A large outbreak of acute gastroenteritis in Shippensburg, Pennsylvania, 1972 revisited: evidence for common source exposure to a recombinant GII.Pg/GII.3 norovirus

Abstract

Historical outbreaks can be an important source of information in the understanding of norovirus evolution and epidemiology. Here, we revisit an outbreak of undiagnosed gastroenteritis that occurred in Shippensburg, Pennsylvania in 1972. Nearly 5000 people fell ill over the course of 10 days. Symptoms included diarrhea, vomiting, stomach cramps, and fever, lasting for a median of 24 h. Using current techniques, including next-generation sequencing of full-length viral genomic amplicons, we identified an unusual norovirus recombinant (GII.Pg/GII.3) in nine of 15 available stool samples from the outbreak. This particular recombinant virus has not been reported in recent decades, although GII.3 and GII.Pg genotypes have been detected individually in current epidemic strains. The consensus nucleotide sequences were nearly identical among the four viral genomes analysed, although each strain had three to seven positions in the genome with heterogenous non-synonymous nucleotide subpopulations. Two of these resulting amino acid polymorphisms were conserved in frequency among all four cases, consistent with common source exposure and successful transmission of a mixed viral population. Continued investigation of variant nucleotide populations and recombination events among ancestral norovirus strains such as the Shippensburg virus may provide unique insight into the origin of contemporary strains.

Keywords: Norwalk agent and related viruses; caliciviruses; gastroenteritis; noroviruses.

Fig. 1.

Features of Shippensburg outbreak with GII.Pg/GII.3 recombinant norovirus. (a) Percent of population affected by university or town association. Figure adapted from initial CDC outbreak report. Percentage of population affected by university association in the college (solid line) and town (dotted line) are displayed. A prominent peak in the university-associated population is seen on 17 November, with a corresponding plateau in the non-university-associated population. Approximately 1500 cases occurred in the college, while an estimated 3500 occurred in the town of Shippensburg and in surrounding areas. Our cases with available onset dates (C2, B24, and C1) have onset indicated by arrows. (b) ORF1-based phylogenetic tree of Shippensburg virus and related strains. Tree was constructed using MEGA version 7.0. Related strains were found via BLAST search, using nucleotides 4242–5110. The Shippensburg B24 strain groups closest with GII.Pg_GII.3/HK71 from 1978. GII3 viruses are represented by blue triangles. The red circle is the Shippensburg B24 strain. Polymerase genotypes are noted on the right. Bootstrap values ⩾75 are noted in the tree. (c) VP1-based phylogenetic tree of Shippensburg virus and related strains. Tree was constructed using MEGA version 7.0. Related strains were found via BLAST search, using nucleotides 5091–5788. The Shippensburg B24 strain groups closest with GII.Pg_GII.3/HK71 from 1978. The GII.Pg polymerase strains are noted by green triangles. Shippensburg B24 is denoted by a red circle. Capsid genotypes are noted on the right. Bootstrap values ⩾75 are noted in the tree. (d) Similarity plot analyses for recombination, Shippensburg strain. A similarity plot was constructed using SimPlot version 3.5.1 with a window size of 200 bases and a step size of 20 bases. Prototype strains of seven GII.Pg and GII.3 combinations were compared with the Shippensburg virus, including: GII.P21/GII.3 (Purple; KM198493, 2010 Vietnam, 30468), GII.P21/GII.3 (Pink; AB365435, 2004 USA, TCH04-577), GII.Pg/GII.3 (Green; JX846924, 1978 Hong Kong, HK71), GII.Pg/GII.1 (Orange; LN854570, 2014 Netherlands, Groningen), GII.Pg/GII.12 (Dark blue; HQ664990, 2010 USA, HS206), GII.Pg/GII.12 (Light blue; KM198503, 2010 Vietnam, C2033), and GII.P16/GII.3 (Black; KF944111, 2011 Russia, Nsk-N1648). GII.Pg viruses (blue and orange lines) have more similarity to the Shippensburg virus in the ORF1, while GII.3 strains (pink and black lines) have greater similarity in ORF2–ORF3. The HK71 virus (GII.Pg/GII.3) has the greatest similarity to Shippensburg for nearly the entire length of the genome. The arrow indicates a possible recombination breakpoint of GII.Pg and GII.3 viruses at the ORF1–ORF2 junction, as shown by the change in percent similarity for the comparison viruses.