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Severe pathogenesis of influenza B virus in pregnant mice
Virology 448 (2014) 74–81
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Severe pathogenesis of influenza B virus in pregnant mice Jeong Cheol Kim a,b,1, Heui Man Kim a,b,1, Young Myong Kang a,b, Keun Bon Ku a,b, Eun Hye Park a,b, Jung Yum a,b, Ji An Kim a,b, Yoo Kyung Kang a, Joo Sub Lee a,b, Hyun Soo Kim c, Sang Heui Seo a,b,n a
Laboratory of Influenza Research, College of Veterinary Medicine, Chungnam National University, Daejeon 305-764, Republic of Korea Institute for Influenza Virus, College of Veterinary Medicine, Chungnam National University, Daejeon 305-764, Republic of Korea c Laboratory of Public Health, College of Veterinary Medicine, Chungnam National University, Daejeon 305-764, Republic of Korea b
art ic l e i nf o
a b s t r a c t
Article history: Received 23 August 2013 Returned to author for revisions 9 September 2013 Accepted 1 October 2013 Available online 20 October 2013
The study on pathogenesis of influenza B virus during pregnancy is limited. Here, we showed using a mouse model that influenza B virus could cause severe disease including death during pregnancy. Infected pregnant mice resulted in 40% mortality, but infected age-matched non-pregnant mice did not show any death. Infected pregnant mice contained high viral loads in lungs with the elevated inductions of inflammatory cytokines and chemokines than infected non-pregnant mice. Infected pregnant mice delivered lower number of neonates than uninfected pregnant mice, suggesting adverse effects of influenza B virus on fetuses. Progesterone which is important for maintaining pregnancy was reduced in uteruses of infected pregnant mice than in those of uninfected pregnant mice. Taken together, our results suggest that influenza B virus can cause severe disease during pregnancy, and that preventive measures including vaccination may be important for protecting women during pregnancy. & 2013 Elsevier Inc. All rights reserved.
Keywords: Pregnancy Influenza B virus Pneumonia Hormone Cytokine Chemokine
Introduction Influenza virus belongs to the family, Orthomyxoviridae, and is classified into three types, A, B, and C (Bouvier and Palese, 2008) and contains negative-sense RNA genome. Influenza A and B viruses contain eight viral RNA segments, and influenza C virus contains seven viral RNA segments (Bouvier and Palese, 2008). Influenza A virus is further divided into subtypes based on hemagglutinin (HA) and neuraminidase (NA). There are 17 subtypes of HA and nine subtypes of NA in influenza A viruses (Bouvier and Palese, 2008; Fouchier et al., 2005; Tong et al., 2012). Influenza A virus has a broad spectrum of host ranges including birds, pigs, horses, and humans. Influenza B and C virus are circulating in humans (Webster et al., 1992). Influenza C virus causes much milder disease in humans than influenza A and B viruses (Rasmussen et al., 2008). By morphology, influenza A and B viruses are not distinguishable, and are spherical or filamentous in shape with host cellderived lipid layers (Bouvier and Palese, 2008). The subtypes of influenza A viruses currently circulating in humans are H1N1 and H3N2. Influenza B virus is not divided into
n Correspondence to: Laboratory of Influenza Research, College of Veterinary Medicine, Chungnam National University, 220 Gung Dong, Yuseong Gu, Daejeon 305-764, Republic of Korea. Fax: þ 82 42 821 6762. E-mail address: [email protected] (S.H. Seo). 1 These authors equally contributed to this work.
0042-6822/$ - see front matter & 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.virol.2013.10.001
subtypes and has two genetically distinct lineages circulating in humans, which are B/Vitoria/2/87-like (Victoria lineage) and B/Yamagata/16/88-like (Yamagata lineage) (Rota et al., 1990). The first influenza A virus was discovered in humans in 1933, and influenza B virus was first found in humans in 1940 (Francis, 1940). Epidemics by influenza B virus could occur at intervals of 2–4 years and the patients infected with influenza B virus showed well-defined discrete illness in all ages of humans (Glezen et al., 1987). Until now, research has been done more on influenza A virus than on influenza B virus (Jackson et al., 2011). The current popular concept is that influenza B virus is less pathogenic to humans than influenza A virus (Rasmussen et al., 2008). However, influenza B virus causes the substantial disease in humans. Among US pediatric deaths by influenza infections between 2004 and 2011, 22–44% of deaths were related to influenza B virus (Glezen et al., 1987). In the United Kingdom, influenza B virus was dominantly circulated in the 2010–2011 season (Glezen et al., 2013), 40 out of 607 deaths associated with influenza virus infections were related to influenza B virus. In Japan and Taiwan, encephalitis in patients infected with influenza B virus was reported (Li et al., 2008; Newland et al., 2003). Pregnant woman is more susceptible to influenza virus infections than non-pregnant humans (Cantu and Tita, 2013). The study on influenza A virus infections in women under pregnancy during 2009 H1N1 pandemic reported the sevenfold increase in the risk of admission to the intensive care unit, the increase in 69% of
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between two was 27.5%. Uninfected non-pregnant mice gained 10.7% and infected non-pregnant mice lost 17.0%. The difference between two was 27.7% (Fig. 1A). Mortality rate of infected pregnant mice was 40% until 7 days p.i. when neonates were born, whereas no morality of infected non-pregnant mice occurred (Fig. 1B). We compared the mean number of neonates delivered by pregnant mice on 10 days p.i. (Fig. 1C). The mean number of neonates delivered by infected pregnant mice was 7.7, and that of neonates delivered by uninfected pregnant mice was 13.8. (Fig. 1C). We measured viral titers in the tissues of mice on 3 and 5 days p.i. Viral titers in nasal turbinates, tracheae, and lungs were higher in infected pregnant mice than in infected nonpregnant mice (Fig. 1D). On 5 days p.i. the mean viral titer in nasal turbinates, tracheae, and lungs of infected pregnant mice was 4.0 EID50/g, 2.5 EID50/g, and 5.0 EID50/g, respectively, and that in nasal turbinates, tracheae, and lungs of infected non-pregnant mice was 2.76 EID50/g, 1.33 EID50/g, and 4.0 EID50/g, respectively (Fig. 1D). No virus was detected in brains, uteruses, and fetuses.
requirement of mechanical ventilation, and 11% of mortality (ANZIC Influenza Investigators, 2010). To date, information on pathogenesis during pregnancy by influenza B virus infection is limited. In this study, using a mouse model we wanted to address whether influenza B virus could cause more severe disease to pregnant mice than non-pregnant female mice, and to find out the underlying mechanism of disease severity of pregnant mice infected with influenza B virus.
Results Clinical signs and viral titers in mice infected with influenza B virus Pregnant mice on day 14 pregnancy and age-matched nonpregnant mice were i.n. infected with influenza B virus to find out the effect of pregnancy on pathogenesis. The change of body weights was similar between infected pregnant mice and infected non-pregnant mice, but the mortality rate is greater in infected pregnant mice than infected non-pregnant mice (Fig. 1). The change of body weights in infected mice were measured based on the weights of mice before infection (Fig. 1A). On 7 days p.i. when the difference of body-weight change is the highest between infected and uninfected mice, uninfected pregnant mice gained 50.9%, while infected pregnant mice gained 23.4%. The difference
Inflammatory responses in sera and lungs of mice infected with influenza B virus To understand the possible cause of severe pathogenesis in pregnant mice infected with influenza B virus, we measured cytokines and chemokines in sera and lungs of mice on 5 days
100
60 Nonpregnant and uninfected 50
Nonpregnant and uninfected
90
Nonpregnant and infected
Nonpregnant and infected
40
Pregnant and uninfected
80
Pregnant and infected
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30
Mortality (%)
Body weight change (%)
75
20 10
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60 50 40 30
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1
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9
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Days post infection
-30
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5
6
7
8
9 10 11 12 13 14
Days post infection
6 Nonpregnant and uninfected
Mean number of neonate
14 12 10 *
8 6 4
Viral titers in tissue (log10EID50/g)
16
*
Nonpregnant and infected
5
Pregnant and uninfected
*
Pregnant and infected
4
3
*
2
1
2 0
Pregnant and uninfected
Pregnant and infected
0 3dpi.
5dpi.
7dpi.
Nasal turbinates
9dpi.
3dpi.
5dpi.
7dpi.
Tracheas
9dpi.
3dpi.
5dpi.
7dpi.
9dpi.
Lungs
Fig. 1. Body-weight change, mortality, viral titers in tissues, and number of neonates of pregnant mice. Pregnant mice (day 14 of pregnancy) or non-pregnant mice (n¼10 per group) were i.n. infected with influenza B virus. Uninfected pregnant and non-pregnant mice were used for controls. The change of body weights (A) was recorded until 14 days p.i., mortality (B) were recorded for 14 days p.i., and the mean number of neonates from pregnant mice (n¼5 per group) (C) were recorded on 10 days p.i., The viral titers (D) in the nasal turbinates, tracheae, and lung tissues from infected pregnant and non-pregnant mice (n¼5 per group) on 3 and 5 days p.i. were measured by log10 EID50/ml. No virus was detected in brains, uteruses, and fetuses. ↓, delivery day. Statistical analysis in neonate number was performed between infected pregnant mice and uninfected pregnant mice. Statistical analysis in viral titers in tissues were performed between infected pregnant mice and infected non-pregnant female mice. nPo0.05. Data are the mean7standard error of mean.
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2.5
Nonpregnant and uninfected Cytokines in lungs (O.D. value: 450nm)
Cytokines in sera (O.D. value: 450nm)
2.5
Nonpregnant and infected 2
Pregnant and uninfected Pregnant and infected
1.5
1
0.5
0
Pregnant and infected 1.5 *
*
1
0.5
4.5
*
Nonpregnant and infected
IL-12 concentration (ng/ml or g)
IL-2 concentration (ng/ml or g)
Pregnant and uninfected
5 Nonpregnant and uninfected
4
3
Nonpregnant and infected 2
0
4.5
3.5
Nonpregnant and uninfected
Pregnant and uninfected Pregnant and infected
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4
* Nonpregnant and uninfected Nonpregnant and infected Pregnant and uninfected
3.5
Pregnant and infected
3 2.5 2 1.5 1 0.5
0 Sera
Lungs
0 Sera
Lungs
Fig. 2. Detection of inflammatory cytokines in mice. Sera and lung tissues of influenza B virus-infected pregnant and non-pregnant mice or uninfected pregnant and nonpregnant mice (n ¼5 per group) were collected on 5 days p.i. Lung tissues were homogenized and suspended in 1 ml of PBS (pH 7.4). A multi-analyte cytokine ELISArray kit was used for screening the inflammatory cytokines in samples of sera (A) and lung tissues (B). Among cytokines, IL-2 and IL-12 which were induced greater in infected mice was quantified using single analyte cytokine ELISArrary kit for IL-2 (C) and IL-12 (D). Statistical analysis in cytokine inductions were performed between infected pregnant mice and infected non-pregnant mice. nPo 0.05. Data are the mean 7 standard error of mean.
p.i. (Figs. 2 and 3). When we screened the induced cytokines in sera and lungs of mice with mutlti-analyte cytokine ELISA kit (Fig. 2A and B), the induction of cytokines which were measured (IL-1α, IL1β, IL-2, IL-4, IL-6, IL-10, IL-12, IL-17α, IFN-γ, TNF-α, G-CSF, GM-CSF) in sera was similar (Fig. 2A), but IL-2 involved in proliferation, and differentiation of T cells and IL-12 stimulating natural killer cells and T lymphocytes were induced greater in lungs of infected pregnant mice than in those of infected non-pregnant mice (Fig. 2B). We quantified IL-2 (Fig. 2C) and IL-12 (Fig. 2D) using a single ELISA kit in sera and lungs of mice. Their inductions in sera were similar between infected pregnant mice and infected nonpregnant mice, but it was induced greater in lungs of infected pregnant mice than in those of infected non-pregnant mice (Fig. 2C and D). The induced amount of IL-2 in the lungs of infected pregnant mice was 3.623 ng/g, and that in the lungs of infected non-pregnant mice was 2.432 ng/g (Fig. 2C). In lungs, the induced amount of IL-12 in infected pregnant mice was 4.532 ng/g, while that of IL-12 in the infected non-pregnant mice was 3.731 ng/g. We measured chemokines involved in attracting inflammatory cells in sera and lungs of mice infected with influenza B virus. We first screened chemokines in sera and lungs from infected mice with mutlti-analyte chemokine ELISA kit which can detect RANTES, MCP-1, MIP-1α, MIP-1β, SDF-1, IP-10, MIG, Eotaxin, TARC, MDC, KC, 6Ckine). MIP-1α (macrophage inflammatory protein-1α) involved in recruiting polymorphonuclear leukocytes, MIG (monokine induced by gamma interferon) which is T-cell chemo-attractant, and MCP-1 (monocyte chemotactic protein-1) which recruits monocytes, memory T cells, and dendritic cells to the sites of infection were induced greater in sera or lungs of infected
pregnant mice than in those of infected non-pregnant mice. We quantified these three chemokines using an each single ELISA kit. The induced amount of MIP-1α was greater in sera of infected pregnant mice than in that of infected non-pregnant mice, but its induction was similar in lungs between infected pregnant mice and infected non-pregnant mice (Fig. 3C). The amount of MIP-1α in infected pregnant mice and infected non-pregnant mice was 2.5 and 1.6 μg/ml, respectively (Fig. 3C). The induced amount of MIG was also greater in sera of infected pregnant mice than infected non-pregnant mice, but its induction was similar in lungs in both mice (Fig. 3D). The amount of MIG in sera of infected pregnant mice was 13.1 μg/ml, while that in sera of infected nonpregnant mice was 10.2 μg/ml. The induction of MCP-1 was greater in sera and lungs of infected pregnant mice than in those of infected non-pregnant mice (Fig. 3E). The induced amount of MCP-1 in sera and lungs of infected pregnant mice was 1.2 μg/ml and 1.7 μg/g, respectively, whereas that in sera and lungs of infected non-pregnant mice was 0.9 μg/ml and 1.0 μg/g, respectively (Fig. 3E). Effect of influenza B virus on hormone production in mice We measured two female hormones, progesterone involved in maintaining pregnancy and estrogen which promotes the development of breast and thickens endometrium of uteruses in pregnant and non-pregnant female mice to understand the influence of influenza B virus infections on hormone production in infected pregnant mice on 5 days p.i. Progesterone production in sera was similar between infected pregnant mice and
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2
Nonpregnant and uninfected Nonpregnant and infected Pregnant and uninfected Pregnant and infected *
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1
Nonpregnant and uninfected Nonpregnant and infected Pregnant and uninfected Pregnant and infected
2 1.5 1 0.5 0
Sera
Lungs
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2
Nonpregnant and uninfected Nonpregnant and infected Pregnant and uninfected Pregnant and infected *
MIG concentration (ng/ml or g)
16 0
Chemokines in lungs (O.D. value: 450nm)
*
2.5
*
Nonpregnant and uninfected *
14
Nonpregnant and infected
12
Pregnant and uninfected
10
Pregnant and infected
8 6 4 2 0
Sera
Lungs
1.5
1
0.5
0
2.5 MCP-1 concentration (ng/ml or g)
Chemokines in sera (O.D. value: 450nm)
2.5
MIP-1 concentration (ng/ml or g)
3
2
Nonpregnant and uninfected Nonpregnant and infected Pregnant and uninfected Pregnant and infected
*
*
1.5 1 0.5 0
Sera
Lungs
Fig. 3. Detection of inflammatory chemokines in mice. Sera and lung tissues of influenza B virus-infected pregnant and non-pregnant mice or uninfected pregnant and nonpregnant mice (n¼ 5 per group) were collected on 5 days p.i. Lung tissues were homogenized and suspended in 1 ml of PBS (pH 7.4). A multi-analyte chemokine ELISArray kit was used for screening the inflammatory chemokines in samples of sera (A) and lung tissues (B). Among chemokines, MIP-1α (C), MIG (D) and MCP-1 (E) which were induced greater in infected pregnant mice were quantified using single analyte chemokine ELISArrary kit for MIP-1α, MIG, and MCP-1. Statistical analysis in chemokine inductions were performed between infected pregnant mice and infected non-pregnant mice. nPo 0.05. Data are the mean 7standard error of mean.
uninfected pregnant mice, but its induction was significantly less in uteruses of infected pregnant mice than in uteruses of uninfected pregnant mice (Fig. 4A). The induced amount of progesterone in uteruses of infected pregnant mice was 39.0 pg/g and that in uteruses of uninfected pregnant mice was 65.0 pg/g. The production of progesterone and estrogen in sera and uteruses of between infected and uninfected non-pregnant female mice was similar (Fig. 4A). Estrogen production was less in sera and uteruses of infected pregnant mice than in those of uninfected pregnant mice (Fig. 4B). Estrogen production was similar in sera and uteruses between infected and uninfected non-pregnant mice though the estrogen amount was greater in uteruses than in sera (Fig. 4B). The estrogen amount in sera of uninfected pregnant and infected pregnant mice was 2.5 pg/ml and 1.2 pg/ ml, respectively, and that in uteruses of uninfected pregnant and infected pregnant mice was 5.5 pg/g, and 3.3 pg/g, respectively (Fig. 4B).
Histopathology and antigen expression in lungs of mice Pregnant mice suffered from higher mortality than nonpregnant mice when they were infected with influenza B virus. We stained lung tissues with H&E to find out possible cause of high mortality in infected pregnant mice (Fig. 5A–D). The lung tissue of infected pregnant mice showed severe interstitial pneumonia with lots of infiltrations of inflammatory cells in the alveolar area (Fig. 5D), while that of infected non-pregnant mice had much milder interstitial pneumonia with a few infiltrations of inflammatory cells in the alveolar area (Fig. 5B). The lung tissues of uninfected non-pregnant female (Fig. 5A) and pregnant mice (Fig. 5C) did not show any signs of interstitial pneumonia. We stained lung tissues with anti-influenza B NP antibody to compare the infectivity of influenza B virus between lungs of infected non-pregnant and infected pregnant mice (Fig. 5E–H). Many cells in lung tissue of infected pregnant mice was stained
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Nonpregnant and uninfected
12
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70 60 50 * 40 30 20
Estrogen concentration (ng/ml or g)
Progesterone concentration (ng/ml or g)
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Pregnant and uninfected Pregnant and infected
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*
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10 0
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Uteruses
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Sera
Uteruses
Fig. 4. Measurement of pregnant hormones in mice. Sera and uterus tissues of influenza B virus-infected pregnant and non-pregnant mice or uninfected pregnant and nonpregnant mice (n¼5 per group) were collected on 5 days p.i. Uterus tissues were homogenized and suspended in 1 ml of PBS (pH 7.4). Mouse-specific progesterone ELISA kit was used for detecting progesterone in sera and uterus (A) and mouse-specific estrogen ELISA kit was used for detecting estrogen in sera and uterus (B). Statistical analysis in hormone inductions were performed between infected pregnant mice and uninfected pregnant mice. nP o0.05. Data are the mean 7 standard error of mean.
(Fig. 5H), but a few cells in lung tissue of infected non-pregnant mice were stained (Fig. 5F). Cells in lung tissues of uninfected nonpregnant (Fig. 5E) and pregnant mice (Fig. 5G) were not stained. When we measured the clinical scores in the H&E-stained lung tissues of mice the clinical score was significantly higher in the lung tissue of infected pregnant mice than in that of infected nonpregnant mice (Fig. 5I). The mean clinical score of infected nonpregnant mice was 1.2, while that of infected pregnant mice was 2.7. Antibody titers in the survived mice Sera were bled on 14 days p.i. to determine antibody difference between infected pregnant and non-pregnant mice by hemagglutination-inhibition (HI) assay. HI titers were similar between infected pregnant mice. HI titers of infected pregnant and non-pregnant mice were 90 and 91, respectively (Fig. 6).
Discussion Influenza A virus is known to cause severe disease during pregnancy. Information on pathogenesis of influenza B virus during pregnancy is largely not known. We studied the disease outcome in mice during pregnancy, which were infected with influenza B virus. Influenza B virus caused much more severe disease in pregnant mice than in non-pregnant mice. Infected pregnant mice resulted in about 40% deaths in contrast to infected non-pregnant mice, which did not show any mortality. Infected pregnant mice suffered from severe interstitial pneumonia. The number of neonates in infected pregnant mice is much lower than that in uninfected pregnant mice. We showed that infected pregnant mice died up to 40%, and that no mortality was observed in infected non-pregnant mice, suggesting that influenza B virus may cause deaths in pregnant women. There has been no published report on mortality of pregnant women infected with influenza B virus alone. However, previous studies showed that season influenza viruses could cause severe pathogenesis in women during pregnancy. Pregnant women infected with seasonal influenza viruses such as H1N1, H3N2, and B virus suffered from a fivefold increase in death compared with infected non-pregnant female women with seasonal influenza viruses (Neuzil et al., 1998; Rasmussen et al., 2008). During pandemics caused by influenza A virus, excess mortality
was high (ANZIC Influenza Investigators, 2010; Freema and Barno, 1959). The mortality rate of pregnant women infected with 1918 H1N1 pandemic influenza virus was up to 27% in Minnesota, USA (Harris, 1919), the death rate of pregnant women infected with 1957 H2N2 pandemic influenza virus was about 20% (Freema and Barno, 1959). The study on mortality of pregnant women in Australia or New Zealand during 2009 H1N1 pandemic reported that seven (11%) out of 64 pregnant women who were admitted to an intensive care unit in hospitals died (ANZIC Influenza Investigators, 2010). Our study showed that about 10% of pregnant mice infected with influenza B virus died between 2 and 5 days p.i. It seems that this mortality was due to the infections rather than anesthesia since viruses were detected in the nasal turbinates, tracheas, and lungs of infected pregnant mice on 3 and 5 days p.i., and no mortality was observed in PBS mock-infected pregnant mice. Our data showed that the number of neonates delivered by pregnant mice infected with influenza B virus was lower than that of neonates delivered by uninfected pregnant mice, indicating that influenza B virus may cause adverse effects on fetuses. The study on adverse effects of 2009 H1N1 pandemic influenza virus on fetuses in the United Kingdom showed a threefold increase in preterm birth in pregnant women admitted to the hospital (Yates et al., 2010). In Australia or New Zealand, of 60 births from pregnant women infected with 2009 H1N1 pandemic influenza virus, four were stillbirths, and three neonates died (ANZIC Influenza Investigators, 2010). Pregnant mice infected with influenza B virus suffered from severe interstitial pneumonia with higher viral titers and increased cytokine and chemokine inductions in lungs and sera compared to infected non-pregnant mice. This result indicates that severe pathogenesis in infected pregnant mice may be due to the elevated viral infections and the enhanced inflammatory responses in lungs and sera. Our data showed that cytokines such as IL-2 and IL-12 involved in promoting the cellular immunity rather than in promoting humoral immunity were induced greater in infected pregnant mice, resulting in the increased pathogenesis in infected pregnant mice. Previous studies showed that the increased pathogenesis in humans and animals infected with influenza A viruses may be due to the elevated production of inflammatory cytokines and chemokines (de Jong et al., 2006; Kang et al., 2011; Kim et al., 2012, 2009). Ferrets infected with 2009 H1N1 pandemic influenza virus
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3.5 *
Histopathology Scores for Inflammation
3 2.5 2 1.5 1 0.5 0 Nonpregnant Nonpregnant and uninfected and infected
Pregnant and uninfected
Pregnant and infected
Fig. 5. Histopathological and antigen staining in lung tissues of mice. Lung tissues of influenza B virus-infected pregnant and non-pregnant mice or uninfected pregnant and non-pregnant mice on 5 days p.i. were stained with H&E (A–D) ( 400), and with mouse influenza B virus anti-nucleoprotein antibody, biotin-labeled goat anti-mouse immunoglobulin and red alkaline phosphatase substrate (E–H) ( 1000). (a)H&E-stained alveoli of uninfected non-pregnant mice. (b) H&E-stained alveoli of infected nonpregnant mice. (C) .H&E-stained alveoli of uninfected pregnant mice. (D) H&E-stained alveoli of infected pregnant mice. (E.)Antigen-stained alveoli of uninfected nonpregnant mice. (F.) Antigen-stained alveoli of infected non-pregnant mice. (G.) Antigen-stained alveoli of uninfected pregnant mice and (H) .Antigen-stained alveoli of infected pregnant mice.Histopathology scores for inflammation in the lung tissues. nPo 0.05.
140
Mean of HI titer
120 100 80 60 40 20 0 Nonpregnant and Nonpregnant and uninfected infected
Pregnant and uninfected
Pregnant and infected
Fig. 6. HI titers of sera from the survived mice. Sera were collected from the survived mice and were treated with receptor-destroying enzyme before HI titers were determined with B/Brisbane/60/2008 and turkey red blood cells. Data are the mean 7 standard error of mean.
showed greater pathogenesis with the elevated inductions of inflammatory cytokines and chemokines than those infected with seasonal H1N1 or influenza B virus (Kang et al., 2011; Kim et al., 2009). In pregnant mice, 2009 H1N1 pandemic influenza virus caused more severe disease with greater inductions of inflammatory cytokines and chemokines in lungs than seasonal H1N1
influenza virus (Kim et al., 2012). Fatal outcomes of humans infected with highly pathogenic H5N1 influenza virus were also associated with high productions of inflammatory cytokines and chemokines (de Jong et al., 2006). The discrepancy between our current study and previous studies may be due to the difference of viruses involved. Our current study used influenza B virus rather than influenza A viruses that were used in previous studies. Our study showed that the induction of progesterone involved in maintaining pregnancy in pregnant mice infected with influenza B virus was reduced in uterus compared to uninfected pregnant mice. The reduction of progesterone in uterus in infected pregnant mice may be responsible for the lower birth rates compared to uninfected pregnant mice. In conclusion, our mouse study on influenza B virus indicates that influenza B virus can cause severe diseases including deaths during pregnancy.
Materials and methods Virus and animal Influenza B virus (B/Brisbane/60/2008) was propagated in the amniotic cavities of 10-day-old hens' eggs.
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Out-bred Female ICR mice were bred in animal facility in Chungnam National University. Mice were freely allowed access to feed and water. About 8-week-old mice were mated (one female to one male) for 16 h. We checked the appearance of a vaginal plug as a copulation, which prompted us to designate day 0 of pregnancy. Animal ethics Animal study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory animals of Korean veterinary quarantine and service. The protocol was approved by Committee on the Ethics of Animal Experiments of Chungnam National University. All animal experiments were performed at the enhanced biosafety level 3 (BSL-3 þ) facility approved by Korean government. Observation of mortality and measurement of body weights in infected mice Pregnant mice (day 14 of pregnancy) or the aged matched nonpregnant mice (n ¼10 per group) were intranasally (i.n.) infected with 106 TCID50 of influenza B virus. The mortality in the infected mice were daily recorded until 14 days post infection (p.i.) when delivery of neonates started. We also measured body weights until 14 days p.i. Viral titration in the tissues of infected mice Tissues (1 g per tissue) of nasal turbinates, tracheae, and lungs were collected from infected pregnant mice or infected nonpregnant mice (n ¼5 per group) on 3 and 5 days p.i. The collected tissues were frozen in liquid nitrogen and were homogenized with a mortar and pestle, and the homogenized tissues were suspended in 1 ml of PBS (pH 7.4) supplemented with 2 anti-biotic-antimycotic solution (Sigma, St. Louis, MO). The suspended samples were serially 10-fold diluted in PBS (pH 7.4) and were inoculated into 10-day-old fertilized eggs. The presence of virus in the infected eggs was determined by hemagglutination (HA) assay. Viral titers were determined by log10 EID50/ml. Counting the delivered neonates by pregnant mice The number of neonates delivered by infected or uninfected pregnant mice (n ¼5) was counted on 10 days p.i., and the mean number of neonates was calculated. Detection of inflammatory cytokines and chemokines in mice The infected pregnant and non-pregnant mice or uninfected pregnant and non-pregnant mice (n ¼5 per group) were euthanized with high doses of anesthetics, Zoletil on 5 days p.i. and their lungs were collected. The collected lungs were frozen in liquid nitrogen and were homogenized with a mortar and pestle. The homogenized lung tissues were suspended in 1 ml of PBS (pH 7.4) and were spun down by a centrifuge (800 g, 10 min). The collected supernatants and sera were used for measuring the induced amount of cytokines and chemokines. Cytokines and chemokines were first screened using a multi-analyte ELISArray kit (SAbioscience, Valencia, CA, USA) and then the differentially expressed cytokines and chemokines between pregnant and non-pregnant female mice were further quantified by singleanalyte ELISA kits. (SAbioscience, Valencia, CA, USA). The reaction was performed as described by the manufacturer. The absorbance of reaction was read at 450 nm by an ELISA plate reader (Tecan, Männedorf, Switzerland). The amount of the individual cytokine in
the single assay was determined based on the standard curve of each cytokine.
Detection of pregnant hormones in mice Mouse-specific estrogen and progesterone ELISA kits (MyBioSource, San Diego, CA USA) were used to measure hormone modulation in infected pregnant and non-pregnant mice or uninfected pregnant and non-pregnant mice (n ¼5 per group). Sera and uterus tissue samples (1 g per group) were collected from the infected mice on 5 days p.i. and from the uninfected mice. Uterus tissues were frozen in liquid nitrogen and were homogenized with a mortar and pestle. The homogenized uterus tissues were suspended in PBS (pH.7.4) (1 g/ml) and were centrifuged to remove the debris (1000 g, 10 min). Optical density (O.D.) in the reacted samples was read at 450 nm using a microtiter plate. The amount of the individual hormone was determined based on the standard curve of each hormone.
Histopathology and antigen detection in lung tissues of mice Infected pregnant and non-pregnant mice or uninfected pregnant and female mice were euthanized on 5 days p.i. The lung tissues were collected and were fixed by submerging in the 10% neutral buffered formalin and were embedded in paraffin. Fivemicrometer sections were cut, and stained with hematoxylin and eosin (H&E) as described (Bancrof and Stevens, 1996). The stained tissues were evaluated under an Olympus DP70 microscope (Olympus Corporation, Tokyo, Japan). Five-micrometer sections were stained with mouse influenza B virus anti-nucleoprotein antibody (Serotech, United Kingdom). Sections were fixed with 100% chilled acetone, and endogenous peroxidase activity in the sections was blocked by 3% H2O2. The blocked tissue sections were labeled with mouse influenza B virus anti-nucleoprotein antibody (1:1000 dilution) and then with biotin-labeled goat anti-mouse immunoglobulin (Vector, USA), VECTASTAIN ABC-AP (Vector, USA), and Vector red alkaline phosphatase substrate (Vector, USA). The stained tissue sections were counterstained with hematoxylin QS (Vector Laboratories, Burlingame, CA). The stained sections were evaluated under an Olympus DP70 microscope (Olympus Corporation, Tokyo, Japan).
Histopathology scores for inflammation in lung legions of mice The five sites in the H&E-stained lung tissues were randomly selected for measuring histopathology scores for inflammation in order to determine histopathology scores in the lung lesions of mice. The scores were designated based on the following criteria: 0, no inflammation; 1, mild, inflammatory cell infiltrates in the perivascular and peribronchiolar compartment; 2, moderate, inflammatory cell infiltrates in the perivascular and peribronchiolar space with modest extension into the alveolar parenchyma; and 3, severe, inflammatory cell infiltrates in the perivascular and peribronchiolar space with a greater magnitude of inflammatory foci in the alveolar parenchyma.
Statistical analysis Groups of data were analyzed by repeated measures ANOVA using pairing of samples with IBMs SPSSs Statistics version 20. A P-value o 0.05 was considered to be statistically significant.
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Acknowledgments This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2012R1A2A2A01002533). References ANZIC Influenza Investigators, 2010. Critical illness due to 2009A/H1N1 influenza in pregnant and postpartum women: population based cohort study. British Medical Journal 340, c1279. Bancrof, J.D., Stevens, A., 1996. Theory and Practice of Histological Techniques, fourth ed. Churchill Living stone, NewYork. Bouvier, N.M, Palese, P., 2008. The biology of influenza viruses. Vaccine 26 (Suppl. 4), D49–D53. Cantu, J., Tita, A.T., 2013. Management of influenza in pregnancy. American Journal of Perinatology 30 (2), 99–103. de Jong, M.D., Simmons, C.P., Thanh, T.T., Hien, V.M., Smith, G.J., Chau, T.N., Hoang, D.M., Chau, N.V., Khanh, T.H., Dong, V.C., Qui, P.T., Cam, B.V., Ha do, Q., Guan, Y., Peiris, J.S., Chinh, N.T., Hien, T.T., Farrar, J., 2006. Fatal outcome of human influenza A (H5N1) is associated with high viral load and hypercytokinemia. Nature Medicine 12 (10), 1203–1207. Fouchier, R.A., Munster, V., Wallensten, A., Bestebroer, T.M., Herfst, S., Smith, D., Rimmelzwaan, G.F., Olsen, B., Osterhaus, A.D., 2005. Characterization of a novel influenza A virus hemagglutinin subtype (H16) obtained from black-headed gulls. Journal of Virology 79, 2814–2822. Francis Jr., T., 1940. A new type of virus from epidemic influenza. Science 92 (2392), 405–408. Freema, D.W., Barno, A., 1959. Deaths from Asian influenza associated with pregnancy. American Journal of Obstetrics Gynecology 78, 1172–1175. Glezen, W.P., Decker, M., Joseph, S.W., Mercready Jr., R.G., 1987. Acute respiratory disease associated with influenza epidemics in Houston, 1981–1983. Journal of Infectious Diseases 155 (6), 1119–1126. Glezen, W.P., Schmier, J.K., Kuehn, C.M., Ryan, K.J., Oxford, J., 2013. The burden of influenza B: a structured literature review. American Journal of Public Health 103 (3), e43–e51. Harris, J.W., 1919. Influenza occurring in pregnant women:statistical study of thirteen hundred and fifty cases. Journal of the American Medical Association 72, 978–980.
81
Jackson, D., Elderfield, R.A., Barclay, W.S., 2011. Molecular studies of influenza B virus in the reverse genetics era. Journal of General Virology 92 (Pt. 1), 1–17. Kang, Y.M., Song, B.M., Lee, J.S., Kim, H.S., Seo, S.H., 2011. Pandemic H1N1 influenza virus causes a stronger inflammatory response than seasonal H1N1 influenza virus in ferrets. Archives of Virology 156 (5), 759–767. Kim, H.M., Kang, Y.M., Song, B.M., Kim, H.S., Seo, S.H., 2012. The 2009 pandemic H1N1 influenza virus is more pathogenic in pregnant mice than seasonal H1N1 influenza virus. Viral Immunology 25 (5), 402–410. Kim, Y.H., Kim, H.S., Cho, S.H., Seo, S.H., 2009. Influenza B virus causes milder pathogenesis and weaker inflammatory responses in ferrets than influenza A virus. Viral Immunology 22 (6), 423–430. Li, W.C., Shih, S.R., Huang, Y.C., Chen, G.W., Chang, S.C., Hsiao, M.J., Tsao, K.C., Lin, T. Y., 2008. Clinical and genetic characterization of severe influenza B-associated diseases during an outbreak in Taiwan. Journal of Clinical Virology 42 (1), 45–51. Neuzil, K.M., Reed, G.W., Mitchel, E.F., Simonsen, L., Griffin, M.R., 1998. Impact of influenza on acute cardiopulmonary hospitalizations in pregnant women. American Journal of Epidemiology 148 (11), 1094–1102. Newland, J.G., Romero, J.R., Varman, M., Drake, C., Holst, A., Safranek, T., Subbarao, K., 2003. Encephalitis associated with influenza B virus infection in 2 children and a review of the literature. Clinical Infectious Diseases 36 (7), e87–e95. Rasmussen, S.A., Jamieson, D.J., Bresee, J.S., 2008. Pandemic influenza and pregnant women. Emerging Infectious Diseases 14 (1), 95–100. Rota, P.A., Wallis, T.R., Harmon, M.W., Rota, J.S., Kendal, A.P., Nerome, K., 1990. Cocirculation of two distinct evolutionary lineages of influenza type B virus since 1983. Virology 175 (1), 59–68. Tong, S., Li, Y., Rivailler, P., Conrardy, C., Castillo, D.A., Chen, L.M, Recuenco, S., Ellison, J.A., Davis, C.T., York, I.A., Turmelle, A.S., Moran, D., Rogers, S., Shi, M., Tao, Y., Weil, M.R., Tang, K., Rowe, L.A., Sammons, S., Xu, X., Frace, M., Lindblade, K.A., Cox, N.J., Anderson, L.J., Rupprecht, C.E., Donis, R.O., 2012. A distinct lineage of influenza A virus from bats. Proceedings of the National Academy of Sciences of the United States of America 109 (11), 4269–4274. Webster, R.G., Bean, W.J., Gorman, O.T., Chambers, T.M., Kawaoka, Y., 1992. Evolution and ecology of influenza A viruses. Microbiological Reviews 56, 152–179. Yates, L., Pierce, M., Stephens, S., Mill, A.C., Spark, P., Kurinczuk, J.J., Valappil, M., Brocklehurst, P., Thomas, S.H., Knight, M., 2010. Influenza A/H1N1v in pregnancy: an investigation of the characteristics and management of affected women and the relationship to pregnancy outcomes for mother and infant. Health Technology Assessment 14 (34), 109–182.
Contents lists available at ScienceDirect
Virology journal homepage: www.elsevier.com/locate/yviro
Severe pathogenesis of influenza B virus in pregnant mice Jeong Cheol Kim a,b,1, Heui Man Kim a,b,1, Young Myong Kang a,b, Keun Bon Ku a,b, Eun Hye Park a,b, Jung Yum a,b, Ji An Kim a,b, Yoo Kyung Kang a, Joo Sub Lee a,b, Hyun Soo Kim c, Sang Heui Seo a,b,n a
Laboratory of Influenza Research, College of Veterinary Medicine, Chungnam National University, Daejeon 305-764, Republic of Korea Institute for Influenza Virus, College of Veterinary Medicine, Chungnam National University, Daejeon 305-764, Republic of Korea c Laboratory of Public Health, College of Veterinary Medicine, Chungnam National University, Daejeon 305-764, Republic of Korea b
art ic l e i nf o
a b s t r a c t
Article history: Received 23 August 2013 Returned to author for revisions 9 September 2013 Accepted 1 October 2013 Available online 20 October 2013
The study on pathogenesis of influenza B virus during pregnancy is limited. Here, we showed using a mouse model that influenza B virus could cause severe disease including death during pregnancy. Infected pregnant mice resulted in 40% mortality, but infected age-matched non-pregnant mice did not show any death. Infected pregnant mice contained high viral loads in lungs with the elevated inductions of inflammatory cytokines and chemokines than infected non-pregnant mice. Infected pregnant mice delivered lower number of neonates than uninfected pregnant mice, suggesting adverse effects of influenza B virus on fetuses. Progesterone which is important for maintaining pregnancy was reduced in uteruses of infected pregnant mice than in those of uninfected pregnant mice. Taken together, our results suggest that influenza B virus can cause severe disease during pregnancy, and that preventive measures including vaccination may be important for protecting women during pregnancy. & 2013 Elsevier Inc. All rights reserved.
Keywords: Pregnancy Influenza B virus Pneumonia Hormone Cytokine Chemokine
Introduction Influenza virus belongs to the family, Orthomyxoviridae, and is classified into three types, A, B, and C (Bouvier and Palese, 2008) and contains negative-sense RNA genome. Influenza A and B viruses contain eight viral RNA segments, and influenza C virus contains seven viral RNA segments (Bouvier and Palese, 2008). Influenza A virus is further divided into subtypes based on hemagglutinin (HA) and neuraminidase (NA). There are 17 subtypes of HA and nine subtypes of NA in influenza A viruses (Bouvier and Palese, 2008; Fouchier et al., 2005; Tong et al., 2012). Influenza A virus has a broad spectrum of host ranges including birds, pigs, horses, and humans. Influenza B and C virus are circulating in humans (Webster et al., 1992). Influenza C virus causes much milder disease in humans than influenza A and B viruses (Rasmussen et al., 2008). By morphology, influenza A and B viruses are not distinguishable, and are spherical or filamentous in shape with host cellderived lipid layers (Bouvier and Palese, 2008). The subtypes of influenza A viruses currently circulating in humans are H1N1 and H3N2. Influenza B virus is not divided into
n Correspondence to: Laboratory of Influenza Research, College of Veterinary Medicine, Chungnam National University, 220 Gung Dong, Yuseong Gu, Daejeon 305-764, Republic of Korea. Fax: þ 82 42 821 6762. E-mail address: [email protected] (S.H. Seo). 1 These authors equally contributed to this work.
0042-6822/$ - see front matter & 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.virol.2013.10.001
subtypes and has two genetically distinct lineages circulating in humans, which are B/Vitoria/2/87-like (Victoria lineage) and B/Yamagata/16/88-like (Yamagata lineage) (Rota et al., 1990). The first influenza A virus was discovered in humans in 1933, and influenza B virus was first found in humans in 1940 (Francis, 1940). Epidemics by influenza B virus could occur at intervals of 2–4 years and the patients infected with influenza B virus showed well-defined discrete illness in all ages of humans (Glezen et al., 1987). Until now, research has been done more on influenza A virus than on influenza B virus (Jackson et al., 2011). The current popular concept is that influenza B virus is less pathogenic to humans than influenza A virus (Rasmussen et al., 2008). However, influenza B virus causes the substantial disease in humans. Among US pediatric deaths by influenza infections between 2004 and 2011, 22–44% of deaths were related to influenza B virus (Glezen et al., 1987). In the United Kingdom, influenza B virus was dominantly circulated in the 2010–2011 season (Glezen et al., 2013), 40 out of 607 deaths associated with influenza virus infections were related to influenza B virus. In Japan and Taiwan, encephalitis in patients infected with influenza B virus was reported (Li et al., 2008; Newland et al., 2003). Pregnant woman is more susceptible to influenza virus infections than non-pregnant humans (Cantu and Tita, 2013). The study on influenza A virus infections in women under pregnancy during 2009 H1N1 pandemic reported the sevenfold increase in the risk of admission to the intensive care unit, the increase in 69% of
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between two was 27.5%. Uninfected non-pregnant mice gained 10.7% and infected non-pregnant mice lost 17.0%. The difference between two was 27.7% (Fig. 1A). Mortality rate of infected pregnant mice was 40% until 7 days p.i. when neonates were born, whereas no morality of infected non-pregnant mice occurred (Fig. 1B). We compared the mean number of neonates delivered by pregnant mice on 10 days p.i. (Fig. 1C). The mean number of neonates delivered by infected pregnant mice was 7.7, and that of neonates delivered by uninfected pregnant mice was 13.8. (Fig. 1C). We measured viral titers in the tissues of mice on 3 and 5 days p.i. Viral titers in nasal turbinates, tracheae, and lungs were higher in infected pregnant mice than in infected nonpregnant mice (Fig. 1D). On 5 days p.i. the mean viral titer in nasal turbinates, tracheae, and lungs of infected pregnant mice was 4.0 EID50/g, 2.5 EID50/g, and 5.0 EID50/g, respectively, and that in nasal turbinates, tracheae, and lungs of infected non-pregnant mice was 2.76 EID50/g, 1.33 EID50/g, and 4.0 EID50/g, respectively (Fig. 1D). No virus was detected in brains, uteruses, and fetuses.
requirement of mechanical ventilation, and 11% of mortality (ANZIC Influenza Investigators, 2010). To date, information on pathogenesis during pregnancy by influenza B virus infection is limited. In this study, using a mouse model we wanted to address whether influenza B virus could cause more severe disease to pregnant mice than non-pregnant female mice, and to find out the underlying mechanism of disease severity of pregnant mice infected with influenza B virus.
Results Clinical signs and viral titers in mice infected with influenza B virus Pregnant mice on day 14 pregnancy and age-matched nonpregnant mice were i.n. infected with influenza B virus to find out the effect of pregnancy on pathogenesis. The change of body weights was similar between infected pregnant mice and infected non-pregnant mice, but the mortality rate is greater in infected pregnant mice than infected non-pregnant mice (Fig. 1). The change of body weights in infected mice were measured based on the weights of mice before infection (Fig. 1A). On 7 days p.i. when the difference of body-weight change is the highest between infected and uninfected mice, uninfected pregnant mice gained 50.9%, while infected pregnant mice gained 23.4%. The difference
Inflammatory responses in sera and lungs of mice infected with influenza B virus To understand the possible cause of severe pathogenesis in pregnant mice infected with influenza B virus, we measured cytokines and chemokines in sera and lungs of mice on 5 days
100
60 Nonpregnant and uninfected 50
Nonpregnant and uninfected
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Nonpregnant and infected
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Body weight change (%)
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-30
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8
9 10 11 12 13 14
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6 Nonpregnant and uninfected
Mean number of neonate
14 12 10 *
8 6 4
Viral titers in tissue (log10EID50/g)
16
*
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5
Pregnant and uninfected
*
Pregnant and infected
4
3
*
2
1
2 0
Pregnant and uninfected
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0 3dpi.
5dpi.
7dpi.
Nasal turbinates
9dpi.
3dpi.
5dpi.
7dpi.
Tracheas
9dpi.
3dpi.
5dpi.
7dpi.
9dpi.
Lungs
Fig. 1. Body-weight change, mortality, viral titers in tissues, and number of neonates of pregnant mice. Pregnant mice (day 14 of pregnancy) or non-pregnant mice (n¼10 per group) were i.n. infected with influenza B virus. Uninfected pregnant and non-pregnant mice were used for controls. The change of body weights (A) was recorded until 14 days p.i., mortality (B) were recorded for 14 days p.i., and the mean number of neonates from pregnant mice (n¼5 per group) (C) were recorded on 10 days p.i., The viral titers (D) in the nasal turbinates, tracheae, and lung tissues from infected pregnant and non-pregnant mice (n¼5 per group) on 3 and 5 days p.i. were measured by log10 EID50/ml. No virus was detected in brains, uteruses, and fetuses. ↓, delivery day. Statistical analysis in neonate number was performed between infected pregnant mice and uninfected pregnant mice. Statistical analysis in viral titers in tissues were performed between infected pregnant mice and infected non-pregnant female mice. nPo0.05. Data are the mean7standard error of mean.
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2.5
Nonpregnant and uninfected Cytokines in lungs (O.D. value: 450nm)
Cytokines in sera (O.D. value: 450nm)
2.5
Nonpregnant and infected 2
Pregnant and uninfected Pregnant and infected
1.5
1
0.5
0
Pregnant and infected 1.5 *
*
1
0.5
4.5
*
Nonpregnant and infected
IL-12 concentration (ng/ml or g)
IL-2 concentration (ng/ml or g)
Pregnant and uninfected
5 Nonpregnant and uninfected
4
3
Nonpregnant and infected 2
0
4.5
3.5
Nonpregnant and uninfected
Pregnant and uninfected Pregnant and infected
2.5 2 1.5 1 0.5
4
* Nonpregnant and uninfected Nonpregnant and infected Pregnant and uninfected
3.5
Pregnant and infected
3 2.5 2 1.5 1 0.5
0 Sera
Lungs
0 Sera
Lungs
Fig. 2. Detection of inflammatory cytokines in mice. Sera and lung tissues of influenza B virus-infected pregnant and non-pregnant mice or uninfected pregnant and nonpregnant mice (n ¼5 per group) were collected on 5 days p.i. Lung tissues were homogenized and suspended in 1 ml of PBS (pH 7.4). A multi-analyte cytokine ELISArray kit was used for screening the inflammatory cytokines in samples of sera (A) and lung tissues (B). Among cytokines, IL-2 and IL-12 which were induced greater in infected mice was quantified using single analyte cytokine ELISArrary kit for IL-2 (C) and IL-12 (D). Statistical analysis in cytokine inductions were performed between infected pregnant mice and infected non-pregnant mice. nPo 0.05. Data are the mean 7 standard error of mean.
p.i. (Figs. 2 and 3). When we screened the induced cytokines in sera and lungs of mice with mutlti-analyte cytokine ELISA kit (Fig. 2A and B), the induction of cytokines which were measured (IL-1α, IL1β, IL-2, IL-4, IL-6, IL-10, IL-12, IL-17α, IFN-γ, TNF-α, G-CSF, GM-CSF) in sera was similar (Fig. 2A), but IL-2 involved in proliferation, and differentiation of T cells and IL-12 stimulating natural killer cells and T lymphocytes were induced greater in lungs of infected pregnant mice than in those of infected non-pregnant mice (Fig. 2B). We quantified IL-2 (Fig. 2C) and IL-12 (Fig. 2D) using a single ELISA kit in sera and lungs of mice. Their inductions in sera were similar between infected pregnant mice and infected nonpregnant mice, but it was induced greater in lungs of infected pregnant mice than in those of infected non-pregnant mice (Fig. 2C and D). The induced amount of IL-2 in the lungs of infected pregnant mice was 3.623 ng/g, and that in the lungs of infected non-pregnant mice was 2.432 ng/g (Fig. 2C). In lungs, the induced amount of IL-12 in infected pregnant mice was 4.532 ng/g, while that of IL-12 in the infected non-pregnant mice was 3.731 ng/g. We measured chemokines involved in attracting inflammatory cells in sera and lungs of mice infected with influenza B virus. We first screened chemokines in sera and lungs from infected mice with mutlti-analyte chemokine ELISA kit which can detect RANTES, MCP-1, MIP-1α, MIP-1β, SDF-1, IP-10, MIG, Eotaxin, TARC, MDC, KC, 6Ckine). MIP-1α (macrophage inflammatory protein-1α) involved in recruiting polymorphonuclear leukocytes, MIG (monokine induced by gamma interferon) which is T-cell chemo-attractant, and MCP-1 (monocyte chemotactic protein-1) which recruits monocytes, memory T cells, and dendritic cells to the sites of infection were induced greater in sera or lungs of infected
pregnant mice than in those of infected non-pregnant mice. We quantified these three chemokines using an each single ELISA kit. The induced amount of MIP-1α was greater in sera of infected pregnant mice than in that of infected non-pregnant mice, but its induction was similar in lungs between infected pregnant mice and infected non-pregnant mice (Fig. 3C). The amount of MIP-1α in infected pregnant mice and infected non-pregnant mice was 2.5 and 1.6 μg/ml, respectively (Fig. 3C). The induced amount of MIG was also greater in sera of infected pregnant mice than infected non-pregnant mice, but its induction was similar in lungs in both mice (Fig. 3D). The amount of MIG in sera of infected pregnant mice was 13.1 μg/ml, while that in sera of infected nonpregnant mice was 10.2 μg/ml. The induction of MCP-1 was greater in sera and lungs of infected pregnant mice than in those of infected non-pregnant mice (Fig. 3E). The induced amount of MCP-1 in sera and lungs of infected pregnant mice was 1.2 μg/ml and 1.7 μg/g, respectively, whereas that in sera and lungs of infected non-pregnant mice was 0.9 μg/ml and 1.0 μg/g, respectively (Fig. 3E). Effect of influenza B virus on hormone production in mice We measured two female hormones, progesterone involved in maintaining pregnancy and estrogen which promotes the development of breast and thickens endometrium of uteruses in pregnant and non-pregnant female mice to understand the influence of influenza B virus infections on hormone production in infected pregnant mice on 5 days p.i. Progesterone production in sera was similar between infected pregnant mice and
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2
Nonpregnant and uninfected Nonpregnant and infected Pregnant and uninfected Pregnant and infected *
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MIG concentration (ng/ml or g)
16 0
Chemokines in lungs (O.D. value: 450nm)
*
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*
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12
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10
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8 6 4 2 0
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1
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Chemokines in sera (O.D. value: 450nm)
2.5
MIP-1 concentration (ng/ml or g)
3
2
Nonpregnant and uninfected Nonpregnant and infected Pregnant and uninfected Pregnant and infected
*
*
1.5 1 0.5 0
Sera
Lungs
Fig. 3. Detection of inflammatory chemokines in mice. Sera and lung tissues of influenza B virus-infected pregnant and non-pregnant mice or uninfected pregnant and nonpregnant mice (n¼ 5 per group) were collected on 5 days p.i. Lung tissues were homogenized and suspended in 1 ml of PBS (pH 7.4). A multi-analyte chemokine ELISArray kit was used for screening the inflammatory chemokines in samples of sera (A) and lung tissues (B). Among chemokines, MIP-1α (C), MIG (D) and MCP-1 (E) which were induced greater in infected pregnant mice were quantified using single analyte chemokine ELISArrary kit for MIP-1α, MIG, and MCP-1. Statistical analysis in chemokine inductions were performed between infected pregnant mice and infected non-pregnant mice. nPo 0.05. Data are the mean 7standard error of mean.
uninfected pregnant mice, but its induction was significantly less in uteruses of infected pregnant mice than in uteruses of uninfected pregnant mice (Fig. 4A). The induced amount of progesterone in uteruses of infected pregnant mice was 39.0 pg/g and that in uteruses of uninfected pregnant mice was 65.0 pg/g. The production of progesterone and estrogen in sera and uteruses of between infected and uninfected non-pregnant female mice was similar (Fig. 4A). Estrogen production was less in sera and uteruses of infected pregnant mice than in those of uninfected pregnant mice (Fig. 4B). Estrogen production was similar in sera and uteruses between infected and uninfected non-pregnant mice though the estrogen amount was greater in uteruses than in sera (Fig. 4B). The estrogen amount in sera of uninfected pregnant and infected pregnant mice was 2.5 pg/ml and 1.2 pg/ ml, respectively, and that in uteruses of uninfected pregnant and infected pregnant mice was 5.5 pg/g, and 3.3 pg/g, respectively (Fig. 4B).
Histopathology and antigen expression in lungs of mice Pregnant mice suffered from higher mortality than nonpregnant mice when they were infected with influenza B virus. We stained lung tissues with H&E to find out possible cause of high mortality in infected pregnant mice (Fig. 5A–D). The lung tissue of infected pregnant mice showed severe interstitial pneumonia with lots of infiltrations of inflammatory cells in the alveolar area (Fig. 5D), while that of infected non-pregnant mice had much milder interstitial pneumonia with a few infiltrations of inflammatory cells in the alveolar area (Fig. 5B). The lung tissues of uninfected non-pregnant female (Fig. 5A) and pregnant mice (Fig. 5C) did not show any signs of interstitial pneumonia. We stained lung tissues with anti-influenza B NP antibody to compare the infectivity of influenza B virus between lungs of infected non-pregnant and infected pregnant mice (Fig. 5E–H). Many cells in lung tissue of infected pregnant mice was stained
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Nonpregnant and uninfected
12
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70 60 50 * 40 30 20
Estrogen concentration (ng/ml or g)
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100
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Pregnant and uninfected Pregnant and infected
8
6
4
*
2
10 0
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Uteruses
0
Sera
Uteruses
Fig. 4. Measurement of pregnant hormones in mice. Sera and uterus tissues of influenza B virus-infected pregnant and non-pregnant mice or uninfected pregnant and nonpregnant mice (n¼5 per group) were collected on 5 days p.i. Uterus tissues were homogenized and suspended in 1 ml of PBS (pH 7.4). Mouse-specific progesterone ELISA kit was used for detecting progesterone in sera and uterus (A) and mouse-specific estrogen ELISA kit was used for detecting estrogen in sera and uterus (B). Statistical analysis in hormone inductions were performed between infected pregnant mice and uninfected pregnant mice. nP o0.05. Data are the mean 7 standard error of mean.
(Fig. 5H), but a few cells in lung tissue of infected non-pregnant mice were stained (Fig. 5F). Cells in lung tissues of uninfected nonpregnant (Fig. 5E) and pregnant mice (Fig. 5G) were not stained. When we measured the clinical scores in the H&E-stained lung tissues of mice the clinical score was significantly higher in the lung tissue of infected pregnant mice than in that of infected nonpregnant mice (Fig. 5I). The mean clinical score of infected nonpregnant mice was 1.2, while that of infected pregnant mice was 2.7. Antibody titers in the survived mice Sera were bled on 14 days p.i. to determine antibody difference between infected pregnant and non-pregnant mice by hemagglutination-inhibition (HI) assay. HI titers were similar between infected pregnant mice. HI titers of infected pregnant and non-pregnant mice were 90 and 91, respectively (Fig. 6).
Discussion Influenza A virus is known to cause severe disease during pregnancy. Information on pathogenesis of influenza B virus during pregnancy is largely not known. We studied the disease outcome in mice during pregnancy, which were infected with influenza B virus. Influenza B virus caused much more severe disease in pregnant mice than in non-pregnant mice. Infected pregnant mice resulted in about 40% deaths in contrast to infected non-pregnant mice, which did not show any mortality. Infected pregnant mice suffered from severe interstitial pneumonia. The number of neonates in infected pregnant mice is much lower than that in uninfected pregnant mice. We showed that infected pregnant mice died up to 40%, and that no mortality was observed in infected non-pregnant mice, suggesting that influenza B virus may cause deaths in pregnant women. There has been no published report on mortality of pregnant women infected with influenza B virus alone. However, previous studies showed that season influenza viruses could cause severe pathogenesis in women during pregnancy. Pregnant women infected with seasonal influenza viruses such as H1N1, H3N2, and B virus suffered from a fivefold increase in death compared with infected non-pregnant female women with seasonal influenza viruses (Neuzil et al., 1998; Rasmussen et al., 2008). During pandemics caused by influenza A virus, excess mortality
was high (ANZIC Influenza Investigators, 2010; Freema and Barno, 1959). The mortality rate of pregnant women infected with 1918 H1N1 pandemic influenza virus was up to 27% in Minnesota, USA (Harris, 1919), the death rate of pregnant women infected with 1957 H2N2 pandemic influenza virus was about 20% (Freema and Barno, 1959). The study on mortality of pregnant women in Australia or New Zealand during 2009 H1N1 pandemic reported that seven (11%) out of 64 pregnant women who were admitted to an intensive care unit in hospitals died (ANZIC Influenza Investigators, 2010). Our study showed that about 10% of pregnant mice infected with influenza B virus died between 2 and 5 days p.i. It seems that this mortality was due to the infections rather than anesthesia since viruses were detected in the nasal turbinates, tracheas, and lungs of infected pregnant mice on 3 and 5 days p.i., and no mortality was observed in PBS mock-infected pregnant mice. Our data showed that the number of neonates delivered by pregnant mice infected with influenza B virus was lower than that of neonates delivered by uninfected pregnant mice, indicating that influenza B virus may cause adverse effects on fetuses. The study on adverse effects of 2009 H1N1 pandemic influenza virus on fetuses in the United Kingdom showed a threefold increase in preterm birth in pregnant women admitted to the hospital (Yates et al., 2010). In Australia or New Zealand, of 60 births from pregnant women infected with 2009 H1N1 pandemic influenza virus, four were stillbirths, and three neonates died (ANZIC Influenza Investigators, 2010). Pregnant mice infected with influenza B virus suffered from severe interstitial pneumonia with higher viral titers and increased cytokine and chemokine inductions in lungs and sera compared to infected non-pregnant mice. This result indicates that severe pathogenesis in infected pregnant mice may be due to the elevated viral infections and the enhanced inflammatory responses in lungs and sera. Our data showed that cytokines such as IL-2 and IL-12 involved in promoting the cellular immunity rather than in promoting humoral immunity were induced greater in infected pregnant mice, resulting in the increased pathogenesis in infected pregnant mice. Previous studies showed that the increased pathogenesis in humans and animals infected with influenza A viruses may be due to the elevated production of inflammatory cytokines and chemokines (de Jong et al., 2006; Kang et al., 2011; Kim et al., 2012, 2009). Ferrets infected with 2009 H1N1 pandemic influenza virus
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3.5 *
Histopathology Scores for Inflammation
3 2.5 2 1.5 1 0.5 0 Nonpregnant Nonpregnant and uninfected and infected
Pregnant and uninfected
Pregnant and infected
Fig. 5. Histopathological and antigen staining in lung tissues of mice. Lung tissues of influenza B virus-infected pregnant and non-pregnant mice or uninfected pregnant and non-pregnant mice on 5 days p.i. were stained with H&E (A–D) ( 400), and with mouse influenza B virus anti-nucleoprotein antibody, biotin-labeled goat anti-mouse immunoglobulin and red alkaline phosphatase substrate (E–H) ( 1000). (a)H&E-stained alveoli of uninfected non-pregnant mice. (b) H&E-stained alveoli of infected nonpregnant mice. (C) .H&E-stained alveoli of uninfected pregnant mice. (D) H&E-stained alveoli of infected pregnant mice. (E.)Antigen-stained alveoli of uninfected nonpregnant mice. (F.) Antigen-stained alveoli of infected non-pregnant mice. (G.) Antigen-stained alveoli of uninfected pregnant mice and (H) .Antigen-stained alveoli of infected pregnant mice.Histopathology scores for inflammation in the lung tissues. nPo 0.05.
140
Mean of HI titer
120 100 80 60 40 20 0 Nonpregnant and Nonpregnant and uninfected infected
Pregnant and uninfected
Pregnant and infected
Fig. 6. HI titers of sera from the survived mice. Sera were collected from the survived mice and were treated with receptor-destroying enzyme before HI titers were determined with B/Brisbane/60/2008 and turkey red blood cells. Data are the mean 7 standard error of mean.
showed greater pathogenesis with the elevated inductions of inflammatory cytokines and chemokines than those infected with seasonal H1N1 or influenza B virus (Kang et al., 2011; Kim et al., 2009). In pregnant mice, 2009 H1N1 pandemic influenza virus caused more severe disease with greater inductions of inflammatory cytokines and chemokines in lungs than seasonal H1N1
influenza virus (Kim et al., 2012). Fatal outcomes of humans infected with highly pathogenic H5N1 influenza virus were also associated with high productions of inflammatory cytokines and chemokines (de Jong et al., 2006). The discrepancy between our current study and previous studies may be due to the difference of viruses involved. Our current study used influenza B virus rather than influenza A viruses that were used in previous studies. Our study showed that the induction of progesterone involved in maintaining pregnancy in pregnant mice infected with influenza B virus was reduced in uterus compared to uninfected pregnant mice. The reduction of progesterone in uterus in infected pregnant mice may be responsible for the lower birth rates compared to uninfected pregnant mice. In conclusion, our mouse study on influenza B virus indicates that influenza B virus can cause severe diseases including deaths during pregnancy.
Materials and methods Virus and animal Influenza B virus (B/Brisbane/60/2008) was propagated in the amniotic cavities of 10-day-old hens' eggs.
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Out-bred Female ICR mice were bred in animal facility in Chungnam National University. Mice were freely allowed access to feed and water. About 8-week-old mice were mated (one female to one male) for 16 h. We checked the appearance of a vaginal plug as a copulation, which prompted us to designate day 0 of pregnancy. Animal ethics Animal study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory animals of Korean veterinary quarantine and service. The protocol was approved by Committee on the Ethics of Animal Experiments of Chungnam National University. All animal experiments were performed at the enhanced biosafety level 3 (BSL-3 þ) facility approved by Korean government. Observation of mortality and measurement of body weights in infected mice Pregnant mice (day 14 of pregnancy) or the aged matched nonpregnant mice (n ¼10 per group) were intranasally (i.n.) infected with 106 TCID50 of influenza B virus. The mortality in the infected mice were daily recorded until 14 days post infection (p.i.) when delivery of neonates started. We also measured body weights until 14 days p.i. Viral titration in the tissues of infected mice Tissues (1 g per tissue) of nasal turbinates, tracheae, and lungs were collected from infected pregnant mice or infected nonpregnant mice (n ¼5 per group) on 3 and 5 days p.i. The collected tissues were frozen in liquid nitrogen and were homogenized with a mortar and pestle, and the homogenized tissues were suspended in 1 ml of PBS (pH 7.4) supplemented with 2 anti-biotic-antimycotic solution (Sigma, St. Louis, MO). The suspended samples were serially 10-fold diluted in PBS (pH 7.4) and were inoculated into 10-day-old fertilized eggs. The presence of virus in the infected eggs was determined by hemagglutination (HA) assay. Viral titers were determined by log10 EID50/ml. Counting the delivered neonates by pregnant mice The number of neonates delivered by infected or uninfected pregnant mice (n ¼5) was counted on 10 days p.i., and the mean number of neonates was calculated. Detection of inflammatory cytokines and chemokines in mice The infected pregnant and non-pregnant mice or uninfected pregnant and non-pregnant mice (n ¼5 per group) were euthanized with high doses of anesthetics, Zoletil on 5 days p.i. and their lungs were collected. The collected lungs were frozen in liquid nitrogen and were homogenized with a mortar and pestle. The homogenized lung tissues were suspended in 1 ml of PBS (pH 7.4) and were spun down by a centrifuge (800 g, 10 min). The collected supernatants and sera were used for measuring the induced amount of cytokines and chemokines. Cytokines and chemokines were first screened using a multi-analyte ELISArray kit (SAbioscience, Valencia, CA, USA) and then the differentially expressed cytokines and chemokines between pregnant and non-pregnant female mice were further quantified by singleanalyte ELISA kits. (SAbioscience, Valencia, CA, USA). The reaction was performed as described by the manufacturer. The absorbance of reaction was read at 450 nm by an ELISA plate reader (Tecan, Männedorf, Switzerland). The amount of the individual cytokine in
the single assay was determined based on the standard curve of each cytokine.
Detection of pregnant hormones in mice Mouse-specific estrogen and progesterone ELISA kits (MyBioSource, San Diego, CA USA) were used to measure hormone modulation in infected pregnant and non-pregnant mice or uninfected pregnant and non-pregnant mice (n ¼5 per group). Sera and uterus tissue samples (1 g per group) were collected from the infected mice on 5 days p.i. and from the uninfected mice. Uterus tissues were frozen in liquid nitrogen and were homogenized with a mortar and pestle. The homogenized uterus tissues were suspended in PBS (pH.7.4) (1 g/ml) and were centrifuged to remove the debris (1000 g, 10 min). Optical density (O.D.) in the reacted samples was read at 450 nm using a microtiter plate. The amount of the individual hormone was determined based on the standard curve of each hormone.
Histopathology and antigen detection in lung tissues of mice Infected pregnant and non-pregnant mice or uninfected pregnant and female mice were euthanized on 5 days p.i. The lung tissues were collected and were fixed by submerging in the 10% neutral buffered formalin and were embedded in paraffin. Fivemicrometer sections were cut, and stained with hematoxylin and eosin (H&E) as described (Bancrof and Stevens, 1996). The stained tissues were evaluated under an Olympus DP70 microscope (Olympus Corporation, Tokyo, Japan). Five-micrometer sections were stained with mouse influenza B virus anti-nucleoprotein antibody (Serotech, United Kingdom). Sections were fixed with 100% chilled acetone, and endogenous peroxidase activity in the sections was blocked by 3% H2O2. The blocked tissue sections were labeled with mouse influenza B virus anti-nucleoprotein antibody (1:1000 dilution) and then with biotin-labeled goat anti-mouse immunoglobulin (Vector, USA), VECTASTAIN ABC-AP (Vector, USA), and Vector red alkaline phosphatase substrate (Vector, USA). The stained tissue sections were counterstained with hematoxylin QS (Vector Laboratories, Burlingame, CA). The stained sections were evaluated under an Olympus DP70 microscope (Olympus Corporation, Tokyo, Japan).
Histopathology scores for inflammation in lung legions of mice The five sites in the H&E-stained lung tissues were randomly selected for measuring histopathology scores for inflammation in order to determine histopathology scores in the lung lesions of mice. The scores were designated based on the following criteria: 0, no inflammation; 1, mild, inflammatory cell infiltrates in the perivascular and peribronchiolar compartment; 2, moderate, inflammatory cell infiltrates in the perivascular and peribronchiolar space with modest extension into the alveolar parenchyma; and 3, severe, inflammatory cell infiltrates in the perivascular and peribronchiolar space with a greater magnitude of inflammatory foci in the alveolar parenchyma.
Statistical analysis Groups of data were analyzed by repeated measures ANOVA using pairing of samples with IBMs SPSSs Statistics version 20. A P-value o 0.05 was considered to be statistically significant.
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Acknowledgments This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2012R1A2A2A01002533). References ANZIC Influenza Investigators, 2010. Critical illness due to 2009A/H1N1 influenza in pregnant and postpartum women: population based cohort study. British Medical Journal 340, c1279. Bancrof, J.D., Stevens, A., 1996. Theory and Practice of Histological Techniques, fourth ed. Churchill Living stone, NewYork. Bouvier, N.M, Palese, P., 2008. The biology of influenza viruses. Vaccine 26 (Suppl. 4), D49–D53. Cantu, J., Tita, A.T., 2013. Management of influenza in pregnancy. American Journal of Perinatology 30 (2), 99–103. de Jong, M.D., Simmons, C.P., Thanh, T.T., Hien, V.M., Smith, G.J., Chau, T.N., Hoang, D.M., Chau, N.V., Khanh, T.H., Dong, V.C., Qui, P.T., Cam, B.V., Ha do, Q., Guan, Y., Peiris, J.S., Chinh, N.T., Hien, T.T., Farrar, J., 2006. Fatal outcome of human influenza A (H5N1) is associated with high viral load and hypercytokinemia. Nature Medicine 12 (10), 1203–1207. Fouchier, R.A., Munster, V., Wallensten, A., Bestebroer, T.M., Herfst, S., Smith, D., Rimmelzwaan, G.F., Olsen, B., Osterhaus, A.D., 2005. Characterization of a novel influenza A virus hemagglutinin subtype (H16) obtained from black-headed gulls. Journal of Virology 79, 2814–2822. Francis Jr., T., 1940. A new type of virus from epidemic influenza. Science 92 (2392), 405–408. Freema, D.W., Barno, A., 1959. Deaths from Asian influenza associated with pregnancy. American Journal of Obstetrics Gynecology 78, 1172–1175. Glezen, W.P., Decker, M., Joseph, S.W., Mercready Jr., R.G., 1987. Acute respiratory disease associated with influenza epidemics in Houston, 1981–1983. Journal of Infectious Diseases 155 (6), 1119–1126. Glezen, W.P., Schmier, J.K., Kuehn, C.M., Ryan, K.J., Oxford, J., 2013. The burden of influenza B: a structured literature review. American Journal of Public Health 103 (3), e43–e51. Harris, J.W., 1919. Influenza occurring in pregnant women:statistical study of thirteen hundred and fifty cases. Journal of the American Medical Association 72, 978–980.
81
Jackson, D., Elderfield, R.A., Barclay, W.S., 2011. Molecular studies of influenza B virus in the reverse genetics era. Journal of General Virology 92 (Pt. 1), 1–17. Kang, Y.M., Song, B.M., Lee, J.S., Kim, H.S., Seo, S.H., 2011. Pandemic H1N1 influenza virus causes a stronger inflammatory response than seasonal H1N1 influenza virus in ferrets. Archives of Virology 156 (5), 759–767. Kim, H.M., Kang, Y.M., Song, B.M., Kim, H.S., Seo, S.H., 2012. The 2009 pandemic H1N1 influenza virus is more pathogenic in pregnant mice than seasonal H1N1 influenza virus. Viral Immunology 25 (5), 402–410. Kim, Y.H., Kim, H.S., Cho, S.H., Seo, S.H., 2009. Influenza B virus causes milder pathogenesis and weaker inflammatory responses in ferrets than influenza A virus. Viral Immunology 22 (6), 423–430. Li, W.C., Shih, S.R., Huang, Y.C., Chen, G.W., Chang, S.C., Hsiao, M.J., Tsao, K.C., Lin, T. Y., 2008. Clinical and genetic characterization of severe influenza B-associated diseases during an outbreak in Taiwan. Journal of Clinical Virology 42 (1), 45–51. Neuzil, K.M., Reed, G.W., Mitchel, E.F., Simonsen, L., Griffin, M.R., 1998. Impact of influenza on acute cardiopulmonary hospitalizations in pregnant women. American Journal of Epidemiology 148 (11), 1094–1102. Newland, J.G., Romero, J.R., Varman, M., Drake, C., Holst, A., Safranek, T., Subbarao, K., 2003. Encephalitis associated with influenza B virus infection in 2 children and a review of the literature. Clinical Infectious Diseases 36 (7), e87–e95. Rasmussen, S.A., Jamieson, D.J., Bresee, J.S., 2008. Pandemic influenza and pregnant women. Emerging Infectious Diseases 14 (1), 95–100. Rota, P.A., Wallis, T.R., Harmon, M.W., Rota, J.S., Kendal, A.P., Nerome, K., 1990. Cocirculation of two distinct evolutionary lineages of influenza type B virus since 1983. Virology 175 (1), 59–68. Tong, S., Li, Y., Rivailler, P., Conrardy, C., Castillo, D.A., Chen, L.M, Recuenco, S., Ellison, J.A., Davis, C.T., York, I.A., Turmelle, A.S., Moran, D., Rogers, S., Shi, M., Tao, Y., Weil, M.R., Tang, K., Rowe, L.A., Sammons, S., Xu, X., Frace, M., Lindblade, K.A., Cox, N.J., Anderson, L.J., Rupprecht, C.E., Donis, R.O., 2012. A distinct lineage of influenza A virus from bats. Proceedings of the National Academy of Sciences of the United States of America 109 (11), 4269–4274. Webster, R.G., Bean, W.J., Gorman, O.T., Chambers, T.M., Kawaoka, Y., 1992. Evolution and ecology of influenza A viruses. Microbiological Reviews 56, 152–179. Yates, L., Pierce, M., Stephens, S., Mill, A.C., Spark, P., Kurinczuk, J.J., Valappil, M., Brocklehurst, P., Thomas, S.H., Knight, M., 2010. Influenza A/H1N1v in pregnancy: an investigation of the characteristics and management of affected women and the relationship to pregnancy outcomes for mother and infant. Health Technology Assessment 14 (34), 109–182.