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Detection of human norovirus GIV.1 in China: A case report
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Case Report
Detection of human norovirus GIV.1 in China: A case report Yuan-yun Ao, Jie-mei Yu, Li-li Li, Miao Jin, Zhao-jun Duan ∗ National Institute for Viral Disease Control and Prevention, China CDC, Beijing 100052, China
a r t i c l e
i n f o
Article history: Received 24 April 2014 Received in revised form 18 July 2014 Accepted 3 August 2014 Keywords: Genogroup IV noroviruses Complete genome Seminested PCR Real-time PCR Phylogenetic analysis
a b s t r a c t Noroviruses (NoVs) are a common cause of acute gastroenteritis (AGE) around the world; however, reports of genogroup IV (GIV) NoVs are rare. Here we report a human GIV genotype 1 (GIV.1) NoV strain (named CHNNGIV2011) identified by 454 high-throughput sequencing from stool samples of children with diarrhea. This is the first documented human GIV.1 NoVs infection in China. The complete genome of the virus is 7525 nucleotides in length. Sequencing and phylogenetic analyses showed that CHNNGIV2011 shared high sequence similarity to other GIV.1 NoVs from all over the world, especially to the recently reported NoV GIV.1 strain Lake Macquarie genome (99.0%). By seminested PCR and real-time PCR, a total of 2 out of 466 samples were positive for GIV.1 CHNNGIV2011 in Lulong County, Hebei Province, China, which supported a low prevalence of GIV.1 NoVs. The positive samples contained 7.2 × 107 and 2.6 × 108 copies/g in feces. In addition, one positive sample was found coinfection with strains NoV GII and salivirus. These findings suggest more study is needed to address the worldwide prevalence and role of GIV NoVs in AGE. © 2014 Elsevier B.V. All rights reserved.
1. Why this case is important
2. Case description
Noroviruses (NoVs) are the major cause of acute gastroenteritis globally. The NoV genome is composed of three open reading frames (ORFs): a large non-structural protein, a major capsid protein (VP1), and a small basic structural protein (VP2). Based on VP1, NoVs have been divided into six genogroups (GI–GVI) [1]. GIV NoVs are further classified into genotype 1 and 2 (GIV.1 and GIV.2). Only GI, GII, and GIV.1 NoVs have been identified in humans [2], among which GI and GII cause most human infections [3]. However, GIV.1 NoVs are rarely identified in humans and their role in AGE has rarely been addressed [4–7]. Unlike NoVs GI and GII, limited sequence information for GIV is available from GenBank; only one complete genome from a human GIV.1 strain has been published [7]. To our knowledge, no GIV NoV has been reported in China. In this study, the complete genome of a human GIV.1 NoV strain (named CHNNGIV2011), which was initially discovered by 454 sequencing of stool samples from children with gastroenteritis, was determined and its local prevalence was investigated.
A total of 466 fecal samples from 466 children under the age of 5 years hospitalized with gastroenteritis were collected at Lulong County People’s Hospital in Lulong County, Hebei Province, China, between April of 2011 and April of 2013. Five fecal samples from children with gastroenteritis were pooled and sequenced by Roche Genome Sequencer FLX Titanium pyrosequencing technology. A customized informatics pipeline [8] with minor modifications identified 599 unique reads that shared sequence similarity to known GIV.1 NoVs. Assembly of the unique reads generated a contig of 7399 bp using Newbler (454 Life Sciences), which showed high nucleotide sequence identity (99.0%) to the NoVs GIV.1 strains in GenBank. Four pairs of primers were initially designed to confirm the contig obtained by 454 sequencing. The 5 and 3 ends of the genome were amplified using a SMART RACE cDNA Amplification Kit (Clontech Laboratories, USA). The complete genome of CHNNGIV2011 obtained was 7525 nucleotides in length, excluding the poly(A) tail, and contained three ORFs: ORF1 (position 5–5068), ORF2 (position 5049–6719), and ORF3 (position 6719–7447). The complete genomic sequence has been deposited in GenBank under accession number KC894731. The primers used are shown in Supplementary Table 1a. Sequence analysis of CHNNGIV2011 showed the highest sequence identity with the complete genome of GIV.1 Lake Macquarie virus [7] (99.0%) and GIV.2 feline NoV [9] (64.0%). The VP1 region of CHNNGIV2011 was 91.6–98.2% identical in nt to that
∗ Corresponding author at: Department of Viral Diarrhea, National Institute for Viral Disease Control and Prevention, China CDC, 100 Ying-Xin Street, Xuan-Wu District, Beijing 100052, China. Tel.: +86 10 6355 2910; fax: +86 10 6355 7757. E-mail addresses: [email protected] (Y.-y. Ao), [email protected] (J.-m. Yu), [email protected] (L.-l. Li), [email protected] (M. Jin), [email protected] (Z.-j. Duan). http://dx.doi.org/10.1016/j.jcv.2014.08.002 1386-6532/© 2014 Elsevier B.V. All rights reserved.
Please cite this article in press as: Ao Y-y, et al. Detection of human norovirus GIV.1 in China: A case report. J Clin Virol (2014), http://dx.doi.org/10.1016/j.jcv.2014.08.002
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Fig. 1. (a) Phylogenetic tree generated by the neighbor-joining method for caliciviruses based on the complete genome of the newly identified GIV.1 strain CHNNGIV2011. CHNNGIV2011 is marked with . Bootstrap values are shown near the branches. (b) Phylogenetic tree generated by the neighbor-joining method for noroviruses targeting the completed capsid (amino acid) sequence. The newly identified GIV.1 strain CHNNGIV2011 is marked with . Bootstrap values are shown near the branches. Table 2 Clinical data for the two GIV.1 norovirus-positive patients.
of reported GIV.1 NoVs, and 98.4–99.1% identical at the aa level. The highly variable P2 domain [10] of the GIV.1 NoVs available in GenBank (including CHNNGIV2011) showed a whole identity of 98.6%. However, the P2 domain of CHNNGIV2011 showed only about 45.0% identical to that of animal GIV.2 NoVs and it is 22 aa shorter. The identities of CHNNGIV2011 to other NoVs in detail are shown in Table 1. Using Clustalx version 1.83 [11] and MEGA4 [12], a phylogenetic analysis [13] of the complete genome of CHNNGIV2011 showed maximum relatedness to Lake Macquarie virus [7] (Fig. 1a), and a phylogenetic analysis of the complete VP1 aa sequence revealed that CHNNGIV2011 and other human GIV.1 NoVs were tightly clustered (Fig. 1b). Two of 466 diarrhea samples were positive for CHNNGIV2011 by the methods of both semi-nested and real-time PCR, as described previously [14,15]. The viruses in the two samples displayed 100% nt identity to each other based on the ORF1/ORF2 region. To construct a standard curve, a 127-base pair DNA fragment corresponding to the ORF1/ORF2 junction in CHNNGIV2011 was synthesized to produce a recombinant plasmid. The viral loads in the two samples were quantified as 7.2 × 107 (ID GR-1113-49) and 2.6 × 108 (ID GR-1113-59) copies per gram of fecal specimen, respectively, by comparison with the standard curves using the obtained quantification cycle value. The probe and primers are shown in Supplementary Table 1b.
Patient ID
GR-1113-49
GR-1113-59
Age (months) Numbers of diarrhea per day Duration of diarrhea (days) Duration of vomiting (days) Duration of hospitalization (days) Temperature Coinfection virus
45 5 6 1 4 36.9
10 7 14 2 4 36.8 NoVs GII and salivirus
The two positive samples were screened for common diarrhea viruses, including rotavirus group A, norovirus GI/GII, sapovirus, astrovirus, enteric adenovirus, enterovirus, and salivirus, using RT-PCR as described previously [16–20]. One of the two positive samples (ID GR-1113-59) was found positive for NoV GII and salivirus [20]. The two NoV GIV-positive patients experienced severe diarrhea: 5–7 numbers of diarrhea per day, diarrhea for 6–14 days and hospitation for 4 days. Among them, the 10-month-old patient (ID GR-1113-59, co-infected with NoV GII and salivirus) had more severe symptoms than the 45-month-old patient (ID GR-1113-49) in terms of number of episodes of diarrhea per day, the duration of diarrhea and vomiting (Table 2).
Table 1 Percent identity of various noroviruses with CHNNGIV2011. GI RdRpa VP1 VP2
60.8–63.9% 61.0–64.7% 50.6–52.3% 44.2–46.0% 42.8–48.8% 26.9–37.1%
GII 65.3–69.1% 69.5–73.5% 55.3–58.1% 49.1–53.7% 49.5–52.3% 40.9–45.8%
GIII 59.4–61.1% 64.0–65.6% 49.0–49.6% 41.7–43.6% 42.0–42.2% 20.0–28.8%
GIV.1 92.0–99.7% 99.6–100% 91.6–98.2% 98.4–99.1% 83.7–98.1% 83.1–98.4%
GIV.2 69.1–70.8% 75.6–76.7% 65.6–66.4% 67.7–69.1% 59.1–60.8% 59.2–62.6%
GV 52.8% 50.8% 50.9% 41.4% 41.8% 17.2%
GVI 68.0–69.9% 75.6–75.9% 57.8–58.6% 54.3–56.2% 49.4–52.8% 45.1–46.7%
NOTE A B A B A B
A: nucleotide sequence; B: amino acid sequence. a The identity of RdRp was analyzed based on the partial RdRp sequence of 750 nt and 250 aa available in the GenBank.
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3. Similar and contrasting cases in the literature and discussion To date, GIV NoVs are rarely reported in humans. Human GIV.1 NoVs were first identified from sporadic cases of gastroenteritis [21]. From then on, they were occasionally detected in NoVs routine screenings of clinical and environmental samples [4–7,22]. Interestingly, in animals, GIV.2 NoVs were also detected [9,23,24]. Data on the clinical role of GIV NoVs are also limited. In the present study, two patients with gastroenteritis were found to be positive for NoV GIV.1 in feces. The complete genomic sequence of the virus from one specimen was obtained. This is the first report on the detection of GIV NoVs in China. Our findings will be useful for understanding the prevalence of GIV NoVs globally. Sequencing and phylogenetic analyses showed CHNNGIV2011 had the highest identity to human GIV.1 NoVs, especially the Lake Macquarie virus [7]. Nt and aa sequence analysis showed little genetic variability in human GIV.1. The P2 domain is the least conserved region of VP1 among norovirus strains, and is thought to play an important role in receptor binding and immune reactivity [10,25]. However the P2 domains of human GIV.1 NoVs are highly conserved. Together, these results indicate the genome of human GIV.1 NoVs are relatively stable. NoVs GI and GII are the most important viral pathogens causing AGE outbreaks [26]; however, NoV GIV.1 outbreaks have only been reported in Australia [7]. Our study demonstrated a low prevalence of NoV GIV.1 as described in other studies [6,22,27,28]. Both the NoV GIV.1-positive patients were hospitalized with gastroenteritis in July of 2011 from Lulong County. Although more evidence is needed, these data suggest that a local NoV GIV outbreak might have existed at that time. In the present study, the two NoV GIV-positive patients experienced severe diarrhea; however, one of the patient had more severe symptoms, possibly due to the difference in age or coinfection with NoV GII and salivirus, as shown in previous reports [29,30]. Similar to previous studies that reported high viral loads in patients with NoV gastroenteritis [31,32], a high viral load was detected in both of the patients with a GIV NoV infection. Based on these findings, it is suggested that a NoV GIV.1 infection can cause severe diarrhea in children under 5 years of age. Further epidemiologic studies are needed to confirm whether NoV GIV.1 is associated with diarrhea. The present study highlights the importance of surveillance programs locally and of studying GIV NoVs globally.
Authors’ contribution Acquisition of data, data analysis, data interpretation and drafting the manuscript (Yuan-yun Ao). Involved in laboratory work (Yuan-yun Ao, Jie-mei Yu and Li-li Li). The conception and design of the study, and revising the manuscript critically for important intellectual content (Zhao-jun Duan, Jie-mei Yu, and Miao Jin). Final approval of the version to be submitted (Zhao-jun Duan, Jie-mei Yu).
Funding This work was partly supported by the Gates Foundation (grant no. OPP1016839), and the National Natural Science Foundation of China (grant no. 81290345).
Competing interests None of the authors has a conflict of interest to report.
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Ethical approval Consent to participate in this study was obtained from the parents of the children. The study protocol was approved by the National Institute for Viral Diseases Control and Prevention (Beijing, China) and the ethics committee of Lulong County People’s Hospital (Hebei Province, China). Acknowledgments We thank David Wang and Guoyan Zhao from the Department of Pathology and Immunology, Washington University School of Medicine (St. Louis, MO, USA), for their help with the bioinformatics analyses. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jcv.2014.08.002. References [1] Green KY. Caliciviridae: the noroviruses. In: Knipe DM, Howley PM, editors. Fields Virology. 6th ed. Philadelphia: Wolters Kluwer Health/Lippincott Williams and Wilkins; 2013. p. 582–608. [2] Zheng DP, Ando T, Fankhauser RL, Beard RS, Glass RI, Monroe SS. Norovirus classification and proposed strain nomenclature. Virology 2006;346:312–23. [3] Kageyama T, Shinohara M, Uchida K, Fukushi S, Hoshino FB, Kojima S, et al. Coexistence of multiple genotypes, including newly identified genotypes, in outbreaks of gastroenteritis due to Norovirus in Japan. J Clin Microbiol 2004;42:2988–95. [4] La Rosa G, Iaconelli M, Pourshaban M, Fratini M, Muscillo M. Molecular detection and genetic diversity of norovirus genogroup IV: a yearlong monitoring of sewage throughout Italy. Arch Virol 2010;155:589–93. [5] Kitajima M, Oka T, Haramoto E, Phanuwan C, Takeda N, Katayama K, et al. Genetic diversity of genogroup IV noroviruses in wastewater in Japan. Lett Appl Microbiol 2011;52:181–4. [6] La Rosa G, Pourshaban M, Iaconelli M, Muscillo M. Detection of genogroup IV noroviruses in environmental and clinical samples and partial sequencing through rapid amplification of cDNA ends. Arch Virol 2008;153:2077–83. [7] Eden JS, Lim KL, White PA. Complete genome of the human norovirus GIV.1 strain Lake Macquarie virus. J Virol 2012;86:10251–2. [8] Finkbeiner SR, Allred AF, Tarr PI, Klein EJ, Kirkwood CD, Wang D. Metagenomic analysis of human diarrhea: viral detection and discovery. PLoS Pathog 2008;4:e1000011. [9] Pinto P, Wang Q, Chen N, Dubovi EJ, Daniels JB, Millward LM. Discovery and genomic characterization of noroviruses from a gastroenteritis outbreak in domestic cats in the US. PLoS ONE 2012;7:e32739. [10] Prasad BV, Hardy ME, Dokland T, Bella J, Rossmann MG, Estes MK. X-ray crystallographic structure of the Norwalk virus capsid. Science 1999;286:287–90. [11] Higgins DG, Thompson JD, Gibson TJ. Using CLUSTAL for multiple sequence alignments. Methods Enzymol 1996;266:383–402. [12] Tamura K, Dudley J, Nei M, Kumar S. MEGA4: Molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol 2007;24:1596–9. [13] Drummond AJ, Rambaut A. BEAST: Bayesian evolutionary analysis by sampling trees. BMC Evol Biol 2007;7:214. [14] La Rosa G, Iaconelli M, Pourshaban M, Muscillo M. Detection and molecular characterization of noroviruses from five sewage treatment plants in central Italy. Water Res 2010;44:1777–84. [15] Trujillo AA, McCaustland KA, Zheng DP, Hadley LA, Vaughn G, Adams SM, et al. Use of TaqMan real-time reverse transcription-PCR for rapid detection, quantification, and typing of norovirus. J Clin Microbiol 2006;44:1405–12. [16] Rahman M, Sultana R, Ahmed G, Nahar S, Hassan ZM, Saiada F, et al. Prevalence of G2P[4] and G12P[6] rotavirus, Bangladesh. Emerg Infect Dis 2007;13:18–24. [17] Yan H, Yagyu F, Okitsu S, Nishio O, Ushijima H. Detection of norovirus (GI, GII), sapovirus and astrovirus in fecal samples using reverse transcription singleround multiplex PCR. J Virol Met 2003;114:37–44. [18] Dey RS, Ghosh S, Chawla-Sarkar M, Panchalingam S, Nataro JP, Sur D, et al. Circulation of a novel pattern of infections by enteric adenovirus serotype 41 among children below 5 years of age in Kolkata, India. J Clin Microbiol 2011;49:500–5. [19] Zoll GJ, Melchers WJ, Kopecka H, Jambroes G, van der Poel HJ, Galama JM. General primer-mediated polymerase chain reaction for detection of enteroviruses: application for diagnostic routine and persistent infections. J Clin Microbiol 1992;30:160–5. [20] Li L, Victoria J, Kapoor A, Blinkova O, Wang C, Babrzadeh F, et al. A novel picornavirus associated with gastroenteritis. J Virol 2009;83:12002–6. [21] Vinje J, Koopmans MP. Simultaneous detection and genotyping of Norwalk-like viruses by oligonucleotide array in a reverse line blot hybridization format. J Clin Microbiol 2000;38:2595–601.
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[22] Muscillo M, Fratini M, Graffeo R, Sanguinetti M, Martella V, Green KY, et al. GIV Noroviruses in wastewaters and in stool specimens from hospitalized patients. Food Environ Virol 2013;5:194–202. [23] Martella V, Campolo M, Lorusso E, Cavicchio P, Camero M, Bellacicco AL, et al. Norovirus in captive lion cub (Panthera leo). Emerg Infect Dis 2007;13:1071–3. [24] Martella V, Lorusso E, Decaro N, Elia G, Radogna A, D’Abramo M, et al. Detection and molecular characterization of a canine norovirus. Emerg Infect Dis 2008;14:1306–8. [25] Lochridge VP, Hardy ME. A single-amino-acid substitution in the P2 domain of VP1 of murine norovirus is sufficient for escape from antibody neutralization. J Virol 2007;81:12316–22. [26] Donaldson EF, Lindesmith LC, Lobue AD, Baric RS. Viral shape-shifting: norovirus evasion of the human immune system. Nat Rev Microbiol 2010;8:231–41. [27] Lim KL, Eden JS, Oon LL, White PA. Molecular epidemiology of norovirus in Singapore, 2004–2011. J Med Virol 2013;85:1842–51.
[28] Estevez A, Arvelo W, Hall AJ, Lopez MR, Lopez B, Reyes L, et al. Prevalence and genetic diversity of norovirus among patients with acute diarrhea in Guatemala. J Med Virol 2013;85:1293–8. [29] Valentini D, Vittucci AC, Grandin A, Tozzi AE, Russo C, Onori M, et al. Coinfection in acute gastroenteritis predicts a more severe clinical course in children. Eur J Clin Microbiol Infect Dis 2013;32:909–15. [30] Partridge DG, Evans CM, Raza M, Kudesia G, Parsons HK. Lessons from a large norovirus outbreak: impact of viral load, patient age and ward design on duration of symptoms and shedding and likelihood of transmission. J Hosp Infect 2012;81:25–30. [31] Chan MC, Sung JJ, Lam RK, Chan PK, Lee NL, Lai RW, et al. Fecal viral load and norovirus-associated gastroenteritis. Emerg Infect Dis 2006;12:1278–80. [32] Rosa G, Pourshaban M, Iaconelli M, Muscillo M. Quantification of norovirus genogroups I and II in environmental and clinical samples using TaqMan realtime RT-PCR. Food Environ Virol 2008;1:15–22.
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Case Report
Detection of human norovirus GIV.1 in China: A case report Yuan-yun Ao, Jie-mei Yu, Li-li Li, Miao Jin, Zhao-jun Duan ∗ National Institute for Viral Disease Control and Prevention, China CDC, Beijing 100052, China
a r t i c l e
i n f o
Article history: Received 24 April 2014 Received in revised form 18 July 2014 Accepted 3 August 2014 Keywords: Genogroup IV noroviruses Complete genome Seminested PCR Real-time PCR Phylogenetic analysis
a b s t r a c t Noroviruses (NoVs) are a common cause of acute gastroenteritis (AGE) around the world; however, reports of genogroup IV (GIV) NoVs are rare. Here we report a human GIV genotype 1 (GIV.1) NoV strain (named CHNNGIV2011) identified by 454 high-throughput sequencing from stool samples of children with diarrhea. This is the first documented human GIV.1 NoVs infection in China. The complete genome of the virus is 7525 nucleotides in length. Sequencing and phylogenetic analyses showed that CHNNGIV2011 shared high sequence similarity to other GIV.1 NoVs from all over the world, especially to the recently reported NoV GIV.1 strain Lake Macquarie genome (99.0%). By seminested PCR and real-time PCR, a total of 2 out of 466 samples were positive for GIV.1 CHNNGIV2011 in Lulong County, Hebei Province, China, which supported a low prevalence of GIV.1 NoVs. The positive samples contained 7.2 × 107 and 2.6 × 108 copies/g in feces. In addition, one positive sample was found coinfection with strains NoV GII and salivirus. These findings suggest more study is needed to address the worldwide prevalence and role of GIV NoVs in AGE. © 2014 Elsevier B.V. All rights reserved.
1. Why this case is important
2. Case description
Noroviruses (NoVs) are the major cause of acute gastroenteritis globally. The NoV genome is composed of three open reading frames (ORFs): a large non-structural protein, a major capsid protein (VP1), and a small basic structural protein (VP2). Based on VP1, NoVs have been divided into six genogroups (GI–GVI) [1]. GIV NoVs are further classified into genotype 1 and 2 (GIV.1 and GIV.2). Only GI, GII, and GIV.1 NoVs have been identified in humans [2], among which GI and GII cause most human infections [3]. However, GIV.1 NoVs are rarely identified in humans and their role in AGE has rarely been addressed [4–7]. Unlike NoVs GI and GII, limited sequence information for GIV is available from GenBank; only one complete genome from a human GIV.1 strain has been published [7]. To our knowledge, no GIV NoV has been reported in China. In this study, the complete genome of a human GIV.1 NoV strain (named CHNNGIV2011), which was initially discovered by 454 sequencing of stool samples from children with gastroenteritis, was determined and its local prevalence was investigated.
A total of 466 fecal samples from 466 children under the age of 5 years hospitalized with gastroenteritis were collected at Lulong County People’s Hospital in Lulong County, Hebei Province, China, between April of 2011 and April of 2013. Five fecal samples from children with gastroenteritis were pooled and sequenced by Roche Genome Sequencer FLX Titanium pyrosequencing technology. A customized informatics pipeline [8] with minor modifications identified 599 unique reads that shared sequence similarity to known GIV.1 NoVs. Assembly of the unique reads generated a contig of 7399 bp using Newbler (454 Life Sciences), which showed high nucleotide sequence identity (99.0%) to the NoVs GIV.1 strains in GenBank. Four pairs of primers were initially designed to confirm the contig obtained by 454 sequencing. The 5 and 3 ends of the genome were amplified using a SMART RACE cDNA Amplification Kit (Clontech Laboratories, USA). The complete genome of CHNNGIV2011 obtained was 7525 nucleotides in length, excluding the poly(A) tail, and contained three ORFs: ORF1 (position 5–5068), ORF2 (position 5049–6719), and ORF3 (position 6719–7447). The complete genomic sequence has been deposited in GenBank under accession number KC894731. The primers used are shown in Supplementary Table 1a. Sequence analysis of CHNNGIV2011 showed the highest sequence identity with the complete genome of GIV.1 Lake Macquarie virus [7] (99.0%) and GIV.2 feline NoV [9] (64.0%). The VP1 region of CHNNGIV2011 was 91.6–98.2% identical in nt to that
∗ Corresponding author at: Department of Viral Diarrhea, National Institute for Viral Disease Control and Prevention, China CDC, 100 Ying-Xin Street, Xuan-Wu District, Beijing 100052, China. Tel.: +86 10 6355 2910; fax: +86 10 6355 7757. E-mail addresses: [email protected] (Y.-y. Ao), [email protected] (J.-m. Yu), [email protected] (L.-l. Li), [email protected] (M. Jin), [email protected] (Z.-j. Duan). http://dx.doi.org/10.1016/j.jcv.2014.08.002 1386-6532/© 2014 Elsevier B.V. All rights reserved.
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Fig. 1. (a) Phylogenetic tree generated by the neighbor-joining method for caliciviruses based on the complete genome of the newly identified GIV.1 strain CHNNGIV2011. CHNNGIV2011 is marked with . Bootstrap values are shown near the branches. (b) Phylogenetic tree generated by the neighbor-joining method for noroviruses targeting the completed capsid (amino acid) sequence. The newly identified GIV.1 strain CHNNGIV2011 is marked with . Bootstrap values are shown near the branches. Table 2 Clinical data for the two GIV.1 norovirus-positive patients.
of reported GIV.1 NoVs, and 98.4–99.1% identical at the aa level. The highly variable P2 domain [10] of the GIV.1 NoVs available in GenBank (including CHNNGIV2011) showed a whole identity of 98.6%. However, the P2 domain of CHNNGIV2011 showed only about 45.0% identical to that of animal GIV.2 NoVs and it is 22 aa shorter. The identities of CHNNGIV2011 to other NoVs in detail are shown in Table 1. Using Clustalx version 1.83 [11] and MEGA4 [12], a phylogenetic analysis [13] of the complete genome of CHNNGIV2011 showed maximum relatedness to Lake Macquarie virus [7] (Fig. 1a), and a phylogenetic analysis of the complete VP1 aa sequence revealed that CHNNGIV2011 and other human GIV.1 NoVs were tightly clustered (Fig. 1b). Two of 466 diarrhea samples were positive for CHNNGIV2011 by the methods of both semi-nested and real-time PCR, as described previously [14,15]. The viruses in the two samples displayed 100% nt identity to each other based on the ORF1/ORF2 region. To construct a standard curve, a 127-base pair DNA fragment corresponding to the ORF1/ORF2 junction in CHNNGIV2011 was synthesized to produce a recombinant plasmid. The viral loads in the two samples were quantified as 7.2 × 107 (ID GR-1113-49) and 2.6 × 108 (ID GR-1113-59) copies per gram of fecal specimen, respectively, by comparison with the standard curves using the obtained quantification cycle value. The probe and primers are shown in Supplementary Table 1b.
Patient ID
GR-1113-49
GR-1113-59
Age (months) Numbers of diarrhea per day Duration of diarrhea (days) Duration of vomiting (days) Duration of hospitalization (days) Temperature Coinfection virus
45 5 6 1 4 36.9
10 7 14 2 4 36.8 NoVs GII and salivirus
The two positive samples were screened for common diarrhea viruses, including rotavirus group A, norovirus GI/GII, sapovirus, astrovirus, enteric adenovirus, enterovirus, and salivirus, using RT-PCR as described previously [16–20]. One of the two positive samples (ID GR-1113-59) was found positive for NoV GII and salivirus [20]. The two NoV GIV-positive patients experienced severe diarrhea: 5–7 numbers of diarrhea per day, diarrhea for 6–14 days and hospitation for 4 days. Among them, the 10-month-old patient (ID GR-1113-59, co-infected with NoV GII and salivirus) had more severe symptoms than the 45-month-old patient (ID GR-1113-49) in terms of number of episodes of diarrhea per day, the duration of diarrhea and vomiting (Table 2).
Table 1 Percent identity of various noroviruses with CHNNGIV2011. GI RdRpa VP1 VP2
60.8–63.9% 61.0–64.7% 50.6–52.3% 44.2–46.0% 42.8–48.8% 26.9–37.1%
GII 65.3–69.1% 69.5–73.5% 55.3–58.1% 49.1–53.7% 49.5–52.3% 40.9–45.8%
GIII 59.4–61.1% 64.0–65.6% 49.0–49.6% 41.7–43.6% 42.0–42.2% 20.0–28.8%
GIV.1 92.0–99.7% 99.6–100% 91.6–98.2% 98.4–99.1% 83.7–98.1% 83.1–98.4%
GIV.2 69.1–70.8% 75.6–76.7% 65.6–66.4% 67.7–69.1% 59.1–60.8% 59.2–62.6%
GV 52.8% 50.8% 50.9% 41.4% 41.8% 17.2%
GVI 68.0–69.9% 75.6–75.9% 57.8–58.6% 54.3–56.2% 49.4–52.8% 45.1–46.7%
NOTE A B A B A B
A: nucleotide sequence; B: amino acid sequence. a The identity of RdRp was analyzed based on the partial RdRp sequence of 750 nt and 250 aa available in the GenBank.
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3. Similar and contrasting cases in the literature and discussion To date, GIV NoVs are rarely reported in humans. Human GIV.1 NoVs were first identified from sporadic cases of gastroenteritis [21]. From then on, they were occasionally detected in NoVs routine screenings of clinical and environmental samples [4–7,22]. Interestingly, in animals, GIV.2 NoVs were also detected [9,23,24]. Data on the clinical role of GIV NoVs are also limited. In the present study, two patients with gastroenteritis were found to be positive for NoV GIV.1 in feces. The complete genomic sequence of the virus from one specimen was obtained. This is the first report on the detection of GIV NoVs in China. Our findings will be useful for understanding the prevalence of GIV NoVs globally. Sequencing and phylogenetic analyses showed CHNNGIV2011 had the highest identity to human GIV.1 NoVs, especially the Lake Macquarie virus [7]. Nt and aa sequence analysis showed little genetic variability in human GIV.1. The P2 domain is the least conserved region of VP1 among norovirus strains, and is thought to play an important role in receptor binding and immune reactivity [10,25]. However the P2 domains of human GIV.1 NoVs are highly conserved. Together, these results indicate the genome of human GIV.1 NoVs are relatively stable. NoVs GI and GII are the most important viral pathogens causing AGE outbreaks [26]; however, NoV GIV.1 outbreaks have only been reported in Australia [7]. Our study demonstrated a low prevalence of NoV GIV.1 as described in other studies [6,22,27,28]. Both the NoV GIV.1-positive patients were hospitalized with gastroenteritis in July of 2011 from Lulong County. Although more evidence is needed, these data suggest that a local NoV GIV outbreak might have existed at that time. In the present study, the two NoV GIV-positive patients experienced severe diarrhea; however, one of the patient had more severe symptoms, possibly due to the difference in age or coinfection with NoV GII and salivirus, as shown in previous reports [29,30]. Similar to previous studies that reported high viral loads in patients with NoV gastroenteritis [31,32], a high viral load was detected in both of the patients with a GIV NoV infection. Based on these findings, it is suggested that a NoV GIV.1 infection can cause severe diarrhea in children under 5 years of age. Further epidemiologic studies are needed to confirm whether NoV GIV.1 is associated with diarrhea. The present study highlights the importance of surveillance programs locally and of studying GIV NoVs globally.
Authors’ contribution Acquisition of data, data analysis, data interpretation and drafting the manuscript (Yuan-yun Ao). Involved in laboratory work (Yuan-yun Ao, Jie-mei Yu and Li-li Li). The conception and design of the study, and revising the manuscript critically for important intellectual content (Zhao-jun Duan, Jie-mei Yu, and Miao Jin). Final approval of the version to be submitted (Zhao-jun Duan, Jie-mei Yu).
Funding This work was partly supported by the Gates Foundation (grant no. OPP1016839), and the National Natural Science Foundation of China (grant no. 81290345).
Competing interests None of the authors has a conflict of interest to report.
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