A Method for Determining Avian Influenza Virus 

Hemagglutinin and Neuraminidase Subtype Association

Open-File Report 2018–1102

U.S. Department of the Interior

U.S. Geological Survey
Cover. Photograph of a blue-winged teal by Andy Ramey, U.S. Geological Survey, on March 29, 

2013, and image of avian influenza virus modified from photograph by Andy Ramey, U.S. Geological 

Survey on March 29, 2013.
A Method for Determining Avian Influenza 

Virus Hemagglutinin and Neuraminidase 

Subtype Association

By Christopher A. Ottinger, Deborah D. Iwanowicz , Luke R. Iwanowicz, Cynthia 

R. Adams, Lakyn R. Sanders, and Christine L. Densmore

Open-File Report 2018–1102

U.S. Department of the Interior

U.S. Geological Survey
U.S. Department of the Interior

RYAN K. ZINKE, Secretary

U.S. Geological Survey

James F. Reilly II, Director

U.S. Geological Survey, Reston, Virginia: 2018

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Suggested citation:

Ottinger, C.A., Iwanowicz, D.D., Iwanowicz, L.R., Adams, C.R., Sanders, L.R., and Densmore, C.L.,2018, A method 

for determining avian influenza virus hemagglutinin and neuraminidase subtype association: U.S. Geological Survey 

Open-File Report 2018–1102, 15 p., https://doi.org/10.3133/ofr20181102.

ISSN 2331–1258 (online)
iii

Contents

Abstract...........................................................................................................................................................1

Background.....................................................................................................................................................1

Methods...........................................................................................................................................................2

Results and Discussion.................................................................................................................................8

Acknowledgments.......................................................................................................................................14

References Cited .........................................................................................................................................14

Figures

1. Typical electropherograms of hemagglutinin subtypes evaluated in this study as

produced by Agilent’s Bioanalyzer DNA 1000 and Agilent’s 2100 Expert software...........8

2. Typical electropherograms of neuraminidase subtypes evaluated in this study as

produced by Agilent’s Bioanalyzer DNA 1000 and Agilent’s 2100 Expert software.........10

3. Electropherograms of hemagglutinin and neuraminidase subtypes from a mixed

sample containing two avian influenza virus HA:NA subtypes ..........................................12

4. Electropherograms of hemagglutinin and neuraminidase subtypes from a mixed

sample containing two avian influenza virus HA:NA subtypes with a shared

hemagglutinin subtype...............................................................................................................12

5. Electropherograms of hemagglutinin and neuraminidase subtypes from pond

sediment spiked with intact virus of the H4:N6 avian influenza virus subtype ................13
iv

Tables

1. Isolates of avian influenza viruses from paired cloacal and oropharyngeal

swabs inoculated into specific-pathogen-free eggs post screening, purified, and

characterized ................................................................................................................................3

2. Primers used for subtyping hemagglutinin and neuraminidase genes of purified and

characterized isolates of avian influenza virus reverse transcribed into cDNA from

purified RNA and amplified using a real-time reverse transcription polymerase

chain reaction ...............................................................................................................................5

3. Sanger subtype-specific sequence data obtained for avian influenza virus

hemagglutinin and neuraminidase subtype target amplicons from this study ..................6

4. Representative nucleotide BLAST data for Sanger sequences obtained for avian

influenza virus hemagglutinin and neuraminidase subtype target amplicons from

this study ........................................................................................................................................7

5. Quantitative real-time reverse transcription polymerase chain reaction linear

regression statistics for hemagglutinin and neuraminidase gene standards used in

the assessment of gene copy counts present in the ribonucleic acid from avian

influenza virus used in this study ...............................................................................................9

6. Copy count of hemagglutinin and neuraminidase genes obtained from gene copy

counts present in the ribonucleic acid from avian influenza virus isolates used in

this study, as determined by quantitative real-time reverse transcription

polymerase chain reaction .........................................................................................................9

7. Specific amplicon size determined for purified and characterized isolates of avian

influenza virus reverse transcribed into complementary deoxyribonucleic acid from

purified ribonucleic acid............................................................................................................11

8. Corrected HA:NA subtype-specific molar ratios determined for amplicons from

purified and characterized isolates of avian influenza virus reverse transcribed into

cDNA from purified RNA ...........................................................................................................11

9. Results from a mixed sample containing two avian influenza virus HA:NA subtypes....11

10. Results from a mixed sample containing two avian influenza virus HA:NA subtypes

sharing a single hemagglutinin subtype .................................................................................13
v

Conversion Factors

International System of Units to U.S. customary units

Multiply By To obtain

Volume

liter (L) 33.814 ounce, fluid (fl. oz)

Mass

gram (g) 0.035274 ounce, avoirdupois (oz)

 

Temperature in degrees Celsius (°C) may be converted to degrees Fahrenheit (°F) as follows:

°F = (1.8 × °C) + 32.

Temperature in degrees Fahrenheit (°F) may be converted to degrees Celsius (°C) as follows:

°C = (°F – 32) / 1.8.

Supplemental Information

Concentrations of chemical constituents are given in nanogram per microliter (ng μL-1) or 

nanomole per liter (nmol/L)

Abbreviations

AIV avian influenza virus

BLAST basic local alignment search tool

BP base pairs

cDNA complementary deoxyribonucleic acid

CV cumulative variance

HA hemagglutinin

HPAIV high pathology avian influenza virus

NA neuraminidase 

NCBI National Center for Biotechnology Information

NF Nuclease free

qPCR quantitative polymerase chain reaction

RNA ribonucleic acid

RT-PCR reverse transcription polymerase chain reaction

RT-qPCR reverse transcription quantitative polymerase chain reaction

USGS U.S. Geological Survey

A Method for Determining Avian Influenza Virus 

Hemagglutinin and Neuraminidase Subtype Association

By Christopher A. Ottinger, Deborah D. Iwanowicz , Luke R. Iwanowicz, Cynthia R. Adams, Lakyn R. Sanders, 

and Christine L. Densmore

Abstract

Methods for grouping specific avian influenza virus 

(AIV) hemagglutinin (HA) and neuraminidase (NA) subtype 

reverse-transcription polymerase chain reaction (RT-PCR) 

products into HA:NA subtypes when egg incubation is technically not feasible were evaluated. These approaches were 

adopted for use as post hoc methods after melt curve analysis. 

The methods are based on ratios obtained from amplicon copy 

count and amplicon molarity and were founded on the premise 

that infectious particles contain an equal copy count of singlestranded ribonucleic acid segments that encode HA or NA, 

and thus subtype-specific amplicons from a single AIV isolate 

should yield a theoretical HA:NA ratio of 1. Single and mixed 

HA:NA AIV subtype samples were evaluated to determine 

whether the calculated HA:NA ratios would approach the 

theoretical value. With these samples, preference was given 

to the molarity methods to better define and correct for the 

effects of multiple potential amplicons in the amplification 

mix. Further, the molarity method was used to evaluate pond 

sediment spiked with intact virus of known HA:NA subtype to 

determine whether the method is sufficiently robust to be used 

with complex samples, such as those acquired from waterfowl 

habitat. This was a proof-of-concept study intended to guide 

future methods development. The methods here are not meant 

to be applied in any other context.

From the analysis of fully characterized isolates of North 

American AIV, the HA:NA molarity-based ratios were found 

to be 1.63 ± 0.75 (mean ± standard deviation) when corrected 

for the difference in amplification strength and the production of multiple amplicons in some reactions using equations 

developed in this study. Copy count HA:NA ratios, obtained 

from HA and NA subtype (RT-qPCR), were 1.146 ± 0.124 

(mean ± standard deviation) when corrected for amplification 

efficiency. Correct associations of HA:NA subtype sample 

composition were made with mixed samples containing 1 HA 

and 2 NA, and 2 HA and 2 NA. When spiked pond sediment 

was evaluated, the molar ratio obtained for the H4 and N6 

identified in the sample was 1.28 with correction and 1.14 

without correction.

Background

Avian influenza virus (AIV) poses global threats to food 

production from domestic animal and human health (Machalaba and others, 2015). Consequently, surveillance programs 

for AIV in waterfowl populations, and recently (2018) 

environmental reservoirs, are of increasing importance (Lang 

and others, 2008). A subset of the known hemagglutinin (HA) 

subtypes is associated with high pathology AIV (HPAIV). 

Although changes in HA gene segments are recognized as 

major components in the transformation of low pathology AIV 

to HPAIV, changes in neuraminidase (NA) gene segments are 

also known to contribute to this transformation (Stech and others, 2015). The role of multiple gene segments in the development of HPAIV emphasizes the importance of recognizing not 

only individual HA and NA subtypes, but also the combined 

HA:NA subtypes in AIV surveillance programs.

Our interest is in the direct molecular-based characterization of AIV from environmental samples to the level 

of HA:NA associated subtypes without the need for virus 

amplification by cell culture. Typical AIV screening utilizes 

quantitative polymerase chain reaction (qPCR) methodology 

that includes the use of subtype-specific TaqMan probes for 

selected virulent subtypes (Payungporn and others, 2006). 

Holistic screening of AIV diversity in the environment may 

yield a better understanding of viral ecology. Methods that 

utilize degenerate primers that amplify the full repertoire of 

known HA and NA subtypes have been developed (Tsukamoto 

and others, 2012) but are problematic in terms of HA:NA 

subtype associations. When single HA and NA subtype genes 

are characterized from a sample, questions remain as to 

whether additional subtypes have been lost during the isolation 

and characterization process. When multiple AIV subtypes 

are present in a sample, the association of HA and NA into 

specific HA:NA subtypes can be impossible without additional information. Here we suggest an alternative method for 

HA:NA subtype association that can assist in this process.

TaqMan- and SYBR Green-chemistry methods have been 

used in combination with reverse transcription quantitative 

polymerase chain reaction (RT-qPCR) to evaluate samples 

for the presence of AIV. The former method is effective for 
2 A Method for Determining Avian Influenza Virus Hemagglutinin and Neuraminidase Subtype Association

diagnostic screening when the exact target AIV subtype 

is known because the inclusion of a probe increases assay 

specificity (Payungporn and others, 2006). The latter method 

has proven useful for surveillance when used with degenerate 

primer sets that target all known HA and NA subtype genes, 

where copy count and melt curve analyses are used to determine specificity (Tsukamoto and others, 2012). Tsukamoto and 

others (2012) produced sample-specific HA and NA subtype 

lists. This product type, produced from PCR-based AIV 

genome copy detection, is like that obtained using antibodybased AIV protein detection (Bowman and others, 2015). 

Product type is the only similarity between these two methods, 

making direct comparisons of HA and NA subtypes ambiguous. Neither method yields HA:NA subtype associations. 

When using TaqMan-chemistry, the grouping of HA and 

NA subtypes into an HA:NA subtype association occurs by 

default because only one HA subtype and one NA subtype are 

targeted using highly specific probes and type-specific methods validation (Payungporn and others, 2006). Alternatively, 

nothing may be detected, or a single subtype may be detected 

resulting in a failed or incomplete analysis. A benefit of probe 

specificity with TaqMan-chemistry is that it increases outcome 

confidence but it also limits detection when subtype sequence 

variation or subtype re-assortment occurs. This limitation 

makes the method less than ideal for environmental surveillance projects intended to detect sequence variation or subtype 

re-assortment. 

When using intercalating dye chemistries such as SYBR 

Green and primer sets for multiple HA and NA subtypes, 

which are degenerate to accommodate type-specific sequence 

variation (Tsukamoto and others, 2012), the confidence associated with high probe specificity is exchanged for breadth of 

the subtype-specific isolate amplification. Unlike the results 

obtained using TaqMan-chemistry, when SYBR Greenchemistry is used with multiple subtype primers, no means for 

subtype association into HA:NA grouping is afforded. Detection of multiple HA and (or) NA subtypes in a single sample 

can make the determination of HA:NA subtypes problematic without additional information. In theory, the subtype 

copy count could be used to associate subtypes into HA:NA 

subtypes because the Influenza A genome consists of single 

copies of the HA and NA genes (Cheung and Poon, 2007). 

The copy count ratio of two subtypes that associate to form an 

HA:NA grouping should approach 1.00. The difficulty with 

this approach is that validation over a broad range of potential 

sample mixtures is required. Here, we suggest an alternative solution for the association of HA and NA subtypes into 

specific HA:NA subtypes and compare the results to results 

obtained from copy count determinations.

Our alternative approach is a modification of RT-qPCR 

melt curve analysis (Tsukamoto and others, 2012) using 

SYBR Green-chemistry and multiple HA and NA primer sets. 

Applying the same reasoning as used in copy count ratios, we 

propose the use of molar ratios. Molar ratios are broadly used 

to describe the composition of complex biomolecules and 

cellular structures, characterize biochemical interactions, and 

differentiate normal versus disease states at the subcellular and 

molecular levels. We are simply applying a classic and well 

described concept in a different way.

In this proof-of-concept study we evaluated the potential for the practical use of subtype-specific amplicon copy 

count and molarity in AIV HA:NA subtype association when 

high throughput sequencing methods are not possible. This 

approach is applicable to environmental samples containing 

single or multiple AIV HA:NA groupings. As such, multiple 

single HA:NA subtypes representing common North American 

subtypes as well as mixtures of these HA:NA subtypes were 

evaluated starting with ribonucleic acid (RNA) from fully 

characterized AIV isolates. As a proof-of-concept, the intent of 

this study is to inform the development of future methods, and 

thus the methods here are not meant to be applied in any other 

context.

This report discusses the methods used in the new 

alternative approach. Results of this approach are presented in 

tables and figures.

Methods

RNA extracted from characterized isolates of multiple 

subtypes was provided by U.S. Geological Survey (USGS) 

Alaska Science Center, Anchorage, Alaska (table 1) (Pearce 

and others, 2009; Pearce and others, 2010; Ramey and others; 

2010a; Ramey and others, 2010b; Pearce and others, 2011; 

Ramey and others, 2011; Reeves and others, 2013). Viral 

RNA was reverse transcribed using the Applied Biosystems 

high-capacity complementary deoxyribonucleic acid (cDNA) 

reverse transcription kit (Life Technologies, Carlsbad, California) with random hexamers from the manufacturer’s protocol.

All HA and NA subtypes were analyzed on an Applied 

Biosystems ViiA 7 real-time qPCR (Life Technologies, Carlsbad, Calif.). PCR with a melt curve analysis was performed as 

described by Tsukamoto and others (2012) but with modifications. The 20-microliter (μL) reaction mixture contained 

Applied Biosystems Power SYBR Green PCR Master Mix 

(Life Technologies, Carlsbad, Calif.), 2 micromoles (μM) of 

forward and reverse primers (table 2), and 1 μL of cDNA. 

The cDNA was not normalized prior to amplification nor were 

starting concentrations measured. The melt curve was used to 

confirm successful amplification of the correct amplicon using 

dissociation temperature as described in Tsukamoto and others (2012). Each reaction was run in duplicate except for the 

control reactions, which were run in triplicate. Replicates were 

pooled prior to purification.

Post-amplification product purification was performed 

using the QIAquick PCR Purification Kit and following the 

manufacture’s protocol (Qiagen, Valencia, Calif.). All purified RT-qPCR) products were evaluated using an Agilent 

Bioanalyzer with Expert Software (Agilent Technologies, 

Santa Clara, Calif.) with Agilent DNA 1000 Kits and following the manufacturer’s protocols. Analysis was performed 
Methods 3

using the default setting, except for height threshold 

which was reduced to 5 fluorescent units. Potential target 

amplicons were identified based on maximum observed 

amplicon molarity determined from subtype-specific reaction products that fell within the assay sensitivity range of 

0.1–50 nanograms per microliter (ng μL-1) (Agilent Technologies) and within the size range of 100–400 base pairs 

(bp; Tsukamoto and others, 2012). Amplicons from all 

reactions were sequenced in both directions using Applied 

Biosystems Big Dye Cycle Sequencing Kit (Foster City, 

Calif.) to confirm nucleotide sequence. The sequencing 

reaction profile used consisted of 30 cycles of 20 seconds at 

96 degrees Celsius (oC), 20 seconds at 58 oC, and 4 minutes at 

60 oC, with a 10-minute extension at 72 °C. The sequencing 

reactions were cleaned with Agencourt CleanSEQ (Beckman 

Coulter Genomics, Beckman Coulter Inc., Brea, Calif.), following manufacturer’s instructions. Sequencing was carried 

out on an Applied Biosystems 3130xl Genetic Analyzer (Foster City, Calif.). Biomatters Limited, Geneious R6 software 

(San Francisco, Calif.) was used to trim and align sequences. 

Finalized sequences were compared with those published for 

the specific isolates from which the target amplicons were 

Table 1. Isolates of avian influenza viruses from paired cloacal 

and oropharyngeal swabs inoculated into specific-pathogen-free 

eggs post screening, purified, and characterized.

[HA, hemagglutinin; NA, neuraminidase; NCBI, National Center for 

Biotechnology Information; H, identifies hemagglutinin gene; N, identifies 

neuraminidase gene]

Isolate HA NA

NCBI 

accession 

number HA

NCBI 

accession 

number NA

Literature cited

3455 H1 N9 HM059985 HM060041 Ramey and 

others, 2010b

48944 H1 N1 GU168283 GU168373 Ramey and 

others,2010a

48982 H1 N1 GU168288 GU168374 Ramey and 

others, 2010a

49176 H1 N1 GU168289 GU168367 Ramey and 

others, 2010a

100756 H1 N1 GQ411892 GQ411894 Pearce and 

others, 2010

103775 H1 N1 HM193551 HM193628 Pearce and 

others, 2011

44927 H10 N7 GU168309 GU168402 Ramey and 

others,2010a

44939 H10 N7 GU168308 GU168401 Ramey and 

others, 2010a

79190 H10 N9 HM059988 HM060036 Ramey and 

others, 2010b

100870 H10 N1 JF323753 JF323778 Ramey and 

others,2011

134560 H10 N7 JX080780 JX081172 Reeves and 

others, 2013

40459 H11 N9 JX080738 JX081129 Reeves and 

others,2013

101124 H11 N9 JF323755 JF323789 Ramey and 

others, 2011

Isolate HA NA

NCBI 

accession 

number HA

NCBI 

accession 

number NA

Literature cited

103717 H11 N9 HM193587 HM193665 Pearce and 

others, 2011

2355 H2 N1 JX080736 JX081127 Reeves and 

others, 2013

2366 H2 N1 JX080744 JX081135 Reeves and 

others, 2013

2390 H2 N5 JX080748 JX081139 Reeves and 

others, 2013

32425 H2 N2 JX080751 JX081142 Reeves and 

others, 2013

32921 H2 N3 JX080765 JX081157 Reeves and 

others, 2013

13526 H3 N8 HM193560 HM193661 Pearce and 

others, 2011

16080 H3 N8 HM193556 HM193660 Pearce and 

others, 2011

37536 H3 N8 JX080757 JX081148 Reeves and 

others, 2013

44303 H3 N8 GU143843 GU168410 Ramey and 

others, 2010a

45315 H3 N6 GU143852 GU168393 Ramey and 

others, 2010a

45321 H3 N8 GU143850 GU168416 Ramey and 

others, 2010a

104839 H3 N6 HM193566 HM193651 Pearce and 

others, 2011

254 H4 N7 JX080760 JX081151 Reeves and 

others, 2013

15913 H4 N6 HM193578 HM193653 Pearce and 

others, 2011

38800 H4 N6 JX080742 JX081133 Reeves and 

others, 2013

49090 H4 N6 GU168299 GU168395 Ramey and 

others, 2010a

60390 H4 N6 HM193576 HM193652 Pearce and 

others, 2011

103889 H4 N6 HM193574 HM193650 Pearce and 

others, 2011

884 H5 N2 JX080782 JX081174 Reeves and 

others, 2013

926 H5 N2 JX080783 JX081175 Reeves and 

others, 2013

945 H5 N2 JX080784 JX081176 Reeves and 

others, 2013

2216 H5 N2 JX080785 JX081177 Reeves and 

others, 2013

3850 H5 N2 JX080756 JX081147 Reeves and 

others, 2013

13618 H6 N2 FJ520080 FJ520139 Pearce and 

others, 2009

52796 H6 N2 GU168302 GU168376 Ramey and 

others, 2010a

56526 H6 N1 FJ520106 FJ520164 Pearce and 

others, 2009

2320 H7 N7 JX080762 JX081154 Reeves and 

others, 2013
4 A Method for Determining Avian Influenza Virus Hemagglutinin and Neuraminidase Subtype Association

obtained using the National Center for Biotechnology Information (NCBI) nucleotide Basic Local Alignment Search Tool 

(BLAST). The size (bp) of confirmed target amplicons was 

used to generate average amplicon sizes for each HA and NA 

subtype evaluated in this study. 

The copy counts for HA and NA genes in each isolate 

were quantified using quantitative RT-qPCR. Briefly, RT-qPCR 

was run using SYBR Green-chemistry with a standard curve 

for each subtype to calculate the copy count. The RT-qPCR 

reaction mixture and amplification conditions were the same 

as those used in melt curve analysis described above. Standards were created using eight dilutions of the purified amplicons from samples of each subtype (table 1) with calculated 

standard concentrations of 108–101 copies/μL. The relation 

between HA and NA copy counts from each isolate was evaluated by calculating HA:NA ratios.

Corrected molar ratios were determined using the following equations: 

H SAM H AS

N AS

N TM

H TM

N SAM

1 1

1

1

1

1

×



 



 



 



×



 



 



 



 (1)

ABS H SAM H AS

N AS

N TM

H TM

1 N 1

1

1

1

× 2 



 



 



 



×



 



 



 



− SAM H AS

N AS

N TM

H TM

N SAM

×



 



×



 



 



 



 1

2

2

1

1

(2)

and

H SAM H AS

N AS

N TM

H TM

ABS N SA

1 1

1

1

1

1

×



 



 



 



×



 



 



 





M H SAM H AS

N AS

N TM

H TM

− ×



 



×



 



 



 









 



2  2

1

1

2

(3)

where

ABS is absolute value, in nanomoles;

H1SAM or N1SAM is specific amplicon molarity of hemagglutinin or neuraminidase subtype under 

consideration;

H1AS or N1AS is amplification strength as measured by total fluorescence at the end of the terminal 

qPCR cycle of hemagglutinin or neuraminidase subtype under 

consideration;

H1TM or N1TM is total molarity observed for all amplicons, including the subtype-specific amplicon 

from subtype-specific product analysis of hemagglutinin or neuraminidase 

subtype under consideration;

H2SAM or N2SAM is similar to H1SAM or N1SAM, except that values are those used to correct for the 

molarity contribution of subtype not considered in ratio;

H2AS or N2AS is similar to H1AS or N1AS, except that values are those used to correct for the 

molarity contribution of subtype not considered in ratio; and 

H2TM or N2TM is similar to H1TM or N1TM, except that values are those used to correct for the 

molarity contribution of subtype not considered in ratio.
Methods 5

Table 2. Primers used for subtyping hemagglutinin and neuraminidase genes of purified and characterized isolates of avian influenza 

virus reverse transcribed into cDNA from purified RNA and amplified using a real-time reverse transcription polymerase chain reaction.

1

[cDNA, complementary deoxyribonucleic acid; RNA, ribonucleic acid; bp, base pairs; H, identifies hemagglutinin gene; N, identifies neuraminidase gene; 

C, cytosine; G, guanine; A, adenine; T, thymine; B, mixed base C/G/T; D, mixed base A/G/T; H, mixed base A/T/C; K, mixed base G/T; M, mixed base A/C; 

R, mixed base A/G; V, mixed base A/C/G; W, mixed base A/T; Y, mixed base C/T]

Subtype Size (bp) Forward sequence Reverse sequence

H1 305 GATRCWCCAGTYCAYAAYT TCTGYGTGCTYTTYTGRT

H2 343 AAMMCTTGAAAAYTGYGA TTCAATYACRGASTTYACYTTG

H3 376 GYATYACTCCWAATGGAAGC ATYCTYCCTTCYACTTCDGM

H4 284 AGYAARTGYCAYACHGACA AGYAARTGYCAYACHGACA

H5 261 CCARTRGGKGCKATAAAYTC GTCTGCAGCRTAYCCACTYC

H6 387 TCARAYYTDCCAATHGAGAA TCARAYYTDCCAATHGAGAA

H10 311 TCARAYYTDCCAATHGAGAA TCAGATTCTATGGATTCGAAC

N1 245 TCARTCTGYATGRYAAYTGG GGRCARAGAGAKGAATTGCC

N2 278 TYTCTMTAACYATTGCRWCARTA GARTTGTCYTTRGARAAVGG

N6 264 AGGAATGACACTATCSGTAGTAAG GAYAGRATRTGCCATGAGTTYAC

N7 261 TCWGGAGTGGCMATAGCACT CACKACCCAYCCTTCAACWTTG

N8 137 CAYATAGYTAGYATYACARTAAC ACAYTRGYATTGTRCCATTG

N9 227 GTRATAGGCAYRATTGCAGT CCTYTRGTCARRTTRTTGAA

1

Post-amplification product purification was performed using the QIAquick PCR.

The equations for corrected molar ratios were derived 

as modifications of the simple HA:NA ratio. Amplification 

strength was used to correct for the production of non-target 

amplicons, a phenomenon associated with SYBR Greenchemistry (Wittwer and others, 1997) that may have a negative 

effect on final target amplicon concentrations. The use of total 

molarity had a similar function correct for the occurrence of 

non-target amplicons in a size range of 100–1,000 bp. Equations used to calculate corrected molarity when two HA:NA 

subtypes with a shared HA or NA subtype occurred (eqs. 2 and 

3) were developed to account for the molarity of one subtype 

being divided between two other subtypes. The equation 

for situations where 1 NA and 2 HA subtypes are detected 

(eq. 3) is provided, but an example is not presented to avoid 

redundancy.

Target amplicon molarity was taken from the standard 

Agilent 2100 Expert software output. Total sample molarity was calculated by obtaining the sum of molarities from 

each double-stranded DNA sample component that was 

100–1,000 bp in size and within the quantitative range of the 

assay. Amplification strength was determined from qPCR 

output for each reaction by measuring total fluorescence at the 

end of cycle 30. Standardized values of amplification strength 

were determined by dividing 1,000,000 by the reaction-specific fluorescence. In this calculation, amplification strength 

decreases as the standardized values increase. To facilitate 

ease of comparison, corrected molar ratios calculated to be 

between 0.01 and 0.99 were converted to a value greater than 

1 by dividing 1 by the calculated value.

Chicken-egg allantoic fluid containing viable H4:N6 

virus was used to produce a “spiked” pond sediment. Total 
6 A Method for Determining Avian Influenza Virus Hemagglutinin and Neuraminidase Subtype Association

RNA was then extracted using a RNA PowerSoil Total RNA 

Isolation Kit (Mo Bio) and following the manufacture’s 

protocol. Initial virus concentrations were in an expected 

range of 105–107 virus per milliliter (mL-1) of allantoic fluid 

(Hon S. Ip, USGS, written commun., 2016). Sediment was 

obtained from a pond used by overwintering waterfowl and 

was a mix of silt, sand, and vegetation. Two spiked sediment 

samples and one negative control sediment sample were 

evaluated. Each sample and the negative control contained 

2.00 ± 0.05 grams (g) of sediment. Total spike volume was 

1 mL. The final concentration of the first spike sample was in 

the range of 105–107 virus per g-1 of sediment. The second 

spiked sample contained a 1:10 dilution of the first spike with 

the dilution performed in RNAase- and DNAase-free molecular grade water (NF water). The negative control sample was 

treated with 1 mL of NF water only. Both alcohol precipitation 

steps used in the RNA extraction process were performed for 

30 minutes at −20 °C. Conversion of extracted RNA to cDNA, 

RT-qPCR amplification, and analysis of PCR products using 

the Bioanalyzer were performed as described above.

Table 3. Sanger subtype-specific sequence data obtained for avian influenza virus hemagglutinin and neuraminidase subtype target 

amplicons from this study.

1

[H, identifies hemagglutinin gene; N, identifies neuraminidase gene; C, cytosine; G, guanine; A, adenine; T, thymine]

Subtype1 Isolate 

identification1 Sequence

H1 3455

GCAACACAAAGTGCCAAACCCCTCACGGGGCCTTGAACAGTAGCCTCCTTTTCAGAATGTGCATCCCATCAC

TATTGGAGAATGTCCCAAATATGTCAAGAGCACCAAACTAAGAATGGCAACAGGACTAAGAAATGTACCATC

CATTCAATCCAGAGGACTATTTGGAGCAATTGCTGGATTCATTGAAGGAGGATGGACAGGTATGATAGATGGATG

GTATGGGTACCACCACCAGAATGAGCAAGGATCAGGATATGCCGCTGACCA

H2 2390

TTGAAAATTGCGAAACTAAATGCCAAACTCCCTTGGGAGCAATAAATACAACATTGCCCTTTCATAATATC

CACCCATTGACCATTGGTGAGTGCCCCAAATATGTAAAATCGGAGAGATTGGTCTTGGCAACAGGATTAAGAAAC

GTCCCTCAGATTGAATCAAGAGGATTGTTTGGGGCAATAGCTGGTTTCATAGAAGGGGGATGGCAAGGAATGGTT

GATGGTTGGTATGGATATCATCACAGCAATGATCAAGGATCCGGGTATGCAGCAGACAAAGAGTCCACT

CAGAAGGCAATTGATGGAATCACC

H3 13526

GCATCCCCAATGACAAGCCCTTTCAGAATGTGAACAAGATCACCTACGGAGCATGTCCTAAGTATGTCAAA

CAGAGCACTCTGAAACTGGCAACAGGAATGAGGAACGTACCTGAGAAACAAACCAGGGGTCTGTTCGGTG

CAATAGCAGGATTTATAGAAAATGGGTGGGAAGGAATGATAGACGGCTGGTATGGTTTCAGACATCAGAACTCT

GAGGGTACAGGACAAGCTGCAGACCTAAAAAGCACACAGGCAGCCATCGACCAGATCAATGGGAAATTGAACC

GAGTAATCGAGAAAACAAATGAGAAGTTTCATCAGATTGAAAAGGAATT

H4 15913

GACAGGGGTTCAATCACTACCACTAAACCCTTTCAGAACATCTCAAGAATATCAATCGGGGACTGTCCCAAATAT

GTCAAACAGGGGTCTTTGAAACTGGCTACAGGGATGAGGAACATCCCTGAGAAGGCAACCAGAGGTCTGTTTG

GTGCAATTGCTGGCTTTATAGAGAATGGCTGGCAAGGTTTAATTGATGGGTGGTATGGGTTTAGGCACCAGAATG

CAGAAGGGACAGGGACGGCTGCAGATCTC

H4

Intact viron 

sediment 

spike

ACCGACAGGGGTTCAATCACTACCACTAAACCCTTTCAGAACATCTCAAGAATATCAATCGGGGACT

GTCCCAAATATGTCAAACAGGGGTCTTTGAGACTGGCTACAGGGATGAGGAACATTCCTGAGAAGGCAAC

CAGAGGTCTGTTTGGTGCGATTGCTGGCTTTATAGAGAATGGCTGGCAAGGTTTAATTGATGGGTGGTATGGGTT

TAGGCACCAGAATGCAGAAGGGACAGGAACGGCTGCAGATCTCAAATCCACTCAGGCAGC

H5 926

ATTGGGGAGTGCCCCAAGTATGTCNAATCGGACAAACTGGTCCTTGCAACAGGACTAAGAAACG

TACCCCAAAGAGAAACAAGAGGCCTATTTGGAGCAATAGCAGGATTCATAGAAGGAGGATGGCAAGGAATGGTT

GATGGATGGTACGGATACCATCATAGCAATGAGCA

H6 52796

CCAATCGAGAACTGTGATGCCACATGCCAGACTATTGCAGGAGTCCTAAGGACTAATAAAACATTTCAGAATGT

GAGTCCTCTGTGGATAGGAGAATGCCCCAAATATGTGAAAAGTGAAAGTTTGAGGCTTGCAACTGGACTGAGA

AATGTTCCACAGATTGAGACTAGAGGACTTTTTGGAGCTATCGCAGGATTTATTGAAGGGGGATGGACTGGAAT

GATAGATGGGTGGTACGGCTATCACCATGAAAATTCTCAAGGCTCAGGATATGCAGCAGACAAAGAAAGCACT

CAAAAGGCTATAGACGGAATTACAAATAAAGTCAATTCCATCATTGACAAAATGAACACACAATTCGAAGCT

GTNCNACCACGAA

H10 134560

AATCTTTCCCCGAGAACTGTGGGTCAATGCCCCAAGTATGTGAACAAAAAGAGCCTGTTGCTTGCTACTG

GAATGAGGAATGTGCCAGAGGTTGTCCAAGGAAGAGGCCTGTTTGGAGCAATAGCTGGATTCATAGAGAATG

GATGGGAAGGAATGGTAGATGGTTGGTATGGTTTCCGACACCAAAATGCCCAAGGCACTGGCCAGGCCGCG

GATTATAAAAGTACTCAGGCAGCTATAGATCAAATAACCGGGAAATTGAACAGACTGATAGAGAAGACAAACA

CAGAGTTCGAATCCATAGAATCTGAA

N1 48982

TGGAATAATAAGTCTGGTGCTACAAANTGGAAATATAATTTCAATATGGGTTAGCCATTCAATCCAAACTGGAAGT

CAGAACTATCCCGAAACATGCAATCAAAGTGTCATTACCTACGAAAACAATACTTGGGTGAATCAGACATACAT

CAACATAAGTAATACCAATTTAATTTCAGAACAGGCTGTAGCTCCAGTAACACTAGCAGGCAATTCCTCTCTCT

N2 52796

CANTATGTTTCCTCATGCAAATTGCCATCCTAGCAACGACTATAACACTGCACTTCAAGCAGAATGAATGCAT

CATCCCCTCGAACAATCAAGTAGTGCCATGTGAGCCAATCATAGTAGAAAGGAACATAACAGAGATAGTGTATT

TAAATAATACCACCATAGAAAGAGAACTTTGTCCTAANTTAACAGANATACAGGGATTGGTCGAAACCACAGTGT

CAGATC
Methods 7

Table 3. Sanger subtype-specific sequence data obtained for avian influenza virus hemagglutinin and neuraminidase subtype target 

amplicons from this study.1—Continued

[H, identifies hemagglutinin gene; N, identifies neuraminidase gene; C, cytosine; G, guanine; A, adenine; T, thymine]

Subtype1 Isolate 

identification1 Sequence

N6 60390

GGGGCTGAACATTGGGCTTCATTTCAAGGTAGGAGACACACCAGAACCAGGACCCCCTAGTACCAATGAGA

CAAACTCCACAACCGCAATAATCAATTACAACACCCAAAACAACTTCACAAATGTGACCAACATTGTGTTGATTA

AAGAAGAAAACAAAATGTTCACNANCCTTTCTAAGCCCTTGTGTGAAGTN

N6

Intact viron 

sediment 

spike

GCTGAACATTGGGCTTCATTTCAAGGTAGGAGACACACCAGAAACAGGAACCCCTAGTACCAATGAGA

CAAACTCCACAACCACAATAATCAACTACAACACCCAAAACAACTTCACAAATGTGACCAACATTGTGTTAATTA

AAAAAGAAAACAAAATGTTCACAAACCTTTCTAAGCCCTTGTGTGAAGTGAACTCATGGCACATCCT

N7 134560

TGGCCATAGCACTGAGTATCCTCAACCTACTAATAGGAATATCCAATGTGGGACTGAATGTCTCACTACACCTA

AAGGGAAGCAGTAATCAGGATAGAAATTGGACATGCACGAGTGTAACACAAAACAATACGACTTTAATTGAAAA

CACATATGTCAACAATACTACTGTCATCAATAAGGAAACAGGGACTGCAAAGCCAAATTATCTAATGCTGAA

CAAGAGCTTATGC

N8 13526 ATAGTTAGCATTACAGTAACAGTGTTAGTTCTCCCTGGAAATGGAAATAATGGGGGTTGCAATGAANCAGT

CATTAGGGAATACAATGAAACAGTAAGAGG-TTGAGAAGG-TAACACAATTTACCAATGTC

N9 10124

GCAGTACTCATAGGAATAGCAAACCTAGGGTTGAACATAGGGCTACATCTGAAACCAAACTGCAACTGCT

CACACTCACAACCTGAAGCAACCAATGCAAGCCAAACAATAATAAACAACTACTATAACGAAACAAACAT

CACCCAAATAAGCAATACCAACATCCAAATGGAGGAGAAAGCAAATAGAGAATT

1

Isolates used in this study. For additional information see table 1.

Table 4. Representative nucleotide BLAST data for Sanger sequences obtained for avian influenza virus hemagglutinin and 

neuraminidase subtype target amplicons from this study.

[BLAST, Basic Local Alignment Search Tool; H, identifies hemagglutinin gene; N, identifies neuraminidase gene; NCBI, National Center for 

Biotechnology Information]

Subtype1 Isolate 

identification1

NCBI accession 

number for 

published data1

BLAST metrics

Sequence 

range

Score Query 

cover

Expected 

(E value) Identities Gaps

H1 3455 HM059985 885−1,152 (268) 496 98% 2E−144 100% 0%

H2 2390 JX080748 871−1,182 (312) 577 99% 9E−169 100% 0%

H3 13526 HM193560 913−1,249 (337) 623 100% 0.0 100% 0%

H4 15913 HM193578 888−1,139 (252) 466 100% 2E−135 100% 0%

H4 Intact virus

sediment spike CY076205.1 896−1,167 (272) 492 99% 2E−135 99% 0%

H5 926 JX080783 914−1,086 (173) 316 100% 1E−90 99% 0%

H6 52796 GU168302 835−1,212 (378) 673 100% 0.0 99% 0%

H10 134560 JX080780 908−1,224 (317) 575 99% 1E−166 99% 0%

N1 48982 GU168374 18−259 (230) 425 99% 6E−123 98% 0%

N2 52796 GU168376 29−252 (219) 405 98% 2E−117 99% 0%

N6 60390 HM193652 84−278 (191) 353 99% 8E−102 99% 0%

N6 Intact virus

sediment spike CY076191.1 96−302 (207) 377 98% 5E−11 99% 0%

N7 134560 JX081172 20−252 (233) 431 100% 4E−125 100% 0%

N8 13526 HM193661 72−206 (135) 241 100% 4E−68 99% 0%

N9 10124 JF323789 75−268 (194) 359 100% 2E−103 100% 0%

1

Isolates used in this study. For additional information see table 1.
8 A Method for Determining Avian Influenza Virus Hemagglutinin and Neuraminidase Subtype Association

Results and Discussion

Development of these assays was intended to be applied 

as a post hoc analysis of the Tsukamoto and others (2012) 

melt curve method. The use of RT-qPCR and molar ratios as 

described here is strictly intended as a component in the characterization of environmental samples that are evaluated using 

molecular methods only. Fragment size analysis provides 

confirmation of amplicon size and an opportunity to determine 

molar ratios in the presence of multiple HA:NA subtypes in a 

single sample. AIV genomic RNA from egg culture was used 

to validate the suggested method. 

Subtype-specific sequence data (table 3) for potential 

target amplicons were consistent with the published data for 

the isolates from which the amplicons were produced (table 4). 

Isolate specific copy count HA:NA ratios 

obtained from HA and NA subtype RT-qPCR were 

1.146 ± 0.124 (mean ± standard deviation). Ratio values ranged from 1.028 to 1.509. Statistical metrics for the 

RT-qPCR, including amplification efficiency (table 5) and 

isolate specific subtype copy counts (table 6), are provided. 

Electropherograms for HA (fig. 1) and NA (fig. 2), including 

amplicon size and molarity values, are typical of Bioanalyzer 

DNA 1000 chip output when evaluated using Agilent 2100 

Expert software to compare amplicons with the included 

standard ladder. Based on the ladder as the sample, the Agilent 

DNA 1000 performance metrics are as follows (Agilent Technologies): sizing accuracy of ± 10 percent, a sizing reproducibility of 5-percent cumulative variance (CV), quantitation 

accuracy of 20 percent CV, and quantitation reproducibility 

Figure 1. Typical electropherograms hemagglutinin subtypes evaluated in this study as produced by Agilent’s Bioanalyzer DNA 

1000 and Agilent’s 2100 Expert software. Analyses produced from purified and characterized isolates reverse transcribed into 

deoxyribonucleic acid from purified ribonucleic acid, amplified by reverse transcription quantitative polymerase chain reaction, and 

then evaluated for amplicon size and molarity. Subtype identification is associated with each graph.

(bp, base pair; FU, fluorescent unit; H, identifies hemagglutinin gene; IM, internal marker; TA, subtype-specific target amplicon)
Results and Discussion 9

Table 5. Quantitative real-time reverse transcription polymerase chain reaction linear regression statistics for hemagglutinin and 

neuraminidase gene standards used in the assessment of gene copy counts present in the ribonucleic acid from avian influenza virus 

used in this study.

[r2, coefficient of determination; H, identifies hemagglutinin gene; N, identifies neuraminidase gene]

Subtype Slope y-intercept r2 Percent amplification efficiency Error

H1 −3.818 41.829 1 82.778 0.016

H2 −3.442 35.125 0.996 95.968 0.043

H3 −3.831 38.908 0.996 82.402 0.055

H4 −3.644 40.997 0.997 88.102 0.054

H5 −3.648 39.442 0.992 87.998 0.068

H6 −3.711 37.791 0.997 85.972 0.045

H10 −3.427 37.992 0.998 95.798 0.034

N1 −3.37 38.073 0.984 98.036 0.121

N2 −4.326 45.098 0.992 70.27 0.09

N6 −3.334 36.952 0.992 99.499 0.062

N7 −0.354 36.199 0.997 92.91 0.045

N8 −3.872 41.275 0.999 81.255 0.019

N9 −3.822 39.307 0.998 82.645 0.036

Table 6. Copy count of hemagglutinin and neuraminidase genes obtained from gene copy counts present in the ribonucleic acid from 

avian influenza virus isolates used in this study, as determined by quantitative real-time reverse transcription polymerase chain reaction.

[H, identifies hemagglutinin gene; N, identifies neuraminidase gene]

Subtype Isolate Mean copy count

H4 254 27.932

N7 254 24.355

H5 884 22.982

N2 884 29.947

H5 926 28.264

N2 926 36.872

H5 945 24.908

N2 945 34.225

H5 2216 28.16

N2 2216 37.395

N7 2320 24.11

H2 2355 28.721

N1 2355 26.394

H2 2390 28.883

H1 3455 25.76

N9 3455 24.175

H5 3850 25.349

N2 3850 32.762

H3 13526 30.406

N8 13526 27.095

H6 13618 31.489

N2 13618 29.411

H4 15913 27.092

N6 15913 25.085

H3 16080 26.392

N8 16080 39.83

H2 32425 32.006

N2 32425 29.052

H2 32921 28.306

H3 37536 29.7

N8 37536 26.286

H4 38800 24.417

N6 38800 23.757

H10 40239 28.224

N2 40239 36.346

N9 40459 26.834

Subtype Isolate Mean copy count

H3 44303 30.933

N7 44927 21.845

H10 44939 23.305

N7 44939 21.544

H3 45315 30.857

N6 45315 23.682

H3 45321 28.468

N8 45321 30.8

H1 48944 27.995

N1 48944 26.777

H1 48982 28.418

N1 48982 26.717

H4 49090 28.631

N6 49090 26.112

H1 49176 28.666

N1 49176 26.861

H6 52796 37.785

N2 52796 34.502

H6 56526 33.872

N1 56526 26.071

H4 60390 23.333

N6 60390 22.483

H10 79190 21.526

N9 79190 24.475

H1 100756 28.669

N1 100870 26.675

N9 101124 23.064

N9 103717 22.247

H1 103775 27.775

N1 103775 26.941

H4 103889 23.022

N6 103889 22.115

H3 104839 28.72

N6 104839 23.809

H10 134560 26.822

N7 134560 25.584
10 A Method for Determining Avian Influenza Virus Hemagglutinin and Neuraminidase Subtype Association

of 15 percent CV within the 25–500 bp range. Reactions 

involving a single HA:NA subtype produced a single amplicon in most of the amplifications. In reactions that did exhibit 

multiple amplicons, the products were less than 100 bp in size 

and thus outside the expected range for the desired amplification products.

Results of analysis of amplicon size indicate that the primary product of reactions involving a single HA:NA grouping 

and targeting a single HA or NA subtype could vary considerably from published values (table 7; Tsukamoto and others, 

2012). Target amplicons that varied from published values by 

more than 10 bp were all smaller than the published values 

and were mostly HA subtypes, except for N6. Departures from 

published values tended to increase with increasing amplicon size (data not shown). Calculated standard deviations of 

amplicon sizes were all low, ranging from 1 to 3.

Corrected molar ratios (eq. 1) obtained for 36 isolates 

from 13 HA:NA subtypes had a mean ± standard deviation (SD) of 1.63 ± 0.75. Corrected molar ratio calculations 

produced values (table 8) closer to theoretical perfect value of 

1 relative to the simple HA:NA ratio for 22 of the 32 isolates 

with an improvement toward 1 of 4.50 ± 7.70 (mean ± SD) 

relative to the simple HA:NA ratio. In the 10 cases where 

the simple ratio produced values closer to the ideal value 

of 1, improvement relative to the corrected molar ratio was 

0.30 ± 0.36. In HA:NA subtypes for which there were at least 

four isolates analyzed, corrected molar ratio mean ± standard deviation values were as follows: H1:N1 1.74 ± 0.13, 

H3:N8 1.41 ± 0.42, H4:N6 1.12 ± 0.07, H5:N2 2.67 ± 1.15.

In mixed samples containing two HA:NA types (fig. 3), 

the lowest corrected molar ratios (eq. 2) created subtype 

associations of H3:N8 and H4:N6 (table 9), which accurately 

reflect the types included in the equation. The H4:N6 combination exhibited the lowest corrected molar ratio (1.09). The 

H3:N8 combination had a lower ratio (2.03) than the alternative H3:N6 (2.13), indicating that, from an H3 perspective, 

H3:N8 would be a more viable combination. Pairings are 

based on the closest fit to the ideal value of 1.00. The combinations of H3:N8 and H4:N6 provide the closest fits for 

the two HA:NA pairing. Single-size appropriate amplicons 

were recognized in each of these reactions. The corrected 

molar ratio for H4:N6 was very close to the mean value for 

H4:N6 isolates evaluated as a single HA:NA subtype. The 

actual isolate used in this mix, isolate 49090, (table 8) exhibited a corrected molar ratio 0.05 less when run as a single 

HA:NA subtype compared to the value obtained when the 

Figure 2. Typical electropherograms of neuraminidase subtypes evaluated in this study as produced by Agilent’s Bioanalyzer 

DNA 1000 and Agilent’s 2100 Expert software. Analyses produced from purified and characterized isolates reverse transcribed into 

deoxyribonucleic acid from purified ribonucleic acid, amplified by reverse transcription quantitative polymerase chain reaction, and 

then evaluated for amplicon size and molarity. Subtype identification is associated with each graph.

(bp, base pair; FU, fluorescent unit; N, identifies neuraminidase gene; IM, internal marker; TA, subtype-specific target amplicon)
Results and Discussion 11

Table 7. Specific amplicon size determined for purified and characterized isolates of avian influenza virus reverse transcribed into 

complementary deoxyribonucleic acid from purified ribonucleic acid.

[SD, standard deviation; bp, base pair]

Subtype Number of 

isolates

Mean ± 

SD amplicon 

size (bp)1

Departure from 

published value 

(bp)1, 2

Hemagglutinin

H1 6 299±1 −6

H2 5 327±2 −16

H3 6 358±2 −17

H4 6 275±1 −10

H5 5 246±1 −15

H6 2 3

361, 365 −24

H10 6 298±3 −13

Subtype Number of 

isolates

Mean ± 

SD amplicon 

size (bp)1

Departure from 

published value 

(bp)1, 2

Neuraminidase

N1 9 239±1 −6

N2 8 269±2 −9

N6 6 252±2 −11

N7 5 260±3 −1

N8 5 137±1 0

N9 4 219±1 −8

1

Value rounded to nearest whole number. Actual amplicon sizes provided for H6.

2

From Tsukamoto and others, 2012; based on amplicon mean size.

3

Mean and standard deviation not determined; 363 used in applicable comparisons.

Table 8. Corrected HA:NA subtype-specific molar ratios determined for amplicons from purified and characterized isolates of avian 

influenza virus reverse transcribed into cDNA from purified RNA.

[cDNA, complementary deoxyribonucleic acid; RNA, ribonucleic acid; HA or H, hemagglutinin; NA or N, neuraminidase]

HA:NA subtype Isolate identification1 Corrected molar ratio2

H1:N1 48944 1.75

H1:N1 48982 1.81

H1:N1 49176 1.75

H1:N1 100756 1.86

H1:N1 103775 1.52

H1:N1 48944 1.75

H1:N1 48982 1.81

H1:N9 3455 1.09

H2:N1 2355 1.05

H2:N1 2366 1.17

H3:N6 45315 2.57

H3:N6 104839 1.13

H3:N8 13526 1.11

H3:N8 16080 1.21

H3:N8 37536 1.28

H3:N8 45321 2.03

H4:N6 15913 1.21

H4:N6 38800 1.14

HA:NA subtype Isolate identification1 Corrected molar ratio2

H4:N6 49090 1.04

H4:N6 60390 1.06

H4:N6 103889 1.15

H4:N7 254 1.54

H5:N2 884 2.38

H5:N2 926 2.02

H5:N2 945 1.34

H5:N2 2216 4.23

H5:N2 3850 3.47

H5:N2 884 2.38

H5:N2 926 2.02

H6:N1 56526 1.33

H6:N2 13618 1.3

H10:N1 100870 1.3

H10:N7 44927 1.19

H10:N7 44939 1.26

H10:N7 134560 2.77

H10:N9 79190 1.02

1

For additional information see table 1.

2

To facilitate ease of comparison, corrected molar ratios calculated to be between 0.01 and 0.99 were converted to a value greater than one as follows: 

1/calculated value.

Table 9. Results from a mixed sample containing two avian influenza virus HA:NA subtypes. Mix produced from purified ribonucleic 

acid extracted from characterized isolates of avian influenza virus which was reverse transcribed into deoxyribonucleic acid, amplified 

by quantitative polymerase chain reaction, and then evaluated for amplicon size and molarity.

[bp, base pair; nmol/L, nanomoles per liter; FU, fluorescent unit; HA:NA, hemagglutinin:neuraminidase; H, identifies hemagglutinin gene; N, identifies 

neuraminidase gene]

Subtype

Predicted 

fragment 

size (bp)

Observed 

fragment 

size (bp)

Observed 

molarity 

(nmol/L)

Amplification 

strength [FU]

Total 

molarity 

(nmol/L)

Potential 

HA:NA 

subtype

Corrected 

molar 

ratio1

H3 358 357 7.5 4.83 7.5 H3:N6 2.13

H4 275 277 26.4 1.92 26.4 H3:N8 2.03

N6 252 258 26.5 2.09 26.5 H4:N6 1.09

N8 137 135 21.4 12.70 27.6 H4:N8 1.23

1

Calculated as per equation 2.
12 A Method for Determining Avian Influenza Virus Hemagglutinin and Neuraminidase Subtype Association

isolate was part of this mixture. The H3:N8 ratio was outside 

one single standard deviation for isolates of this HA:NA subtype but was identical to the value obtained from the isolate 

45321, which was used in the mixture (table 8).

A second mixed sample containing two HA:NA subtypes 

was evaluated (fig. 4). In this mix, only three subtypes were 

present (H4, N6, N7), requiring the use of a different equation for calculating corrected molar ratios (eq. 3). Here, where 

H4 was the only HA subtype, the corrected molar ratios with 

N6 and N7 (table 10) were within one standard deviation of 

the mean from the 31 isolates evaluated individually. The 

H4 and N7 subtype-specific amplifications produced multiple 

amplicons with a larger contribution of these additional products to total molarity observed for H4. The H4:N6 corrected 

ratio was outside one standard deviation of ratios obtained for 

H4:N6 isolates evaluated in isolation. The corrected ratio for 

Figure 3. Electropherograms of hemagglutinin and neuraminidase subtypes from a mixed sample containing two avian influenza 

virus HA:NA subtypes. Analytes produced from purified and characterized isolates reverse transcribed into deoxyribonucleic acid 

from purified ribonucleic acid, amplified by quantitative polymerase chain reaction, and then evaluated for amplicon size and molarity. 

Subtype identification is associated with each graph.

(HA:NA, hemagglutinin: neuraminidase; bp, base pair; FU, fluorescent unit; H, identifies hemagglutinin gene; N, identifies neuraminidase gene; IM, internal 

marker; TA, subtype-specific target amplicon)

Figure 4. Electropherograms of hemagglutinin and neuraminidase subtypes from a mixed sample containing two avian influenza virus 

HA:NA subtypes with a shared hemagglutinin subtype. Analytes produced from purified and characterized isolates reverse transcribed 

into deoxyribonucleic acic from purified ribonucleic acid, amplified by quantitative polymerase chain reaction, and then evaluated for 

amplicon size and molarity. Subtype identification is associated with each graph.

(HA:NA, hemagglutinin: neuraminidase; FU, fluorescent unit; H, identifies hemagglutinin gene; N, identifies neuraminidase gene; TA, subtype-specific target 

amplicon; IM, internal marker; bp, base pair)
Results and Discussion 13

H4:N7 was slightly lower than the ratio obtained when this 

HA:NA subtype was evaluated alone (table 8).

To evaluate the efficacy of the method as it relates to 

environmental samples, we spiked sediment acquired from a 

pond used by migratory waterfowl using intact virions of the 

H4:N6 subtype. Our intent here was to simulate actual methods that would be used in environmental surveillance. Analysis of amplified products for H4 and N6 revealed single products in the 100–1,000 bp range (fig. 5). Amplification strengths 

for H4 and N6 were 1.71 and 2.19, respectively, which are 

consistent with amplification strength for these subtypes when 

evaluated directly from allantoic fluid (tables 9 and 10). Molar 

ratios determined for the spiked H4:N6 were 1.28 (corrected) 

and 1.14 (not corrected). These values are like those obtained 

for H4:N6 from allantoic fluid (table 8), indicating that the 

inclusion of an environmentally relevant substrate had little 

effect on analysis outcome. The identities of the H4 and N6 

subtypes were confirmed by sequencing (tables 3 and 4).

As indicated in process results, the use of RT-qPCR was 

more effective at establishing ratios closer to the ideal value of 

1, as shown by the lower ratio mean ± standard deviation values obtained from the copy count, relative to those produced 

using corrected molar ratios. Further, the use of amplification 

efficiency provides a simpler means of ratio correction for 

AIV RNA obtained from egg cultures.

The relatively high concentrations of RNA from other 

organisms obtained from direct extraction of environmental 

samples and the potential variation in the concentration and 

sequence of this RNA complicates the determination of amplification efficiency by essentially forcing an efficiency determination for each HA and NA subtype in each sample. The 

use of molar ratios was an attempt to circumvent this issue. 

Correction of molar ratios from the simple HA:NA equation 

was necessary because of relative differences in the observed 

degree of amplification (matrix effect) and because of the production of multiple non-specific products in some reactions. 

The requirement for determining some measure of amplification efficiency was not eliminated using molarity measures.

Our objective was to evaluate the use of molarity in 

AIV HA:NA subtype grouping, using robust conditions while 

still using purified and fully characterized isolates to facilitate methods development. Our final goal was to provide a 

method application for processes in which RNA extractions 

are performed directly from samples without intervening viral 

proliferation steps. The difference in sequence-confirmed 

target amplicon size relative to those reported by Tsukamoto 

and others (2012) contributed to the potential variation 

Table 10. Results from a mixed sample containing two avian influenza virus HA:NA subtypes sharing a single hemagglutinin subtype. 

Mix produced from purified ribonucleic acid extracted from characterized isolates of avian influenza virus which was reverse 

transcribed into deoxyribonucleic acid, amplified by quantitative polymerase chain reaction, and then evaluated for amplicon size and 

molarity.

[bp, base pair; nmol/L, nanomoles per liter; FU, fluorescent unit; HA:NA, hemagglutinin:neuraminidase; H, identifies hemagglutinin gene; N, identifies 

neuraminidase gene; --, no additional information intended]

Subtype

Predicted 

fragment 

size (bp)

Observed 

fragment 

size (bp)

Observed 

molarity 

(nmol/L)

Amplification 

strength [FU]

Total 

molarity 

(nmol/L)

Potential 

HA:NA 

subtype

Corrected 

molar 

ratio1

H4 275 267 47.8 2.15 51.5 H4:N6 2.04

N6 252 248 32.9 2.32 32.9 H4:N7 1.21

N7 260 254 14.5 4.04 15.0 -- --

1

Calculated using equation 3.

Figure 5. Electropherograms of hemagglutinin and neuraminidase subtypes from pond sediment spiked with intact virus of the H4:N6 

avian influenza virus subtype. Analytes produced from reverse transcribed deoxyribonucleic acid from purified virus ribonucleic 

acid, amplified by quantitative polymerase chain reaction, and then evaluated for amplicon size and molarity. Subtype identification is 

associated with each graph.

(bp, base pair; FU, fluorescent unit; H, identifies hemagglutinin gene; N, identifies neuraminidase gene; TA, subtype-specific target amplicon; IM, internal 

marker; bp, base pair)
14 A Method for Determining Avian Influenza Virus Hemagglutinin and Neuraminidase Subtype Association

that was encountered. The isolates used by Tsukamoto and 

others (2012) were not evaluated, and a different type of 

qPCR instrument and different reagents were used. The reason 

for the observed difference in amplicon size was not determined. We can, however, point out that even when all other 

things are equal, there can be variation in amplicon size within 

a subtype (table 7) that supersedes known instrument accuracy and reproducibility tolerances. Amplicon size is directly 

related to molarity and thus to corrected molarity ratios.

Simple HA:NA copy ratios produced from RT-qPCR and 

molarity can accurately associate AIV HA and NA subtypes 

into HA:NA groupings. Isolates from some HA:NA subtypes 

can yield ratios that approach the theoretically perfect value 

of 1.00, whereas isolates from other HA:NA subtypes consistently produce higher ratios, indicating the presence of 

HA:NA subtype isolate dependency. Further, the same or 

very similar ratios can be obtained from isolates whether they 

are analyzed individually or in an amalgam. The method is 

not perfect in that we could not account for the increase in 

corrected molar ratios involving the H4:N6 subtype in mixed 

samples. Some primer sets used in this study clearly produce 

amplicons besides those intended, as seen in the electropherograms and in total product molarity. The current equations 

(eqs. 1–3) take such products into account but do not completely compensate for their effects on the ratio outcome. 

Despite the imperfections, the method is sufficiently robust 

to be applicable to complex samples, such as pond sediment. When selecting the RT-qPCR and molarity approaches 

to group HA and NA subtypes into HA:NA subtypes, we 

recognized that the same objectives can be accomplished 

with next generation sequencing technologies, particularly 

using amplicon resequencing. Such an approach has a distinct 

advantage in that sequence data are generated directly, thus 

avoiding the need for follow-up Sanger sequencing to confirm 

amplicon identity. The results obtained in this study, while 

promising, are limited in scope and do not completely address 

the full spectrum of potential issues involved with the association of HA:NA subtypes. Future methods development for the 

identification of HA:NA subtypes would likely benefit from 

the inclusion of next-generation sequencing technologies to 

accelerate the development process and further characterize 

process requirements.

Acknowledgments

We extend our sincere appreciation to Dr. Andy Ramey 

of the USGS Alaska Science Center for providing us with the 

avian influenza virus (AIV)-isolate ribonucleic acid (RNA) 

used in this study and to Dr. Hon Ip of the USGS National 

Wildlife Health Center for the isolated virus he provided to 

Dr. Ramey, making our receipt of the RNA possible. We also 

thank Dr. Ip for the intact AIV used in our sediment spike 

experiment.

References Cited 

Bowman, A.S., Nolting, J.M., Massengill, R., Baker, J., 

Workman, J.D., and Slemons, R.D., 2015, Influenza A virus 

surveillance in waterfowl in Missouri, USA, 2005–2013: 

Avian Diseases, v. 59, p. 303–308. [Also available at 

https://doi.org/10.1637/11002-121014-Reg.]

Cheung, T.K.W., and. Poon, L.L.M., 2007, Biology of influenza A virus, in Lal, S.K., ed., Biology of emerging viruses: 

SARS, Avian and human influenza, metapneumovirus, 

Nipah, West Nile, and Ross River Virus: Annals of the New 

York Academy Science, v. 1102, p. 1–25.

Lang, A.S., Kelly, A., and Runstadler, J.A., 2008, Prevalence 

and diversity of avian influenza viruses in environmental 

reservoirs: Journal of General Virology, v. 89, p. 509–519. 

[Also available at https://doi.org/10.1099/vir.0.83369-0.]

Machalaba, C.C., Elwood, S.E., Forcella, S., Smith, K.M., 

Hamilton, K., Jebara, K.B., Swayne, D.E., Webby, R.J., 

Mumford, E., Mazet, J.A.K., Gaidet, N., Daszak, P., and 

Karesh, W.B., 2015, Global avian influenza surveillance in 

wild birds: A strategy to capture viral Diversity: Emerging 

Infectious Diseases, v. 21, p. e1–e7, accessed January 25, 

2018, at https://wwwnc.cdc.gov/eid/article/21/4/14-1415_

article. 

Payungporn, S., Chutinimitkul, S., Chaisingh, A., 

Damrongwantanapokin, S., Buranathai, C., Amonsin, A., 

Theamboonlers, A., and Poovorawan, Y., 2006, Single 

step mutilplex real-time RT-PCR for H5N1 influenza A 

virus detection: Journal of Virological Methods, v. 131, 

p. 143–147. [Also available at https://doi.org/10.1016/j.

jviromet.2005.08.004.]

Pearce, J.M., Ramey, A.M., Flint, P.L., Koehler, A.V., Fleskes, 

J.P., Franson, J.C., Hall J.S., Derksen, D.V., and Ip, H.S., 

2009, Avian influenza at both ends of a migratory flyway: 

Characterizing viral genomic diversity to optimize surveillance plans for North America: Evolutionary Applications, 

v. 2, p. 457–468. [Also available at https://doi.org/10.1111/

j.1752-4571.2009.00071.x.]

Pearce, J.M., Ramey, A.M., Ip, H.S., and Gill R.E., 2010, 

Limited evidence of trans-hemispheric movement of avian 

influenza viruses among contemporary North American 

shorebird isolates: Virus Research, v. 148, p. 44–50. [Also 

available https://doi.org/10.1016/j.virusres.2009.12.002.]

Pearce, J.M., Reeves, A.B., Ramey, A.M., Hupp, J.W., Ip, 

H.S., Bertram, M., Petrula, M.J., Scotton, B.D., Trust, B., 

Meixell, K.A., and Runstadler, J.A., 2011 Interspecific 

exchange of avian influenza virus genes in Alaska: The 

influence of trans-hemispheric migratory tendency and 

breeding ground sympatry: Molecular Ecology, v. 20, 

p. 1015–1025. [Also available at https://doi.org/10.1111/

j.1365-294X.2010.04908.x.]
References Cited 15

Ramey, A.M., Pearce, J.M., Ely, C.R., Sheffield Guy, L.M., 

Irons, D.B., Derksen, D.V., and Ip, H.S., 2010a, Trans-

mission and reassortment of avian influenza viruses at 

the Asian—North American interface: Virology, v. 406, 

p. 352–359. [Also available at https://doi.org/10.1016/j.

virol.2010.07.031.]

Ramey, A.M., Pearce, J.M., Flint, P.L., Ip, H.S., Derksen, 

D.V., Franson, J.C., Petrula, M.J., Scotton, B.D., Sowl, 

K.M., Wege, M.L., and Trust, K.A., 2010b, Intercontinental 

reassortment and genomic variation of low pathogenic avian 

influenza viruses isolated from Northern Pintails (Anas 

acuta) in Alaska: Examining the evidence through space 

and time: Virology, v. 401, p.179–189.

Ramey, A.M., Pearce, J.M., Reeves, A.B., Franson, J.C., 

Petersen, M.R., and Ip, H.S., 2011, Evidence for limited 

exchange of avian influenza viruses between seaducks 

and dabbling ducks at Alaska Peninsula coastal lagoons: 

Archives of Virology, v. 156, p. 1813–1821. [Also available 

at https://doi.org/10.1007/s00705-011-1059-z.]

Reeves, A.B., Pearce, J.M., Ramey, A.M., Ely, C.R., Schmutz, 

J.A., Flint, P.L., Derksen, D.V., Ip, H.S., and Trust, K.A., 

2013, Genomic analysis of avian influenza virus from 

waterfowl in western Alaska, USA: Journal of Wildlife 

Disease, v. 49, p. 600–610. [Also available at https://doi.

org/10.7589/2012-04-108.]

Stech O., Veits, J., Abdelwhab, E.S.M., Wessels, U., Metten-

leiter, T.C., and Stech, J., 2015, The neuraminidase stalk 

deletion serves as major virulence determinant of H5N1 

influenza viruses in chicken: Scientific Reports, v. 5., Article 

number 13493, accessed January 25, 2018, at https://www.

nature.com/articles/srep13493.

Tsukamoto K., Javier, C.P., Shishido, M., Noguchi, D., Pearce, 

J., Kang, H.-M., Jeong, O.M., Lee, Y-J., Nakanishi, K., and 

Ashizawa, T., 2012, SYBR Green-based real-time reverse 

transcription-PCR for typing and subtyping of all hemag-

glutinin and neuraminidase genes of avian influenza viruses 

and comparison to standard serological subtyping tests: 

Journal of Clinical Microbiology, v. 50, p. 37–45. [Also 

available at https://doi.org/10.1128/JCM.01195-11.]

Wittwer, C.T., Herrmann, M.G., Moss, A.A., and Rasmussen, 

R.P., 1997, Continuous fluorescence monitoring of rapid 

cycle DNA amplification: Biotechniques, v. 22, p. 130–138. 

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Ottinger and others—A Method for Determining Avian Influenza Virus Hemagglutinin and Neuraminidase Subtype Association—Open-File Report 2018–1102 

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