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What's New with Pandemic Flu
Clinical Microbiology N e w s l e t
CMN
Stay Current... Stay Informed.
t e r
What’s New with Pandemic Flu
Vol. 38, No. 4 February 15, 2016 www.cmnewsletter.com
I n Th is
CMN
Issu e
27 What’s New with Pandemic Flu 32 Leptospiral Infective Endocarditis with Concurrent Dengue Infection
Deborah A. Wildoner, R.N., Fourth Mesa Laboratory Consultants, Oakland Park, Florida
Abstract The term “bird flu” refers to avian influenza viruses. The recently emerged avian H7N9 influenza viruses replicate efficiently in birds and in mammals. Although H7N9 viruses rarely infect humans, they were the cause of sporadic human infections in China in 2014. In this review, we focus on the avian H7N9 influenza virus, summarize the characteristic biological features, and assess its pandemic potential. We also review information about highly pathogenic avian influenza viruses as possible sources of pandemic outbreaks.
Introduction The first influenza pandemic of the 21st century arrived in late March 2009 and was eventually named the 2009 influenza A H1N1 pandemic (H1N1pdm09) virus. One of its predecessors, the pandemic of 1918 to 1920, caused an estimated 50 to 100 million human deaths [1]. The 1957 (H2N2) and 1968 (H3N2) pandemics caused an estimated 2 million and 1 million deaths respectively. The 2009 pandemic was also very deadly. The first positive human samples were identified from patients in California and Texas [2,3]; however, the virus originated in Mexico prior to detection in the United States. A postoutbreak report from the World Health Organization in 2010 noted 19,000 deaths worldwide, but a recent analysis [4] estimated 10 times that number of deaths. The influenza virus genome is composed of 8 negative-sense single-stranded RNA segments, each encoding at least one protein and totaling at least 11 proteins in all.
Corresponding author: Deborah A. Wildoner, R.N., Fourth Mesa Laboratory Consultants, 60 NW 48th Court, Oakland Park, FL 33309-4071. Tel.: 570-578-4626. E-mail: [email protected]
The A/California/04/2009 (H1N1) pandemic strain was the first pandemic strain for which a complete genome became publically available through GenBank [2,3]. Among the newest threats for the next predicted worldwide pandemic are a variety of “avian influenza viruses.” Because humans lack protective antibodies
against these viruses, H5N1 or H7N9 viruses that can be transmitted to humans could spread worldwide, resulting in an influenza pandemic. Most humans infected with H7N9 influenza virus exhibit general influenza-like symptoms, including fever and cough. More than half of the infections typically progress to severe pneumonia, acute respiratory distress syndrome, and multiorgan failure [5-9]. Most H7N9 virus-infected patients possess one or more underlying medical conditions, such as chronic obstructive pulmonary disease, diabetes, hypertension, obesity, and/or chronic lung and heart disease [5,6,9,10], suggesting that these comorbidities may increase the risk of severe H7N9 virus infection.
Zoonoses Influenza A viruses are primarily sustained in poultry, wild waterfowl, pigs, horses, and humans; however, reports of influenza virus have also occurred for marine mammals, dogs, and other mammalian species, such as bats [10]. These viruses possess mammalian-adapting amino acid changes that likely contribute to their ability to infect mammals. Influenza A viruses are composed of two viral surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA), by which viral subtypes are characterized. To date, 18 HA and 11 NA subtypes have been identified [10], but only 3 HA subtypes (H1, H2, and H3) and 2 NA
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subtypes (N1 and N2) are identified in humans. Human strains cause annual epidemics, and in the past two centuries, sporadic pandemics have occurred. Most influenza A viral subtypes have been detected in waterfowl, which are considered the natural host of influenza A viruses [10]. Viral replication occurs in the intestinal tract, which spreads the virus to other birds via the fecal-oral route, in contrast to mammalian replication, which occurs primarily in the respiratory tract [10]. Despite the broad host range and widespread occurrence of influenza A viruses, their transmission from avian to mammalian species, or vice versa, is rare. Influenza A viruses circulating in humans (“human influenza viruses”) rarely infect avian species, and those circulating in avian species (the “avian influenza viruses”) rarely infect humans [10]. Despite this rarity, avian influenza A viruses of the H5N1 and H7N9 subtypes have recently caused hundreds of human infections. Fortunately, sustained human-tohuman transmission of these viruses has not yet been reported; nonetheless, it is a possibility. Adaptive mutations and/or reassortment with circulating human viruses may enable H5N1 or H7N9 viruses to efficiently infect humans and facilitate humanto-human transmission. Human infections with avian H7N9 influenza viruses occurred in China in 2013–2014, and two waves of human H7N9 infection have been observed [10]. The first wave started with a human case of H7N9 influenza virus infection in Shanghai in February 2013 [9]. In April 2013, the number of human cases of H7N9 virus infections increased significantly, reaching 125 confirmed cases in China. Most cases were reported in the eastern part of China [10]. Epidemiological data suggest that associated contact with poultry or live bird markets was the likely source of many, but not all, of the human infections [10]. The association prompted the closure of live poultry markets in several provinces in mid-April 2013, which likely led to the rapid decline in new human H7N9 cases during the following 2 weeks [10]. In autumn 2013, after reopening of poultry markets, the second wave of human infections began [10]. Human H7N9 virus infections spiked in January and February of 2014, with more than 30 new cases over the next several weeks. Since February 2014, the number of new human cases has declined, although new infections continue to be reported [10]. The second wave continued and had a more extensive geographic spread. While most human cases during the first wave were reported in eastern China, the majority of human H7N9 virus infections during the second wave occurred in the southern province of Guangdong [10]. As of May 2014, a total of 440 human infections with H7N9 viruses have been confirmed (425 of the cases occurred in China, whereas the remaining 15 were exported cases [10]).
Epidemiology Epidemiological studies report that H7N9 virus infections have affected mainly middle-aged or older individuals [5,6,10]. Twothirds of the infected individuals have been male [5,6,8,10]. The high number of cases among elderly men may be associated with the fact that elderly men have frequent work-related or non-job-
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related contact with poultry. The following evidence suggests that contact with live poultry is the source of human H7N9 virus infections [10]: (i) active surveillance in live bird markets isolated H7N9 viruses with close homology to the viruses isolated from humans; (ii) most H7N9 patients were exposed to live poultry before their illnesses; (iii) a worker who worked with infected poultry developed a mild illness that was confirmed as H7N9 virus infection; (iv) serological surveillance of poultry workers revealed antibodies to H7N9 viruses in >6% of the individuals tested, whereas no antibodies to H7N9 viruses were detected in the general population; and (v) an association between human H7N9 infections and poultry in live bird markets was underscored by the rapid decline in human H7N9 infections upon closure of these markets in midApril 2013. The H7N9 seropositivity in poultry workers shows that subclinical human infections occurred, and several studies suggested that a significant number of mild unidentified cases of human H7N9 infections occurred [10]. Several surveillance studies identified H7N9 virus-positive individuals who exhibited only mild to moderate influenza symptoms and recovered quickly [10]. Although sustained H7N9 virus transmission among humans was not reported, the potential for human-to-human transmission cannot be ruled out in several family clusters, implying that close contacts in household settings, and perhaps also genetic factors, may be risk factors for infection with H7N9 viruses [10].
Origins of H7N9 Influenza Viruses Phylogenetic analyses revealed that the novel H7N9 viruses were likely to have emerged via reassortment of at least four avian influenza A virus strains [10]. The H7N9 NA gene is closely related to those of the avian H2N9 and/or H11N9 influenza viruses found in wild migratory birds along the East Asian flyway. The H7N9 HA gene belongs to the Eurasian lineage of avian influenza viruses and is closely related to those of avian H7N3 viruses isolated from ducks in eastern China in 2010–2011 [10]. The remaining six viral genes likely originated from two subgroups of an H9N2 sub-lineage circulating in eastern China [9, 10]. This genetic heterogeneity suggests that several reassortment events occurred during the generation and ongoing evolution of H7N9 viruses [10].
Host Range How does avian influenza virus overcome the normal host restriction barriers to infect humans? Two viral proteins play a major role in the host range of influenza viruses: the surface glycoprotein, HA, which determines host-specific receptor binding, and PB2, which is the polymerase subunit that determines the replicative ability. Influenza virus infections are initiated when the HA glycoprotein binds to receptors on host cells. Avian influenza viruses preferentially bind to the major sialyloligosaccharide, which is expressed in the intestinal tract of waterfowl, sialic acid-a2,3galactose (SAa2,3 Gal) [11]. In contrast, human influenza viruses preferentially bind the predominant sialyloligosaccharide species expressed on epithelial cells in the upper respiratory tract of humans, sialic acid-a2,6-galactose (SAa2,6 -Gal) [11]. Typically,
avian influenza viruses exhibit low affinity for human-type receptors; therefore, a shift of HA receptor-binding specificity is a critical step that needs to occur for avian influenza viruses to replicate and transmit efficiently in humans. H7N9 viruses may require additional adaptive mutations to efficiently bind to the human-type receptors [12]. There is a possibility that the binding of H7N9 virions to human-type receptors might be affected by other viral components (i.e., the NA) [10,12]. There is a leucine residue at position 226 of HA, which is characteristic of human influenza viruses, and it is encoded by most avian H7N9 virus isolates, indicating that it is most likely of avian origin and did not arise during H7N9 virus replication in humans. A leucine or isoleucine residue at position 226 of HA in H7N9 viruses likely emerged during virus replication in poultry and may now facilitate infection of mammalian cells [10,12]. The PB2 protein is one of the three subunits of the viral polymerase complex, and it catalyzes viral replication and transcription in the nuclei of infected cells. Found in most human influenza viruses, a lysine residue at position 627 of PB2 allows efficient replication of avian influenza viruses in mammals. In contrast, glutamic acid at this position is found in most avian influenza viruses, and this restricts avian influenza virus replication in mammals. Currently, the mechanism through which the amino acids direct viral replication is thought most likely to be temperature sensitive and to involve interactions with other viral and/or host proteins [10]. Since avian body temperature is 41°C, most avian influenza viruses replicate more efficiently at 41°C, and this differs from temperatures in the human lung and upper respiratory tract, generally 37°C and 33°C, respectively [10]. The placement of a lysine amino acid at position 627 of PB2 confers efficient replication on avian influenza viruses at 33°C and 37°C and allows robust replication in mammals. The PB2-627K mutation has also been shown to increase the transmissibility of avian influenza viruses [10]. Sequence analyses have shown that H7N9 viruses isolated from avian hosts possess glutamic acid at position 627 of PB2, whereas many human H7N9 viruses encode lysine at this position, suggesting that the mutation likely emerges during virus replication in humans. The same mechanism occurred in the human cases of H7N7 avian influenza virus infection in the Netherlands in 2003 [13]. Of note, there are also other mutations that can confer replication in humans. Some of the human H7N9 virus isolates that encode PB2-627E have an aspartic acid-to-asparagine mutation at position 701 of PB2, a mutation known to improve avian virus replication in mammalian cells. Another human H7N9 isolate lacking PB2627K acquired a glutamine-to-lysine mutation at position 591 of PB2. A basic amino acid at this position compensates for the lack of PB2-627K in pandemic 2009 H1N1 viruses [10]. Together, these findings demonstrate that the H7N9 viruses currently circulating in birds do not encode strong determinants of mammalian adaptation in PB2 but that mutations arise easily during H7N9 virus replication in humans. There are also other viral proteins that contribute to H7N9 virulence, although to a lesser extent than the major virulence determi-
nants. Previous studies suggested a potential role for PA, another subunit of the viral polymerase complex, in the adaptation and pathogenicity of avian influenza viruses in a mammalian host [10].
Risk Assessment of Human-to-Human Transmission Influenza viruses transmit from human to human through direct and indirect contact via aerosols, respiratory droplets, and fomites; therefore, the primary concern with H7N9 viruses is that they may gain efficient human-to-human transmissibility and cause a pandemic. Several research groups have evaluated the transmissibility of H7N9 viruses in two animal models, namely, ferrets and guinea pigs although the efficiency of transmission is lower than that of human influenza viruses [10,12]. The pandemic potential of novel H7N9 viruses appears to be greater than that of highly pathogenic H5N1 viruses, and in patients treated with NA inhibitors, oseltamivir-resistant H7N9 variants have been detected [14,15] and may be able to replicate efficiently [16]. Combined with the emergence of partially humanadapted strains, the oseltamivir-resistant H7N9 viruses would pose a significant pandemic threat.
Sequence Analysis Elucidating evolutionary trajectories of individual viral segments enables researchers to determine how frequently segments jump between different host species. In addition, sequence analysis can identify key reassortment events and thereby track down the geographic origin of pandemic viruses. As scientists continue to better understand how influenza viruses evolve and reassort, it may be possible to design better novel vaccines and antiviral drugs and to predict the efficiency of existing treatments [10].
HPAI Most of the poultry outbreaks in the United States have been caused by highly pathogenic avian influenza (HPAI) (H5N2) viruses. HPAI H5 viruses infect the avian gastrointestinal and respiratory tracts and can spread rapidly, causing high mortality in infected poultry. Many birds have died, and millions of chickens and turkeys in several states have been destroyed in an attempt to control the outbreaks. Signs of HPAI in poultry can include sudden death; lack of energy, appetite, and coordination; purple discoloration or swelling of various body parts; diarrhea; nasal discharge; coughing; sneezing; and reduced egg production, or soft-shelled or misshapen eggs [17]. Three subtypes of HPAI H5 viruses have been found: the H5N1, H5N2, and H5N8 viruses, which have been detected in U.S. birds. Preliminary studies suggest that the HPAI H5 viruses causing the poultry outbreaks are not well adapted for infecting humans, and no human infections have been identified in the U.S. The H5N1 virus detected in the U.S. is a reassortant virus with genes from HPAI H5 Eurasian viruses and low-pathogenic North American viruses. The H5N2 viruses detected in the U.S. are similar to H5N2 viruses first detected in early December 2014 on poultry farms in British Columbia, Canada, and are reassortant viruses that combine genes from Eurasian H5 viruses and North American N2 viruses. No human cases of infection have been associated
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with either the North American or the Eurasian lineage of H5N2 viruses. The H5N8 viruses detected in the U.S. are similar to viruses that were first reported on duck farms in China in 20092010. During 2014, similar H5N8 viruses were found in wild birds and poultry in Japan and Korea. In November 2014, H5N8 viruses were reported in England, the Netherlands, Germany, and Italy in poultry and wild birds. No human cases of infection have been associated with these H5N8 viruses [17]. Sporadic cases of human respiratory illness with high mortality from infections with other closely related HPAI H5 viruses (e.g., H5N1 and H5N6) have occurred outside the U.S. HPAI H5 virus infection of humans can start with signs and symptoms of uncomplicated seasonal influenza, such as fever, upper respiratory tract symptoms, and myalgia. Symptoms can progress to the lower respiratory tract. Outside the U.S., severe pneumonia, multi-organ failure, encephalitis, and septic shock have been reported with both H5N1 and H5N6 viruses. While a rare sign of seasonal influenza, conjunctivitis has been reported as a sign of avian influenza virus infection, and atypical presentations of fever and diarrhea preceding pneumonia have been reported. Other signs and symptoms include cough, runny nose, sore throat, headache, muscle aches, eye redness, difficulty breathing, and shortness of breath [17]. Most human infections with H5N1 or H5N6 viruses have occurred in persons who were not using appropriate personal protective equipment and who had exposures, such as direct physical contact with infected birds or surfaces contaminated by the viruses, close proximity to infected birds (within approximately 6 feet), or visiting a live poultry market. High-risk contact includes direct contact with birds (e.g., handling, slaughtering, defeathering, butchering, or preparation for consumption), direct contact with surfaces contaminated with feces or bird parts (carcasses, internal organs, etc.), or prolonged exposure to birds in a confined space. Those exposed should monitor their health for 10 days after exposure and report any symptoms to a physician and the local and state public health departments. Human infection with avian influenza viruses does not occur from eating properly cooked poultry or poultry products [17]. For a list of avian influenza A H5 virus infections identified in birds in the U.S., see the reports at the CDC website [17].
Specimen Collection for HPAI H5 and H7N9 Viruses Specimen collection and processing for patients who may be infected with novel HPAI influenza A viruses are detailed below. Clinicians and public health personnel should consider the following recommendations for surveillance and testing of HPAI H5 viruses [17]. • Consider the possibility of infection with novel influenza
A viruses in patients with medically attended influenza-like infection and acute respiratory infection who have had recent contact (<10 days prior to illness onset) with sick or dead birds in any of the following categories: - Domestic poultry, such as chickens, turkeys, and ducks - Wild aquatic birds, such as ducks, geese, and swans
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- Captive birds of prey, such as falcons, that had contact with wild aquatic birds • Collect respiratory specimens with appropriate infection
control precautions and send specimens to the state or local health department for immediate testing. At state health departments, initiate a public health investigation and notify the CDC. Specimens to be tested for novel influenza A viruses should be sent first to the state or local public health laboratory for testing. Laboratories should liaison with the CDC for any confirmatory testing for any novel influenza A virus, such as H7N9 or H5N1 virus, other avian H5 viruses, or variant influenza viruses, such as H3N2v. • The duration of viral shedding for HPAI viruses is unknown,
but recommendations are based on what is known about seasonal influenza virus infections. Obtain specimens for novel influenza A virus testing as soon as possible after illness onset, ideally within 7 days of onset, but if HPAI virus infection is suspected, specimens can be tested after 7 days from onset. Prolonged shedding of influenza virus in the lower respiratory tract has been documented for critically ill patients with HPAI H5N1 virus and avian influenza A H7N9 virus infections. • If possible, multiple respiratory specimens from different
sites should be obtained from the same patient on at least two consecutive days. Always consider diagnostic testing for other respiratory pathogens, since novel influenza A virus infections of humans are very rare, even in exposed persons. • Collect specimens as soon as possible after illness onset.
- Nasopharyngeal swab - Nasal aspirate or wash • Two swabs may be combined into one viral transport medium
(e.g., a nasal or nasopharyngeal swab combined with an oropharyngeal swab. • For patients with lower respiratory tract illness, a lower respi-
ratory tract specimen, such as an endotracheal aspirate or bronchoalveolar lavage fluid, may be preferred, because these specimens have a higher yield for detecting avian influenza A H5N1 and H7N9 viruses and also may facilitate detection of other novel avian influenza A viruses. • Standard, contact, and airborne precautions are recommended
for patient management and collection of respiratory specimens. Practitioners should employ infection control precautions consistent with those recommended for novel influenza A viruses known to cause severe disease in humans. Specimens should be placed into sterile viral transport medium and immediately placed on refrigerant gel packs or at 4°C for transport to the laboratory [17]. • Clinical specimens sent to state public health laboratories
should be shipped in the appropriate packaging. If clinical specimens will be examined within 72 hours after collection, keep the specimen at 4°C (2 to 8°C) and ship on refrigerant gel packs. If storage is longer, store frozen at -70°C or less and ship on dry ice [17].
• Testing of symptomatic human cases of novel influenza A
virus infections should be referred to the nearest public health laboratory because the performances of current FDA-cleared tests have not been demonstrated with most novel influenza A viruses. Existing, commercially available FDA-cleared molecular assays may fail to detect novel influenza A viruses or may detect with results that indicate “influenza A positive” but with no subtype detected. For these assays, a novel influenza A virus may give an influenza A “unsubtypeable” result. • Some diagnostic assays may detect the presence of some novel
influenza A viruses; however, a negative result should not be used to rule out influenza when testing possible human cases [17]. • Clinicians and laboratorians using molecular assays that are
capable of detecting all currently circulating influenza A seasonal influenza subtypes who encounter an unsubtypeable result should contact the CDC and their state or local public health laboratory for additional testing [17]. Rapid influenza diagnostic tests and immunofluorescence assays are antigen detection tests that also have unknown sensitivity and specificity to detect human infection with novel influenza A viruses. Therefore, negative results from either type of test do not exclude novel influenza virus infection, especially in patients with signs and symptoms suggestive of influenza. A negative test result could be a false negative and should not be used as a final diagnostic test for influenza, including novel influenza A virus infection. These tests may give a positive influenza A virus result for a specimen containing novel influenza A virus but cannot identify the subtype and cannot distinguish a novel influenza A virus from a seasonal influenza A virus. Testing at state health laboratories for any patient with suspected avian influenza A virus infection is recommended [17].
Summary To date, the novel H7N9 and HPAI H5 influenza viruses have not caused a pandemic in humans due to their inability to support sustained human-to-human transmission. The viruses exhibit efficient replication but limited transmissibility in mammals. H7N9 has acquired amino acid changes that enable adaptation to mammals, and it may reassort with circulating human viruses. The viruses can also easily acquire resistance to the NA inhibitor oseltamivir. Perhaps most importantly for its potential to create a pandemic event, humans lack protective immunity to H7N9 infection and often show weak antibody responses when infected with these viruses. To develop countermeasures against avian influenza virus infections in humans, it will be important to develop a better understanding of the mechanisms of pathogenicity, the levels of transmissibility, and the ability of humans to develop an immune response to the avian influenza viruses. It will be important to continue surveillance in avian and human populations so that scientists can provide warning of future pandemics.
References [1] Johnson NP, Mueller J. Updating the accounts: global mortality of the 1918-1920 “Spanish” influenza pandemic. Bull Hist Med 2002;76:105-15. [2] Garten RJ, Davis CT, Russell CA, Shu B, Lindstrom S, Balish A, et al. Antigenic and genetic characteristics of swine-origin 2009 A(H1N1) influenza viruses circulating in humans. Science 2009;325:197-201. [3] Smith GJ, Vijaykrishna D, Ellis TM, Dyrting KC, Leung YH, Bahl J, et al. Characterization of avian influenza viruses A (H5N1) from wild birds, Hong Kong, 2004-2008. Emerg Infect Dis 2009;15:402-7. [4] Simonsen L, Spreeuwenberg P, Lustig R, Taylor RJ, Fleming DM, Kroneman M, et al. Global mortality estimates for the 2009 Influenza Pandemic from the GLaMOR project: a modeling study. PLoS Med 2013;10:e1001558. [5] Lu S, Li T, Xi X, Chen Q, Liu X, Zhang B, et al. Prognosis of 18 H7N9 avian influenza patients in Shanghai. PLoS One 2014;9:e88728. [6] Gao HN, Lu HZ, Cao B, Du B, Shang H, Gan JH, et al. Clinical findings in 111 cases of influenza A (H7N9) virus infection. N Engl J Med 2013;368:2277-85. [7] Liu S, Sun J, Cai J, Miao Z, Lu M, Qin S, et al. Epidemiological, clinical and viral characteristics of fatal cases of human avian influenza A (H7N9) virus in Zhejiang Province, China. J Infect 2013;67:595-605. [8] Li Q, Zhou L, Zhou M, Chen Z, Li F, Wu H, et al. Epidemiology of human infections with avian influenza A(H7N9) virus in China. N Engl J Med. 2014;370:520-32. [9] Centers for Disease Control and Prevention. Emergence of avian influenza A(H7N9) virus causing severe human illness—China, February-April 2013. MMWR Morb Mortal Wkly Rep 2013;62:36671. [10] Watanabe T, Watanabe S, Maher EA, Neumann G, Kawaoka Y. Pandemic potential of avian influenza A (H7N9) viruses. Trends Microbiol 2014;22:623-31. [11] de Graaf M, Fouchier RA. Role of receptor binding specificity in influenza A virus transmission and pathogenesis. EMBO J. 2014;33:823-41. [12] Tharakaraman K, Jayaraman A, Raman R, Viswanathan K, Stebbins NW, Johnson D, et al. Glycan receptor binding of the influenza A virus H7N9 hemagglutinin. Cell 2013;153:1486-93. [13] Jonges M, Welkers MR, Jeeninga RE, Meijer A, Schneeberger P, Fouchier RA, et al. Emergence of the virulence-associated PB2 E627K substitution in a fatal human case of highly pathogenic avian influenza virus A(H7N7) infection as determined by Illumina ultradeep sequencing. J Virol 2014;88:1694-702. [14] Wu Y, Bi Y, Vavricka CJ, Sun X, Zhang Y, Gao F, et al. Characterization of two distinct neuraminidases from avian-origin humaninfecting H7N9 influenza viruses. Cell Res 2013;23:1347-55. [15] Hu Y, Lu S, Song Z, Wang W, Hao P, Li J, et al. Association between adverse clinical outcome in human disease caused by novel influenza A H7N9 virus and sustained viral shedding and emergence of antiviral resistance. Lancet 2013;381:2273-9. [16] Hai R, Schmolke M, Leyva-Grado VH, Thangavel RR, Margine I, Jaffe EL, et al. Influenza A(H7N9) virus gains neuraminidase inhibitor resistance without loss of in vivo virulence or transmissibility. Nat Commun 2013;4:2854. [17] Centers for Disease Control and Prevention. 2015. http://www.cdc. gov/flu/avianflu/index.htm, accessed 12/15/2015.
Clinical Microbiology Newsletter 38:4,2016 | ©2016 Elsevier
31
CMN
Stay Current... Stay Informed.
t e r
What’s New with Pandemic Flu
Vol. 38, No. 4 February 15, 2016 www.cmnewsletter.com
I n Th is
CMN
Issu e
27 What’s New with Pandemic Flu 32 Leptospiral Infective Endocarditis with Concurrent Dengue Infection
Deborah A. Wildoner, R.N., Fourth Mesa Laboratory Consultants, Oakland Park, Florida
Abstract The term “bird flu” refers to avian influenza viruses. The recently emerged avian H7N9 influenza viruses replicate efficiently in birds and in mammals. Although H7N9 viruses rarely infect humans, they were the cause of sporadic human infections in China in 2014. In this review, we focus on the avian H7N9 influenza virus, summarize the characteristic biological features, and assess its pandemic potential. We also review information about highly pathogenic avian influenza viruses as possible sources of pandemic outbreaks.
Introduction The first influenza pandemic of the 21st century arrived in late March 2009 and was eventually named the 2009 influenza A H1N1 pandemic (H1N1pdm09) virus. One of its predecessors, the pandemic of 1918 to 1920, caused an estimated 50 to 100 million human deaths [1]. The 1957 (H2N2) and 1968 (H3N2) pandemics caused an estimated 2 million and 1 million deaths respectively. The 2009 pandemic was also very deadly. The first positive human samples were identified from patients in California and Texas [2,3]; however, the virus originated in Mexico prior to detection in the United States. A postoutbreak report from the World Health Organization in 2010 noted 19,000 deaths worldwide, but a recent analysis [4] estimated 10 times that number of deaths. The influenza virus genome is composed of 8 negative-sense single-stranded RNA segments, each encoding at least one protein and totaling at least 11 proteins in all.
Corresponding author: Deborah A. Wildoner, R.N., Fourth Mesa Laboratory Consultants, 60 NW 48th Court, Oakland Park, FL 33309-4071. Tel.: 570-578-4626. E-mail: [email protected]
The A/California/04/2009 (H1N1) pandemic strain was the first pandemic strain for which a complete genome became publically available through GenBank [2,3]. Among the newest threats for the next predicted worldwide pandemic are a variety of “avian influenza viruses.” Because humans lack protective antibodies
against these viruses, H5N1 or H7N9 viruses that can be transmitted to humans could spread worldwide, resulting in an influenza pandemic. Most humans infected with H7N9 influenza virus exhibit general influenza-like symptoms, including fever and cough. More than half of the infections typically progress to severe pneumonia, acute respiratory distress syndrome, and multiorgan failure [5-9]. Most H7N9 virus-infected patients possess one or more underlying medical conditions, such as chronic obstructive pulmonary disease, diabetes, hypertension, obesity, and/or chronic lung and heart disease [5,6,9,10], suggesting that these comorbidities may increase the risk of severe H7N9 virus infection.
Zoonoses Influenza A viruses are primarily sustained in poultry, wild waterfowl, pigs, horses, and humans; however, reports of influenza virus have also occurred for marine mammals, dogs, and other mammalian species, such as bats [10]. These viruses possess mammalian-adapting amino acid changes that likely contribute to their ability to infect mammals. Influenza A viruses are composed of two viral surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA), by which viral subtypes are characterized. To date, 18 HA and 11 NA subtypes have been identified [10], but only 3 HA subtypes (H1, H2, and H3) and 2 NA
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subtypes (N1 and N2) are identified in humans. Human strains cause annual epidemics, and in the past two centuries, sporadic pandemics have occurred. Most influenza A viral subtypes have been detected in waterfowl, which are considered the natural host of influenza A viruses [10]. Viral replication occurs in the intestinal tract, which spreads the virus to other birds via the fecal-oral route, in contrast to mammalian replication, which occurs primarily in the respiratory tract [10]. Despite the broad host range and widespread occurrence of influenza A viruses, their transmission from avian to mammalian species, or vice versa, is rare. Influenza A viruses circulating in humans (“human influenza viruses”) rarely infect avian species, and those circulating in avian species (the “avian influenza viruses”) rarely infect humans [10]. Despite this rarity, avian influenza A viruses of the H5N1 and H7N9 subtypes have recently caused hundreds of human infections. Fortunately, sustained human-tohuman transmission of these viruses has not yet been reported; nonetheless, it is a possibility. Adaptive mutations and/or reassortment with circulating human viruses may enable H5N1 or H7N9 viruses to efficiently infect humans and facilitate humanto-human transmission. Human infections with avian H7N9 influenza viruses occurred in China in 2013–2014, and two waves of human H7N9 infection have been observed [10]. The first wave started with a human case of H7N9 influenza virus infection in Shanghai in February 2013 [9]. In April 2013, the number of human cases of H7N9 virus infections increased significantly, reaching 125 confirmed cases in China. Most cases were reported in the eastern part of China [10]. Epidemiological data suggest that associated contact with poultry or live bird markets was the likely source of many, but not all, of the human infections [10]. The association prompted the closure of live poultry markets in several provinces in mid-April 2013, which likely led to the rapid decline in new human H7N9 cases during the following 2 weeks [10]. In autumn 2013, after reopening of poultry markets, the second wave of human infections began [10]. Human H7N9 virus infections spiked in January and February of 2014, with more than 30 new cases over the next several weeks. Since February 2014, the number of new human cases has declined, although new infections continue to be reported [10]. The second wave continued and had a more extensive geographic spread. While most human cases during the first wave were reported in eastern China, the majority of human H7N9 virus infections during the second wave occurred in the southern province of Guangdong [10]. As of May 2014, a total of 440 human infections with H7N9 viruses have been confirmed (425 of the cases occurred in China, whereas the remaining 15 were exported cases [10]).
Epidemiology Epidemiological studies report that H7N9 virus infections have affected mainly middle-aged or older individuals [5,6,10]. Twothirds of the infected individuals have been male [5,6,8,10]. The high number of cases among elderly men may be associated with the fact that elderly men have frequent work-related or non-job-
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related contact with poultry. The following evidence suggests that contact with live poultry is the source of human H7N9 virus infections [10]: (i) active surveillance in live bird markets isolated H7N9 viruses with close homology to the viruses isolated from humans; (ii) most H7N9 patients were exposed to live poultry before their illnesses; (iii) a worker who worked with infected poultry developed a mild illness that was confirmed as H7N9 virus infection; (iv) serological surveillance of poultry workers revealed antibodies to H7N9 viruses in >6% of the individuals tested, whereas no antibodies to H7N9 viruses were detected in the general population; and (v) an association between human H7N9 infections and poultry in live bird markets was underscored by the rapid decline in human H7N9 infections upon closure of these markets in midApril 2013. The H7N9 seropositivity in poultry workers shows that subclinical human infections occurred, and several studies suggested that a significant number of mild unidentified cases of human H7N9 infections occurred [10]. Several surveillance studies identified H7N9 virus-positive individuals who exhibited only mild to moderate influenza symptoms and recovered quickly [10]. Although sustained H7N9 virus transmission among humans was not reported, the potential for human-to-human transmission cannot be ruled out in several family clusters, implying that close contacts in household settings, and perhaps also genetic factors, may be risk factors for infection with H7N9 viruses [10].
Origins of H7N9 Influenza Viruses Phylogenetic analyses revealed that the novel H7N9 viruses were likely to have emerged via reassortment of at least four avian influenza A virus strains [10]. The H7N9 NA gene is closely related to those of the avian H2N9 and/or H11N9 influenza viruses found in wild migratory birds along the East Asian flyway. The H7N9 HA gene belongs to the Eurasian lineage of avian influenza viruses and is closely related to those of avian H7N3 viruses isolated from ducks in eastern China in 2010–2011 [10]. The remaining six viral genes likely originated from two subgroups of an H9N2 sub-lineage circulating in eastern China [9, 10]. This genetic heterogeneity suggests that several reassortment events occurred during the generation and ongoing evolution of H7N9 viruses [10].
Host Range How does avian influenza virus overcome the normal host restriction barriers to infect humans? Two viral proteins play a major role in the host range of influenza viruses: the surface glycoprotein, HA, which determines host-specific receptor binding, and PB2, which is the polymerase subunit that determines the replicative ability. Influenza virus infections are initiated when the HA glycoprotein binds to receptors on host cells. Avian influenza viruses preferentially bind to the major sialyloligosaccharide, which is expressed in the intestinal tract of waterfowl, sialic acid-a2,3galactose (SAa2,3 Gal) [11]. In contrast, human influenza viruses preferentially bind the predominant sialyloligosaccharide species expressed on epithelial cells in the upper respiratory tract of humans, sialic acid-a2,6-galactose (SAa2,6 -Gal) [11]. Typically,
avian influenza viruses exhibit low affinity for human-type receptors; therefore, a shift of HA receptor-binding specificity is a critical step that needs to occur for avian influenza viruses to replicate and transmit efficiently in humans. H7N9 viruses may require additional adaptive mutations to efficiently bind to the human-type receptors [12]. There is a possibility that the binding of H7N9 virions to human-type receptors might be affected by other viral components (i.e., the NA) [10,12]. There is a leucine residue at position 226 of HA, which is characteristic of human influenza viruses, and it is encoded by most avian H7N9 virus isolates, indicating that it is most likely of avian origin and did not arise during H7N9 virus replication in humans. A leucine or isoleucine residue at position 226 of HA in H7N9 viruses likely emerged during virus replication in poultry and may now facilitate infection of mammalian cells [10,12]. The PB2 protein is one of the three subunits of the viral polymerase complex, and it catalyzes viral replication and transcription in the nuclei of infected cells. Found in most human influenza viruses, a lysine residue at position 627 of PB2 allows efficient replication of avian influenza viruses in mammals. In contrast, glutamic acid at this position is found in most avian influenza viruses, and this restricts avian influenza virus replication in mammals. Currently, the mechanism through which the amino acids direct viral replication is thought most likely to be temperature sensitive and to involve interactions with other viral and/or host proteins [10]. Since avian body temperature is 41°C, most avian influenza viruses replicate more efficiently at 41°C, and this differs from temperatures in the human lung and upper respiratory tract, generally 37°C and 33°C, respectively [10]. The placement of a lysine amino acid at position 627 of PB2 confers efficient replication on avian influenza viruses at 33°C and 37°C and allows robust replication in mammals. The PB2-627K mutation has also been shown to increase the transmissibility of avian influenza viruses [10]. Sequence analyses have shown that H7N9 viruses isolated from avian hosts possess glutamic acid at position 627 of PB2, whereas many human H7N9 viruses encode lysine at this position, suggesting that the mutation likely emerges during virus replication in humans. The same mechanism occurred in the human cases of H7N7 avian influenza virus infection in the Netherlands in 2003 [13]. Of note, there are also other mutations that can confer replication in humans. Some of the human H7N9 virus isolates that encode PB2-627E have an aspartic acid-to-asparagine mutation at position 701 of PB2, a mutation known to improve avian virus replication in mammalian cells. Another human H7N9 isolate lacking PB2627K acquired a glutamine-to-lysine mutation at position 591 of PB2. A basic amino acid at this position compensates for the lack of PB2-627K in pandemic 2009 H1N1 viruses [10]. Together, these findings demonstrate that the H7N9 viruses currently circulating in birds do not encode strong determinants of mammalian adaptation in PB2 but that mutations arise easily during H7N9 virus replication in humans. There are also other viral proteins that contribute to H7N9 virulence, although to a lesser extent than the major virulence determi-
nants. Previous studies suggested a potential role for PA, another subunit of the viral polymerase complex, in the adaptation and pathogenicity of avian influenza viruses in a mammalian host [10].
Risk Assessment of Human-to-Human Transmission Influenza viruses transmit from human to human through direct and indirect contact via aerosols, respiratory droplets, and fomites; therefore, the primary concern with H7N9 viruses is that they may gain efficient human-to-human transmissibility and cause a pandemic. Several research groups have evaluated the transmissibility of H7N9 viruses in two animal models, namely, ferrets and guinea pigs although the efficiency of transmission is lower than that of human influenza viruses [10,12]. The pandemic potential of novel H7N9 viruses appears to be greater than that of highly pathogenic H5N1 viruses, and in patients treated with NA inhibitors, oseltamivir-resistant H7N9 variants have been detected [14,15] and may be able to replicate efficiently [16]. Combined with the emergence of partially humanadapted strains, the oseltamivir-resistant H7N9 viruses would pose a significant pandemic threat.
Sequence Analysis Elucidating evolutionary trajectories of individual viral segments enables researchers to determine how frequently segments jump between different host species. In addition, sequence analysis can identify key reassortment events and thereby track down the geographic origin of pandemic viruses. As scientists continue to better understand how influenza viruses evolve and reassort, it may be possible to design better novel vaccines and antiviral drugs and to predict the efficiency of existing treatments [10].
HPAI Most of the poultry outbreaks in the United States have been caused by highly pathogenic avian influenza (HPAI) (H5N2) viruses. HPAI H5 viruses infect the avian gastrointestinal and respiratory tracts and can spread rapidly, causing high mortality in infected poultry. Many birds have died, and millions of chickens and turkeys in several states have been destroyed in an attempt to control the outbreaks. Signs of HPAI in poultry can include sudden death; lack of energy, appetite, and coordination; purple discoloration or swelling of various body parts; diarrhea; nasal discharge; coughing; sneezing; and reduced egg production, or soft-shelled or misshapen eggs [17]. Three subtypes of HPAI H5 viruses have been found: the H5N1, H5N2, and H5N8 viruses, which have been detected in U.S. birds. Preliminary studies suggest that the HPAI H5 viruses causing the poultry outbreaks are not well adapted for infecting humans, and no human infections have been identified in the U.S. The H5N1 virus detected in the U.S. is a reassortant virus with genes from HPAI H5 Eurasian viruses and low-pathogenic North American viruses. The H5N2 viruses detected in the U.S. are similar to H5N2 viruses first detected in early December 2014 on poultry farms in British Columbia, Canada, and are reassortant viruses that combine genes from Eurasian H5 viruses and North American N2 viruses. No human cases of infection have been associated
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with either the North American or the Eurasian lineage of H5N2 viruses. The H5N8 viruses detected in the U.S. are similar to viruses that were first reported on duck farms in China in 20092010. During 2014, similar H5N8 viruses were found in wild birds and poultry in Japan and Korea. In November 2014, H5N8 viruses were reported in England, the Netherlands, Germany, and Italy in poultry and wild birds. No human cases of infection have been associated with these H5N8 viruses [17]. Sporadic cases of human respiratory illness with high mortality from infections with other closely related HPAI H5 viruses (e.g., H5N1 and H5N6) have occurred outside the U.S. HPAI H5 virus infection of humans can start with signs and symptoms of uncomplicated seasonal influenza, such as fever, upper respiratory tract symptoms, and myalgia. Symptoms can progress to the lower respiratory tract. Outside the U.S., severe pneumonia, multi-organ failure, encephalitis, and septic shock have been reported with both H5N1 and H5N6 viruses. While a rare sign of seasonal influenza, conjunctivitis has been reported as a sign of avian influenza virus infection, and atypical presentations of fever and diarrhea preceding pneumonia have been reported. Other signs and symptoms include cough, runny nose, sore throat, headache, muscle aches, eye redness, difficulty breathing, and shortness of breath [17]. Most human infections with H5N1 or H5N6 viruses have occurred in persons who were not using appropriate personal protective equipment and who had exposures, such as direct physical contact with infected birds or surfaces contaminated by the viruses, close proximity to infected birds (within approximately 6 feet), or visiting a live poultry market. High-risk contact includes direct contact with birds (e.g., handling, slaughtering, defeathering, butchering, or preparation for consumption), direct contact with surfaces contaminated with feces or bird parts (carcasses, internal organs, etc.), or prolonged exposure to birds in a confined space. Those exposed should monitor their health for 10 days after exposure and report any symptoms to a physician and the local and state public health departments. Human infection with avian influenza viruses does not occur from eating properly cooked poultry or poultry products [17]. For a list of avian influenza A H5 virus infections identified in birds in the U.S., see the reports at the CDC website [17].
Specimen Collection for HPAI H5 and H7N9 Viruses Specimen collection and processing for patients who may be infected with novel HPAI influenza A viruses are detailed below. Clinicians and public health personnel should consider the following recommendations for surveillance and testing of HPAI H5 viruses [17]. • Consider the possibility of infection with novel influenza
A viruses in patients with medically attended influenza-like infection and acute respiratory infection who have had recent contact (<10 days prior to illness onset) with sick or dead birds in any of the following categories: - Domestic poultry, such as chickens, turkeys, and ducks - Wild aquatic birds, such as ducks, geese, and swans
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- Captive birds of prey, such as falcons, that had contact with wild aquatic birds • Collect respiratory specimens with appropriate infection
control precautions and send specimens to the state or local health department for immediate testing. At state health departments, initiate a public health investigation and notify the CDC. Specimens to be tested for novel influenza A viruses should be sent first to the state or local public health laboratory for testing. Laboratories should liaison with the CDC for any confirmatory testing for any novel influenza A virus, such as H7N9 or H5N1 virus, other avian H5 viruses, or variant influenza viruses, such as H3N2v. • The duration of viral shedding for HPAI viruses is unknown,
but recommendations are based on what is known about seasonal influenza virus infections. Obtain specimens for novel influenza A virus testing as soon as possible after illness onset, ideally within 7 days of onset, but if HPAI virus infection is suspected, specimens can be tested after 7 days from onset. Prolonged shedding of influenza virus in the lower respiratory tract has been documented for critically ill patients with HPAI H5N1 virus and avian influenza A H7N9 virus infections. • If possible, multiple respiratory specimens from different
sites should be obtained from the same patient on at least two consecutive days. Always consider diagnostic testing for other respiratory pathogens, since novel influenza A virus infections of humans are very rare, even in exposed persons. • Collect specimens as soon as possible after illness onset.
- Nasopharyngeal swab - Nasal aspirate or wash • Two swabs may be combined into one viral transport medium
(e.g., a nasal or nasopharyngeal swab combined with an oropharyngeal swab. • For patients with lower respiratory tract illness, a lower respi-
ratory tract specimen, such as an endotracheal aspirate or bronchoalveolar lavage fluid, may be preferred, because these specimens have a higher yield for detecting avian influenza A H5N1 and H7N9 viruses and also may facilitate detection of other novel avian influenza A viruses. • Standard, contact, and airborne precautions are recommended
for patient management and collection of respiratory specimens. Practitioners should employ infection control precautions consistent with those recommended for novel influenza A viruses known to cause severe disease in humans. Specimens should be placed into sterile viral transport medium and immediately placed on refrigerant gel packs or at 4°C for transport to the laboratory [17]. • Clinical specimens sent to state public health laboratories
should be shipped in the appropriate packaging. If clinical specimens will be examined within 72 hours after collection, keep the specimen at 4°C (2 to 8°C) and ship on refrigerant gel packs. If storage is longer, store frozen at -70°C or less and ship on dry ice [17].
• Testing of symptomatic human cases of novel influenza A
virus infections should be referred to the nearest public health laboratory because the performances of current FDA-cleared tests have not been demonstrated with most novel influenza A viruses. Existing, commercially available FDA-cleared molecular assays may fail to detect novel influenza A viruses or may detect with results that indicate “influenza A positive” but with no subtype detected. For these assays, a novel influenza A virus may give an influenza A “unsubtypeable” result. • Some diagnostic assays may detect the presence of some novel
influenza A viruses; however, a negative result should not be used to rule out influenza when testing possible human cases [17]. • Clinicians and laboratorians using molecular assays that are
capable of detecting all currently circulating influenza A seasonal influenza subtypes who encounter an unsubtypeable result should contact the CDC and their state or local public health laboratory for additional testing [17]. Rapid influenza diagnostic tests and immunofluorescence assays are antigen detection tests that also have unknown sensitivity and specificity to detect human infection with novel influenza A viruses. Therefore, negative results from either type of test do not exclude novel influenza virus infection, especially in patients with signs and symptoms suggestive of influenza. A negative test result could be a false negative and should not be used as a final diagnostic test for influenza, including novel influenza A virus infection. These tests may give a positive influenza A virus result for a specimen containing novel influenza A virus but cannot identify the subtype and cannot distinguish a novel influenza A virus from a seasonal influenza A virus. Testing at state health laboratories for any patient with suspected avian influenza A virus infection is recommended [17].
Summary To date, the novel H7N9 and HPAI H5 influenza viruses have not caused a pandemic in humans due to their inability to support sustained human-to-human transmission. The viruses exhibit efficient replication but limited transmissibility in mammals. H7N9 has acquired amino acid changes that enable adaptation to mammals, and it may reassort with circulating human viruses. The viruses can also easily acquire resistance to the NA inhibitor oseltamivir. Perhaps most importantly for its potential to create a pandemic event, humans lack protective immunity to H7N9 infection and often show weak antibody responses when infected with these viruses. To develop countermeasures against avian influenza virus infections in humans, it will be important to develop a better understanding of the mechanisms of pathogenicity, the levels of transmissibility, and the ability of humans to develop an immune response to the avian influenza viruses. It will be important to continue surveillance in avian and human populations so that scientists can provide warning of future pandemics.
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