Influenza a (h1n1) - worldwide (13)

A ProMED-mail post
<http://www.promedmail.org>
ProMED-mail is a program of the
International Society for Infectious Diseases
<http://www.isid.org>

In this update:
[1] & [2] Diversity of H1 viruses in pigs in China
[3] Role of bacterial infection

******
[1] Diversity of H1 viruses in pigs in China
Date: Wed 6 May 2009
From: Chen Jiming <jmchen66@yahoo.cn>


Part 1. Detection and differentiation of H1 subtype swine influenza
viruses plus the diversity of the influenza viruses circulating in
pigs in China
-----------------------------------------------
1. Background

[Combining] evidence of BLAST [searches] in GenBank and phylogenetic
analyses, all the 8 gene segments of the current A(H1N1) influenza
virus spreading in humans should be from H1N1 subtype swine influenza
viruses (1, 2, 3), and the virus has been identified [as] infectious
and pathogenic [in] pigs in Canada (3). Therefore, it was possible
that the virus [originated] from pig populations somewhere in North
America, and also, it is possible that the virus remains in some pig
populations somewhere in North America, though we do not exactly
[know] where they are, as we have done little on this issue. So the
detection and differentiation of H1 subtype swine influenza viruses
circulating in pigs are very important for risk analysis and also for
pandemic control. However, it is very difficult to design a simple
pathogenic method to detect and identify the viruses in pigs because
the diversity of H1 subtype influenza viruses is very complicated in
pigs. Currently, there are no reports [of] methods to detect and
differentiate the viruses in pigs, and it is important to share some
advance [information] on this aspect in a rapid communication [via]
ProMED-mail to facilitate the pathogenic surveillance in pigs.

2. Genetic diversity of H1 subtype influenza virus

There are 3 main clusters of H1 subtype swine influenza viruses
circulating in pigs in the world, designated as h1.3.2, h1.1.3 and
h1.2.5 by a newly proposed universal numeral nomenclature system for
all influenza viruses (3). They correspond to classical, avian-like
and human-like swine influenza viruses, respectively (3). Sometimes
the cluster h1.1.3 was designated as the Eurasian lineage due to [the
fact] that almost all of the viruses within the cluster were isolated
from Europe and Asia (1). Another cluster, h1.3.1, corresponding to
the classical swine influenza viruses circulating in the world in the
1930s and 1940s, largely has disappeared from the world.

According to the viral HA gene sequences, the current A(H1N1) swine
influenza virus spreading in humans is in the cluster of h1.3.2
(classical) and highly homogenous to some North American strains
isolated after the year 1999 (1, 3). As for N1 subtype swine
influenza viruses, there are 2 main clusters circulating in pigs,
designated as n1.3.2 and n1.1.7 by the aforementioned newly proposed
universal numeral nomenclature system. They correspond to classical
and avian-like (Eurasian) swine influenza viruses, respectively (3).
Different from the viral HA gene [situation], the current A(H1N1)
swine influenza virus spreading in humans is situated in the cluster
of n1.1.7 (avian-like) and highly homogenous to many Eurasian strains
and at least 2 North American strains isolated in the past decade (1, 2, 3).

[On the basis] of BLAST [searches] in GenBank plus the above
background, 6 gene segments, including the HA segment of the current
A(H1N1) swine influenza virus spreading in humans, should be from the
previous h1.3.2 (classical) swine influenza viruses, and the other 2
(NA and M) are believed to be from h1.1.3 (avian-like) swine influenza viruses.

Beside the aforementioned viruses, sometimes some human or avian
influenza viruses also have been isolated in pigs.

3. Detection and differentiation of H1 swine influenza viruses
circulating in pigs

With deep discussions among 5 animal influenza experts in China (the
original discussion materials are available upon request), the
following 3 agreements on the detection and differentiation of swine
influenza viruses were reached.

Firstly, it is possible to detect the HA gene of all the 3 clusters
of swine influenza virus within the H1 subtype and the HA gene of the
current A(H1N1) swine influenza virus spreading in humans using a
single RT-PCR system with a common upper primer and 2 differentiation
down primers (one for all H1 swine influenza viruses and the other
for the current A(H1N1) swine influenza virus spreading in humans).
Their sequences are shown in the following, respectively:

H1S-3: 5'-TAAGCAAAAGCAGGGGAAAATAAAA-3'
H1R-1143: 5'-TGGTGATAACC(G/A)TACCATCCATCT-3'
H1Rm-610: 5'-CACGAGGACTTCTTTCCCTTTATCAT-3'

The putative positive amplicons of the pair, H1S-3 and H1R-1143, are
of 1143 bp in length and cover the HA1 domain, which is important for
antigenic, pathogenic and host tropism analysis. The putative
positive amplicons of the pair, H1S-3 and H1m-610 are of 610 bp in length.

Secondly, the experts agreed that it is important to detect the viral
NA gene to clarify [whether] the virus is similar to the current
A(H1N1) swine influenza virus spreading in humans. Therefore, if we
obtain suspicious samples through the aforementioned detection of the
viral HA gene, we can use the following primer to amplify and
sequence the viral NA gene (corresponding to all H1 swine influenza viruses).

NAS-5A 5'-GCAAAAGCAGGAGTTTAAAATGAA-3'
NAR-1108A 5'-GTTCTCCCTATCCAAACACCAT-3'

Thirdly, the experts recognized that the sequences of the viral HA
and NA genes of any H1 subtype swine influenza viruses isolated in
pigs in China are important for molecular epidemiological analysis
and risk assessment. Therefore, it is of little value to design a
pair of primers to detect whether a suspicious sample contains the NA
gene similar to the current A(H1N1) swine influenza virus spreading in humans.

It is not difficult to evaluate the quality and the specificity of
the aforementioned primers through BLAST [searches] in GenBank and
some primer-design software tools. Some related evaluation materials
are also available upon request. The kit based on these primers [will
be] available soon.

Currently, another kit has been approved for the detection and
differentiation of subtype H1 swine, which detects the viral HA gene
using the following primers:

H1-762U: 5'-TATCAACAATAAGAA-3'
H1-762L: 5'-CAAACATCCAGAAGA-3'
H1-292U: 5'-CATTAATGATAAAGG-3'
H1-292 L: 5'-TCCAGCATTTCTTTC-3'

The pair H1-762U and H1-762L are designed for the detection of all H1
subtype swine influenza viruses (the amplicon is 762 bp in length),
and the pair H1-292U and H1-292L are designed for the detection of
the current A(H1N1) swine influenza virus circulating in humans (the
amplicon is 292 bp in length).

The kit also [includes] a pair of screening primers for the detection
of the viral conserved matrix gene to check whether there are
influenza viruses of any subtypes in the samples.

The kit has 2 flaws in design, presumably due to very tight time
limitations and very complicated work. 1st, all the primers for the
detection and differentiation of the viral HA gene are too short (all
15 bp in length) and of too low Tm values (25.4-38.4). Therefore, the
specificity of the primers and the effective combination of the
primers to the corresponding regions cannot be guaranteed. 2nd, the
upper primer H1-762U is located at a highly variable region within
the targeted gene. The BLAST [search] in GenBank using the primer
H1-762U as the query sequence identified only one H1 subtype
influenza virus, i.e. A/swine/Shanghai/1/2007(H1N2), among the top
5000 sequences homogenous to the primer! Therefore,
proof-in-principle, it is probable that most of subtype H1 swine
influenza viruses circulating in pigs cannot be identified using this
kit. We will provide some experimental data soon to confirm or revise
the above inferences.

4. The diversity of the influenza viruses circulating in pigs in China

Many domestic and international people are interested in the
diversity of the influenza viruses circulating in pigs in China.
Through the dozens of related publications (most in Chinese), in the
past decade, most of the influenza viruses circulating in pigs in
China were common H1N1 (cluster: h1.3.2 or classical) and H3N2
(cluster: h3.1.5) swine influenza viruses (3). The viruses within
cluster h1.1.3 (avian-like) were isolated from pigs only in 2007 in
Shanghai and Zhejiang in China. Meanwhile, H9N2 subtype avian
influenza viruses have been isolated from pigs in China by several
different research groups at least since 2003. H5N1 subtype avian
influenza viruses were isolated from pigs only once in 2005 and never
after has China implemented compulsory H5 vaccination in poultry. A
few human H3N2 and H1N1 subtypes of influenza viruses were also
isolated from pigs in China.

Moreover, it seemed that re-assortment of the gene segments of these
influenza viruses from different origins was rather frequent in pigs
in China (5), and a re-assortment subtype (H1N2) swine influenza
virus has been isolated by several groups since 2004 (6). The current
A(H1N1) swine influenza virus spreading in humans has never been
isolated in China [despite] surveillance by several research groups
in the past decade in China. However, with the experience of the
recent emergence of a dangerous human influenza virus, presumably
directly from pigs in North America, it is of high significance to
carry out rigid surveillance on the influenza viruses circulating in
the pigs in China for a long time.

The current A(H1N1) influenza viruses spreading in humans are very
dangerous for China, which [has] a very large population and limited
hygiene resources. The morbidity and mortality of the epidemic, if
spreading in China, could be as high as in Mexico, since both are
developing countries, and consequently, thousands of young people in
China could die due to the infection. Therefore, it is rational for
China to take some rigid measures to prevent the disease from
spreading to China, according with International Health Regulations
and the Code of the World Organisation for Animal Health (OIE).

References:

1. Swine Influenza A(H1N1) infection in 2 children--Southern
California, March-April 2009. MMWR Morb Mortal Wkly Rep. 2009 Apr
24;58(15):400-2.
2. ProMED-mail. Influenza A (H1N1) - worldwide, ProMED-mail 2009; 30
Apr: 20090430.1636.
<http://www.promedmail.org>. Accessed 4 May 2009.
3. Panorama phylogenetic diversity and distribution of Type A
influenza virus. PLoS ONE. 2009;4(3):e5022.
4. ProMED-mail. Influenza A (H1N1): animal health (04), infected
swine, Canada, ProMED-mail 2009; 2 May: 20090502.1653.
<http://www.promedmail.org>. Accessed 4 May 2009.
5. Genetic evolution of swine influenza A (H3N2) viruses in China
from 1970 to 2006. J Clin Microbiol. 2008 Mar;46(3):1067-75.
6. Genetic characterization of novel reassortant H1N2 influenza A
viruses isolated from pigs in southeastern China. Arch Virol. 2006
Nov;151(11):2289-99.

--
Ji-Ming Chen
Director
The Laboratory of Animal Epidemiological Surveillance
China Animal Health and Epidemiology Center
Qingdao, 266032, P. R. China
<jmchen66@yahoo.cn>

******
[2] Diversity of H1 viruses in pigs in China
Date: Wed 6 May 2009
From: Chen Jiming <jmchen66@yahoo.cn>


Part 2. Detection and differentiation of H1 swine influenza
-----------------------------------------------
In my earlier email to ProMED-mail (see part [1] above), 2 RT-PCR
methods for detection and differentiation of H1 swine influenza
viruses were described and compared through theoretical analysis.
Now, we have some experimental evidence to confirm and revise the
theoretical inferences. We extracted the RNA of 2 standard common H1
swine influenza viruses from allantoic fluid and serially diluted
them. Then, we amplified them using the 2 methods, respectively. The
results of the detection of the 2 viruses were consistent with each
other and clearly demonstrated that the pair of primers, H1S-3,
H1R-1143, in the 1st method could amplify the RNA effectively and
specifically as expected at least after 1:3125 dilution, and the pair
of primers, H1-726U and H1-726L, in the 2nd method could not amplify
the RNA effectively and specifically even at the dilution of 1:5. In
addition, the results of the amplification using the differentiation
pair of primers demonstrated that neither of the 2 methods could
differentiate an H1 swine influenza virus similar to the current
A(H1N1) swine influenza virus spreading in humans. Therefore, we can
just differentiate the virus through sequencing the amplicons using
the 1st method with the pair of primers H1S-3, H1R-1143.

--
Ji-Ming CHEN, PhD
Head of the Laboratory of Animal Epidemiological Surveillance
China Animal Health and Epidemiology Center
Qingdao, 266032, P. R. China
<jmchen66@yahoo.cn>

[ProMED-mail welcomes the opportunity to pass on to the wider world
these 2 communications on techniques for the detection and
differentiation of H1 subtype swine influenza viruses and the
information derived relating to the diversity of the influenza
viruses circulating in pigs in China. Readers requiring further
information on these diagnostic reagents and procedures should
contact Professor Ji-Ming Chen directly. - Mod.CP]

*****
[3] Role of bacterial infection
Date: Wed 6 May 2009
From: Maria Salvato <msalvato@ihv.umaryland.edu>


I have been following the ProMed "swine flu" updates and noticed an
interesting omission. I am a virologist who works on RNA viruses
(including flu), and I feel that the reports are "viro-centric," i.e.
heavily biased towards blaming everything on influenza and not enough
speculation about co-infections. I certainly agree that there should
be more funding for basic respiratory disease research and am pleased
that President Obama asked for an additional USD 1.5 billion to help
out, but I do not think spending it all on influenza vaccine trials is wise.

Three observations make it highly likely that the "flu" fatalities
are due to bacterial co-infections. 1st, a study from Baylor (Ramilio
et al, Blood, 2007) profiling humans with flu-symptoms found that
greater than 30 percent of the fatalities were in people with
bacterial-influenza co-infections. Knowing the inadequacy of the
diagnostics, I would assume the number of co-infections is much
higher than 30 percent, possibly even 90 percent in fatalities. 2nd,
the index case in Mexico was a small boy who got severely sick, but
his doctor gave him an antibiotic (usually for bacterial infections),
and he survived. The fact that he got better 2 days after an
antibiotic may indicate that he had a bacterial infection in addition
to a flu infection. 3rd, one of the earliest Mexican deaths was a
diabetic woman in her 40's. A lung biopsy contained influenza.
Several people around her had respiratory diseases, but none of them
were positive for influenza! This means that it was possible she too
had both flu and bacterial infections that lead to her death.

It is well known that flu infections make it easier to maintain
respiratory bacterial infections and visa versa. To safeguard against
flu-related deaths, doctors should prescribe more antibiotics.
However, since the cost of antibiotics is high and since
over-prescribing antibiotics is dangerous, diagnostics that
discriminate between viral and bacterial infections should be used.
For example, there is already an FDA approved pregnancy-test-type
device [produced by Rapid Pathogen Screening (RPS), Inc.] that
determines whether teardrops from a person with conjunctivitis arise
from bacterial or viral infection (it uses antibodies to MxA and
C-reactive proteins, which are early host responses to either viral
or bacterial infections). I have no personal interest in that
company, but I have strong interests in exploiting the host's
responses to disease in detection.

It is not surprising to me that molecular biologists are now finding
changes in circulating H3N2 viruses as well as H1N1. The currently
available diagnostics are poor, and much of what was blamed on H1N1
could have been H3N2 or another virus or bacteria or a co-infection.
Much more funding should go to developing host-response diagnostics,
since there are about 100 known host proteins that can discriminate
acute bacterial from acute viral infections. Expression of those
proteins in the plasma should allow 1st responders to discriminate
the serious cases from the light ones.

--
Communicated by:
Maria Salvato, PhD
Professor, Inst of Human Virology
Univ of MD School of Medicine
Baltimore, MD 21201
<msalvato@ihv.umaryland.edu>

[Antibiotics should be used only with probable bacterial infection.
What is also important is the use of appropriate antibiotics, e.g.
anti-staphylococcal agents if staphylococcal rather than
pneumococcal.pneumonia is suspected. For example in the 1957
pandemic, _S. aureus_ was 2nd only to the pneumococcus as a cause of
secondary bacterial pneumonia, but many other organisms also caused
secondary bacterial pneumonia. One issue that has been discussed is
the need to have an adequate supply of antibacterial agents available
as well as antivirals in the presence of a pandemic. The concern of
many infectious diseases experts in the United States is that we do
not have such a supply and that there are inadequate antibiotics in
the pipeline to deal with emerging bacterial resistance. - Mod.DK]