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The National Rotavirus Reference Centre has conducted rotavirus surveillance by means of a collaborative laboratory based initiative started in June 1999. The serotypes of rotaviruses that lead to the hospitalisation of children with acute diarrhoea were determined from June 1999 to May 2000. We examined 1126 rotavirus specimens using a combination of monoclonal antibody immunoassay, reverse transcription-polymerase chain reaction, and hybridisation. The four most common serotypes G1-G4 were represented. More than 50% of isolates tested were serotype G1, with serotype G1 being represented in most centres Australia-wide. Serotype G9 rotaviruses were identified for the first time in Australia, and were second in importance with 10% of samples tested. The significant presence of G9 viruses throughout Australia suggests the emergence of a new serotype and has implications for current rotavirus vaccine strategies that target serotypes G1-G4. Commun Dis Intell 2000;24:195-198.

Introduction

Rotaviruses are the most important cause of severe gastroenteritis in young children worldwide. The pathogen is believed to be responsible for the annual admission of up to 10,000 children to hospitals nationwide.¹ A national rotavirus surveillance programme was commenced in June 1999 to undertake the surveillance and characterisation of rotavirus strains causing annual epidemics of severe diarrhoea in young children throughout Australia. The programme was designed to monitor the antigenic variation of rotaviruses prior to and after the anticipated rotavirus vaccine release in Australia. The study was reliant on the cooperation and participation of sentinel laboratories from all States and the Northern Territory. It was designed to supplement data from existing notification schemes, by reporting the serotypes circulating in Australian urban centres. The following report covers the period June 1999 to May 2000.

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Methods

A network of laboratories from each State and the Northern Territory undertake rotavirus detection by enzyme immunoassay (EIA) or latex agglutination. Rotavirus-positive specimens were collected, stored frozen and forwarded to the Royal Children's Hospital (RCH) in Melbourne, together with relevant age and sex details. Representative faecal specimens were then tested using an in-house monoclonal antibody (MAb) based serotyping EIA. The EIA incorporates a panel of MAbs specific for the common group A human rotavirus serotypes (serotypes G1, G2, G3, and G4). Specimens with an absorbance value greater than 0.2 were considered positive for that serotype. Northern hybridisation analysis with G type specific DNA probes using stringent hybridisation conditions was also employed to confirm serotype specificities. Selected strains unable to be assigned a serotype, were genotyped by reverse transcriptase/ polymerase chain reaction using serotype specific primers.

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Results

Number of isolates

In all 1545 rotavirus positives were sent to the Royal Children's Hospital. Specimens received from New South Wales made up 30.5% of all specimens received (Newcastle, Narrabri and Sydney). Twenty-five percent of specimens were received from Western Australia (Perth and northern Western Australia), 16% from Victoria (Melbourne and Horsham), 14% from South Australia (Adelaide), 7% from Queensland (Brisbane and Townsville), 7% from the Northern Territory (Darwin, Gove and Alice Springs) and 0.5% from Tasmania (Hobart). Representative specimens were incorporated into the serotyping EIA (1126 specimens). Specimens that were not confirmed to be positive for rotavirus, or had insufficient specimen for testing, were excluded.

Seasonal occurrence

The overall nationwide rotavirus reports peaked in August (Figure 1). The peak month of activity varied between centres, with temperate regions experiencing a noticeable winter peak between July and October. The rotavirus seasons of combined centres in Victoria, Tasmania and New South Wales peaked in August, followed by Queensland in September (Figure 2). Western Australia shared its rotavirus peak with Alice Springs and Adelaide in October. The tropical Northern Territory rotavirus season peaked in January 2000.

Figure 1. Rotavirus reports, Australia, June 1999 to May 2000, by month



Figure 2. Seasonal peaks of rotavirus reports, Australia by region, June 1999 to May 2000, by month

Age group

The age distribution of rotavirus-positive patients showed the peak incidence occurred in children aged between 1 and 2 years (Figure 3). The male:female ratio was 1.18:1.

Figure 3. Rotavirus positive children, Australia, June 1999 to May 2000, by age group in months


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Serotype distribution

The serotypes circulating in Australia from June 1999 to May 2000 are shown in Figure 4. Serotype G1 accounted for 58 per cent of specimens nationally, and was responsible for more than 60% of rotavirus serotypes in some centres; Hobart (100%), Darwin (80%), Northern Western Australia (69%) and Perth (80%) (Table 1). Second in importance to G1 were G9 rotaviruses (10%). The G9 rotaviruses appeared in Sydney and Melbourne in June 1999 and were present in 12 of the 18 centres studied (all centres except Darwin, Townsville, Horsham, Gove, northern Western Australia and Hobart). Serotype G2, G3 and G4 viruses accounted for less than 5 per cent of all serotypes detected. Serotype G2 viruses were detected along the east coast of Australia, Narrabri, Adelaide, Perth and northern Western Australia. Serotype G3 viruses were present in Melbourne and Adelaide only. Serotype G4 viruses were found in all States except Tasmania and the Northern Territory. A serotype could not be assigned to 27 per cent of specimens. Specimens containing mixtures of rotavirus G types were detected in less than 1 per cent of samples tested.

Figure 4. Rotavirus isolates, Australia, June 1999 to May 2000, by serotype

1. NR = non-reactive

Table 1. Rotavirus positive specimens, Australia, June 1999 to May 2000 by typing centre and serotype

Centre	Serotype
	G1	G2	G3	G4	G9	Mix	NR1	Total
Brisbane	31	2		2	5	1	34	75
Townsville		3					16	19
South-eastern Sydney	123			1	31		52	207
Western Sydney	46	1		2	20	1	23	93
Newcastle	2			2	7		5	16
Narrabri		2			2		19	23
Melbourne2	117	1	5	8	26	5	72	234
Melbourne3	3						1	4
Horsham	6			2				8
Hobart	5							5
Adelaide	52	5	2	2	3	2	11	77
Perth	199	4		3	7	1	32	246
WA PathCentre	18	4					4	26
Darwin4	22						2	24
Darwin5	6						5	11
Gove	2						1	3
Alice Springs	20				12		23	55
Total	652	22	7	22	113	10	300	1,126

1. NR = Non reacting to G1, G2, G3, G4 and G9 monoclonal antibodies.
2. Royal Children's Hospital
3. Southern Cross Pathology
4. Royal Darwin Hospital
5. Western Diagnostic Pathology

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Discussion

The surveillance study was marked by the appearance of G9 rotaviruses in Australia for the first time.² The virus was responsible for at least ten percent of acute diarrhoea rota-viral hospital admissions. Retrospective analysis showed two G9 viruses were present in Perth and Melbourne in 1997. The subsequent spread of G9 viruses appears to be almost nationwide with the increase in numbers making G9 viruses second in importance to G1 viruses. G9 viruses were found in all States except Tasmania. G9 viruses have been reported in India (1993-1994),³ Bangladesh (1987 to 1997),⁴ Malawi (1997-1998),⁵ the USA (1996-1997)⁶ and the UK (1996).⁷ The rapid emergence of G9 as a major infecting serotype has important implications for rotavirus vaccine strategies. Current candidate vaccines target only serotype G1-G4 infections. Ongoing surveillance is warranted to obtain a clearer picture of the importance of the spread of G9 viruses.

Serotype G1 viruses were the most prevalent serotype in Australia during the sampling period. This G1 dominance was consistent with recent studies undertaken in Australia (1993-1996) (Personal communication, Professor Ruth Bishop, Department of Gastroenterology, Royal Children's Hospital. Parkville Victoria), the UK (1996)⁷ and the USA (1996-1997)⁶. The virus was present in all centres tested except Townsville and Narrabri.

Centres that showed evidence of more than one serotype generally had larger populations. There appeared to be more serotypes circulating and presenting as hospital admissions in the bigger centres. Smaller population centres such as Hobart, Townsville and Darwin had only one serotype detected and appear to have insufficient population size to sustain multiple G types. Serotype G2, G3 and G4 viruses were present in small numbers and appeared sporadically.

Antigenic similarities between G4 and G9 viruses were noted. There were a number of specimens that reacted to both G4 and G9 serotyping MAbs. The use of Northern hybridisation and RT/PCR analysis clarified any serological cross reactivities.

Several specimens were unable to be assigned a serotype by EIA. These non-reactive (NR) specimens, generally contained minimal amounts of viral capsid protein and were unable to be detected by the serotyping MAbs. Electrophoretic analysis of the RNA from some of them showed that the viruses circulating in Townsville, Brisbane and Narrabri all shared the same electrophoretic profile. RT/PCR typing of limited numbers showed the samples were serotype G2. Type G2 viruses were under represented in the serotyping EIA suggesting genetic changes in the epitopes targeted by G2-specific MAbs. Specimens received from remote regions of northern Western Australia shared serotype similarities with specimens collected from Perth children. This coexistence of virus serotypes in geographically diverse locations suggests rapid spread of virus strains across the country. The incorporation of remote locations into the study helped to gain a better insight into the pattern of spread.

Ongoing surveillance of seasonal rotavirus serotype patterns is warranted, in particular to monitor the spread of new or emerging serotypes. Such information will influence the strategy for development of second and third generation rotavirus vaccines, and will show whether Australia's requirements differ from those of other parts of the world. This is particularly important now that the first G1-G4 targeted vaccine has been withdrawn from sale in the USA due to its apparent association with intussusception - a form of bowel obstruction.

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Acknowledgements

Rotavirus positives were collected from numerous centres throughout Australia. The significant time and effort involved in the collection, storage, packaging, compiling data and forwarding of specimens was much appreciated. Without the contribution of the following key people the study would not have been possible.

Western Australia

C Farrar and members of Virology Department
Princess Margaret Hospital for Children
SUBIACO WA 6008

Dr D Smith, Dr D Harnett and members of Division of Microbiology
PathCentre
The Queen Elizabeth Medical Centre
NEDLANDS WA 6009

Northern Territory

J De Boer and members of Microbiology Department
Royal Darwin Hospital
CASUARINA NT 0810

B Truscott and members of Pathology Department
Western Diagnostic Pathology
TIWI NT 0810

F Morey, and members of Microbiology Department
Alice Springs Hospital
ALICE SPRINGS NT 0871

A Reed and members of Pathology Department
Gove District Hospital
NHULUNBUY NT 0880

New South Wales

A Kesson, I Tam and members of Department of Virology
The New Children's Hospital
WESTMEAD NSW 2145

Dr C McIver and members of Microbiology Department
The Prince of Wales Hospital
RANDWICK NSW 2031

F Groeneveld and members of Microbiology Laboratory
Sydpath
NARRABRI NSW 2390

Dr John Ferguson and members of Pathology Department,
John Hunter Hospital,
NEW LAMBTON NSW 2305

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South Australia

A Lawrence and members of Microbiology Department
Women's and Children's Hospital
72 King William Road
NORTH ADELAIDE SA 5006

Victoria

Southern Cross Pathology
Monash Medical Centre
CLAYTON VIC 3168

Dr Roger Schnagl
School of Microbiology
La Trobe University
BUNDOORA VIC 3083

Dr Robert Alexander and members of Pathology Department
Royal Children's Hospital
PARKVILLE VIC 3052

P Chondros
Clinical Epidemiology and Biostatistics Unit
Royal Children's Hospital
PARKVILLE VIC 3052

S Lloyd Jones
Melbourne Pathology
COLLINGWOOD VIC 3066

Angela Clarke and members of Pathology Department
Wimmera Base Hospital
HORSHAM VIC 3400

Queensland

L Davis and members of Microbiology Division
Queensland Health Pathology Service
Royal Brisbane Hospital
HERSTON QLD 4029

F Francis and members of Pathology Department
Townsville General Hospital
TOWNSVILLE QLD 4180

Tasmania

R Peterson and members of Microbiology Department
Royal Hobart Hospital
HOBART TAS 7001

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Author affiliations

1. Corresponding author: Paul Masendycz, National Rotavirus Reference Centre, Murdoch Children's Research Institute, Royal Children's Hospital, Flemington Road, Parkville, Victoria, Australia 3052. Telephone: (03) 9345 5069 Fax: (03) 9345 6240 E-mail:   masendyp@cryptic.rch.unimelb.edu.au

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References

1. Carlin JB, Chondros P, Masendycz P, Bugg H, Bishop RF, Barnes GL. Rotavirus infection and rates of hospitalisation for acute gastroenteritis in young children in Australia, 1993-1996. Med J Aust 1998;169:252-256.

2. Palombo EA, Masendycz PJ, Bugg HC, Bogdanovic-Sakran N, Barnes GL, Bishop RF. Emergence of serotype G9 human rotaviruses in Australia. J Clin Microbiol 2000;38:1305-1306.

3. Ramachandran M, Das BK, Vij A, et al. Unusual diversity of human rotavirus G and P genotypes in India. J Clin Microbiol 1996;34:436-439.

4. Unicomb LE, Podder G, Gentsch JR, et al. Evidence of high-frequency genomic reassortment of group A rotavirus strains in Bangladesh: emergence of type G9 in 1995. J Clin Microbiol 1999;37:1885-1891.

5. Cunliffe NA, Gondwe JS, Broadhead RL, et al. Rotavirus G and P types in children with acute diarrhea in Blantyre, Malawi, from 1997 to 1998: predominance of novel P(6)G8 strains. J Med Virol 1999;57:308-312.

6. Ramachandran M, Gentsch JR, Parashar UD, et al. Detection and characterisation of novel rotavirus strains in the United States. J Clin Microbiol 1998;36:3223-3229.

7. Cubitt WD, Steele AD, Iturriza M. Characterisation of rota-viruses from children treated at a London hospital during 1996: emergence of strains G9P2A(6) and G3P2A(6). J Med Virol 2000;61:150-154.


This article was published in Communicable Diseases Intelligence Volume 24, No 7, July 2000.