D'Aguilar are shown in green, purple, brown, and blue, respectively.The percentage bootstrap values calculated from 1000 replications are indicated around the internal nodes.The scale represents 0.10% sequence divergence.

Figure 1 .
Figure 1.Phylogenetic profile showing the relationships among the Palyam serogroup virus (PALV) strains based on the complete coding region of genome segment 2. The Japanese PALV strains sequenced in this study are underlined.Serotypes Chuzan, Marrakai, Bunyip Creek, and D'Aguilar are shown in green, purple, brown, and blue, respectively.The percentage bootstrap values calculated from 1000 replications are indicated around the internal nodes.The scale represents 0.10% sequence divergence.

Pathogens 2024 ,
13, 550 14 of 16 virus detection is recommended.Since much remains unknown about the risk of clinical disease due to PALV infection in cattle, it is expected that this assay will help to further clarify the actual status of clinical cases caused by PALV.

Figure 3 .
Figure 3. Proposed use of the real-time RT-PCR assay for the detection of targeted viruses.Samples are tested with combinations of Pan-PALV-1 and β-actin sets and Pan-PALV-2 and β-actin sets in multiplex assays (first test).When samples tested positive for Palyam serogroup virus, the samples were then subjected to the second test with a combination of Chuzan-and D'Aguilar-specific sets in a multiplex assay.

Figure 3 .
Figure 3. Proposed use of the real-time RT-PCR assay for the detection of targeted viruses.Samples are tested with combinations of Pan-PALV-1 and β-actin sets and Pan-PALV-2 and β-actin sets in multiplex assays (first test).When samples tested positive for Palyam serogroup virus, the samples were then subjected to the second test with a combination of Chuzan-and D'Aguilar-specific sets in a multiplex assay.

Table 1 .
Characteristics of Japanese PALV strains used for whole-genome sequencing in this study.
a Identified by phylogenetic analysis based on Seg-2 and calculation of nucleotide and amino acid identities of Seg-2/VP2 among PALV strains.

Table 2 .
Primers and probes used for the development of the real-time RT-PCR assay.

Table 4 .
Ct values of the real-time PCR assay obtained by testing PALV strains and other arboviruses using the four primer/probe sets.

Table 5 .
Ct values of the real-time PCR assay obtained by testing spiked samples and MEM mixed with Chuzan virus, D'Aguilar virus or Bunyip Creek virus.
a No Ct.

Table 6 .
Ct values of the real-time PCR assay obtained by testing field-collected bovine samples.
a All the samples tested negative using the CHUV-specific set.b No Ct.