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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Virology. Author manuscript; available in PMC 2010 August 1.
Published in final edited form as:
PMCID: PMC2710411
NIHMSID: NIHMS118819

ZOONOTIC POTENTIAL OF HIGHLY PATHOGENIC AVIAN H7N3 INFLUENZA VIRUSES FROM PAKISTAN

Abstract

H5 and H7 avian influenza viruses can become highly pathogenic in chickens after interspecies transmission. These viruses have transmitted directly to humans from birds in Eurasia and Africa (H5N1), the Netherlands (H7N7), and Canada (H7N3). Here we report antigenic, sequence, and phylogenetic analyses of H7N3 viruses isolated from chickens in Pakistan from 1995 to 2002. We compared the pathogenic and zoonotic potential of the Pakistani viruses in avian and mammalian hosts. In chickens, all of the isolates showed high pathogenicity with poor transmissibility to contact birds. Viral shedding from the trachea and cloaca was equivalent, but cloacal shedding occurred longer; dissemination of virus into the tissues was widespread. In contrast, the viruses replicated poorly in 6-week-old mallard ducks. In mammalian hosts, of the two Pakistani H7N3/02 viruses that caused weight loss, one also caused 40% mortality in mice without prior adaptation, and preliminary experiments in ferrets showed significant virus multiplication in the lungs, intestine, and conjunctiva. We conclude that the H7N3/02 isolates from Pakistan show limited antigenic drift and have evolved slowly during their 8-year circulation in chickens; however, these viruses have the potential to infect mammals.

Keywords: highly pathogenic, H7N3, avian influenza, Pakistan 1995-2002

INTRODUCTION

Between 1959 and 2002, eleven outbreaks of highly pathogenic avian influenza of the H7 subtype were reported worldwide, primarily H7N7 and H7N3 strains (Capua and Alexander, 2004). The designation of avian influenza as highly pathogenic (HP) or low pathogenic (LP) refers to the virus’ virulence in young adult chickens (Senne et al., 1996) and usually cannot be extrapolated to pathogenicity or transmissibility in mammals. Prior to the outbreaks of H7N7 in the Netherlands in 2003, humans had not been considered at high risk of infection with H7 subtypes, though isolated cases of H7N7 conjunctivitis had been reported (Banks, Speidel, and Alexander, 1998; Campbell, Webster, and Breese, 1970; DeLay, Casey, and Tubiash, 1967; Kurtz, Manvell, and Banks, 1996; Taylor and Turner, 1977; Webster et al., 1981). Due to the insensitivity of hemagglutination inhibition (HI) assays to detect antibodies to avian influenza viruses in mammals, antibody responses to H7 influenza viruses had not been consistently detected in humans (Campbell, Webster, and Breese, 1970; Webster et al., 1981). Between February 28 and May 7, 2003, an extensive outbreak of highly pathogenic avian influenza of the H7N7 subtype occurred among poultry in the Netherlands (Fouchier et al., 2004; Koopmans et al., 2004). During that outbreak, more than 80 human H7N7 infections were also detected, consisting primarily of conjunctivitis but also including the death of a previously healthy veterinarian (Fouchier et al., 2004).

Between February 17 and May 18, 2004, an outbreak of avian influenza caused by an H7N3 virus occurred among poultry in the Fraser Valley of British Columbia, Canada, that resulted in two human infections with conjunctivitis (Hirst et al., 2004; Tweed et al., 2004). The source of the H7N3 virus was not defined, but it was phylogenetically related to H7N3 viruses detected in healthy migratory shorebirds from the Americas (Pasick et al., 2005). The available evidence supported the hypothesis that the Fraser Valley H7N3 became highly pathogenic by nonhomologous recombination with insertion of seven amino acids from its M gene.

The first documented outbreak of H7 highly pathogenic avian influenza (HPAI) in Pakistan was in 1995. That outbreak caused widespread disease and mortality in chickens in the Northern part of the country (Naeem and Hussain, 1995). Among the isolates obtained at that time were A/Chicken/Pakistan/CR2/95, A/Chicken/Pakistan/447/95, and A/Chicken/Pakistan/16/99/95; all of which belong to the H7N3 subtype. These viruses were phylogenetically distinct from the same subtype in Eurasian and Australian lineages (Banks et al., 2000). The current knowledge about the H7N3 viruses from Pakistan and their phylogenetic characteristics is limited.

H7 influenza viruses have a propensity to cause conjunctivitis in humans (Belser et al., 2007); both the HP and LP strains of H7 influenza have this property (Meijer et al., 2006). In contrast, the H5 influenza viruses do not. No studies of the effects of the Pakistani H7N3 influenza viruses in mammalian models have been reported. Here we characterize the HPAI H7 viruses from Pakistan, their zoonotic potential in mammalian models, and their propensity to infect by the conjunctival route.

RESULTS

Characteristics of Disease Outbreaks in the Field

The first outbreaks of H7N3 in Pakistan started in December 1994 and continued until April, 1995. A total of 45 flocks in one poultry estate were affected out of 80 flocks in that vicinity. The area is a very isolated, mountainous region in the northern part of Pakistan. During this period the disease primarily affected broiler-breeding stocks and a few of the commercial broiler flocks. Each of the H7N3 isolates was isolated from disease outbreaks on large chicken farms. In the case of broiler breeders, the flock age varied between 10-65 weeks, with typical signs of avian influenza, including facial edema, cyanotic combs, and high morbidity. The mortality ranged between 40-80%. In the case of broiler flocks (3-5 week age), the clinical signs included facial swelling and variable mortality between 30-50%. No vaccine had been used at that time.

The next outbreaks were recorded in 1998 among broiler-breeders in an isolated area, about 100 km away from the previous outbreak area. Only 3 farms were affected, resulting in heavy mortality ranging between 50-70%. The outbreak of 2001-2 started in December-2001 and primarily affected a different area (Chakwal), about 150 km away from the outbreak areas of 1995 & 1998, where only commercial layers are raised. The outbreak was confined to 5 farms in close proximity, resulting in a drop in egg production from 80 to 10% and mortality ranged between 30-40%. This outbreak was successfully contained by adopting culling and ring vaccination approach, where water based autogenous vaccine was used in the surrounding farms.

The National Reference Laboratory for Poultry Diseases at the National Agricultural Research Centre in Islamabad, Pakistan, identified five HPAI H7N3 viruses and provided those isolates for our detailed analyses. Two of the H7N3 viruses were first detected in 1995, [A/Chicken/Pakistan/34668/95 (H7N3) and A/Chicken/Pakistan/34669/95 (H7N3)]; one H7N3 virus was detected in 1998, [A/Chicken/Pakistan/C-1998/98 (H7N3)]; and two were from 2002 [A/Chicken/Pakistan/NARC-68/02 (H7N3) and A/Chicken/Pakistan/NARC-72/02 (H7N3)].

Antigenic Relations among the H7N3 Isolates

To determine the antigenic relationships of the H7N3 viruses from Pakistan with reference H7 viruses and the extent of antigenic variation among the five Pakistani strains, we examined them in HI tests with monospecific antiserum, post infection (PI) sera, and monoclonal antibodies to the hemagglutinin (HA) of H7 reference viruses (Table 1). Each of the isolates from Pakistan reacted to high HI titers with monospecific reference antiserum to A/FPV/Rostock/34 (H7) and cross-reacted to lower titers with the H7N7 isolates from the Netherlands [A/Netherlands/219/03 (H7N7)] and Canada [A/Canada/RV444/04 (H7N3)]. With PI ferret and chicken sera, the Pakistani H7N3 isolates were antigenically closely related to each other and to the reference viruses; the maximal difference being four fold or less. Antigenic analysis with monoclonal antibodies indicated that the A/Chicken/Pakistan/34668/95 (H7N3) was antigenically distinguishable from the other H7N3 viruses with four monoclonal antibodies (4/2, 14/2, 55/2, 71/6), but overall, the other H7N3 viruses were antigenically closely related and showed minimal antigenic drift between 1995 and 2002.

Table 1
Antigenic Analysis of Pakistan H7N3 isolates in hemagglutination inhibition tests

Genomic Sequence Analysis

Due to the limited number of completed sequences available for HP H7 influenza viruses from Asia, we sequenced all eight gene segments for each of the H7N3 Pakistani viruses used in this study.

HA Genes

The HA genes of the five Pakistani isolates studied here, as well as those from A/Chicken/Pakistan/CR2/95 and A/Chicken/Pakistan/447/95, were compared for sequence identity. Strains with higher sequence identities are generally thought to share a relatively recent common ancestor. With the exception of A/Chicken/Pakistan/34668/95, the strains exhibited an average of 99.6% identity at the nucleotide level and 99.1% identity at the amino acid level. In contrast, A/Chicken/Pakistan/34668/95 exhibited an average of 90.1% nucleotide similarity and 93.4% amino acid similarity to the other Pakistani strains. This difference was clearly visible when we examined the H7 sequence and phylogenetic topology (Figs. 1a). A/Chicken/Pakistan/34668/95 was clustered with Group 2, whereas the other strains were clustered with Group 1. Given this phenomenon, and the previously mentioned antigenic differences, A/Chicken/Pakistan/34668/95 was checked repeatedly for the presence of a contaminant; however, no such finding was determined. Additionally, given that A/Chicken/Pakistan/34668/95 clusters with the other Pakistani strains for the other seven gene segments (Supplementary Data); we believe that this anomaly is purely the result of reassortment with a strain whose HA was more similar to those isolated in Group 2.

figure nihms-118819-f0001figure nihms-118819-f0002

The deduced amino acid sequences (Data not shown) of the HA cleavage site showed variability. The 1995 isolates (34668 and 34669) had motifs PEIPKGR*GLF and PETPKRKRKR*GLF, the latter of which is identical to a previously reported H7 isolate: A/Chicken/Pakistan/447/95 (AF202226). The 1998 virus and the two 2002 isolates had the same cleavage site motif PETPKRRKR*GLF as A/Chicken/Pakistan/CR2/95 (AF202230), with 99% identity on the nucleotide and amino acid level. The motif PETPKRRNR*GLF, which was reported for another 1995 isolate, A/Chicken/Pakistan/16/95 (Banks et al., 2000), was not observed in the recent isolates.

The HAs of the 1995 isolates have no differences in glycosylation patterns at five potential sites at positions 12, 28, and 231 of the HA1 and at positions 406 and 478 of HA2 (H7 numbering). The 1998 virus contained four of the five sites mentioned above, with the exception of the potential site at position 28. The two 2002 H7N3 isolates, as well as C1998 and 34669/95, showed an additional glycosylation site at position 123 that was not observed in the other 1995 viruses.

Phylogenetic comparison of all complete H7 genes in Genbank to the H7N3 viruses from Pakistan establish that, with the exception of A/Chicken/Pakistan/34668/95, these viruses form a tight cluster within Group 1 (Figure 1a). Only one isolate, the partially sequenced A/Peregrine Falcon/UAE/188/2384/98 (Manvell et al., 2000), clustered with this isolated clade with up to 98% nucleic acid and amino acid sequence identity to the Pakistani isolates, for the sequence that is available for that segment.

A comparison of the viruses isolated in 1995 that cluster within Group 1, indicates the presence of four non-synonymous substitutions, three of which were present in A/Chicken/Pakistan/CR2/95: A150D, K388E, D440N (H7 numbering). A/Chicken/Pakistan/34669/95 had a single substitution, A125T. When compared to the 1995 Group 1 strains, A/Chicken/Pakistan/C-1998/98, has seven novel substitutions (N28D, I38F, R40S, T358I, T395I, I495M, and D508E), as well as three previously noted ones (A125T, K388E and D440N) whereas the two viruses isolated in 2002 had one novel substitution (L379I) and four previously determined substitutions (A125T, A150D K388E, D440N). The latter substitutions found in the 2002 isolates did not overlap any of the novel ones found in the 1998 strain; rather they were all previously detected in the 1995 strains. This finding indicates that A/Chicken/Pakistan/C-1998/98 either underwent a divergence in the lineage or accumulated a random set of mutations. Analysis of additional temporal and geographical strains would be needed for a more definitive explanation.

NA Genes

We compared the neuraminidase (NA) sequences of the Pakistani H7N3 isolates with all completed available N3 sequences and gave special attention to changes in the NA protein that are indicative of adaptation to land-based poultry (i.e., deletions in the NA stalk and reduction in glycosylation) (Banks et al., 2000; Matrosovich et al., 1999) (Fig. 1b). The NA sequence of one Pakistani H7N3 isolate included in this study, A/Chicken/Pakistan/34669/95 (H7N3), was previously made available in GenBank (AY207504). All the isolates showed greater than 99% identity to this isolate’s NA gene. Overall, the H7N3 Pakistani HPAI isolates acquired five amino acid changes in NA: A/Chicken/Pakistan/34668/95 had H84L; A/Chicken/Pakistan/NARC-C1998/98 had a deletion from amino acids 52 to 67; A/Chicken/Pakistan/NARC-68/02 had V263I; and A/Chicken/Pakistan/34669/95 (AY207504) had retained the non-Pakistani consensus I78 and had I291V. We found a single amino acid change at position N67S in the stalk region of the N3 that was not seen in the wild bird isolates.

There was 89-94% similarity between the NA segments of the Pakistani strains and those of the A/Mallard/Italy/208/00 (H5N3) (AY586414) and A/Mallard/Netherlands/12/00 (H7N3) (CY005846) (data not shown). Analysis of the NA glycosylation sites showed that all of the isolates, except A/Chicken/Pakistan/C-1998/98, had eight potential glycosylation sites at positions, 14, 57, 66, 72, 143, 146, 308, and 435. Thus, these isolates showed no loss of potential glycosylation sites, as seen in the land-based isolates from the Italian lineage. A/Chicken/Pakistan/C-1998/98, however, was missing the potential sites at position 57 and 66, due to the aforementioned sequence deletion.

Analysis of the conserved amino acids coding for the hemadsorbing (HB) site of NA, a second sialic acid—binding pocket of unknown function, on the NA surface showed that the residues involved in the hemadsorption activity of the N3 strains were all conserved (Kobasa et al., 1997). Similarly, no changes were observed in the 18 amino acids that constitute the enzymatic site of the molecule and have been found to be highly conserved in all NA subtypes analyzed thus far (Colman, Varghese, and Laver, 1983).

Phylogenetic analysis of the NA genes of the Pakistani isolates showed that they belong to the Eurasian lineage of influenza viruses. The NA genes are part of an isolated clade in the overall polyphyletic Eurasian clade; separated by some distance from the H7N3 isolates from Italy and the H7N3 viruses from China (Fig. 1b). Based on the sequence identity and phylogenetic topology, we conclude that all of these H7N3 isolates in chickens share a recent common ancestor, with regard to the NA gene, which possibly was derived from other Eurasian groups that have diversified and evolved within the region.

Internal Protein Genes

The internal segments had high levels of sequence identity (>98.7%) among the Pakistani strains, which allowed them to form tight clusters on their respective phylogenetic trees (Data not shown).

Pathogenicity and Transmission of H7N3 Viruses in Birds

Chickens

To determine the pathogenicity of the H7N3 influenza virus isolates from Pakistan, we determined the intravenous pathogenicity index (IVPI) in chickens (Alexander, 2004). Each of the viruses examined had high IVPI values ranging from 2.22 to 2.94, indicating that they are HP influenza viruses (Table 2). All infected chickens showed swelling of the head with cyanotic combs and wattles, respiratory distress, and neurological signs and seizures. All of the birds died.

Table 2
Pathogenicity and transmission of Pakistani H7N3 viruses in chickens

One of the features noted in the field was that although the H7N3 viruses were highly pathogenic, they did not spread throughout the entire flock. This phenomenon was most noticeable among younger chickens. To determine whether the H7N3 viruses transmitted more efficiently among older chickens than younger ones, we inoculated two viruses, A/Chicken/Pakistan/NARC-68/02 and A/Chicken/Pakistan/NARC-72/02 into 6-week-old and 33-week-old birds by the oral/tracheal route. Tracheal and cloacal sampling of the infected chickens indicated peak viral titers at day 5 PI; the average tracheal titer was 103.5 50% egg infectious doses (EID50), and the average cloacal titer was 104.3 EID50 (Data not shown). Cloacal shedding always lasted longer than tracheal shedding, and the two viruses shed virus in tracheal samples for 5 days. Contact birds of the same age were introduced into the cages one day after initial infection. Both viruses infected the 6-week-old and 33-week-old birds, and most of the infected birds died. Of the two viruses tested for transmission neither A/Chicken/Pakistan/NARC-68/02 nor A/Chicken/Pakistan/NARC-72/02 transmitted to 6-week-old birds despite the fact that the inoculated birds died. In the 33-week-old birds, A/Chicken/Pakistan/NARC-68/02 transmitted to the contact birds, but A/Chicken/Pakistan/NARC-72/02 did not.

Virus titers were determined in the 33-week-old birds at day 3 PI: all of the viruses studied showed evidence of generalized infection, with virus in the brain (results not shown). The highest titer (6.0 log10 EID50.) was detected in the kidneys of 33-week-old birds infected with the H7N3 isolates from 2002 (Table 2). Duration of virus shedding was determined in 6 week old chickens infected by the intranasal and oral routes with low doses of virus (104 EID50). The duration of virus shedding varied among the isolates from 5 to 14 days. Although these H7N3 influenza viruses are highly pathogenic, we should note that under experimental conditions, they did not transmit efficiently in young or old chickens.

Mallard Ducks

We infected groups of three 6-week-old mallard ducks and placed two contact ducks in the cages 8 hours PI. The viruses tested in this fashion included A/Chicken/Pakistan/34668/95, A/Chicken/Pakistan/C1998/98, and A/Chicken/Pakistan/NARC-72/02. A dose of 106 EID50, which caused 100% lethality in chickens, was administered.

None of the infected or contact ducks showed disease signs, and only one of the infected ducks in each group had detectable virus (data not shown). The only virus that was detected in the contact ducks was A/Chicken/Pakistan/34668/95, which was detected at the lowest level of detection in only one bird (EID 50 < 10 0.5). Serum samples collected from the ducks had no detectable antibodies (data not shown).

Pathogenicity and Transmission of H7N3 Viruses in Mammalian Hosts

Mice

HP H7N3 isolates pose potential threats of transmission to humans; thus, we infected groups of 6-week-old BALB-c mice to assess the pathogenic potential of these viruses in this mammalian host. We used 106 EID50/0.1 mL, a dose we found to be invariably fatal in chickens. Each of the H7N3 isolates from Pakistan replicated in the lungs of mice for more than 6 days without evidence of generalized infection; none of the viruses were found in the brain (Table 3). The 1995 and 1998 H7N3 isolates from Pakistan did not cause morbidity or weight loss in the mice. However, the 2002 H7N3 isolates caused some mortality and disease symptoms including ruffled fur, weight loss, and conjunctivitis in the infected animals. The more pathogenic of the two viruses was A/Chicken/Pakistan/3466/NARC-68/02 (H7N3), which caused 40% mortality and had the highest titers in the lungs of mice (105.1 EID50/mL). The mouse lethal dose 50 for the A/Chicken/Pakistan/NARC-69/02 and A/Chicken/Pakistan/NARC-68/02 were 2.37 and 3.16 log 10 respectively. None of the Pakistani H7N3 viruses possessed a lysine at residue 627 of the PB2 gene, which was previously associated with high pathogenicity in mice (Hatta et al., 2001).

Table 3
Pathogenicity of Pakistani influenza A (H7N3) viruses in mice

Ferrets

Studies on the pathogenesis of Eurasian H7 influenza viruses in mice and ferrets has raised concern about the zoonotic potential of these viruses and demonstrated ocular infection and replication in the eyes of mice (Belser et al., 2007). Preliminary studies to determine the pathogenicity of the Pakistani H7N3 viruses in ferrets, and the capacity of those viruses to replicate in the ferret eye were done with the A/Chicken/Pakistan/NARC-68/02 (H7N3) strain, the virus that caused 40% mortality and ocular infection in mice.

Ferrets (3-4 months old) were infected either by the ocular or tracheal route under anesthesia. The animals were observed for signs of infection (i.e., body temperature, weight loss, and ocular signs). Virus shedding from the nares, eyes, and rectum were also monitored. The ferrets infected by the ocular route showed mild depression with reduced food intake for the first 48 hours PI, but overall, the infection by either route caused only moderately severe signs of infection (Table 4). After intraocular inoculation, the A/Chicken/Pakistan/NARC-68/02 (H7N3) virus replicated in the nasal tract (>105.0 log EID50). The eyes of the infected ferrets were puffy with reddening of the conjunctiva from days 3 to 5 PI, but control ferrets that received mock eye rubs showed no puffiness or reddening; virus was also detected in rectal swabs. The ferrets inoculated by the intranasal route shed virus in nasal secretions, with a peak of more than 105.0 log EID50 on day 3 PI, with continued shedding past day 5 PI; no virus was detected in eye or rectal swabs. The virus shed from the eyes and rectum of ferrets was sequenced and shown to be identical to the infecting virus (data not shown), indicating that this was not a minor virus population selected after eye inoculation.

Table 4
A/Chicken/Pakistan/NARC-68/02 (H7N3) virus replication in ferrets varied based on the inoculation route

DISCUSSION

HPAI viruses can cause systemic disease in poultry that is associated with rapid mortality approaching 100%. To date, only H5 and H7 subtypes have shown this property, and each HP lineage has emerged from a nonpathogenic precursor (Alexander, 2007). HPAI H7 viruses are not normally present in wild-bird populations but arise from LP avian influenza viruses introduced into poultry flocks from wild birds (Alexander, 2007; Rohm et al., 1995). Human infection caused by avian influenza is a concern because of the potential for pandemic candidates to emerge either directly through adaptive mutation or indirectly through genetic reassortment with human influenza viruses (Fouchier et al., 2004). Pandemics in humans during the previous century are believed to have evolved by reassortment between avian and human strains (Kawaoka, Krauss, and Webster, 1989) or by direct transfer of avian influenza viruses to humans, as is proposed to have occurred with the emergence of the 1918 Spanish influenza (Taubenberger, 2006).

In the present study, we characterized a number of H7N3 influenza viruses isolated from chickens in Pakistan from 1995 to 2002. Intravenous Pathogenicity Index analysis indicated that the H7N3 viruses were highly pathogenic in chickens but were not uniformly transmitted to either young or older chickens. This finding was surprising because the viruses shed as long as 5 days from the trachea and as long as 14 days from the cloaca. Determination of the minimal infectious dose of A/Chicken/Pakistan/NARC-68/02 (H7N3) in chickens was 104.68 EID50 (data not shown), suggesting that a high dose of virus is required for infection of chickens.

Serological analysis in HI tests with PI sera from chickens and ferrets showed that the H7N3 viruses were antigenically homogenous, i.e., they are more closely related to A/Rostock/45/34 (H7N1) and are distinguishable from A/Netherlands/219/03 (H7N7) and A/Canada/RV444/04 (H7N3) with PI chicken antisera. Although the H7N3 influenza viruses from chickens in Pakistan were isolated from 1995 to 2002, they showed little antigenic diversity; the only virus that was distinguishable with monoclonal antibodies to H7 was A/Chicken/Pakistan/34668/95 (H7N3). The other H7N3 viruses were uniformly reactive with the PI sera and monoclonal antibodies. Results from studies in mallard ducks are noteworthy, because the virus replicated poorly (if at all), suggesting that these viruses are probably not being perpetuated in ducks in Pakistan.

In vivo studies in mice showed that H7N3 Pakistani isolates all replicated and caused conjunctivitis, but only the A/Chicken/Pakistan/NARC-68/02 (H7N3) caused any mortality (40%). Preliminary studies with this isolate in ferrets demonstrated that after intraocular inoculation, the virus replicated in the eye and spread to the respiratory and intestinal tracts. In contrast, after intratracheal inoculation, the virus did not spread to the eyes or the intestinal tract.

The outbreak of H7N3 in chickens in Pakistan started in the northern part of the country in 1995, when it caused widespread disease and mortality. Although phylogenetic analysis of the HA gene indicates that these strains are most closely related to A/Peregrine Falcon/UAE/188/2384/98 (H7N3) (Manvell et al., 2000), it is unlikely that birds of prey were involved in the introduction of the H7N3 viruses. To control the outbreak, an autologous inactivated vaccine was prepared and used; quarantine and controlled marketing were also employed (Naeem and Hussain, 1995). The spread to central and southern parts of Pakistan occurred during the subsequent 8 years, and there has been no detectable selection of antigenic-drift variants.

In the recent past, HP H7N3 influenza viruses have emerged in Chile and two provinces of Canada, British Columbia and Saskatchewan, by nonhomologous recombination with inserts of amino acids at the cleavage site of the HA gene (Berhane et al., ; Pasick et al., 2005; Suarez et al., 2004). Examination of the connecting peptide of the HA in the Pakistani H7N3 isolates revealed three patterns: The isolates from 1995 have connecting peptides of PEIPKGR*GLF and PETPKRKRKR*GLF, and the remaining isolates have connecting peptides of PETPKRRKR*GLF. Because the HA genes have been conserved over the past 8 years, continuing evolution has probably not occurred at the connecting-peptide region.

Previous studies have shown that in addition to the lower respiratory tract epithelium, avian influenza viruses have a predilection for α-2, 3—linked sialic acid (SA) receptors that are present on human ocular and lachrymal epithelial cells, (Olofsson et al., 2005; van Riel et al., 2007). Furthermore, Koopmans et al. (2004) showed a higher detection rate for H7N7 viruses in eye swabs than in throat swabs on the second day of illness in humans (Koopmans et al., 2004). Thus, the eye may serve as a route of entry for avian H7 influenza viruses to infect human or mammalian hosts. The ability to multiply in ocular tissues may facilitate eventual receptor switching of α-2, 3-linked SA to α-2, 6—linked receptors, with few mutations needed in HA. Studies of respiratory syncytial virus (RSV) and Influenza A viruses have shown replication in murine lungs after inoculation by the ocular route (Bitko, Musiyenko, and Barik, 2007). The probable route of this spread could be via the lachrymal duct, with the draining tear fluids providing the vehicle for the microbes to reach the nasal cavity and establish a nasal infection. The role of the nasal-lachrymal immune cells in this process is uncertain. There are no reported incidences of ocular signs associated with human influenza subtypes such as H1 and H3, possibly due to receptor-type specificity. There has been limited ocular sampling associated with influenza in general, which might explain the absence of cases caused by other influenza subtypes. Further studies are required to determine whether ocular infection applies to other influenza subtypes besides H7.

The remarkable difference between the highly pathogenic H7N3 influenza viruses that have been perpetuated in the domestic poultry in Pakistan for more than 8 years and the HP H5N1 that has been perpetuated in Asia for over a decade is that the H7N3 viruses from Pakistan have behaved more like classical H5 and H7 viruses. They also have shown minimal evolution after acquiring the HP trait and are antigenically highly conserved. In contrast, the Eurasian HP H5N1 virus has evolved rapidly and developed into at least 10 antigenically different clades with multiple subclades (Peiris, de Jong, and Guan, 2007). One possible explanation for the diversity of the H5N1, as compared with the H7N3 virus, is the failure of H7N3 influenza viruses from Pakistan to transmit to other hosts. Although the in vivo experiments in mice and ferrets showed that the Pakistani H7N3 viruses have some capacity for replication, their conserved evolutionary pattern suggests that they have less pandemic potential than other HP H7 viruses.

MATERIALS AND METHODS

Viruses

All of the isolates in this study were obtained during disease outbreaks in Pakistan from 1995 to 2002. Influenza viruses were propagated in 10- to 11-day-old embryonated chicken eggs for 30 to 36 hours. The eggs were chilled, and the allantoic fluid containing the virus was harvested. The viruses were identified using an HA test according to standard procedure (Palmer et al., 1975). The virus stocks were stored at −70 °C in the St. Jude Virus Repository until further analyzed. All of the experiments were conducted under biosafety level 3+ conditions approved for work with these viruses.

Antigenic Characterization

Subtype identification of influenza viruses was performed using HI and neuraminidase inhibition (NI) assays with a panel of reference antigens and antisera, as described previously (Palmer et al., 1975). A more detailed analysis of HA antigenic reactivity was carried out by an HI test using hyperimmune sera to FPV/Rovstock/45/34 (H7N1), A/Netherlands/219/03 (H7N7), A/Canada/RV444/04 (H7N3), and representative isolates from outbreaks in Chile, Italy, and Canada. The HI test was performed with chicken red blood cells, according to standard procedures and using 0.5% suspension in PBS (phosphate-buffered saline). HI titers were read after 30 minutes (Palmer et al., 1975).

Gene Sequencing & Phylogenetic Analysis

We extracted viral RNA from infected allantoic fluid of embryonated chicken eggs by using an RNEasy Mini Kit (Qiagen, Valencia, CA). After reverse transcription, cDNA was amplified by polymerase chain reaction (PCR) analysis, and the PCR products were sequenced using synthetic oligonucleotides produced by the Hartwell Center for Bioinformatics and Biotechnology (St. Jude Children’s Research Hospital, Memphis, Tennessee). Template DNA was sequenced using BDTv3.1 (Applied Biosystems, Inc. Foster City, California). Samples were analyzed on an AB 3730xl DNA sequencer.

Gene sequences used to construct phylogenetic trees were either obtained from the Influenza Virus Resource Database (http://www.ncbi.nlm.nih.gov/genomes/FLU/FLU.html), or they were from this study. For HA and NA segments, phylogenies showing the relationship among all 311 H7 or 188 N3 strains were initially constructed (data not shown). A subset of those strains was chosen to construct the final topologies. For the internal segments, 191 viruses that had either an H7 or N3 segment and whose genome was completely sequenced were used to construct the resulting phylogenies. Non-H7 and Non-N3 strains were specifically not included in the analysis of the internal gene segments because we wanted to compare the same strains across all internal segments. Select isolates for each segment, representative of the larger topologies, were subsequently chosen for the smaller phylogenies. For all trees, the sequences were aligned, and the ends were trimmed to equal lengths using BioEdit sequence alignment editor software, version 7.0.9.0. All Bayesian trees were subjected to the GTR+I+G model of evolution, as selected by MrModeltest (Nylander, 2004) and were constructed using MrBayes version 3.1.2 (Huelsenbeck and Ronquist, 2001). All trees were rooted using segments from an equine host as the outgroup, except for NA, which used a swine host. The nucleotide sequences obtained from this study are available from GenBank under accession numbers AFxxxxxx to AFxxxxxx.

Avian Host Studies

Chickens

The IVPI of the H7N3 viruses was determined by intravenous inoculation of 1 mL of 1/10 dilution of the virus in ten 6-week-old white leghorn chickens per virus. For viral shedding and transmission studies, two 6-week-old white leghorn chickens were inoculated with 1 mL of virus-infected allantoic fluid (containing 104 EID50) via the intranasal, intraocular, or intratracheal route. The contact birds were introduced to the inoculated birds’ cage 1 day PI.

The same strategy was repeated in 33-week-old white leghorn chickens to verify organ distribution and elucidate any relationship between age of the bird and transmission fitness of the viruses. All birds were observed daily for weight loss, other disease signs, and mortality. Tracheal and cloacal swabs were collected from all birds on days 3, 5, 7, and 10 PI, and virus was titrated in 10-day-old embryonated chicken eggs. Sera samples were collected from all birds before inoculation and on day 13 PI, and HI assays (Palmer et al., 1975) were used to analyze seroreactivity/seroconversion to corresponding viruses.

Virus Titration in Organs

On day 3 PI, lungs, trachea, brain, spleen, small and large intestine, kidneys, liver, bursa, and cardiac tissue were collected for virus titration in 10-day-old embryonated chicken eggs.

Mallard Ducks

The pathogenicity and transmission of H7N3 viruses from Pakistan was tested in 6-week-old mallard ducks; three birds were inoculated with 1 mL of virus-infected allantoic fluid (containing 106 EID50 of each virus, which was a lethal dose for chickens) via the intranasal, intraocular, or intratracheal route. Two contact birds were introduced to the inoculated birds’ cage 1 day PI. Tracheal and cloacal swabs were collected from all birds on days 3, 5, 7, and 10 PI, and sera samples were collected for the HI assay.

Mammalian Host Studies

BALB-c Mice

We evaluated the replication and pathogenicity of five Pakistani H7N3 isolates in groups of 10 six week-old BALB-c mice by intranasal inoculation of 106 EID50 /50 ul PBS. Additionally we determined the MLD50 of A/Chicken/Pakistan/NARC-68/02 and A/Chicken/Pakistan/NARC-72/02 via the intranasal route in groups of fifteen BALB-c mice using serial dilutions (101-107 EID50). The animals were observed for 3 weeks. Morbidity and mortality were scored daily. Organs (lungs, brain, spleen, and blood) were collected from three mice per virus on days 3, 6, and 9 PI for virus titration in embryonated chicken eggs.

Ferrets

The observation of unilateral and bilateral conjunctivitis in mice inoculated intranasally prompted us to study the most virulent mouse H7N3 virus in 3- to 5-month-old ferrets (Marshall Farms, North Rose, NY) to analyze the potential for viral dissemination after intraocular inoculation. The ferrets were seronegative by HI assay for exposure to currently circulating H1N1, H3N2, and H7N3 influenza viruses. The more recent isolate that had shown the highest mortality and weight loss in mice, A/Chicken/Pakistan/NARC-68/02, was used for this study. There is no precedent for ocular studies of influenza isolates in mammalian hosts; thus, we devised a protocol based on a similar treatment/vaccine regimen used in a mouse study of RSV. Under inhalational isoflurane anesthesia, three ferrets were inoculated with 100 μL of 106 EID50 virus into the lower conjunctival sac. The eye was then closed and rubbed for 25 to 30 s (Bitko, Musiyenko, and Barik, 2007). The same dose of the virus was inoculated intranasally in three other ferrets. The animals were observed for activity level and food/water intake. Clinical signs of infection, relative inactivity indexes, body weight and temperature were recorded daily.

Body temperature was measured by a subcutaneously implanted temperature transponder. Each ferret’s temperature was recorded for 3 days before inoculation, and the values were averaged to obtain a baseline value (range, 38.8-39.2 °C). A body temperature of more than three standard deviations was considered significant. All studies were conducted under applicable laws and guidelines and after approval from the St. Jude Animal Care and Use Committee.

Virus Titration in the Upper Respiratory Tract

On days 3, 5, and 7 PI, ferrets were anesthetized with ketamine (25 mg/kg body weight) injected intramuscularly. Into each nostril, 0.5 mL of sterile PBS containing antibiotics was introduced which immediately induced sneezing and the discharge was collected in sterile containers. Conjunctival and rectal swabs were collected at the same intervals. Viruses were detected by inoculation of 100 to 105 dilutions of nasal washes and 100 to 104 dilutions of conjunctival and rectal swabs in 10-day-old embryonated chicken eggs. Pre- and postinfection blood samples were collected for serology analysis.

ACKNOWLEDGEMENTS

We thank our colleagues at the National Agricultural Research Centre (Islamabad, Pakistan) for their continuous and invaluable collaboration. We are also grateful to Kelly Jones, Jennifer McLaren, David Walker, and the late Cedric Proctor for their excellent technical support and to Angela McArthur for editorial assistance. This work was funded, in part, by the National Institute of Allergy and Infectious Diseases, the National Institutes of Health, under contract number HHSN 266200700005C, and the American Lebanese Syrian Associated Charities (ALSAC). We thank James Knowles for manuscript preparation.

Footnotes

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