The Detection and Characterization of Herpes Simplex Virus Type 1 in Confirmed Measles Cases

Based on measles surveillance in Shanghai, People’s Republic of China, from 2006 to 2015, we found that measles virus isolates from 40 throat swab samples exhibited atypical cytopathic effects in Vero/hSLAM cells, which was found to be a result of coinfection with measles virus (MeV) and human herpes simplex virus type 1 (HSV-1). Serological and molecular approaches were used to confirm and characterize the coinfections in these patients. Among the 40 measles cases, measles-specific IgM was detected in 37 cases, while measles-specific IgG was detected in 27 cases. HSV-1-specific IgM and IgG were detected in 7 and 34 cases, respectively, suggesting that most of the MeV infections were primary, but that HSV-1 infection was due to the reactivation of latent virus in most cases. The titers of HSV-1 IgG in patients with either measles or measles-HSV-1 coinfection were significantly higher than those in the healthy group (P = 0.0026 and P < 0.0001, respectively); however, there was no significant difference in the titers of HSV-1 IgG in the MeV and MeV-HSV-1 coinfection patients (P = 0.105). Nucleic acids from MeV and HSV-1 were detected in 40 and 39 throat swabs, respectively. Twenty five MeV RNA sequences were genotyped, and all represented genotype H1, which is the endemic genotype in China. Sequences from the glycoprotein G gene of HSV-1 were used to classify the isolates into two distinct phylogenetic groups: 34 belonged to group A and 3 belonged to group B.

www.nature.com/scientificreports www.nature.com/scientificreports/ from clinical specimens and/or viral isolates. MeV infection can cause cell membrane fusion and induce the formation of multinucleated giant cells (syncytia) in susceptible cells 11 . Such a pronounced cytopathic effect (CPE) is often used to indicate the presence of MeV in cell culture. During serial passages of cultures from a throat swab specimen from a suspected measles case, we observed changes in the types of CPE observed in infected Vero/ hSLAM cells. Syncytia were observed in cells inoculated with the first two passages of virus; however, beginning with the third passage, the infected cells exhibited cell rounding with a decrease in syncytia. Human herpes simplex virus type 1 (HSV-1) infection was confirmed in this culture by real-time PCR. A retrospective study was conducted to investigate coinfection of MeV and HSV-1 based on samples collected from 2006 to 2015. Forty coinfected cases were identified from a total of 4921 cell cultures obtained from throat swabs. were confirmed in Shanghai. The measles incidence was between 0.35/100,000 and 6.79/100,000 annually. The ratio of male to female cases was 1.28:1. Most cases occurred in populations aged 8-11 months, 20-29 years and 30-39 years. Adults aged >20 years accounted for 50% of all cases. Measles immunization has been documented since 2009 when the MSS was established. The majority of measles cases occurred in individuals without an immunization history or who had an unknown history. According to the MSS, a total of 186 cases were identified in individuals who had received at least one dose of measles-containing vaccine (MCV), who accounted for 2.48% to 13.25% of the cases between 2009 and 2015. Among these cases, 70.43% had received one dose of MCV, 24.73% had received two doses of MCV, and 4.84% had received more than two doses of MCV.

Measles surveillance in
From 1253 MeV isolates during this period, 40 cases of MeV and HSV-1 coinfection were identified. The average age was 23 (the age ranged from 6 months to 53 years old). Twenty-four were adults older than 20 years old. Three had documented MCV (SH11300, SH15370, and SH15185), whereas the remaining had an unknown or no history of MCV or disease (Table 1). All 40 patients reported fever and rash, 33 (82.5%) experienced coughing, and 24 (60%) experienced catarrhal symptoms.
Detection of viral nucleic acids. Viral nucleic acids were detected by real-time PCR/RT-PCR from throat swab samples or infected Vero/hSLAM cultures, which were divided in to 3 groups, the coinfection group (n = 40), measles group (n = 463) and healthy group (n = 192). Among the 40 MeV and HSV-1 coinfection cases, MeV RNA was detected in 40 (100.0%) throat swabs by RT-qPCR. There were no significant differences in the MeV RNA copy numbers between the coinfection group and the measles group (P = 0.5033). HSV-1 nucleic acids were detected in 39 of the 40 throat swabs by qPCR, and the copy number of HSV-1 DNA in the coinfection group was significantly higher than that in the measles group (P = 0.0028).
Of the 463 throat swabs from MeV cases, 17 (3.67%) were positive for HSV-1 DNA, whereas 3 (1.56%) swabs from the healthy group were positive. There was no significant difference in the positive HSV-1 DNA rate between these two groups (P = 0.1316).
Serological results. Serum samples from the 40 MeV and HSV-1 coinfection cases were tested for IgM and IgG antibodies (Table 1), the OD value of IgM and IgG and judgement were supplied in detail as Supplementary  Table S1. Thirty-seven (92.5%) were positive for MeV IgM, and 27 (67.5%) were positive for MeV IgG. Although three (SH11300, SH12649, and SH15370) were negative for MeV IgM, MeV RNA was detected in the corresponding throat swabs. Of the 40 cases, seven (17.5%) and 34 (85.0%) were positive for HSV-1 IgM and IgG, respectively. Five (SH06064, SH08035, SH11300, SH12538 and SH151187) were negative based on both HSV-1 IgG and IgM. Among these five patients, four were children under 5, while SH151187 represented a patient older than 50. Table 2 shows the titers of HSV-1 IgG antibody in the healthy group, coinfection group, and measles group. Statistically significant differences were found between the measles group and the healthy group (P = 0.0026), the healthy group and the coinfection group (P < 0.0001), but no significant difference was found between the measles group and the coinfection group (p = 0.105).
Virus isolation and laser scanning confocal microscopy results. A total of 1253 MeV isolates were obtained from 4921 throat swabs between 2006 and 2015. The proportion of MeV and HSV-1 coinfection ranged from 0.0-11.1% each year. All MeV isolates produced syncytium formation in infected Vero/hSLAM cells during the first three passages. Forty MeV isolates exhibited another type of CPE characterized by larger and rounder cells in cultures as early as the 3 rd passage, which became more prominent in the subsequent passages. These cultures were confirmed to be coinfected with MeV and HSV-1 by the detection of viral nucleic acids.
Laser scanning confocal microscopy was used to demonstrate the dynamics of MeV and HSV-1 infection in coinfected Vero/hSLAM cells (Fig. 1). In cells infected with SH13390 at passage 3, MeV infection, detected by antibody of anti-MeV matrix (M) protein, as indicated by green fluorescence, was readily observed in some cells, whereas very few HSV-1-infected cells, detected by antibody of anti-HSV-1 ICP0, as indicated by red fluorescence, were found. In the 6 th passage of the infected culture, significant increases in MeV and HSV-1 infection were observed. In HSV-1-infected cells, the red fluorescence appeared as granules in the cytoplasm. As passaging continued, the number of cells stained with red fluorescence increased, while the number of cells stained with green fluorescence decreased. Eventually, at the 17 th passage, only cells with red fluorescence were detected (data not shown). www.nature.com/scientificreports www.nature.com/scientificreports/ Supplementary Table S2), and all were genotype H1a (Fig. 2). The nucleic acid sequences of these 25 MeV exhibited 97.8-100.0% identity, and there was 98.0-100% amino acid sequence identity. The nucleotide sequence identity among the sequences of the MeV viruses and the reference sequence of genotype H1 (GeneBank: AF045212) was 97.3-98.0% (96.0-98.0% for the amino acid sequences). The overall nucleotide sequence identity between We sequenced 37 HSV-1 isolates from the 40 coinfected cultures (GenBank accession numbers: MN166405-MN166441; More sequence information of HSV-1 isolates were provided as Supplementary  Table S3). All contained 4-10 guanine-adenine-guanine (GAG) trinucleotide tandem repeats beginning at nt 235 (GenBank: JN555585, using the same numbering scheme) in the gG region. Point mutations in the GAG repeats were detected in some viruses. Based on phylogenetic analysis, these 37 HSV-1 isolates were divided into two lineages. The majority (34/37) of the sequences belonged to group A, and the remaining sequences belonged to group B (Fig. 3). The group A HSV-1 isolates had 98.8-100.0% nucleic acid identity and 97.0-100.0% amino acid sequence identity, while all three group B isolates had identical sequences. The nucleic acid identity between groups A and B was 96.5-97.3%, and the amino acid sequence identity was 94.0-95.7%. In the region between nt 267-703, the viruses in group A had completely different bases than the viruses in group B at 17 loci ( Table 3)  www.nature.com/scientificreports www.nature.com/scientificreports/ commonly associated with HSV-2 and HIV infection, and coinfection of Lyme borreliosis and HSV-1 has also been reported [15][16][17] . In addition, since both HSV and VZV can establish latent infections in sensory neurons after primary infection, a rare case of coinfection due to the reactivation of both HSV and VZV has been reported 18 . In this study, nucleic acids from the rubella virus, dengue fever virus, EBV, B19, Adv, HEV, HHV6, CMV or VZV were not detected in any of the clinical throat swabs or cell cultures from the measles or coinfection groups.

Sequence analysis of
Based on the serological testing and immunization records, the majority of the codetection cases (37/40, 92.5%) were primary measles infections. SH11300, SH15185 and SH15370 had received at least one dose of MCV. SH11300 was negative for MeV IgM and IgG. This case had received a dose of MCV 5 days before the onset of rash. Unfortunately, we were not able to determine whether the infection was a coincidental event or a vaccine-associated measles case due to a lack of sequence information. SH15370, which was only positive for MeV IgG, was classified as secondary vaccine failures. SH15185, which was only positive for IgM and the patient was 1.5 years old, might be a primary vaccine failure 19,20 . SH12649, which was lack of immunization records and positive only to IgG test, couldn't be confirmed as immune failures, suggesting prior exposure to MeV or vaccine. In the same way, there were 25 cases testing positive to both IgM and IgG and it may be due to the window period of immunological test under primary infections 21 . SH11300, SH12649 and SH15370 were MeV IgM negative but were confirmed measles cases based on RT-qPCR testing. Sera were collected from these three cases within 2 days after the onset of rash.
The rates of HSV-1 IgG detection in the coinfection (85%) and measles groups (73%) were significantly higher than in the healthy group (57%). The HSV-1 IgG detection rates are lower than the reported seroprevalence (92%) in China 22 . Among the 40 coinfection patients, serological results suggested that 13 of these cases were likely to be primary infections of HSV-1, and 10 of these patients were under the age of 5. Upon comparison of the HSV-1 IgG titers, the titers in the MeV group and the coinfection group were significantly higher than in the healthy    Table 3. Analysis of the loci in recombined regions in the HSV-1 gG gene. The nine loci between nt 324 and nt 429 were those where recombination occurred between groups A and B in AF1171219.
www.nature.com/scientificreports www.nature.com/scientificreports/ the nucleic acid sequence of the whole gG gene, the phylogenetic analysis showed that most of the HSV-1 isolates identified in this study were in group A and that three isolates were in group B. This distribution is different from that found in European countries 26,27 . The nt 267-703 region in the HSV-1 gG gene has been identified as a recombination hotspot between groups A and B 23 . It has been noticed that the grouping of HSV-1 differs when different segments of the gG gene sequence are used for phylogenic analysis, suggesting that recombination has occurred within this region between the two groups 23,28 . Using sequence analysis and SimPlot, we did not find any evidence of recombination events in this region in the 37 HSV-1 isolates. In this study, we found 17 loci in the gG gene where the bases were completely different between group A and group B. More sequences are needed to determine whether these loci also serve as recombination hotspots.
Fatal cases of coinfection with MeV and other pathogens have been reported 12 . It is not clear whether MeV infection increases susceptibility to HSV-1 infection or triggers reactivation. Because of the high prevalence of HSV-1 in the population and the ability of HSV-1 to cause latent infections, the number of coinfection cases may be underestimated. Additional research is needed to investigate coinfections of MeV and other pathogens and to determine whether these co-infections affect the disease severity and/or require special treatment. In this study, the measles group consisted of cases confirmed by either MeV IgM testing or MeV RNA testing by real-time RT-PCR assay in 2015. The MeV and HSV-1 coinfection group included confirmed measles cases with HSV-1 infection that exhibited atypical CPE in infected cells after three passages. In addition, sera and throat swabs were collected from individuals who did not have clinical presentation or signs of measles at the time of sample collection to serve as controls ("healthy group"). The total numbers of sera samples in the measles, coinfection, and healthy groups were 741, 40 and 741, respectively, and the numbers of throat swab specimens collected for these three groups were 463, 40 and 192, respectively.

Methods
To obey ethical guidelines and ensure data protection, the serum and throat swab samples used in this study were collected according to the guidelines of the national and Shanghai municipal measles and rubella surveillance programs and did not involve human experimentation. This study strictly followed the relevant ethical requirements and strictly protected the data security and privacy of the participants.
Detection of viral nucleic acids. Viral nucleic acid from throat swab samples or infected Vero/hSLAM cultures was extracted using the QIAamp Viral RNA/DNA Mini Kit (QIAGEN; Hilden, Germany) according to the manufacturer's instructions. Viral nucleic acid was detected by real-time PCR/RT-PCR using a detection kit for MeV and rubella virus (RuV) (BioPerfectus Technologies, Taizhou, Jiangsu, China), human herpes virus type 6 (HHV6) and varicella-zoster virus (VZV) (Tianlong Biotechnology Co. Suzhou, Jiangsu, China) according to the manufacturer's instructions. The detection of other viral febrile respiratory illnesses, such as human cytomegalovirus (CMV) 29 , Epstein-Barr virus (EB) 30 , human parvovirus B19 (B19) 31 , human adenovirus (Adv), human enterovirus (HEV) 32 and herpes simplex virus type 1 33 , were carried out using the One Step PrimeScript™ RT-PCR Kit (Perfect Real Time) or Premix Ex Taq™ (Probe qPCR) (TaKaRa; Dalian, China) for RNA or DNA viruses as reported previously.
Serological tests. Serological specimens were collected within 5 days after the onset of measles infection.
Detection of MeV IgM was carried out using an IgM ELISA test kit (Haitai Biological Pharmaceutical Co.; Zhuhai, Guangdong, China). MeV IgG, HSV-1 IgM, and HSV-1 IgG were detected using reagent kits manufactured by Institute Virion/Serion GmbH (Würzburg, Germany). All procedures and the interpretation of the results were performed according to the manufacturer's instructions. Specifically, if the results of two repeated experiments were consistent for the cases within the cut off value, it would be judged as negative.
Virus isolation. The Vero/hSLAM cell line was used for MeV isolation from clinical specimens 11 . Vero/ hSLAM cells were kindly shared by Dr. Yanagi at Kyushu University, Japan, through the WHO Global Measles and Rubella Laboratory Network (GMRLN). Procedures used for the maintenance of cells, sample handling and virus infection were performed as described in the Manual for the Laboratory-based Surveillance of Measles, Rubella, and Congenital Rubella Syndrome (WHO) 34 .
Indirect immunofluorescence assay (IFA) and laser scanning confocal microscopy. A throat swab sample, SH13390, from a confirmed MeV and HSV-1 coinfected case was used to infect Vero/hSLAM cells, which was followed by ten cell passages. Infected cells from each passage were subjected to IFA. Briefly, the cells were washed with 0.01 M phosphate-buffered solution (PBS) and fixed with 4% polyoxymethylene