Abstract
During the winter of 1996 to 1997 two cases of influenza C were confirmed, one by isolation and the second by serology (fourfold increase in hemagglutination inhibition antibodies). The cases of influenza C occurred during an outbreak of influenza A (H3N2) and B viruses. The positive isolation was from one of three throat washings sent to the laboratory, and the other case was from a group of 51 students participating in a study of influenza virus vaccination. It seems, therefore, that influenza C virus should also be considered when examining patients with respiratory infections during the influenza season.
Influenza C virus was first isolated in the forties (1947), with sporadic cases reported (10).
Influenza C viruses occur primarily in a pattern of sporadic cases or in limited outbreaks of mild illness involving children or young adults (4, 8).
In a seroepidemiological study carried out in France in 1992, 61 to 70% of the population was found to have been previously exposed to the virus, the highest rates for positive samples being found in the 16- to 30-year-old group. The results indicated intense circulation of influenza C virus in the population (7).
During the influenza season attention is paid to influenza A and B viruses in isolation efforts, as well as in serology studies. During the influenza virus outbreak in the winter of 1996 to 1997, cases of influenza C were found by isolation or by serology, as described in this communication.
Case reports. (i) Patient 1.
A 27-year-old female came to the clinic on 26 January 1997 with a high fever (39.5°C) that started on that day. She complained of diffuse pains, mainly lower abdominal, nausea with vomiting, runny nose, sore throat, coughing, weakness and fever blisters on her lips. A 5-ml volume of throat washing in saline was sent to the laboratory, together with two other samples from patients suffering from similar symptoms.
(ii) Patient 2.
A 25-year-old female medical student was among a group of 51 students participating in a study of intranasal influenza virus vaccine efficacy for the 1996-to-1997 period (6). After vaccination in the middle of November, the group was under surveillance for respiratory infections until the end of March. Blood samples for hemagglutination inhibition (HI) antibody tests were taken from those complaining of respiratory symptoms. Nine vaccinees complained of symptoms ranging from mild, without fever, to more severe, with fever.
Isolation of virus.
A 0.1-ml volume of throat washing was inoculated into the amniotic sacs, and a 0.2-ml volume was inoculated into the allantoic cavities, of 10-day-old embryonated eggs, which were incubated for 72 h at 34°C. The first two passages (three and five eggs) were negative, while on the third passage, three of five showed respective hemagglutination titers of 1:16, 1:4, and 1:32. The blind passages were performed with samples from a pool of infected amniotic allantoic fluids. Hemagglutination was stable at 4°C but eluted rapidly, within 20 min, at room temperature. MDCK cells were simultaneously infected, with no isolation success following three consecutive passages. However, virus from the third passage from eggs replicated in MDCK cells, as evidenced by the hemagglutination of supernatants and hemadsorption at 4°C. The titer was 103.5 50% tissue culture infective doses (TCID50) in the presence of trypsin (2 μg/ml) and only 101.5 TCID50 without trypsin. Cytopathic effect, described for some influenza C virus strains (2), was not observed. The virus isolated from eggs was inhibited by antisera to C/Taylor/1233/47 (1:320) and C/Johannesburg 1/66 (1:320) (kindly provided by N. Versanon of the Central Viral Laboratory, Tel Hashomer, Israel).
The identity of isolate C/Jerusalem/2/97 was further confirmed by A. R. Douglas and A. Hay (World Health Organization Collaborative Center for Reference and Research on Influenza, National Institute for Medical Research, Ridgeway, Mill Hill, London, United Kingdom), and the isolate was found to be similar to C/Johannesburg/1/66.
The isolate was insensitive to inhibitors of normal horse serum but was inhibited by normal rat serum (1:320 to 1:640 dilution). The inhibiting substance could not be removed from the rat serum by heating for 30 min at 58°C.
There can be no question of contamination from other sources, since this is our first encounter with the influenza C virus.
Serological testing.
Sera from all nine volunteers complaining of respiratory infections were evaluated for rise in HI antibodies against the three recently isolated influenza A virus strains (A/Texas/36/91 [H1N1], A/Nanchang/933/95 [H3N2], and B/Harbin/7/94), and against the influenza C virus isolate. HI testing was performed by the standard microtiter technique (11) in twofold dilutions, starting at 1:5. The serum was pretreated with receptor destroying enzyme (Sigma Chemical Co., St. Louis, Mo.) for 18 h at 37°C and then was inactivated (56°C for 30 min) and tested against four hemagglutination units of each of the influenza virus strains. Of the nine cases tested, three had fourfold rises in HI antibodies, one to B/Harbin, one to A/Nanchang, and one to C/Jerusalem/2/97 (1:5 to 1:20). For the last case, the student had low fever for 3 to 4 days and a runny nose at the end of January. She was immune to Texas/91 and A/Nanchang/95. She had no rise in HI antibodies to B/Harbin, to which she was not immune.
Discussion.
During the winter, infection with influenza C virus coincides with influenza A (H3N2 and H1N1) and B virus activity. The influenza C cases may exhibit symptoms with a severity similar to that caused by influenza A or B virus, and it is therefore important to better understand the epidemiology of this virus and its exact role in acute viral respiratory infections.
In France a high prevalence of antibodies, as well as significant titers, indicates intense circulation of influenza C virus, especially among young adults (7). In Japan 18 influenza C virus strains infecting humans were found with monoclonal antibodies to hemagglobulin esterase glycoprotein and were included in three distinct groups closely related to C/Yamagata/26/81, C/Aichi/1/81, and C/Mississippi/80 (5).
Influenza C virus was thought to infect humans only but was isolated from swine in China (1). The close relationship of strains from humans to isolates from pigs that was found and the possibility of interspecies transmission of the influenza C virus between humans and pigs (5) further indicate the potential involvement of the virus in respiratory infections. The finding of persistent infection in chicken lungs in vitro (3), as well as the fact that frequent genetic reassortment between influenza C virus strains occurs in nature (9), calls for broadening this study.
It is possible that because of more efficient control of influenza A and B viruses by vaccination and/or antiviral drugs, influenza C virus could have a better chance of persisting, leading to more cases being identified.
Acknowledgments
We are indebted to Avi Izhak from Rishon LeZion for sending us samples.
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