molecular epidemiology of norovirus infections in … · 1 was from a nursing home. 13 involved...

Molecular Epidemiology of Norovirus Infections in Sporadic Cases and Outbreaks of

Gastroenteritis in Hong Kong

Derek WongCS Lau

Louis TongJanice Lo

Wilina Lim

Government Virus UnitPathology Service

Department of Health

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NorovirusesFirst discovered in 1968 in Norwalk County, OhioBelong to the Calicivirus family, formerly known as Norwalk-like virusesNaked single-stranded RNA viruses, virions 27-40 nmTwo Genogroups, with a number of subgroups within each genogroup.Distributed globally, associated with epidemic outbreaks of gastroenteritis. However, It is now clear that noroviruses account for more endemic infections than epidemic infections.

Outbreaks occur regularly in institutions such as nurseries, hospitals, elderly homes, and cruise ships. Associated with consumption of shellfish, other contaminated foods such as salads, and faecallycontaminated water.

Norwalk Virus particles Sapporo VirusBaculovirus-expressed

Virus-like particles

Electron micrographs of Caliciviruses



Prevalence RatePreviously it was thought that rotaviruses accounted for the majority of cases of viral gastroenteritis.

Rotavirus 70%Adenovirus 15%Astrovirus 6%Noroviruses 3%Caliciviruses 1%Others 5%

Now it is thought that noroviruses could account for up to 90% of all cases of viral gastroenteritis, and 7% of deaths due to viral gastroenteritis.


Situation in Hong Kong1988-1999 – Approx 10 cases per year diagnosed by direct Electron Microscopy.

No outbreaks of gastroenteritis was associated with noroviruses. DH didn’t even provide a separate category for viral gastroenteritis.

RT-PCR then became available in the GVU in 2000.

2001 – noroviruses responsible for 45 outbreaks of gastroenteritis. and Noroviruses was the leading cause of infective gastroenteritis in the Acute Diarrhoeal Diseases Surveillance Programme.

There is now a active program for monitoring Norovirus contamination of imported oysters.


Study DesignSpecimens (July 2001 and June 2002)

987 specimens from patients enrolled in the Acute Diarrhoeal Diseases Surveillance Programme of the Department of Health122 specimens collected from individuals involved in 44 norovirusoutbreaks reported to the Department of Health, Hong Kong Government.

MethodsRT-PCR – viral RNA was extracted from stools and then subjected to RT-PCR using primers directed against the RNA polymerase region of noroviruses.DNA Sequencing – was carried out on all PCR products.Phylogenetic analysis – DNA sequences obtained were compared to sequences of known standard strains of noroviruses.


Acute Diarrhoeal Diseases Surveillance Programme

1. Acute watery diarrhoea (passage of 3 or more loose or watery stools in 24 hours) with or without dehydration.

2. Non-infectious causes of acute diarrhoea such as ingestion of medicine (including herbal medicine) before onset of symptoms, food allergy and irritable bowel syndrome excluded.

3. Collection of stool within 72 hours of onset of symptoms.4. Patient does not require immediate referral for admission

to hospital.


Results – Acute Diarrhoeal Diseases Surveillance

Rotavirus was seen in 29 (2.9%) of specimens of which 2 were also positive for norovirus RNA. There were no significant differences between sexes.

no. NLV %

Jul-Sep 377 28 7.43

Oct-Dec 223 31 13.9

Jan-Mar 178 23 12.9

Apr-Jun 209 16 7.66

987 98 9.93

no. NLV %

<5 87 21 24.1

5-14 71 9 12.7

14-24 84 4 4.76

25-59 496 38 7.66

>60 249 26 10.4

Total 987 98 9.93


Results – Outbreak CasesOf the 44 norovirus outbreaks in the study,

20 were from kindergartens or nurseries 5 were from schools1 was from a nursing home.13 involved catered meals consumed at hotels, restaurants or food stalls.5 involved meals taken at home.

The majority of outbreaks (29 outbreaks, 65.9%) occurred in the 6-month period between July and December 2001 with 90 (73.8%) of specimens involved. Overall, 101 (82.8%) out of 122 specimens were positive for norovirusRNA by RT-PCR. In two outbreaks, strains from different genetic clusters. Otherwise, individual strains within other outbreaks were identical to each other.


Genetic Diversity of Noroviruses in Hong KongThe distribution of clusters and genogroups was similar between the surveillance and outbreak cases. For the first six months of the study period, the predominant strain was the 95/96-US subset that belonged to the Bristol cluster of Genogroup II. This strain was associated with pandemic infection.In the later six months of the study, Genogroup I and strains belonging to other clusters of Genogroup II were more commonly seen.

OB2-4

OB2-6

OB2-2

OB2-1

OB2-7

OB2-3

OB2-5

DOUG4770

OB27-2

536/96/Net

BRISTOL

LORDSDALE

OB27-1

OB35-01

OB35-02

HAW AII

SMV

TORONTO

MEXICO

OB40-2

OB27-3

JENA

SOV

OB40-1

OB39-1

NORW ALK

DSV

OB44-1

OB44-2

NAPIER

GW YNEDD52

25

100

80

35

29

81

75

48

10066

100

90

50

26

2298

44

7359

79

39

79

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ConclusionsThis study demonstrate that Noroviruses is probably the most important cause of sporadic cases of acute gastroenteritis in all age groups in Hong Kong.

The positive rate for Norovirus infections is highest among those less than 5 years old with symptoms of acute gastroenteritis.

The pandemic US 95/96 subset accounted for the majority of isolates in the 6 month period of July-December 2001. After which, its incidence dropped off dramatically.

This data would help us better investigate and control norovirus outbreaks and coordinate with other countries through the exchange of molecular data e.g. the Calicinet.


Hospital Cases of GE

no. NLV % no. NLV % no. NLV %

<5 495 99 20 208 40 19.2 287 59 20.56

5-14 127 14 11 47 7 14.9 80 7 8.75

14-24 31 3 9.68 16 2 12.5 15 1 6.667

25-59 56 5 8.93 25 3 12 31 2 6.452

>60 26 2 7.69 16 2 12.5 10 0 0

Total 735 123 16.7 312 54 17.3 423 69 16.31

Norwalk Virus particles Sapporo VirusBacculovirus-expressed

Virus-like particles

Electron micrographs of Caliciviruses



Situation in Hong Kong1988-1999 – Approx 10 cases per year diagnosed by direct Electron Microscopy.

No outbreaks of gastroenteritis was associated with NLV. DH didn’t even provide a separate category for viral gastroenteritis.

RT-PCR then became available, was set up by Mr. Louis Tong following an attachment with Dr. Owen Caul in Bristol.

2001 – NLV responsible for 45 outbreaks of gastroenteritis.

NLV was the leading cause of infective gastorentertis in the Acute Diarrhoeal Diseases Surveillance.

Summary on Causative Agents for Food Poisoning Outbreaks in 2000

Causative Agents Months

Bacteria Chemicals Biotoxins Others* Total

Jan - Mar 80 (266) 3 (7) 10 (25) 22 (110) 115 (408)

Apr - Jun 97 (376) 7 (13) 11 (34) 11 (61) 126 (484)

Jul - Sep 189 (949) 11 (19) 16 (34) 14 (30) 230 (1032)

Oct - Dec 93 (375) 38 (88) 5 (17) 11 (48) 147 (528)

Total 459 (1966) 59 (127) 42 (110) 58 (249) 618 (2452) Summary on Causative Agents for Food Poisoning Outbreaks in 2001


Bacteria Chemicals Biotoxins Viruses Others* Total

Jan - Mar 106 (447) 7 (12) 5 (16) 23 (212) 7 (18) 148 (705)

Apr - Jun 152 (608) 13 (29) 12 (31) 14 (156) 5 (20) 196 (844)

Jul - Sep 175 (715) 15 (28) 13 (34) 1 (3) 5 (10) 209 (790)

Oct - Dec 96 (312) 9 (22) 3 (4) 7 (22) 3 (9) 118 (369)

Total 529 (2082) 44 (91) 33 (85) 45 (393) 20 (57) 671 (2708)


2001 2002

Acute Diarrhoeal Diseases Surveillance –Notification Rate


Acute Diarrhoeal Diseases Surveillance

Laboratory results

2001

Text version

Bacteria Viruses

No. positive No. positive Months No. of

specimens* Vibrio parahaemolyticus

Salmonella spp.

Campylobacter spp. Others Total

No. of specimens*

Norwalk-like

viruses Rotavirus Others Total

Jul-Sep 351 20 18 11 1 50 378 22 1 0 23

Oct-Dec 214 8 9 6 0 23 222 30 6 2 38

Total 565 28 27 17 1 73 600 52 7 2 61


Summary on Causative Agents for Food Poisoning Outbreaks in 2000


Bacteria Chemicals Biotoxins Others* Total

Jan - Mar 80 (266) 3 (7) 10 (25) 22 (110) 115 (408)

Apr - Jun 97 (376) 7 (13) 11 (34) 11 (61) 126 (484)

Jul - Sep 189 (949) 11 (19) 16 (34) 14 (30) 230 (1032)

Oct - Dec 93 (375) 38 (88) 5 (17) 11 (48) 147 (528)

Total 459 (1966) 59 (127) 42 (110) 58 (249) 618 (2452) Summary on Causative Agents for Food Poisoning Outbreaks in 2001


Bacteria Chemicals Biotoxins Viruses Others* Total

Jan - Mar 106 (447) 7 (12) 5 (16) 23 (212) 7 (18) 148 (705)

Apr - Jun 152 (608) 13 (29) 12 (31) 14 (156) 5 (20) 196 (844)

Jul - Sep 175 (715) 15 (28) 13 (34) 1 (3) 5 (10) 209 (790)

Oct - Dec 96 (312) 9 (22) 3 (4) 7 (22) 3 (9) 118 (369)

Total 529 (2082) 44 (91) 33 (85) 45 (393) 20 (57) 671 (2708)



Clinical FeaturesIncubation Period 24-48 hours after exposure.

Vomiting is the main symptom

Mild diarrhoea with pale stool and no cells

Pyrexia low grade or absent

Spontaneous and rapid recovery.


Laboratory Diagnosis - 1Electron Microscopy - classical means of diagnosis of NLV infection, the viruses are non-cultivable. Require samples with large number of virus particles and also highly skilled observers.

Direct EM – after Ammonium persulphate precipitaion or differential centrifugation.IEM – classical IEM or SPIEM using human convalescent sera.

Antigen Detection – a number of RIAs and EIAs had been developed using NLV-specific polyclonal or monoclonal IgG. However, none are shown to detect all strains of NLV and are not as sensitive as RT-PCRAntibody Detection – a number of serological assays are available for the detection of rising titres of IgG or IgM using recombinant capsid proteins.


Laboratory Diagnosis - 2RT-PCR

Have become a major means of diagnosing NLV infection. Only in-house methods are available.Sensitivity and specificity affected by the RNA extraction method, primers used, and post-PCR analysisPrimers

Generally require two sets of primers, one for genogroup 1, and the other for genogroup 2. The RNA polymerase region is usually used, other regions such as the capsid carry less sensitivity.

Post-PCR analysishybridization with oligonucleotide probes is complicated by the very high genetic variability of NLV. Requires multiple probes.DNA sequencing


ConclusionsNorwalk-like Virus is an emerging pathogen whose significance in causing human disease has just been recognized.

Research in NLV had been hampered by the lack of a culture system and the high genetic variability of the virus.

It is one of the few viral infections where PCR is the method of choice or gold standard for diagnosis.

In a few short years, it had gone from a very unimportant pathogen to a situation where there is potential overreaction.

Clearly there is a need for monitoring NLV in food, water and sewage but it is unclear what the standards should be.


Viral GastroenteritisIt is thought that viruses are responsible for up to 3/4 of all infective diarrhoeas. Viral gastroenteritis is the second most common viral illness after upper respiratory tract infection.In developing countries, viral gastroenteritis is a major killer of infants who are undernourished. Rotaviruses are responsible for half a million deaths a year.


Prevalence RatePreviously it was thought that rotaviruses accounted for the majority of cases of gastroenteritis.

Rotavirus 70%Adenovirus 15%Astrovirus 6%Noroviruses 3%Calciviruses 1%Others 5%

Now it is thought that NLV could account for up to 90% of all cases of viral gastroenteritis, and 7% of deaths due to viral gastroenteritis.

molecular epidemiology of norovirus infections in … · 1 was from a nursing home. 13 involved...

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