Influenza D Virus: Serological Evidence in the Italian Population from 2005 to 2017

Influenza D virus is a novel influenza virus, which was first isolated from an ailing swine in 2011 and later detected in cattle, suggesting that these animals may be a primary natural reservoir. To date, few studies have been performed on human samples and there is no conclusive evidence on the ability of the virus to infect humans. The aim of this serological study was to assess the prevalence of antibodies against influenza D virus in human serum samples collected in Italy from 2005 to 2017. Serum samples were analysed by haemagglutination inhibition and virus neutralization assays. The results showed that the prevalence of antibodies against the virus increased in the human population in Italy from 2005 to 2017, with a trend characterized by a sharp increase in some years, followed by a decline in subsequent years. The virus showed the ability to infect and elicit an immune response in humans. However, prevalence peaks in humans appear to follow epidemics in animals and not to persist in the human population.


Introduction
Influenza D virus (IDV), a novel influenza virus, was first isolated from an ailing swine in 2011 in Oklahoma, USA [1,2]. Although the viral genome shows approximately 50% overall homology with influenza C virus (ICV), no cross-reactivity with antibodies directed against human ICV and no re-assortment with human ICVs have been observed so far. Moreover, attempts to detect viable recombinant progeny involving ICV and IDV have not been successful [1,[3][4][5].

Influenza Viruses
Influenza D/bovine/Oklahoma/660/2013 virus was originally isolated from the bovine herds of Oklahoma.
Influenza C/Victoria/2/2012 virus was originally isolated in 2012 from a nasopharyngeal swab of a child with clinical symptoms of acute respiratory infection and was submitted for virus isolation to the Victorian Infectious Diseases Reference Laboratory, Melbourne, Australia. The virus was isolated after cultivation in Madin Darby Canine Kidney (MDCK) cells at 33 • C, together with RT-PCR diagnosis and sequence confirmation.
Cells were seeded in a T175 cm 2 culture flask at a density of 1 × 10 6 cells/mL with UltraMDCK SFM. After 18-20 h, the cell monolayer was washed twice with sterile Dulbecco's phosphate buffered saline (DPBS). After the DPBS had been carefully removed, cells were infected with 3.5 mL (IDV) or 10mL (ICV) of UltraMDCK SFM (without Trypsin) containing the respective virus at a multiplicity of infection of 0.001. After 1 h of incubation at 33 • C in a humidified atmosphere with 5% CO 2 , 50 mL of UltraMDCK SFM containing a final concentration of 2 µg of trypsin acetylated (IDV) and 0.5 µg of TPCK (ICV) was added to the flask. The infected cells were incubated at 33 • C in a humidified atmosphere with 5% CO 2 for 36 h. At the end of the incubation period, additional acetylated trypsin was added up to a final concentration of 1 µg/mL and the flask was incubated at 37 • C in a humidified atmosphere with 5% CO 2 for another 36-48 h. The cytopathic effect (CPE) was monitored every day, along with the hemagglutination (HA) titre of the supernatant. At 90% of the CPE, the culture medium was harvested, centrifuged at 4 • C in order to remove the cell debris, and stored at −80 • C.

Serum Samples
Archived human serum samples from adults (≥18 years old) were obtained from the Serum Bank of the Laboratory of Molecular Epidemiology, Department of Molecular and Developmental Medicine, University of Siena, Siena, Italy.
The samples were anonymously collected in Tuscany (Central Italy) and Apulia (Southern Italy) and stored in compliance with Italian ethics law. The only information available was the age of each subject and the year of sampling. A total of 1281 serum samples collected from 2005 to 2017 (approximately 100 serum samples for each year) were randomly selected, though a balanced distribution between males and females and among age-groups in each year was ensured, according to the availability of serum samples for each year (Table S1).
Human serum without IgA, IgM and IgG was used as a negative control (Sigma-Aldrich, S5393).

Haemagglutination Inhibition Assay
The HI assay was performed as described in Hause et al. [1]. All serum samples, including positive and negative controls, were pre-treated with receptor-destroying enzyme from Vibrio Cholerae (Sigma Aldrich, Milano, Italy) (ratio 1:5) followed by heat inactivation for 1 h at 56 • C. Serum samples were tested in duplicate by using turkey red blood cells (0.35%). The antibody titre was expressed as the reciprocal of the highest serum dilution that showed complete inhibition of agglutination. Since the starting dilution was 1:10, when the titre was below the detectable threshold, the results were conventionally expressed as 5 for calculation purposes [31].

Virus Neutralization Assay
The MDCK cell cultures were grown at 37 • C in 5% CO 2 and pre-incubated in a 96-well plate for 4 h.
Serum samples, including positive and negative controls, previously heat-inactivated at 56 • C for 30 min and tested in duplicate, were two-fold diluted with EMEM culture medium supplemented with 0.5% fetal bovine serum in a 96-well plate and mixed with an equal volume of virus (100 TCID50/well). After 1 h incubation at 37 • C in 5% CO 2 , the mixture was added to the MDCK cell suspension (1.5 × 10 5 cells/mL). Plates were read for HA activity in the supernatant after three days of incubation at 37 • C in 5% CO 2 .

Statistical Analysis
According to the definition of seropositivity used for other newly emerging viruses, all positive serological responses (HI titres ≥1:10) were classified as seropositive, while non-detectable HI responses (<1:10) were regarded as negative and arbitrarily expressed as a value of 5 [31]. In addition, positive titres were classified in positive (≥1:10, ≥1:20), and highly positive (≥1:40). For the purpose of direct comparison of HI and virus neutralization (VN) assays, the proportions for each category of titres were calculated by applying the total number of serum samples tested in the HI and VN assays. Calculation of the 95% confidence intervals for the proportions was based on the Clopper-Pearson exact method [32].
For positive and highly positive titres, the Pearson's Chi-squared test was used to verify the significant differences among the proportions of titres between the two assays across years, and to make yearly-based comparisons of the observed proportions for each assay. The Marascuilo procedure was used for the post-hoc analysis, which accounts for multiple comparisons. Relative changes of proportions were evaluated in relation to the value of the proportion measured for 2005, the first year tested, which was used as the base year for the analysis. The Hodrick-Prescott filter, with a smoothness penalty parameter λ = 1600, was implemented over the time series of the relative changes in proportions in order to estimate their trend components [33]. All the analyses were made as two-sided tests and conducted at a significance level of 5%. RStudio (version 1.1.463) was used for all the statistical analyses.

Investigations on Potential Cross-Reactivity between IDV-and ICV-Positive Serum Samples
The specificity of the HI assay with respect to the potential cross-reactivity between IDV and ICV was evaluated by testing both viral antigens against IDV and ICV specific hyper-immune antisera generated in swine and rooster. As shown in Table 1, no cross-reaction between IDV and ICV was observed. The anti-serum specific for the D/bovine/Oklahoma/660/2013 strain showed a high HI titre (1:10,240) as did the anti-serum specific for the C/Victoria/2/2012 (HI titre 1:640). No HI titres were detectable when the hyper-immune antisera were tested against the respective heterologous influenza C or D strain. These results demonstrated the specificity of the IDV HI assay and were therefore used for the analysis of the presence of IDV-specific antibodies in human serum samples. The IDV hyperimmune serum sample was used as positive control in HI and VN assays showing a titre range of 2560-10,240 and 1280-5120, respectively.

Analysis of Human Serum Samples for the Presence of IDV-Specific Antibodies
A total of 1281 human serum samples, collected randomly from adults in the Italian regions of Tuscany and Apulia from 2005 to 2017, were tested by HI assay in order to detect the presence of antibodies against IDV. The results clearly show that IDV specific HI antibodies were present in at least a small subset of serum samples taken in every single year between 2005 and 2017, although IDV was isolated and described for the first time in 2011 ((A) in Table 2). IDV antibodies displayed low levels, between 5.  10) were then tested in an IDV specific VN assay to confirm the positive HI titres and subsequently the specificity of the HI assay for IDV ((B) in Table 2).

Pearson's Chi-Squared Test for Multi-Proportions
Differences between the HI and VN assays proportions in homologous class of titres were not significant. By contrast, the analysis of proportions among years showed significant differences for both assays in each class of titres (Table S2). Comparisons of the titre proportions over years were conducted on each class of titres except the negative class, as this class is overtly complementary to the positive (≥1:10) class of titres (Table S3). In the HI assay, the proportions of titres ≥1:10 in the first three years (2005)(2006)(2007)  Our analysis of the relative changes in proportions provided insights into the dynamics of the proportions of titres. Figure 1 shows the normalized proportions of negative, positive and highly positive HI titres and their corresponding trend curves.

Discussion
The detection and isolation of IDV in pigs with influenza-like clinical signs and respiratory distress in 2011, and the evidence of a seroprevalence of 1.3% of humans [1] in combination with the circulation of IDV in pigs in Northern Italy which was confirmed in 2015 [25], have raised the question of whether IDV antibodies can be found in the Italian population since this potential outbreak. This hypothesis is supported by a study conducted in Florida by White et al., which reported a seroprevalence of 94% among workers exposed to cattle [30].
The results of the present study indicate that the findings of studies conducted in various countries also apply to Italy. The low seroprevalence (5.1%) of IDV specific antibodies found in the human serum samples from the first year of the study (2005) suggests that IDV may have circulated at least in Italy already before 2005. This finding is supported by a study by Luo et al. [13], which suggested that Nebraska beef cattle had been exposed to IDV since at least 2003, and that the virus may have already circulated at least 8 years before its detection.
The potential IDV circulation in pigs in Northern Italy in 2015 [25], which was confirmed by PCR analysis, positive virus isolation, and increased IDV specific HI antibody responses in animals from the farms affected, may have been associated with an increase in IDV HI-specific antibodies in humans from 25.7% in 2015 to 33.9% in 2016. These data also indicate that there could have been undetected IDV circulation in pigs and/or cattle already in 2007 and reflect a confirmed outbreak in

Discussion
The detection and isolation of IDV in pigs with influenza-like clinical signs and respiratory distress in 2011, and the evidence of a seroprevalence of 1.3% of humans [1] in combination with the circulation of IDV in pigs in Northern Italy which was confirmed in 2015 [25], have raised the question of whether IDV antibodies can be found in the Italian population since this potential outbreak. This hypothesis is supported by a study conducted in Florida by White et al., which reported a seroprevalence of 94% among workers exposed to cattle [30].
The results of the present study indicate that the findings of studies conducted in various countries also apply to Italy. The low seroprevalence (5.1%) of IDV specific antibodies found in the human serum samples from the first year of the study (2005) suggests that IDV may have circulated at least in Italy already before 2005. This finding is supported by a study by Luo et al. [13], which suggested that Nebraska beef cattle had been exposed to IDV since at least 2003, and that the virus may have already circulated at least 8 years before its detection.
The potential IDV circulation in pigs in Northern Italy in 2015 [25], which was confirmed by PCR analysis, positive virus isolation, and increased IDV specific HI antibody responses in animals from the farms affected, may have been associated with an increase in IDV HI-specific antibodies in humans from 25.7% in 2015 to 33.9% in 2016. These data also indicate that there could have been undetected IDV circulation in pigs and/or cattle already in 2007 and reflect a confirmed outbreak in cattle in 2011 [7], since the seroprevalence of HI specific antibodies in humans increased from 9.8% in 2007 to 37.9% in 2008 and from 19.6% in 2012 to 41.0% in 2013.
These data clearly show that seroprevalence against IDV increased in Italy from 2005 to 2017. This increase was not constant over the years, it shows sharp rises in some years followed by drops in subsequent years. The seroprevalence peaks detected in humans appear to follow IDV epidemics in animals, as an epidemic in cattle in France in 2011 [7] and an outbreak in pigs in Italy in 2015 [25] have been reported. Moreover, as the titres did not remain high in the years following the increase, but dropped to lower levels, we could speculate that a spill over event from an animal reservoir occurred, and that the virus does not circulate primarily in humans. However, IDV proved able to elicit an immune response in humans. The background seropositivity rate in the population was higher in 2017 (11.9%) than in 2005 (5.1%), although this difference did not prove statistically significant. Follow-up studies over the next couples of years should be performed in order to determine whether the titres drop further to levels below 10% over the years or remain at a higher level than that observed in 2005. In addition, cattle and pigs should be carefully observed and analysed with respect to potential new outbreaks, which could be followed by an increase in IDV-specific antibodies in humans in the subsequent year.
Although IDV was first isolated from a diseased pig in 2011 [1], this virus is notable for being the first influenza virus identified in cattle [3]. This is supported by evidences of past infection in cattle from the same farm as the diseased pig. Finally, additional studies revealed cows to be the primary reservoir [3,[9][10][11][12]. Since then, IDV has been isolated from cattle and pigs in several countries, including China, the United States and France [3][4][5][6][7][8], and antibodies against the virus have been found in sheep, goats, horses and camelids in China, Ethiopia, France, Japan, Mongolia, Ireland and the United States [19][20][21][22][23][24].
The origin and ecology of IDV remain unknown. Although it is not yet known how or when IDV first emerged, analysis of archived serum samples by SJCEIRS (St. Jude Children's Research Hospital Center of Excellence for Influenza Research and Surveillance) suggests that IDV has been circulating in cattle since at least 2004 [2,9,10,13]. IDV infections in cattle also tend to be associated with other respiratory infections, particularly pneumonia, but the significance of this observation is not known [8].
To date, no indications that IDV can cause disease in humans have been found, but several groups have already signalled its potential threat as an emerging pathogen in specific target groups, such as cattle-workers [5], or as a considerable public health risk [2,5,13,26,34,35]. This eventuality is supported by a number of findings by different groups. One of the main risks stems from the ability of the virus to infect and to be transmitted to a number of domestic mammal species, such as cattle, pigs, goats, sheep and camelids, and also to wild animals, such as feral pigs [22]. In addition, it has been shown that IDV can infect ferrets, the gold standard for influenza studies in animals [1,26], and guinea pigs, as shown by transmission studies [26,36].
Seroprevalence studies in humans have shown that the risk of transmission from infected cattle to humans may be very high. Indeed, seroprevalence rates of 91% (HI assay) and 97% (VN assay) have been documented in cattle-workers, as opposed to 18% (on VN assay) in control subjects without contact with cattle [26,30]. The results of the present study show seroprevalence peaks in humans that seem to follow IDV epidemics in animals (outbreak in pigs in Italy in 2015 [25]). However, further studies on the circulation of IDV in animals in the same years would be useful to support this hypothesis. The increased risk of transmission to humans is supported by the finding that the IDV hemagglutinin-esterase-fusion glycoprotein exhibits an open receptor-binding cavity, which forms the basis for its broad cell tropism and, consequently, its broad host tropism [37]. A valuable tool for studies on IDV replication kinetics and cell tropism has been provided by Holwerda et al. [38], who used "primary well-differentiated human airway epithelial cells" as an in vitro respiratory epithelium model of humans.
At least three antigenic lineages of IDV have been identified-D/Oklahoma, D/660 and D/Japan-and HI analysis have shown up to a 10-fold loss in cross-reactivity against heterologous antiserum [8,39,40]. Serological analysis in this study has been conducted using D/660-like strain; however, the use of a single IDV strain may ultimately lead to an underestimation of the true seroprevalence in Italian population, leaving the conclusions of this study unchanged.
Despite the solid results yielded by a considerable number of studies, our current knowledge of IDV is still limited, and neither potential threats to exposed individuals nor public health issues can be fully excluded. Consequently, additional research on IDV and diligent observation of IDV prevalence in the various animal hosts and in potentially affected individuals should be conducted in the future.