Development and validation of a microarray for the confirmation and typing of norovirus RT-PCR products

Noroviruses are implicated in many worldwide institutional, food and waterborne outbreaks each year. Genetic typing of isolates is valuable for monitoring outbreak spread as well as variation in circulating strains. Microarrays have the potential to provide rapid genotype information for norovirus samples. The NoroChip v3.0 provides an oligonucleotide hybridization platform to screen for over 600 potential interactions in each experiment. The NoroChip v3.0 was developed at Health Canada and validated in seven international partner laboratories. Each laboratory validated the NoroChip v3.0 using norovirus amplicons routinely characterized in their testing protocols. Fragments from the capsid region (region C) and a 2.4 kb amplicon spanning polymerase and capsid sequences (region AD) were validated in six of the partner laboratories and provided correct genogroup typing information (GI or GII) when hybridized to the NoroChip v3.0. Results indicate that the current limiting factor for implementing the NoroChip v3.0 as a strain typing tool is the difficulty obtaining a long, specific amplicon from all circulating norovirus strains. Data obtained with the longer region AD amplicon provided the best discrimination between norovirus strains.     

珠海市冬春季节儿童病毒性腹泻的流行病学特征

目的 探讨珠海市冬春季节儿童诺如病毒(NV)、扎如病毒(SV)和星状病毒(AstV)等3种重要病毒性腹泻的流行病学特征.方法 采集2009年11月21日至2010年4月3日在珠海市妇幼保健院就诊的腹泻儿童粪便样本,选取轮状病毒和腺病毒筛查阴性的样本,采用逆转录-聚合酶链反应(RT-PCR)技术检测NV、SV和AstV的特异基因片段,并对诺如病毒检测阳性的样本进行分子分型.结果 3种病毒季节总检出率为21.49％,其中2009年12月的检出率最高,为29.05％,而2010年2月检出率最低,为12.20％,87.96％的阳性样本来自0～30月龄患儿.NV、SV及AstV的季节检出率分别为14.70％、2.75％和4.04％,3种病毒中以NV和SV各月份阳性检出率差别较大,而AstV较为一致,12 ～ 18月龄患儿NV检出率最高(34.09％),60 ～ 120月龄患儿SV检出率最高(12.5％),24 ～30月龄患儿AstV检出率最高(16.67％).诺如病毒经分子分型后均为GⅡ型.结论 NV是引起2009年冬春季节珠海市儿童病毒性腹泻的主要病原之一,SV与AstV也是重要的病原,应加强对婴幼儿NV、SV和AstV等3种重要病毒性腹泻的监测和防控工作.     

粪便标本中诺如病毒GⅡ拷贝数定量检测方法的建立和应用

目的 构建诺如病毒GⅡ（Norovirus Genogroup Ⅱ,NoV GⅡ）拷贝数标准质粒及其检测体系。方法 将合成高度保守的NoV GⅡ基因序列片段克隆至pUC57载体上,构建NoV GⅡ标准质粒,采用聚合酶链反应（PCR）进行验证。通过实时荧光定量PCR扩增曲线结果绘制Ct值与病毒拷贝数的标准曲线,求得其相应的标准曲线方程。采用建立的标准质粒及其检测体系对2018年12月昆明市儿童医院4份临床粪便标本进行检测。结果 经PCR验证,NoV GⅡ拷贝数的标准质粒构建正确。采用实时荧光定量PCR的扩增曲线获得Ct值与病毒拷贝数相对应的标准曲线方程为Y=-3.972X+39.03,R²=0.991,4份粪便标本原液NoV GⅡ病毒拷贝数分别为30 443.45、9 468.40、53 176.69、4 493.12 copies/μL。结论 成功建立用于检测粪便标本中NoV GⅡ病毒拷贝数的标准质粒及其检测体系,可为疾病的预防控制和相关试验研究提供有效的定量检测方法。     

Super-infections and relapses occur in chronic norovirus infections

BackgroundNorovirus causes chronic infections in immunocompromised patients with considerable associated morbidity. It is not known whether chronic infections involve super- or re-infections or relapses.ObjectivesTo retrospectively investigate whether longitudinal sampling in chronically infected patients demonstrates persistent infection with the same virus, or super- or re-infection.Study designNorovirus full genomes were generated from 86 longitudinal samples from 25 paediatric patients. Consensus sequences were used for phylogenetic analysis and genotyping.ResultsSuper-infections occurred in 17% of chronically infected patients who were continuously PCR positive; including two with mixed norovirus infections. The median duration of infection was 107 days longer in those with super-infections; however this was not statistically significant. A third of patients with interrupted norovirus shedding continued to be infected with the same virus despite up to 2 months of PCR negative stools, classified as a relapse. The majority (67%) of patients with interrupted shedding were re-infected with a different genotype.ConclusionsChronically infected patients who are continuously PCR positive are most likely to remain infected with the same virus; however super-infections do occur leading to mixed infection. Patients with interrupted shedding are likely to represent re-infection with a different genotype, however relapsing infections also occur.Our findings have implications for infection control as immunosuppressed patients remain susceptible to new norovirus infections despite current or recent infection and may continue to be infectious after norovirus is undetectable in stool. The relevance to children without co-morbidities remains to be determined.     

The impact of calicivirus mixed infection in an oyster-associated outbreak during a food festival

BackgroundDespite calicivirus food-borne outbreaks posing major public health concern worldwide, little information is at present available about the impact of caliciviruses mixed infection in an oyster-associated outbreak in China.ObjectivesTo investigate the clinical and epidemiologic characteristics of an oyster-associated calicivirus outbreak initiated by a food festival in Shanghai, China, in April 2014.Study designMolecular epidemiological studies based on nucleotide sequencing and phylogenetic analysis of calicivirus strains from patients.ResultsA total of 65 of the 78 (83%) cases from this outbreak were associated with raw oyster consumption. Forty-six calicivirus strains were identified from 25 stool specimens with norovirus (NoV) GII.4 Sydney_2012, GII.13, GI.2, GI.5 and sapovirus (SaV) GI.2 being predominant genotypes and with a prevalence of triple-, double- and single-infection being 20%, 48% and 28%, respectively. Meanwhile, 13 putative NoV recombinants were indicated by the phylogenetic inconsistency between capsid and polymerase genotype, mainly including GII.Pe/GII.4 Sydney_2012. Molecular epidemiological investigation showed possible multiple route transmission in the field. The clinical and epidemiologic characteristics of the mixed point-source calicivirus outbreak also conformed to Kaplan’s criteria.ConclusionsThis is the first reported oyster-associated calicivirus outbreak with a high prevalence of mixed infection during a food festival described in China. Our investigation underscores the importance of early surveillance and comprehensive etiologic identification of mixed point-source outbreaks and the need for reliable standards of monitoring oysters to prevent and control calicivirus food-borne outbreaks in China.     

Norovirus shedding among food and healthcare workers exposed to the virus in outbreak settings

BackgroundNoroviruses (NoV) are highly contagious and the leading cause of nonbacterial outbreaks of gastroenteritis worldwide. Individuals who are infected asymptomatically may act as reservoirs and facilitate the transmission of NoV, but the likelihood of workers of becoming infected in outbreak settings has not been systematically studied.ObjectivesWe evaluated the occurrence of norovirus infections among workers exposed to the virus in different outbreak settings.Study designWe screened feces from food handlers and healthcare workers related with gastroenteritis outbreaks, and shedding concentrations over time were calculated from serial samples of infected individuals. Sequence analyses of the capsid P2 domain and region C were used to evaluate linkage between asymptomatic employees and outbreak cases.ResultsOf all employees, 59.1% were positive for NoV, and more than 70% of them were asymptomatic. Asymptomatic infections were significantly more frequent in foodborne compared to person-to-person transmitted outbreaks; and in restaurants and hotels, compared to nursing homes and healthcare institutions. Mean viral loads were similar between symptomatic and asymptomatic individuals, starting at 7.51 ± 1.80 and 6.49 ± 1.93 log₁₀ genome copies/g, respectively, and decreasing to 5.28 ± 0.76 and 4.52 ± 1.45 log₁₀ genome copies/g after 19 days.ConclusionsThe likelihood of becoming infected when a NoV outbreak occurs at the work place is high and similar between food handlers and healthcare workers, but asymptomatic infections are more frequently identified among food handlers. Since shed amounts of viruses in the absence of symptoms are also high, reinforcement of hygiene practices among workers is especially relevant to reduce the risk of virus secondary transmissions.     

Norovirus detection from sera of young children with acute norovirus gastroenteritis

BackgroundAntigenemia and viremia are common in rotavirus infection but only few studies have shown norovirus (NoV) RNA in the blood circulation.ObjectivesTo detect NoV RNA from serum of NoV-infected children and study if NoV RNAemia correlates with clinical severity of acute gastroenteritis.Study designSerum specimens were collected from 176 Finnish children with acute NoV gastroenteritis. Semi-nested PCR was optimized to detect NoV capsid RNA from sera. NoV positive samples were further analyzed by sequencing.ResultsNoV RNA was found in 11/176 (6.3%) of serum specimens. NoV GII.4 was found in 8 cases and GII.3, GII.6 and GII.7 in one case each. The genotypes detected in serum were identical to findings in stools in all cases. Most of the NoV RNA detections in serum were in young children less than 18 months of age. The clinical features of NoV serum-positive cases were not different from NoV serum-negative cases.ConclusionNoV RNA in serum is an uncommon finding limited to young children.     

Immunogenicity of a bivalent virus-like particle norovirus vaccine in children from 1 to 8 years of age: A phase 2 randomized,double-blind study

BackgroundYoung children can suffer severe consequences of norovirus gastroenteritis. We performed a dose-finding study of a bivalent virus-like particle (VLP) vaccine candidate (TAK-214) in healthy 1–8-year-old children.MethodsIn this phase 2 study two age cohorts (1–3 and 4–8 years of age inclusive, N = 120 per cohort) of children enrolled from Finland, Panama and Colombia were initially randomized 1:1:1:1 to four groups which were further split into two equal subgroups, to receive one or two intramuscular doses of four TAK-214 formulations containing 15/15, 15/50, 50/50 or 50/150 μg of GI.1/GII.4c genotype VLPs and 0.5 mg Al(OH)₃ at 28 days interval. ELISA Pan-Ig and histoblood group antigen-blocking (HBGA) antibodies against each VLP were measured on days 1, 29, 57 and 210. Parents/guardians recorded solicited local and systemic adverse events (AE) and any unsolicited or serious AEs (SAE).ResultsAll formulations were well-tolerated across both age cohorts and dosage groups with no vaccine-related SAEs reported. Solicited AEs were mostly mild-to-moderate, resolved quickly, and did not increase after the second dose. Pan-Ig and HBGA responses induced after one dose were only slightly increased by the second dose. Across dose groups at Day 29 after one dose GI.1 Pan Ig seroresponse rates (SRR) were 82–97% and 81–96% and GII.4c SRR were 79–97% and 80–91% in 1–3 and 4–8 year-olds, respectively. Respective rates were to 92–93% and 73–92% for GI.1, and 77–100% and 62–83% for GII.4c at Day 57 following two doses. HBGA responses had similar profiles. Both Pan Ig and HBGA geometric mean titers persisted above baseline up to Day 210.ConclusionsAll dosages of TAK-214 displayed acceptable reactogenicity in 1–8-year-old children and induced robust, durable immune responses after one dose which are further increased after two doses.