Municipal wastewater treatment plants as pathogen removal systems and as a contamination source of noroviruses and Enterococcus faecalis

Municipal wastewater treatment plants play a crucial role in reducing the microbial and pathogen load of human wastes before the end-products are discharged to surface waters (final effluent) or land spread (biosolids). This study investigated the occurrence frequency of noroviruses, Enterococcus faecalis and Enterococcus faecium in influent, final effluent and biosolids from four secondary wastewater treatment plants in northwestern Ireland (plants A-D) and observed the seasonal and spatial variation of the plant treatment efficiencies in the pathogen removals. It was noted that norovirus genogroup II was more resistant to the treatment processes than the norovirus genogroup I and other active viral particles, especially those in the discharge effluents. The percolating biofilm system at plant D resulted in better effluent quality than in the extended aerated activated sludge systems (plants A and B); primary biosolids produced at plant D may pose a higher health risk to the locals. The spread of norovirus genogroup II into the environment, irrespective of the wastewater treatment process, coincides with its national clinical predominance over norovirus genogroup I. This study provides important evidence that municipal wastewater treatment plants not only achieve pathogen removal but can also be the source of environmental pathogen contamination.     

Prevalence and genetic characterization of caliciviruses among children hospitalized for acute gastroenteritis in the United States.

Human calicivirus was the first recognized viral agent causing gastroenteritis in humans. Norovirus (NV) and Sapovirus (SV), two genera within the Caliciviridae family, cause epidemic and endemic acute gastroenteritis in children and adults. The role of these viruses as a cause of sporadic acute gastroenteritis in young children requiring hospitalization is not well established. The aim of this study was to assess the prevalence and genetic diversity of caliciviruses among children hospitalized with symptoms of acute gastroenteritis. Stool samples were collected over 2 years from symptomatic children (N=1840) up to 5 years of age at three pediatric hospitals in the US. Overall, 156 (8.5%) samples were CV-positive, 131 (7.1%) confirmed by sequencing to be NV and 25 (1.4%) confirmed to be SV. Sequences of RT-PCR-amplified polymerase gene segments were analyzed using distance, maximum likelihood and parsimony algorithms. Phylogenetic analysis of 97 NV sequences showed that seven strains were in genogroup I, 86 strains were in genogroup II and four strains were not in genogroup I, II, or III, likely representing three new NV genogroups IV, VI and VII. Genogroup I and genogroup II strains were in 12 new genetic clusters, three in genogroup I and nine in genogroup II. Within genogroups I and II, most (98%) NV strains were in genetic clusters with no known prototype in GenBank. Phylogenetic analysis of 24 SV strains showed that half grouped with the London/92 strain in one genogroup and the remainder in three other proposed genogroups, one novel. In conclusion, NV and SV were frequent causes of hospitalization for acute gastroenteritis in young children and infecting strains were highly diverse, including newly recognized genogroups and genetic clusters within known genogroups.     

浙江省2008-2009年三起诺如病毒胃肠炎暴发的病原分子特征分析

目的 分析浙江省3起病毒性胃肠炎暴发疫情的诺如病毒分子特征.方法 收集监测期间病毒性胃肠炎暴发疫情患者的粪便标本,采用荧光定量RT-PCR方法检测诺如病毒,并选择部分阳性标本扩增部分多聚酶区(RdRp)和衣壳蛋白区,同时采用3′RACE(rapidamplification of cDNA 3′ends)扩增诺如病毒基因组3′末端,获得完整的开放读码框架(ORF)2和ORF3序列.结果 3起暴发疫情共检测标本62份,诺如病毒阳性41份,其中诺如病毒Ⅰ(G Ⅰ)基因组阳性27例,Ⅱ基因组(GⅡ)阳性9例,G Ⅰ+GⅡ阳性5例.结果 显示,引起浙江省2008-2009年3起病毒性胃肠炎暴发疫情的诺如病毒具有病毒基因型的多样性,包括G Ⅰ.8、GⅡ.b、GⅠ.2与GⅠ.6重组株、GⅠ.8和GⅡ-b混合感染.结论 诺如病毒是浙江省病毒性腹泻暴发疫情的重要病原体,呈现出病毒基因型的多样性,并在省内首次检测到诺如病毒的重组和混合感染毒株.

Abstract：

Objective To study the molecular characteristic of norovirus in 3 outbreaks of gastroenteritis in Zhejiang province. Methods During January 2008 and December 2009, fecal specimens of patients were collected from 3 outbreaks of acute viral gastroenteritis. Noroviruses were detected by Real-time RT-PCR. Part of the positive samples were randomly selected and detected by RT-PCR. PCR products were sequenced. Sequence analysis was undertaken based on partial sequence of RNA dependent RNA polymerase(RdRp)and capsid protein gene. Some positive samples were amplified by 3' RACE(rapid amplification of cDNA 3' ends), 3200 bp in length. The exact whole ORF2, ORF3 and 3' untranslation regions(UTR)gene of norovims were identified. Results There were in total 3 outbreaks of viral gastroenteritis caused by norovirus being reported. A total of 62 stools were obtained from cases with acute gastroentefitis. Noroviruses were detected in 41 cases including 27 strains of genogroup Ⅰ norovirus and 9 strains of genogroup Ⅱ norovirus, 5 strains of genogroup Ⅰ + Ⅱ norovirus. Four genotypes including G Ⅰ .8, G Ⅱ .b, G Ⅰ .2/0 Ⅰ .6 recombination together with co-infection of G Ⅰ .8 and G Ⅱ .b were detected. Conclusion Norovirus was confirmed as the major cause of outbreaks of viral gastroenteritis in Zhejiang province and multiple genotype of norovirus were identified from the outbreaks. It was the first time to have found a recombinant of G Ⅰ .6 capsid and G Ⅰ .2 polymerase norovims as well as the co-infection of G Ⅰ .8 and G Ⅱ .b norovirus in the same sample.     

Norovirus in captive lion cub (Panthera leo)

African lions (Panthera leo) are susceptible to viral diseases of domestic carnivores, including feline calici-virus infection. We report the identification of a novel enteric calicivirus, genetically related to human noroviruses of genogroup IV, in a lion cub that died of severe hemorrhagic enteritis.     

Intergenogroup recombination in sapoviruses

Sapovirus, a member of the family Caliciviridae, is an etiologic agent of gastroenteritis in humans and pigs. Analyses of the complete genome sequences led us to identify the first sapovirus intergenogroup recombinant strain. Phylogenetic analysis of the nonstructural region (i.e., genome start to capsid start) grouped this strain into genogroup II, whereas the structural region (i.e., capsid start to genome end) grouped this strain into genogroup IV. We found that a recombination event occurred at the polymerase and capsid junction. This is the first report of intergenogroup recombination for any calicivirus and highlights a possible route of zoonoses because sapovirus strains that infect pig species belong to genogroup III.     

Japanese Encephalitis Outbreak, India, 2005

An outbreak of viral encephalitis occurred in Gorakhpur, India, from July through November 2005. The etiologic agent was confirmed to be Japanese encephalitis virus by analyzing 326 acute-phase clinical specimens for virus-specific antibodies and viral RNA and by virus isolation. Phylogenetic analysis showed that these isolates belonged to genogroup 3.     

Characterization of enterotoxigenic Escherichia coli strains isolated from the massive multi-pathogen gastroenteritis outbreak in the Antofagasta region following the Chilean earthquake, 2010

In March 2010, a massive outbreak of gastroenteritis started in the region of Antofagasta (northern Chile). The outbreak was mainly attributed to Norovirus genogroup II although ETEC strains were also isolated with high frequency from clinical samples. We review this outbreak and determined that ETEC was an underestimated etiologic agent.     

New Viruses in Idiopathic Human Diarrhea Cases,the Netherlands

Emerging viral infections can be identified by using a viral metagenomics approach for clinical human material. Diarrhea samples of patients with unexplained gastroenteritis from the Netherlands were analyzed by using viral metagenomics. Novel circular DNA viruses, bufaviruses, and genogroup III picobirnaviruses were identified. These data expand our knowledge of the human virome.     

Fecal viral concentration and diarrhea in norovirus gastroenteritis

Fecal viral concentrations of 40 patients infected with norovirus genogroup GII.4 correlated with diarrhea duration and frequency of vomiting. Higher viral concentration and older age were independently associated with prolonged diarrhea (> or =4 days). These findings provide information on the pathogenesis and transmission of norovirus infections.     

宫颈病变进展与高危型人乳头瘤病毒负荷量相关性研究

目的探讨高危型人乳头瘤病毒(HPV)的病毒负荷量与宫颈病变的发生及进展的关系,宫颈病变经LEEP或冷刀锥切手术后疗效评估。方法选择妇产科门诊就诊宫颈病变患者4712例,采用HC-Ⅱ方法检测其HPV-DNA含量,阴道镜下定位活检,病理学结果为确诊标准。其中CINⅠ～Ⅲ263例先后进行LEEP或冷刀锥切治疗手术,术后6、12月对所有HC-Ⅱ阳性患者进行随访HC-Ⅱ及细胞学检查,并以HC-Ⅱ阳性宫颈炎患者行同期相检查为对照进行疗效评价。结果4712例患者中高危型病毒负荷量随着宫颈病变程度增高而增高,它们之间显著正相关(H=1984.714,P0.05),慢性宫颈炎与其他3个组差异有统计学意义(P0.05),而CIN组间差异无统计学意义(P0.05)。CIN患者经LEEP或冷刀锥切手术后术前HC-Ⅱ阳性CINⅠ、CINⅡ、CINⅢ的术后HPV平均持续感染率分别为0%、0.8%、15.21%,较之保守治疗的慢性宫颈炎组HPV平均感染率36.2%,疗效差异有统计学意义(P0.05)。结论高病毒负荷量尤其是持续性高病毒负荷量与宫颈病变发生及进展呈正相关;经LEEP或冷刀锥切术后的宫颈病变患者,疗效明显优于持续性高病毒负荷量而采取保守治疗的患者。     

Analysis of GII.P7 and GII.6 noroviruses circulating in Italy during 2011–2016 reveals a replacement of lineages and complex recombination history

Noroviruses are important human enteric pathogens and monitoring their genetic diversity is important for epidemiological surveillance, vaccine development, and understanding of RNA viruses evolution. Epidemiological investigations have revealed that genogroup II, genotype 6 noroviruses (GII.6) are common agents of gastroenteritis. Upon sequencing of the ORF2 (encoding the viral capsid), GII.6 viruses have been distinguished into three variants. Sentinel hospital-based surveillance in Italy revealed that GII.6 noroviruses were the second most common capsid genotype in 2015, mostly in association with a GII.P7 ORF1 (encoding the viral polymerase). Upon molecular characterization of the ORF1 and ORF2, the GII.P7_GII.6 epidemic viruses circulating in 2014–2015 (variant GII.6b) were different from those that circulated sporadically in 2011–2013 (variant GII.6a). Analysis of the ORF1 (GII.P7) and ORF2 (GII.6) sequences available in the databases unveiled marked genetic diversity and peculiarities in the phylogenetic segregation patterns, suggesting multiple recombination events. Phylogenetic analyses suggest that recent GII.P7_GII.6b viruses were circulating as early as 2008, and formed a genetically homogenous group that emerged globally.     

感染性腹泻患者标本中检出GⅡ型诺如病毒

目的为确定引起腹泻的病原体,对腹泻患者标本进行诺如病毒核酸检测。方法应用实时荧光RT—PCR法对5份感染性腹泻患者粪便标本进行GI型和GII诺如病毒RNA检测。结果5份患者粪便标本中,1份检出GII型诺如病毒核酸。结论大连地区的感染性腹泻病例中存在GII诺如病毒感染。     

昆明地区高危HPV病毒载量与宫颈病变和宫颈癌相关性

目的了解昆明地区高危人乳头瘤病毒(human papilloma virus,HPV)载量与宫颈病变和宫颈癌相关性。方法选取昆明地区妇女两万余例,用杂交捕获Ⅱ(second-generation Hybrid Capture,HC-II)方法检测高危型HPV-DNA,选取3608例进行阴道镜下多点活组织检查送病理检查,宫颈上皮内瘤变(cervical intraepithelial neoplasia,CIN)患者以宫颈锥切术后病理检查结果为准,宫颈癌患者以术后病理检查为准,分析HPV病毒载量与宫颈病变级别的相关性。结果宫颈炎2420例(67.07%),CINⅠ448例(12.42%)、CINⅡ376例(10.42%)、CINⅢ280例(7.76%)、宫颈癌84例(2.33%)。经秩和检验,5组间的HPV病毒载量比较,差异有统计学意义(H=185.597,P<0.05);CINⅠ与宫颈炎、宫颈癌和CINⅡ的病毒载量比较,差异均无统计学意义(P>0.05);余下各组两两比较差异有统计学意义(P<0.05)。年龄和HPV病毒载量均是宫颈病变和宫颈癌的危险因素。CINⅡ以上病变随着病毒载量的升高,阳性率逐渐增高。而宫颈癌主要集中在中度级别病毒载量之间发病。结论不同宫颈病变级别间HPV病毒载量有差别,HPV病毒载量可作为诊断CINⅡ以上病变的指标。     

Can genotype mismatch really affect the level of protection conferred by Newcastle disease vaccines against heterologous virulent strains?

Newcastle disease (ND), caused by virulent class II avian paramyxovirus 1 (Newcastle disease virus, NDV), occurs sporadically in poultry despite their having been immunized with commercial vaccines. These vaccines were all derived from NDV strains isolated around 70 years ago. Since then, class II NDV strains have evolved into 18 genotypes. Whether the vaccination failure results from genotype mismatches between the currently used vaccine strains and field-circulating velogenic strains or from an impaired immune response in the vaccination remains unclear. To test the first hypothesis, we performed a heterologous genotype II vaccine/genotype XI challenge in one-day old specific pathogen free (SPF) chicks and reproduced viral shedding. We then produced two attenuated strains of genotype II and XI by reverse genetics and used them to immunize two-week old SPF chickens that were subsequently challenged with velogenic strains of genotypes II, VII and XI. We found that both vaccines could induce antibodies with hemagglutination inhibition titers higher than 6.5 log₂. Vaccination also completely prevented disease, viral shedding in swabs, and blocked viral replication in tissues from different genotypes in contrast to unvaccinated chickens that died shortly after challenge. Taken together, our results support the hypothesis that, in immunocompetent poultry, genotype mismatch is not the main reason for vaccination failure.     

Multivalent norovirus vaccines induce strong mucosal and systemic blocking antibodies against multiple strains

Noroviruses are important agents of human gastroenteritis characterized by extensive sequence variation in the major capsid structural protein that likely encodes critical antigenic determinants of protective immunity. The lack of an infection model has limited detailed characterizations of viral antigenic relationships and identification of the essential components for protective immunity. This information would contribute to efficacious vaccine design against a broad array of norovirus strains. To understand the extent of heterotypic norovirus antibody specificity to inter- and intra-genogroup strains and its applicability to vaccine design, we collected sera from humans infected with different norovirus strains and from mice inoculated with alphavirus vectors expressing strain-specific recombinant norovirus-like particles (VLPs). We used VLPs that were assembled from Norwalk virus (NV), Hawaii virus (HV), Snow Mountain virus (SM) and Lordsdale virus (LV) as antigens to define and compare heterotypic antibody responses in humans and mice. We also examined if these heterotypic antibodies could block specific binding of ABH histo-blood group antigens, putative receptors for norovirus binding and entry, to norovirus VLPs. Furthermore, we examined the effect of multivalent inocula on the specificity, titer, and ligand blockade properties of systemic and mucosal norovirus-specific antibodies in mice. Our studies suggest that infection with one of several different genogroup I (GI) strains in humans induces heterotypic antibodies that block NV binding to ABH antigens, although comparable findings were not evident following infection with genogroup (GII) strains. Additionally, inoculating mice with vaccine cocktails encoding multiple norovirus VLPs enhances heterotypic and ligand attachment-blocking antibody responses against the LV strain not included in the cocktail. These data suggest that multivalent vaccination may provide better protection from a broader range of noroviruses than monovalent vaccination.