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A first report of rotavirus B from Zambian pigs leading to the discovery of a novel VP4 genotype P[9]

Virology Journal volume 21, Article number: 263 (2024) Cite this article

Abstract

Background

Rotavirus B (RVB) causes diarrhea in humans and pigs. Although various RVB strains were identified in humans and various animals globally, little is known about the epidemiology RVB infection in Africa. In this study, we attempted to examine the prevalence of RVB infection in pig populations in Zambia.

Methods

Metagenomic analyses were conducted on pig feces collected in Zambia to detect double stranded RNA viruses, including RVB. To clarify the prevalence of RVB infection in pig populations in Zambia, 147 fecal samples were screened for the RVB detection by RT-qPCR. Full genome sequence of a detected RVB was determined by Sanger sequencing and genetically analyzed.

Results

The metagenomic analyses revealed that RVB sequence reads and contigs of RVB were detected from one fecal sample collected from pigs in Zambia. RT-qPCR screening detected RVB genomes in 36.7% (54/147) of fecal samples. Among 54 positive samples, 13 were positive in non-diarrheal samples (n = 48, 27.1%) and 41 in diarrheal samples (n = 99, 41.4%). Genetic analyses demonstrated that all the segments of ZP18-18, except for VP4, had high nucleotide sequence identities (80.6–92.6%) with all other known RVB strains detected in pigs. In contrast, the VP4 sequence of ZP18-18 was highly divergent from other RVB strains (< 64.6% identities) and formed a distinct lineage in the phylogenetic tree. Notably, the VP8 subunit of the VP4 showed remarkably low amino acid identities (33.3%) to those of known RVB strains, indicating that the VP8 subunit of ZP18-18 was unique among RVB strains. According to the whole genome classification for RVB, ZP18-18 was assigned to a genotype constellation, G18-P[9]-I12-R4-C4-M4-A8-N10-T5-E4-H7 with the newly established VP4 genotype P[9].

Conclusions

This current study updates the geographical distribution and the genetic diversity of RVB. Given the lack of information regarding RVB in Africa, further RVB surveillance is required to assess the potential risk to humans and animals.

Background

Rotaviruses (RVs) cause gastroenteritis in children and young animals worldwide and RVs infections are serious issues in both public health and livestock production [1]. RVs, belonging to the genus Rotavirus in the family Sedoreoviridae, are currently classified into nine species (A–J, except for E) on the basis of antigenic and genetic characteristics of the intermediate capsid protein VP6 [1, 2]. RV A, B, C, and H (RVA, RVB, RVC, and RVH) infections have been reported in pig populations, and RVA infection is the most common and prevalent among those infections. Meanwhile, RVB has been reported to cause diarrhea in suckling and weaning pigs [3, 4]. RVB-associated diarrhea outbreaks in piglets in China and Brazil were reported in 2022 and 2017, respectively [5, 6]. Thus, RVB infection in piglets may potentially become a serious issue for animal health and pig production.

RVs belong to segmented double-stranded RNA (dsRNA) viruses with eleven genome segments encoding six structural proteins (VP1–VP4, VP6, and VP7) and five nonstructural proteins (NSP1–NSP5) [1]. The RVB NSP1 segment exclusively contains two overlapping ORFs (NSP1-1 and NSP1-2) unlike other RVs [7, 8]. RV virion has triple-layered particle (TLP) composed of the outer layer (VP7 and VP4), the intermediate layer (VP6), and the inner core (VP1, VP2 and VP3) [1]. VP7 and VP4, the outer capsid protein, play a role in infection of host cells and immune responses of infected cells, and are target for neutralizing antibodies [1]. The spike protein VP4 is proteolytically cleaved into VP8 and VP5, and the VP8 domain interacts with host cell receptors [1, 9].

To well-understand genetic diversity of RVs and genome reassortment events among RVs, the Rotavirus Classification Working Group (RCWG, https://rega.kuleuven.be/cev/viralmetagenomics/virus-classification/rcwg) has developed a comprehensive genotype classification system using all eleven segments of RVs. The genotype constellation is defined as follows: Gx-P[x]-Ix-Rx-Cx-Mx-Ax-Nx-Tx-Ex-Hx, representing each genotype of VP7-VP4-VP6-VP1-VP2-VP3-NSP1-NSP2-NSP3-NSP4-NSP5, respectively [10]. Additionally, RVs are designated based on a standardized criteria proposed by the RCWG as follows: RV group / species origin / country where the virus was collected / virus strain name / year of sample collection / G and P genotypes [11]. Recently, Shepherd et al. established a classification system for all eleven genes of RVB strains with percent identity cutoff values of 76–83%, and classified these as 26 G, 5 P, 13 I, 5 R, 5 C, 5 M, 8 A, 10 N, 6 T, 5 E, and 4 H genotypes [12]. According to this classification, each one genotype constellation, G1-P[1]-I1-R1-C1-M1-A1-N1-T1-E1-H1 and G2-P[2]-I2-R2-C2-M2-A2-N2-T2-E2-H2 have been reported for murine and human RVB, respectively. Bovine and caprine RVB showed 1–2 genotypes for eleven gene segments as follows: bovine RVB, G3/5-P[3]-I3-R5-C3/5-M3-A4/5-N3/4-T3-E3-H3/5; caprine RVB G3-P[3]-I3-R3-C3-M3-A3/4-N3-T3-E3-H3. In contrast, porcine RVBs have shown 22 G, 2 P, 10 I, 1 R, 1 C, 2 M, 3 A, 6 N, 3 T, 1 E, 3 H genotypes to date, indicating a more heterogeneous evolution compared to human and other animal RVBs [12]. Although 5 P genotypes for the VP4 gene have been classified as mentioned above, three novel VP4 genotype candidates were recently proposed following the identification of new porcine RVB strains, indicating an increased genetic diversity of the VP4 gene for RVB [5, 13, 14].

RVB was initially found in severe gastroenteritis patients in China in the 1980s and continues to be responsible for diarrhea in humans in Asian countries [15,16,17,18]. In addition to humans, RVB has been sporadically detected in mammalian animals, including pigs, rats, cattle, goats, and sheep [19,20,21,22]. To date, RVB genotype constellations show no cross-species reassortment events between humans and domestic animals in contrast to RVA [12]. RVB host specificity is broader than that of RVA. RVB strains have been identified in pigs in various countries such as Japan, India, Vietnam, Australia, Russia, Italy, Brazil, and the United States [4, 8, 23,24,25,26,27,28]. Recently, the RVB genome was detected from pigs in South Africa using virome analyses for clarifying the presence of RVB [29]. However, little information has been reported on the prevalence of RVB infection in other African countries.

We have previously performed a surveillance of RVA infection in pigs in Zambia [30], and recognized that RVB could also be detected in pig feces by metagenomic analyses. Therefore, we attempted to examine the prevalence of RVB infection in pig populations in Zambia to better understand the distribution of this virus in Africa and have identified RVB strains expressing a novel VP4 genotype.

Methods

Sample collection

In our previous study, collection of fecal samples from pigs was carried out at five farms where the owners consented to sampling in 2018; farms A and E were in Lusaka, farm B in Chilanga, farm C in Kafue, and farm D in the Chibombo districts [31]. In total, 47 and 100 fecal samples were obtained from 0–12-week-old pigs with or without diarrhea, respectively, and stored at − 80 °C in the laboratory until analyses.

Metagenomic analyses for dsRNA viruses

Metagenomic data of pig feces in Zambia was obtained using an Illumina HiSeq X Ten (Illumina, San Diego, CA, USA) for RVA surveillance in our previous study [30]. Briefly, viral dsRNA was isolated from 10% fecal suspensions and subjected to the library preparation and metagenomic sequencing. CLC Genomics Workbench software (CLC bio, Hilden, Germany) was used for analyzing the metagenomic data. Raw paired-end sequence reads were trimmed and mapped to the host (Sus scrofa) reference genome for removing host-derived rRNA reads. The cleaned reads were analyzed by de novo assembly, and the obtained assembled contigs were subjected to a blastn program with viral nucleotide sequences downloaded from a Reference sequence data (RefSeq) in the National Centre for Biotechnology Information (NCBI) Virus database (https://www.ncbi.nlm.nih.gov/labs/virus/vssi/#/). We identified RVB genomes from one of six fecal samples, and two contigs (230 bp and 218 bp in length) showed 91.67% and 95.45% identities to the reference genome encoding the RNA dependent RNA polymerase from the human RVB strain Bang373 (NC_021541), respectively. Accordingly, the obtained contigs were analyzed by a local blastx with various amino acid sequences of 11 RVB proteins downloaded from the NCBI database. The contigs showing low E-values (the cutoff value: 1.0 × 10− 18) on the blastx, were re-analyzed by BLAST search (https://blast.ncbi.nlm.nih.gov/Blast.cgi) at NCBI to identify accurately the RVB sequences. Finally, 28 contigs were identified from one fecal sample as RVB genomes, including nine of eleven segments (Table S1). To obtain viral genome sequences of the remaining two segments, all the trimmed reads were additionally mapped to the remaining segment nucleotide sequences of RVB strains genetically close to the obtained contigs (Table S2).

RT-qPCR screening in pig feces

To screen for the detected RVB, total RNA was extracted from fecal suspensions of pigs using QIAamp Viral RNA Mini Kit (QIAGEN, Hilden, Germany). RT-qPCR primers and hydrolysis probes were designed based on the obtained VP6 contig sequence of the detected RVB as follows: RVB-VP6-qF (5′- GAACATGACGGGAGGCAATA-3′), RVB-VP6-qR (5′- TCTGAATCCATGCCTGACATAC-3′), and RVB-VP6-probe (5′-FAM-AGCGACATT-ZEN-AGGTAGATGGTCCGGT-IBFQ-3′). The probes were double-quenched using 5′ 6-FAM fluorophore, 3′ IBFQ quencher and the inner ZEN quencher (Integrated DNA Technologies, Coralville IA). RT-qPCR was carried out using a One Step PrimeScript III RT-qPCR Mix, with UNG (Takara) with the LightCycler 96 System (Roche Diagnostics, Basel, Switzerland). Briefly, 2 µL of extracted RNA were added to RVB primers, and then were subjected to denaturation at 95 °C for 5 min. The denaturation mixtures were then added to the RT-qPCR-reaction mixture containing the RVB probe and One Step PrimeScript III RT-qPCR Mix with UNG at a final volume of 20 µL. One-step RT-PCR cycling was performed as follows: 52 °C for 5 min and 95 °C for 10 s, followed by 45 cycles of 95 °C for 5 s and 60 °C for 30 s. Samples with a sigmoidal curve < 39 were considered RVB positive.

Sanger sequencing of the detected RVB

The nucleotide sequences of eleven genome segments of the detected RVB were determined by conventional RT-PCR and Sanger sequencing. The purified viral dsRNA was denatured at 95 °C for 5 min, and was then subjected to conventional RT-PCR, using SuperScript IV Reverse Transcriptase (Life Technologies, Carlsbad, CA) and Ex Premier DNA Polymerase (Takara, Shiga, Japan) with specific primers for the sequence reads or contigs and newly designed universal primers for porcine RVB (Table S3). Amplicons were purified with the KAPA HyperPure Beads (KAPA BIOSYSTEMS, Woburn, MA), and sequenced using the Big Dye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems, Foster City, CA). The determined sequences were deposited in the DNA Data Bank of Japan (DDBJ) under accession numbers LC818970-LC818980.

Genetic comparison and phylogenetic analysis

The determined sequences of the eleven segments were subjected to a BLAST search to identify the genotype and the closest RVB strain. Identity comparison analyses were carried out between RVB strains using GENETYX-MAC Ver.22 with the global homology program based on the Lipman-Pearson model (GENETYX Corporation, Tokyo, Japan). Phylogenetic analyses based on the nucleotide sequences of the eleven genome segments and the amino acid sequence of VP8 were conducted using MEGA X software [32] with the MUSCLE method for a multiple sequence alignment. Phylogenetic trees were constructed using the maximum likelihood method based on the best fit models as follows: GTR + G + I for VP1, VP2, VP3, VP4, VP6, VP7, and NSP2, GTR + G for NSP1, NSP3, and NSP5, TN93 + G for NSP4, WAG + G for VP8. Tree reliability was assessed using 1,000 bootstrap replicates. The reference RVB strains and their accession numbers used in this study are listed in Supplemental Table S4.

Statistical analysis

Differences in the prevalence rates of RVB infection were analyzed by the chi-squared test using R version 4.2.0. p < 0.05 denoted statistical significance.

Results

Detection of the RVB genome in pigs in Zambia

In our previous study, the VP6 gene of RVA was detected from 34 of 147 fecal samples collected from pigs in Zambia using RT-PCR screening, and six RVA-positive feces were subjected to metagenomic analyses to obtain the complete genomes of the detected RVAs [30]. Of these, two feces showed RVA genome sequences for all eleven segments while the others yielded only a few RVA sequences with poor coverage. Accordingly, we re-analyzed the metagenomic data for other viruses, and sequence reads and contigs of RVB were detected from one of them (Sample ID, ZP18-18). To clarify the prevalence of RVB infection in Zambia, we subjected 147 fecal samples to RT-qPCR newly designed base on the partial RVB sequence of metagenomic data (Table 1). Of the total samples, 36.7% (54/147) were positive for the RVB genome. The RVB genome was detected in 27.1% (13/48) and 41.4% (41/99) of asymptomatic and diarrheal samples respectively, and no significant difference in the detection rate of RVB genome between asymptomatic and diarrheal pigs was found (p = 0.13). In addition, RVB-positive samples were obtained from 25.5% (12/47) and 42.0% (42/100) of suckling (0–4-week-old) and weaning/fattening pigs (5–12-week-old) with no significant difference (p = 0.08), respectively. Sorting by farms, 28.2% of fecal samples (11/39) in farm A, 16.7% (5/30) in farm B, 53.4% (31/58) in farm C, and 43.8% (7/16) in farm E were positive for the presence of RVB genomes while all four samples collected in farm D were negative.

Table 1 Prevalence of RVB infection in pigs in Zambia
Full size table

Sequencing for the full genome sequence of the detected RVB

Metagenomic analyses for the fecal sample (ZP18-18) revealed that partial RVB segment sequences were achieved for VP1, VP2, VP3, VP4, VP6, VP7, NSP1, NSP2, and NSP3 by de novo assembly while there were some reads of NSP4 and NSP5 which were not sufficient to assemble the sequence consensus accurately. As above mentioned, metagenomic data of the remaining five sample shows no RVB sequence although these samples were positive for RVB genome by the RT-qPCR screening. For filling in the sequence gaps, the terminus, and low-coverage regions, the RVB segment genome was amplified by conventional RT-PCR, and the complete genome sequence of the detected RVB strain (ZP18-18) was determined.

Genomic characterization of detected RVB strain

To identify detailed genome information on ZP18-18, we carried out BLAST search and phylogenetic analyses using the nucleotide sequence of the eleven RVB segments (Table 2). Except for VP4, all segments of ZP18-18 showed relatively high nucleotide sequence identities (80.6–92.6%) and amino acid sequence identities (85.6–98.0%) to known RVB strains detected from pigs in various countries, exceeding the cutoff values for genotype assignment [12]. Based on the VP1, VP2, VP3, VP6, VP7, NSP1, NSP2, NSP3, NSP4, and NSP5 phylogenies, ZP18-18 clustered with various known porcine RVB strains (Figs. 1, 2 and 3). In contrast, the VP4 gene of ZP18-18 showed low nucleotide (65.4%) and amino acid sequence identities (57.7%) to known RVB strains (Table 2). The partial nucleotide sequence of ZP18-18 (corresponding to position 687–2244, VP5 region) showed the highest nucleotide identity (80.1%) to RVB/Pig-wt/VNM/14177_18/2012/G24PX (a nucleotide sequence of its VP8 was unavailable). The BLAST analyses for VP8 nucleotide and amino acid sequence of ZP18-18 showed no significant similarity and remarkably low identities (33.3%) to known RVB strains, respectively. Phylogenetic analyses of VP8 from RVBs revealed that ZP18-18 formed a distinct branch among RVB strains (Fig. S1). Pairwise identities based on the complete sequences of VP4 between ZP18-18 and other known RVB strains, including murine, human, bovine, caprine, and porcine RVBs, were less than 65.4% (Table S5). Phylogenetic analyses of the complete VP4 sequence demonstrated that ZP18-18 formed an independent lineage from other known genotype groups but shared a common origin with all porcine RVB strains (Fig. 4). These results suggest that VP4 of ZP18-18 was highly divergent from those of known RVBs. Based on the cut-off values for genotyping VP4 (80%) [12], the sequence showed identities below the proposed threshold with known RVB strains, suggesting that this could belong to a novel genotype P[9]. Recently, new RVB strains, 06-2017-Medj and HNLY-2022 strains were identified in pigs in Croatia and China, respectively, and the VP4 of each strain could be considered to belong to novel genotypes based on the cut-off values [5, 13]. Since each study tentatively proposed the same number as the VP4 genotype, P[6], we propose to re-number the VP4 genotype as follows: 2017-Medj, P[6]; HNLY-2022, P[8]. Additionally, the VP4 and VP7 genes of new RVB strains identified in Spain were highly divergent from those of other RVB strains, showing candidates for new VP4 and VP7 genotypes in a previous study [14]. Since the study did not number the novel genotypes, we provisionally named the novel VP4 and VP7 genotypes as follows: the VP4 genes of B304 and B377 strains, P[7]; the VP7 genes of B377 and B422 strains, G28; the VP7 genes of P1C and P2B strains, G29 (Figs. 1A and 4). Based on the classification system for RVB, ZP18-18 was assigned to a new genotype constellation, G18-P[9]-I12-R4-C4-M4-A8-N10-T5-E4-H7, including a new VP4 genotype (Table 3). Finally, based on the nomenclature guideline of RCWG, the ZP18-18 was formally named as RVB/Pig-wt/ZMB/ZP18-18/2018/G18P[9].

Fig. 1
figure 1

Phylogenetic analysis of the VP7 and VP6 genes. Phylogenetic trees based on the nucleotide sequence of VP7 (A) and VP6 genes (B) were constructed using the maximum likelihood method with 1,000 bootstrap replicates. Bootstrap values greater than 70% are shown on the interior branch nodes, and scale bars indicate the number of substitutions per site. Black circles represent the detected RVB strains in this study. The genotype of the detected RVB strain are indicated in red

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Fig. 2
figure 2

Phylogenetic analysis of VP1, VP2, VP3, and NSP1 genes. Phylogenetic trees based on the nucleotide sequence of VP1 (A), VP2 (B), VP3 (C), NSP1 genes (D) were constructed using the maximum likelihood method with 1,000 bootstrap replicates. Bootstrap values greater than 70% are shown on the interior branch nodes, and scale bars indicate the number of substitutions per site. Black circles represent the detected RVB strains in this study. The genotypes of the detected RVB strain are indicated in red

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Fig. 3
figure 3

Phylogenetic analysis of the NSP2, NSP3, NSP4, and NSP5 genes. Phylogenetic trees based on the nucleotide sequence of NSP2 (A), NSP3 (B), NSP4 (C), and NSP5 genes (D) were constructed using the maximum likelihood method with 1,000 bootstrap replicates. Bootstrap values greater than 70% are shown on the interior branch nodes, and scale bars indicate the number of substitutions per site. Black circles represent the detected RVB strains in this study. The genotypes of the detected RVB strain are indicated in red

Full size image
Fig. 4
figure 4

Phylogenetic analysis of the VP4 gene of RVB. A phylogenetic tree based on the nucleotide sequence of VP4 gene was constructed using the maximum likelihood method with 1,000 bootstrap replicates. Bootstrap values greater than 70% are shown on the interior branch nodes, and scale bars indicate the number of substitutions per site. A black circle represents the detected RVB strains in this study. The genotype of the detected RVB strain is indicated in red

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Table 2 Genotype constellation of ZP18-18 strain with reference RVB strains exhibiting the highest sequence identities
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Table 3 Genome constellations for representative RVB strains
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Discussion

In this study, we identified a novel RVB strain in pigs in Zambia and revealed that RVB infection was common in the Zambian pig population. Although RVBs have been identified in humans and animals worldwide [4, 8, 23,24,25,26,27, 29], information on RVB infection in African countries is lacking, and this is the first report of RVB detection and surveillance in pigs in Zambia. In our RT-qPCR screening, 36.7% of samples were RVB-positive, and the detection rate was higher in the weaning/fattening stage than in the suckling stage. Although no significant difference in the prevalence of RVB infection between these age/stage groups was found, similar prevalence patterns were also observed in the pig populations in Japan and the United States [3, 33]. These results suggested that the prevalence of RVB infection in pigs may have similar trends to that of RVA infection, and seem to increase with age, implying that suckling pigs may be protected by maternal antibodies through breast milk. It has been previously reported that RVB was associated with diarrhea in suckling and weaned pigs [3, 6]. Our screening showed higher frequencies of RVB detection in diarrheal pigs than those in asymptomatic pigs in Zambia, and this result was observed in all the investigated farms, except for farm D where the RVB genome was not detected. Although there was no significant difference in the frequencies of RVB detection in fecal samples between asymptomatic and diarrheal pigs due to the insufficient sample sizes (p = 0.13), RVB detection rate does seems to be associated with diarrhea in Zambian pigs.

We determined the complete genome sequence of a representative RVB strain in Zambia. Full genome sequences of all the RVB segments are required to fully understand the evolutionary history and to identify the reassortment events among RVB strains. Although many partial RVB genomes have been identified using RT-qPCR-based approaches, genetic information of RVB strains with the complete genome constellation is limited (Table 3). Genetic comparison with porcine RVB strains showed a common consensus genome constellation backbone (R4-C4-M4-A8-N10-T5-E4/5/6-H7), and the genetic backbone of ZP18-18 is identical to RVB/Pig-wt/USA/KS1/2012/G16P[4]. In contrast, porcine RVB strains showed a large genetic diversity within the VP6 and VP7 genes [3, 34], and various VP7/VP4/VP6 genotype combinations have been observed. Phylogenetic analyses based on the complete VP4 sequence revealed that ZP18-18 formed a distinct lineage from other known RVB strains, and VP8 of ZP18-18 is notably unique among RVB strains. The spike protein VP4 plays a pivotal role in viral entry, and VP4 is proteolytically cleaved into VP8 and VP5 subunits. The VP8 is located in the globular head domain of VP4 and interacts with cellular receptor for RVs, and serves as a neutralizing antigen, which can result in large sequence divergences [9]. Since BLAST analysis revealed that VP5 sequence of ZP18-18 showed relatively high nucleotide identity with porcine RVB strains in Viet Nam and China, it seems reasonable to speculate that RVB with the novel genotype P[9] may be present in other Asian countries. The discovery of the novel P[9] genotype supports the high genetic diversity of the VP4 gene for porcine RVBs compared to RVB of other hosts. Pigs play an important role in RVB evolution, and the genetic diversity of porcine RVB may result in difficulties in the development of effective RVB vaccines. As for RVA, a live RVA vaccine with one genotype produced no heterologous clinical protection against other genotypes [35]. In addition, there are no reports of the isolation of RVB, indicating that serological phenotypes of RVB strains are not completely understood at this time. It has been reported that a subunit RVB vaccine targeting conserved antigenic sites in RVB might be one method which could be employed for the control of RVB infection [36]. Thus it is possible that our novel P[9] RVB genome information may contribute to development of new RVB vaccines.

Recently, the first full-length genome sequence of an RVB strain (UFS-BOC050) was detected from pig stool samples in South Africa [29]. Although BLAST analyses based on the eleven genome segments of ZP18-18 did not show UFS-BOC050 as the closest strain, UFS-BOC050 possessed the common consensus genome constellation backbone (R4-C4-M4-A8-N10-T5-E4/5/6-H7) which is the same as ZP18-18 (Table 3). The segments of UFS-BOC050 showed 82.1–88.5% nucleotide sequence identities to known RVB strains detected from pigs in USA, Spain, Japan, China, and Vietnam [29], and the genome sequences of ZP18-18 showed similar patterns of low identities to closest strains (Table 2). These results highlight the lack of sequence data for RVB strains in Africa. Unlike RVA, genome information of RVB strains is still very limited. According to the NCBI Virus database, and as note above only 1,844 genome sequences for RVB strains worldwide have been deposited, in contrast to 119,904 genome sequences for RVA strains. Further studies with the accumulation of more RVB genome data are needed to better understand the evolutionary history of RVB strains including potential reassortment events among the virus strains. As this is the first investigation on RVB in Zambia, we determined the whole genome sequence of ZP18-18 as a reference sequence, and this should be helpful for future RVB research in Africa and elsewhere. Further epidemiological studies for analyzing the detected RVB strains in Zambia are warranted to better clarify the genetic diversity of these viruses.

Conclusion

We have clarified in our preliminary studies the geographical distribution of RVB in Zambia and determined the full genome sequence of the detected RVB strain. The VP4 of the Zambian RVB strain was highly divergent from other known RVB strains, resulting in the identify of a novel VP4 genotype P[9]. Considering the limited sequence data of RVB, continued RVB surveillance worldwide is needed for well-understanding the genetic diversity of RVB.

Data availability

Sequence data from this article have been deposited with the DDBJ/EMBL/GenBank Data Libraries under Accession No. LC818970-LC818980. The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.

Abbreviations

RV:

Rotavirus

RVA:

Rotavirus A

RVB:

Rotavirus B

RVC:

Rotavirus C

RVH:

Rotavirus H

dsRNA:

Double-stranded RNA

TLP:

Triple-layered particle

RCWG:

Rotavirus Classification Working Group

NCBI:

National Centre for Biotechnology Information

DDBJ:

DNA Data Bank of Japan

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Acknowledgements

We would like to thank Ms. Sakiko Ueda from the Women’s Future Development Organization, Tokyo University of Agriculture and Technology for the technical assistance of data analyses. We also would like to thank students at the International Institute for Zoonosis Control, Hokkaido University, and technicians at School of Veterinary Medicine, the University of Zambia for collecting samples.

Funding

This work was supported by the Japan Agency for Medical Research and Development (AMED) [grant number JP243fa627005], the Japan Program for Infectious Diseases Research and Infrastructure from AMED (JP23wm0125008), and AMED and the Japan International Cooperation Agency (JICA) within the framework of the Science and Technology Research Partnership for Sustainable Development (SATREPS) [grant number JP23jm0110019].

Author information

Author notes

  1. Yongjin Qiu

    Present address: Laboratory of Parasitology, Department of Disease Control, Graduate School of Infectious Diseases, Faculty of Veterinary Medicine, Hokkaido University, Sapporo, 060-0818, Japan

Authors and Affiliations

  1. Laboratory of Veterinary Public Health, Faculty of Agriculture, Tokyo University of Agriculture and Technology, Fuchu, 183-8509, Tokyo, Japan

    Hayato Harima & Kanako Ishihara

  2. Division of International Research Promotion, International Institute for Zoonosis Control, Hokkaido University, Sapporo, 001-0020, Japan

    Yongjin Qiu, Joseph Ndebe, Masahiro Kajihara & Naganori Nao

  3. Division of Molecular Pathobiology, International Institute for Zoonosis Control, Hokkaido University, Sapporo, 001-0020, Japan

    Michihito Sasaki & Yasuko Orba

  4. Institute for Vaccine Research and Development, Hokkaido University, North 21 West 11, Kita-ku, Sapporo, 001-0021, Japan

    Michihito Sasaki, Yasuko Orba, William W Hall & Hirofumi Sawa

  5. Department of Disease Control, School of Veterinary Medicine, the University of Zambia, Lusaka, 10101, Zambia

    Joseph Ndebe, Kapila Penjaninge, Edgar Simulundu, Ayato Takada & Hirofumi Sawa

  6. Macha Research Trust, Choma, 20100, Zambia

    Edgar Simulundu

  7. Technical office, International Institute for Zoonosis Control, Hokkaido University, Sapporo, 001- 0020, Japan

    Aiko Ohnuma

  8. Division of Risk Analysis and Management, International Institute for Zoonosis Control, Hokkaido University, Sapporo, 001-0020, Japan

    Keita Matsuno

  9. One Health Research Center, Hokkaido University, Sapporo, 060-0818, Japan

    Keita Matsuno, Naganori Nao, Yasuko Orba, Ayato Takada & Hirofumi Sawa

  10. International Collaboration Unit, International Institute for Zoonosis Control, Hokkaido University, Sapporo, 001-0020, Japan

    Keita Matsuno, Yasuko Orba, Ayato Takada, William W Hall & Hirofumi Sawa

  11. Division of Global Epidemiology, International Institute for Zoonosis Control, Hokkaido University, Sapporo, 001-0020, Japan

    Ayato Takada

  12. Africa Center of Excellence for Infectious Diseases of Humans and Animals, the University of Zambia, Lusaka, 10101, Zambia

    Ayato Takada, Bernard M. Hang’ombe & Hirofumi Sawa

  13. National Virus Reference Laboratory, School of Medicine, University College Dublin, Dublin, Ireland

    William W Hall

  14. Department of Para-clinical Studies, School of Veterinary Medicine, the University of Zambia, Lusaka, 10101, Zambia

    Bernard M. Hang’ombe

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  1. Hayato Harima
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Contributions

HH conceived and designed this research; MS and KM advised for the method of viral detection; HH, YQ, JN, KP, and ES carried out the field activity for collecting fecal samples; HH, YQ, JN, KP, and AO performed the experiments; HH, Y.Q, and AO obtained and analyzed the data; HH visualized the data for the manuscript; HH wrote the original draft; WWH assisted in writing and editing the manuscript; AT and HS acquired the funding for this research; BMH and HS supervised this research activity. All authors edited and approved the final manuscript.

Corresponding author

Correspondence to Hirofumi Sawa.

Ethics declarations

Ethics approval and consent to participate

The study proposal presented in this study was approved by the University of Zambia Biomedical Research Ethics Committee (UNZABREC) (REF. NO. 1382–2020). Verbal consent was obtained from farm owners before sampling. Since fecal specimens analyzed in this study were obtained from fresh stools on the floor in pens, no animal experiments were involved.

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Not applicable.

Competing interests

The authors declare no competing interests.

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Harima, H., Qiu, Y., Sasaki, M. et al. A first report of rotavirus B from Zambian pigs leading to the discovery of a novel VP4 genotype P[9]. Virol J 21, 263 (2024). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12985-024-02533-5

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Virology Journal

ISSN: 1743-422X