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Characterisation of a G2P[4] Rotavirus Outbreak in Western Australia, Predominantly Impacting Aboriginal Children

In May, 2017, an outbreak of rotavirus gastroenteritis was reported that predominantly impacted Aboriginal children ≤4 years of age in the Kimberley region of Western Australia. G2P[4] was identified as the dominant genotype circulating during this period and polyacrylamide gel electrophoresis revealed the majority of samples exhibited a conserved electropherotype. Full genome sequencing was performed on representative samples that exhibited the archetypal DS-1-like genome constellation: G2-P[4]-I2-R2-C2-M2-A2-N2-T2-E2-H2 and phylogenetic analysis revealed all genes of the outbreak samples were closely related to contemporary Japanese G2P[4] samples. The outbreak samples consistently fell within conserved sub-clades comprised of Hungarian and Australian G2P[4] samples from 2010. The 2017 outbreak variant was not closely related to G2P[4] variants associated with prior outbreaks in Aboriginal communities in the Northern Territory. When compared to the G2 component of the RotaTeq vaccine, the outbreak variant exhibited mutations in known antigenic regions; however, these mutations are frequently observed in contemporary G2P[4] strains. Despite the level of vaccine coverage achieved in Australia, outbreaks continue to occur in vaccinated populations, which pose challenges to regional areas and remote communities. Continued surveillance and characterisation of emerging variants are imperative to ensure the ongoing success of the rotavirus vaccination program in Australia.


Introduction
Group A rotaviruses, belonging to the Reoviridae virus family, remain one of the main aetiological agents of acute gastroenteritis in infants and young children worldwide, estimated to have caused 128,500 deaths and 258,173,300 episodes of diarrhea among children <5 years of age in 2016 [1]. The substantial decrease in the global burden of rotavirus disease over the last decade can be attributed to varied public health measures, such as improved sanitation, as well as the inclusion of rotavirus vaccines into the National Immunisation Programs (NIPs) of over 100 countries worldwide [2]. In Australia, the live-attenuated vaccines Rotarix ® (monovalent, human G1P [8] strain) and RotaTeq®(pentavalent, humanbovine reassortant vaccine comprising G1P [5], G2P [5], G3P [5], G4P [5], and G6P [8] strains) were introduced into the NIP in mid-2007, with a state-based vaccine selection method in place up until mid-2017, after which a national tender process was initiated, with all states and territories now using Rotarix [3,4].

Genotyping
Cases were designated to the month of May based on the optimal date of onset (ODOO). Of the 122 cases from the month of May, a stool sample was available for 70 and were sent for genotype analysis at the National Rotavirus Reference Centre (NRRC), Murdoch Children's Research Institute in Melbourne, Australia. The predominant genotype identified was G2P [4] (94%, 66/70) ( Table 2), with the majority of G2P [4] cases in the KIMB region (61%, 40/66). In the KIMB region, Aboriginal people were disproportionately represented, accounting for 90% of cases (36/40) ( Table 2).  (3/19), and vaccination status was unknown for two cases (10%).

Sequence Analysis of G2P[4] Samples
A total of 38 G2P [4] samples were analysed using polyacrylamide gel electrophoresis to visualise the electropherotype pattern. The majority of samples had a highly similar electropherotype, indicating that a relatively conserved strain was circulating during the outbreak (data not shown).
Across the NSP1, NSP2, NSP3, NSP4, and NSP5 gene trees, the 2017 outbreak samples exhibited the same pattern across all trees: clustering with same group of contemporary Japanese samples, and falling within conserved clades comprised of G2P [4] strains from Belgium and Hungary that were detected in 2012, and Australian 2010 samples (Figure 5g-k).
The 2017 outbreak samples were not closely related to the samples RVA/Humanwt/AUS/V233/1999/G2P [4], RVA/Human-wt/AUS/336190/2004/G2P [4] and RVA/ Human-wt/AUS/V203/2009/G2P [4], which were associated with prior outbreaks in the Northern Territory, often clustering in separate lineages or distinct clades. This suggests that the current outbreak variant was not derived from the prior G2P [4] outbreak variants that had undergone genetic drift or reassortment over the intervening years but were more closely related to a G2P [4] variant that has been detected in Japan, Hungary, and other regions of the world. It may be derived from the Australian 2010 G2P [4] variant that has undergone moderate genetic drift during global circulation.

Comparison of the Outbreak Samples to the G2 VP7 Gene Component of the RotaTeq Vaccine
The VP7 gene of the 2017 outbreak samples possessed 93.37-99.48% nt and 94.79-95.01% aa similarity with the G2 VP7 gene of RotaTeq. The amino acid differences between the outbreak samples and RotaTeq were analysed and 16 residues differed between the G2 component of RotaTeq and the two outbreak samples RVA/Human-wt/AUS/WAPC2824/2017/G2P [4] and RVA/Human-wt/AUS/WAPC2769/2017/G2P [4]. RVA/Human-wt/AUS/WAPC2784/ 2017/G2P [4] had 17 residues that differed. The altered residues that fell between amino acid 78 and 312 were mapped to the surface of the VP7 monomer to highlight mutations in proximity to the VP7 antigenic epitopes 7-1a, 7-1b, and 7-2 [25] (Figure 6). Mutations were observed in all three samples in antigenic epitope regions: positions A87T and D96N in antigenic region 7-1a, and S213D in region 7-1b. Additionally, the mutation D145G in the antigenic epitope region 7-2 was observed in RVA/Human-wt/AUS/WAPC2784/2017/G2P [4]. The three outbreak samples exhibited the residues D96N and S213D, which are amino acid changes that have been shown to escape neutralisation with monoclonal antibodies [26].

Discussion
Rotavirus was gazetted as a notifiable disease in WA in 2006 in part to monitor the effectiveness of the rotavirus vaccine when it was added to the childhood immunisation schedule in Australia in mid-2007 [11]. The introduction of rotavirus vaccines has lessened the once prominent seasonality of rotavirus infection in Australia [27,28]. Following campylobacteriosis and salmonellosis, rotavirus was the third most commonly notified enteric infection in the population of WA in 2017 [29]. A large increase in rotavirus notifications was noted in the second quarter of 2017, with the highest number of cases noted in May, indicating an outbreak occurring prior to the onset of winter (Figure 1).
A total of 236 rotavirus notifications were recorded in the second quarter of 2017, compared to the five-year second quarter average of 100.8 notifications, highlighting the scale of the outbreak. The five-year second quarter average was somewhat skewed by an outbreak in the second quarter of 2015 ( Figure 1) that affected all WA regions, and predominantly affected non-Aboriginal people. Multiple outbreaks related to child care and aged care facilities were noted during this time, with the predominant strain identified as G12P [8] [30].
In contrast to 2015, the increase in the second quarter of 2017 was noted to disproportionally affect young Aboriginal children in the KIMB region, which is in the north of the state. A number of towns and Aboriginal communities in the KIMB region were affected. The KIMB PHU investigated the increase in notifications, with assistance from local government environmental health officers. Several public health interventions were implemented as a result of their investigations, including the distribution of a public health alert to local hospitals and Aboriginal medical service providers, liaising with environmental health officers and community health staff to provide public health advice for affected communities, and an interview on local radio.
It is noteworthy that an increased burden of rotavirus disease was reported elsewhere in Australia for 2017. Multiple outbreaks were recorded across Australia, due to equine-like G3P [8] in New South Wales and G8P [8] in New South Wales and Victoria [3]. In addition to the WA outbreak herein described, outbreaks due to G2P [4] were also reported in the Northern Territory (NT) and South Australia [3]. It is thought that the 2017 G2P [4] outbreak began in the NT and subsequently spread to rural and remote regions of WA adjacent to the border between these states [31]. A companion study described the weak protective effect of either Rotarix or RotaTeq vaccination in the setting of this outbreak [31]. Suboptimal vaccine-effectiveness, particularly in the second year of life, has been reported in other high-burden, low-resource settings [32]. There are varied factors that could contribute to a reduced vaccine response, such as poor infant nutrition, the intestinal microbiota, co-morbid infections, as well as high levels of maternally derived anti-rotavirus antibodies [33].
The inclusion of rotavirus vaccines into the Australian NIP in 2007 has resulted in a considerable and sustained decrease in rotavirus morbidity across most of Australia, with a 71% decline in rotavirus-coded hospitalisations of children aged <5 years reported [34]. However, the observed decrease in hospitalisations has been less in Aboriginal and Torres Strait Islander children; they remain at greater risk of severe rotavirus disease requiring hospitalisation than their non-Indigenous counterparts [34]. Following rotavirus vaccine introduction in WA, significant declines in rotavirus-coded hospitalisation rates have been observed in all children aged <5 years, up to 79% among non-Aboriginal and up to 66% among Aboriginal children [35]. During the outbreak peak in May 2017 (122 cases), over a third of cases were hospitalised as a result of their infection, with Aboriginal people representing two thirds of these hospitalisations. As would be expected with rotavirus infection, the vast majority of cases hospitalised were ≤4 years of age, and Aboriginal children accounted for 71% of hospitalisations in this age group. Compared to their non-Indigenous counterparts, the paediatric Aboriginal population exhibit a greater burden of disease due to infections, and large, biannual rotavirus outbreaks have been reported in the Northern Territory [18][19][20][21][22]. Continued surveillance is critical to elucidate the complex factors that contribute to the occurrence of these outbreaks.
When the vaccination status of cases from the May peak was compared to hospitalisation status, vaccination did not appear to impact on whether a case was hospitalised. Fully or partially vaccinated children represented 75% of hospitalised cases (27/36) compared to unvaccinated eligible children accounting for 8% of hospitalised cases (3/36). Vaccination status was unknown for 3% of cases (1/36) and the remaining 14% of cases (5/36) were ineligible to have been vaccinated based on age. In May 2017, RotaTeq was the vaccine prescribed in the WA vaccination schedule and the vast majority of cases who were either fully or partially vaccinated were vaccinated with RotaTeq. Whilst a genotype-specific vaccine effectiveness has not been estimated for children in WA, the vaccine effectiveness of three doses of RotaTeq has been estimated at 82% (95% CI: 59-92) [36].
Full genome sequencing was performed on representative samples from the outbreak. These samples were found to be most closely related to Japanese G2P [4] strains detected in 2016 and 2017 across all genes in the genome. In one associated paper, these closely related samples were reported as minor G2P [4] variants circulating in the Mie prefecture in 2017 [37]. However, this variant was also detected in Tokyo in 2017, where G2P [4] was the dominant genotype, accounting for 40% of samples [38]. The outbreak samples also consistently clustered with Hungarian G2P [4] from 2012, where this genotype accounted for 13.5% of the samples genotyped in 2012 [39]. The WA outbreak samples clustered within a clade that also included G2P [4] strains from Australia that were circulating in 2010. These samples were collected during 2010-2011 when there was a substantial increase in G2P [4] strains in Australian states using the RotaTeq vaccine; G2P [4] strains replaced G1P [8] as the dominant genotype for the first time since vaccine introduction [23]. Overall, this suggests that the strain circulating during the 2017 WA outbreak is a global variant that was previously detected in Australia and has continued to be successfully transmitted in various regions around the world for over almost a decade. Based on the available sequencing, the majority of samples exhibit a relatively conserved genome that has not undergone substantial reassortment, with the diversity observed indicative of genetic drift over the years. It is highly likely that this variant represents a re-introduction into Australia rather than reflecting genetic drift that has only occurred in the Australian population. The 2017 variant was not closely related to G2P [4] strains that had caused prior outbreaks in the Northern Territory in 1999, 2004, and 2009 [18].
The VP7 gene of the 2017 WA outbreak samples was compared to the G2 VP7 gene component of the RotaTeq vaccine. A total of 16 residues differed between the G2 component of RotaTeq and the two outbreak samples RVA/Human-wt/AUS/WAPC2824/2017/G2P [4] and RVA/Human-wt/AUS/WAPC2769/2017/G2P [4], and RVA/Human-wt/AUS/WAPC2784/ 2017/G2P [4] had 17 residues that differed. However, this is not unexpected as the RotaTeq G2 VP7 gene is derived from a strain that was circulating in 1992; global strains have undergone extensive genetic drift over the intervening years. Three of these altered residues in all three outbreak samples were observed in antigenic epitopes at positions A87T and D96N in antigenic region 7-1a, and S213D in region 7-1b [25]. Altered residue D145G in region 7-2 was only observed in RVA/Human-wt/AUS/WAPC2784/2017/G2P [4]. Residues D96N and S213D have been shown to escape neutralisation with monoclonal antibodies [26]. The observed altered residues A87T, D96N, and S213D have been observed in the majority of G2P [4] strains circulating globally over the last two decades [40]. In particular, mutations A87T, D96N, D145G, and S213D were observed in G2P [4] strains associated with outbreaks in children in Indonesia in 2018 and a nosocomial outbreak in adults within a German hospital [41,42]. Genetic drift in VP7 antigenic epitope regions could adversely impact the effectiveness of the RotaTeq vaccine against G2P [4] strains. However, large-scale studies combining genetic and antigenic characteristics of circulating variants are required to further elucidate this. It is possible that genetic drift between circulating variants and the vaccine strain, in combination with host-related facts that impact vaccine effectiveness in this population contribute to the occurrence of these outbreaks.
A limitation of this study was that a stool sample was available for 70/122 cases from the May peak. Not genotyping all samples could result in the proportion of the different genotypes being over-or underestimated. However, it does not alter the result that G2P [4] was the dominant genotype in the KIMB region as 41/53 samples were available and genotyped. The 70 samples available for genotyping were representative of the age distribution of rotavirus cases in WA during this period. However, more samples from Aboriginal cases were genotyped compared to non-Aboriginal cases (76% vs. 45%) and this could have overestimated the proportion of G2P [4] cases reported. Similarly, more samples were genotyped from the remote areas of the KIMB and PILB regions, which may also have overestimated the proportion of G2P [4] cases seen. Given this study largely focuses on the KIMB region, it is unlikely that this had any major impact on the overall results of the study.

Notification Data
Data on WA cases of rotavirus were obtained from the WA Notifiable Infectious Disease Database (WANIDD). The notifications contained in WANIDD are received from medical practitioners and pathology laboratories under the provisions of the Public Health Act 2016 and subsequent amendments, and are retained in WANIDD if national case definitions are met. Rotavirus was listed as a notifiable disease in WA in July 2006 [11]. Data was extracted from WANIDD by optimal date of onset (ODOO) for the time period 01/01/2012 to 31/12/2017 and exported to Microsoft ® Excel 365 (Microsoft ® , Version 1808, Redmond, WA, USA). The ODOO is a composite of the 'true' date of onset provided by the notifying doctor or obtained during case follow-up, the date of specimen collection for laboratory notified cases, and when neither of these dates is available, the date of notification by the doctor or laboratory, or the date of receipt of notification, whichever is earliest. Notification data are broken down by regions that are based on Public Health Unit (PHU) boundaries, reflecting WA Health administrative regions: Central/Wheatbelt

Vaccination Status
Records of vaccine administration were submitted to the Australian Immunisation Register (AIR) (curated by Services Australia, Australian Government). The AIR includes vaccines administered under the national immunisation program, school programs, and privately. CDCD staff accessed AIR to determine the rotavirus vaccine status of notified cases.

Rotavirus Positive Faecal Samples
A total of 122 faecal samples collected from children and adults presenting to hospital or general practice clinics with severe gastroenteritis in Western Australia during May, 2017 were determined to be rotavirus positive by a local diagnostic laboratory. Seventy de-identified rotavirus positive specimens were sent to the National Rotavirus Reference Centre (NRRC) at the Murdoch Children's Research Institute. A further 27 samples did not have adequate remaining volume and were not sent for genotyping. There is no agreement with private pathology laboratories to forward samples for genotyping. As a result, 25/122 (20%) of samples were not genotyped. Where possible, metadata, including date of collection, date of birth, gender, and postcode, were collected. Samples were stored at −80 • C until analysis, allocated a unique laboratory code, and entered into a REDCap database.

Conformation of Vaccine-Line Strains
Sequencing of VP6 and VP7 genes was performed for suspect RotaTeq samples with mixed G types or were P non-typeable as previously described [18].

Polyacrylamide Gel Electrophoresis
The 11 segments of rotavirus dsRNA were separated on 10% w/v polyacrylamide gel with 3% w/v polyacrylamide stacking gel at 25 mA for 16 h. The genome migration patterns (electropherotypes) were visualised by silver staining according to the established protocol [46].

Whole Genome Sequencing
Each of the 11 genes were reverse transcribed and amplified by PCR using the OneStep RT-PCR Kit (QIAGEN, Hilden, Germany) using gene-specific sense and antisense primers (primer sequences available upon request). RNA was denatured and reverse transcribed for 30 min at 45 • C, followed by PCR activation for 15 min at 95 • C. Then, 40 cycles of amplification for 10 s at 94 • C, 1 min at 55 • C, and 3 min at 68 • C, followed by a final extension for 10 min at 68 • C were performed. The amplicons were gel purified using the Wizard ® SV Gel and PCR Clean-Up System (Promega, Madison, WI, USA) according to the manufacturer's instructions.
The purified products were pooled in equimolar concentrations and subjected to standard library construction for Illumina sequencing using the Nextera XT DNA Library Preparation Kit following the manufacturer's recommendations for dual-indexed barcoding (Illumina Inc., San Diego, CA, USA). Normalised samples were pooled and sequenced using 500-cycle (2 × 250-bp paired-end) MiSeq reagent kits (v2; Illumina Inc., San Diego, CA, USA).

Sequence Assembly
Raw reads were trimmed for quality and adapters using BBDuk Adapter/Quality Trimming Version 38.37, duplicate reads were removed using Dedupe Duplicate Read Remover version 38.37 and pair-end reads were merged using BBMerge Paired Read Merger version 38.37, all performed within Geneious Prime. Reads were mapped to reference rotavirus genomes using the Bowtie2 mapper within Geneious Prime [47].

Assignment of Genotypes
The genotypes of each of the 11 genome segments were determined using the online RotaC v2.0 rotavirus genotyping tool (http://rotac.regatools.be, accessed on 18 January 2021) in accordance with the recommendations of the Rotavirus Classification Working Group (RCWG) [8].

Phylogenetic Analysis
Nucleotide similarity searches were performed using the BLAST server on the Gen-Bank database at the National Center for Biotechnology Information, USA (www.ncbi. nlm.nih.gov, accessed on 18 January 2021). The nucleotide and amino acid sequences of each gene were compared with sequences available in the GenBank database that possessed the entire open reading frame. Multiple nucleotide and amino acid alignments were constructed using the Multiple Sequence Comparison by Log Expectation (MUSCLE) algorithm in Geneious Prime [48].

Accession Numbers
The nucleotide sequences for genes described in this study have been deposited in GenBank under the accession numbers MW275246-MW275278.

Conclusions
This G2P [4] outbreak disproportionately impacted Aboriginal children ≤4 years of age in the remote Kimberley region of Western Australia. The G2P [4] variant circulating was closely related to contemporary Japanese G2P [4] samples, suggesting a global variant that exhibited the altered residues A87T, D96N, and S213D compared to the G2 component of the RotaTeq vaccine, residues that have been observed in the majority of G2P [4] strains circulating globally over the last two decades. Despite national vaccine coverage of 85.4%, outbreaks continue to occur in vaccinated populations in Australia, in particular impacting Aboriginal populations. These outbreaks pose particular challenges to regional areas and remote communities. Continued surveillance and characterisation of emerging variants are imperative to ensure the ongoing success of the rotavirus vaccination program in Australia.